tag:blogger.com,1999:blog-72599340020905675832024-03-16T08:09:31.082+07:00BAHAN KULIAH DAN MAKALAH KESEHATANKUMPULAN BAHAN KULIAH KESEHATAN MASYARAKAT, KEPERAWATAN DAN KEBIDANAN, MAKALAH, ASKEP, ASKEB, SKRIPSI, KTI DLLRafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.comBlogger409125tag:blogger.com,1999:blog-7259934002090567583.post-49656772254474329492011-09-01T13:02:00.000+07:002011-09-01T13:05:41.954+07:00ASUHAN KEPERAWATAN DENGAN POST HERNIOTOMY AKIBAT HERNIA INGUINALIS LATERAL<br />
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<span lang="IN">ASUHAN KEPERAWATAN DENGAN POST HERNIOTOMY AKIBAT HERNIA INGUINALIS
LATERAL</span></div>
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<br /></div>
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<b style="mso-bidi-font-weight: normal;"><span lang="IN">A.<span style="font: 7pt "Times New Roman";"> </span></span></b><b style="mso-bidi-font-weight: normal;"><span lang="IN">Konsep Dasar</span></b></div>
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<span lang="IN">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Definisi</span></div>
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<span lang="IN">a.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia </span></div>
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<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia adalah menonjolnya suatu
organ atau struktur organ dari tempatnya yang normal melalui sebuah defek
kongenital atau yang didapat (C.Long,
Barbara, 1996 : 246 ).</span></div>
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<span lang="IN">2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia adalah penonjolan isi perut
dari rongga yang normal melalui suatu defek pada fasia muskuloaponeurotik
dinding perut, baik secara kongenital atau didapat,yang memberi jalan keluar
pada setiap alat tubuh selain yang biasa melalui dinding tersebut (Mansjoer
dkk, 2002 : 313 ).</span></div>
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<span lang="IN">3)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia merupakan protrusi atau
penonjolan isi suatu rongga melalui defek atau bagian lemah dari dinding rongga
bersangkutan. Pada hernia abdomen, isi perut menonjol melalui defek atau
bagian lemah dari lapisan muskulo-aponeurotik
dinding perut (Sjamsuhidayat, 2004: 523 ).</span></div>
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<span lang="IN">Dari beberapa pengertian diatas dapat
disimpulkan bahwa hernia adalah
penonjolan suatu organ atau struktur organ yang normal melalui kongenital atau
yang didapat karena kelemahan otot
perut.</span></div>
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<span lang="IN">b.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia Inguinalis Lateral</span></div>
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<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia inguinalis lateral adalah
hernia yang melalui anulus inguinalis internus yang terletak disebelah lateral
vasa epigastrika inferior, menyusuri kanalis inguinalis dan keluar kerongga
perut melalui anulus inguinalis eksternus ( Mansjoer dkk, 2002 : 314 )</span></div>
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<span lang="IN">2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia inguinalis lateral
merupakan penonjolan yang keluar dari rongga peritoneum melalui anulus
inguinalis internus yang terletak lateral dari pembuluh epigastrika inferior,
kemudian hernia masuk kedalam kanalis inguinalis dan jika cukup panjang,
menonjol keluar dari anulus inguinalis eksternus ( Sjamsuhidayat, 2004 : 527 ).</span></div>
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<span lang="IN">3)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernia inguinalis yaitu
berkenaan dengan lipat paha, saluran
tubuler melalui bagian bawah dinding anterior abdomen dan letaknya sejajar
serta sedikit diatas ligamentum inguinale.</span></div>
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<span lang="IN">Dari ketiga definisi diatas dapat
disimpulkan bahwa yang dimaksud dengan Hernia inguinalis lateral adalah hernia
yang melalui anulus inguinalis internus yang terletak lateral dari pembuluh
epigastrika inferior kemudian hernia masuk ke dalam kanalis inguinalis dan jika
cukup panjang, menonjol keluar dari anulus inguinalis eksternus. </span></div>
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<span lang="IN">c.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Herniotomi </span></div>
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<span lang="IN">Herniotomi adalah pembesaran kantong
hernia sampai ke lehernya, kantong dibuka dan isi hernia dibebaskan kalau ada
perlekatan, kemudian direposisi kantong hernia dijahit-ikat setinggi mungkin
lalu dipotong. (Sjamsuhidayat, 2004 : 531 )</span></div>
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<span lang="IN">d.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Post herniotomi</span></div>
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<span lang="IN">Keadaan setelah dilakukan pembedahan
hernia sampai kelehernya, kantong dibuka dan isi hernia dibebaskan kalau ada
perlekatan, kemudian direposisi kantong hernia dijahit-ikat setinggi mungkin
lalu dipotong</span></div>
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<span lang="IN">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Anatomi fisiologi Region
Inguinalis</span></div>
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<span lang="IN">Kanalis inguinalis dibatasi
dikraniolateral oleh anulus inguinalis internus yang merupakan bagian terbuka
dari fasia transpersalis dan aponeurosis m.tranversus abdominis. Dimedial
bawah, diatas tuberkulum tubkum, kanal ini dibatasi oleh anulus inguinalis
eksternus, bagian terbuka dari aponeurosis m.oblikus eksternus. Atapnya adalah
aponeurosis m.oblikus eksternus, dan didasarnya terdapat ligamentum inguinale.
Kanal berisi tali sperma pada lelaki, dan ligamentum rotundum pada perempuan. </span></div>
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<span lang="IN">Hernia inguinalis indirek, disebut juga
hernia inguinalis lateralis, karena keluar dari peritonium melalui anulus
inguinalis internus yang terletak lateral dari pembuluh epigastrika inferior,
kemudian hernia masuk ke dalam kanalis inguinalis dan jika cukup panjang,
menonjol keluar dari anulus inguinalis eksternus. Apabila hernia ini berlanjut,
tonjolan akan sampai ke skrotum, ini disebut hernia skrotalis (Sjamsuhidayat,
2004 :526).</span></div>
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<span lang="IN">a.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pada Pria </span></div>
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<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Fenikulus spermaticus</span></div>
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<span lang="IN">2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Vasa spermatika </span></div>
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<span lang="IN">3)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Proccesus vaginalis peritoni </span></div>
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<span lang="IN">b.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pada wanita </span></div>
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<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Ligamentum Rotundum </span><br />
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<a href="http://img191.imageshack.us/img191/8967/dindingabdomendilihatda.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="http://img191.imageshack.us/img191/8967/dindingabdomendilihatda.jpg" /></a></div>
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<span lang="IN">Gambar 2.1</span></div>
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<span lang="IN">Dinding abdomen dilihat dari depan/<i style="mso-bidi-font-style: normal;">
(Region kanalis inguinalis)</i></span></div>
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<span lang="IN">(Sumber: Sjamsuhidayat, 2004: 527).</span></div>
<span lang="IN"> </span></div>
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<span lang="IN">Kanalis inguinalis adalah kanal yang
normal pada fetus. Pada bulan ke-8
kehamilan terjadi desensus testis melalui kanal tersebut. Penurunan testis
tersebut akan menarik peritoneum kedaerah skrotum sehingga terjadi penonjolan
peritoneum yang disebut dengan prosesus vaginalis peritonei. Pada bayi yang
sudah lahir, umumnya prosesus ini telah mengalami obliterasi sehingga isi
rongga perut tidak dapat melalui kanalis tersebut namun dalam beberapa hal,
seringkali kanalis ini tidak menutup. Karena testis kiri turun terlebih dahulu,
maka kanalis inguinalis kanan lebih sering terbuka.Bila kanalis kiri terbuka
maka biasanya yang kanan juga terbuka. Dalam keadaan normal, kanalis yang terbuka ini akan menutup pada
usia 2 bulan (Mansjoer dkk, 2002 : 314).</span></div>
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<span lang="IN">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Etiologi</span></div>
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<span lang="IN">Hernia ingunalis dapat terjadi karena
anomali kongenital atau karena sebab yang didapat. Pada bayi dan anak, hernia
lateralis disebabkan oleh kelainan bawaan berupa tidak menutupnya prosesus
vaginalis peritoneum sebagai akibat proses penurunan testis ke skrotum.Insiden
hernia meningkat dengan bertambahnya umur mungkin karena meningkatnya penyakit
yang meninggikan tekanan intra abdomen dan berkurangnya kekuatan jaringan
penunjang.</span></div>
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<span lang="IN">Faktor yang dipandang berperan kausal
adalah :</span></div>
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<span lang="IN">1).<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Adanya prosesus vaginalis yang
terbuka</span></div>
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<span lang="IN">2).<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Peninggian tekanan didalam rongga
perut</span></div>
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<span lang="IN">3).<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Kelemahan otot dinding perut
karena usia. </span></div>
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<br /></div>
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<span lang="IN">4.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Patofisiologi</span></div>
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<span lang="IN">Pada hernia inguinalis lateral bahwa
apabila ada defek integritas dinding otot pada ligamen inguinal disertai dengan
adanya tekanan intra abdominal (tekanan intra abdominal ini disebabkan
kegemukan, hamil, mengangkat benda berat, mengejan saat defekasi, atau trauma
benda tumpul.</span></div>
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<span lang="IN">Herniotomi harus dilakukan apabila cincin
hernia memutuskan suplai darah pada segmen hernia. putusnya suplai darah ini
karena cin cin hernia menjepit segmen hernia ( Luckman & Sorensens, 2000:
1658).</span></div>
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<span lang="IN">5.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Manifestasi Klinis</span></div>
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<span lang="IN">Pasien mengatakan turun berok, burut,
atau klingsir, atau mengatakan adanya benjolan diselangkangan atau kemaluan.
Benjolan tersebut bisa mengecil atau menghilang pada waktu tidur, dan bila
menangis, mengejan, atau mengangkat benda berat dan bila posisi pasien berdiri
dapat timbul kembali.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 9.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Bila telah terjadi kompliksi dapat
ditemukan nyeri. Keadaan umum pasien biasanya bak, bila benjolan tidak nampak,
pasien dapat disuruh mengejan dengan menutup mulut dalam keadaan berdiri. bila
ada hernia maka akan tampak benjolan. (Mansjoer et al, 2000: 314).</span></div>
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<span lang="IN">6.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Manajemen medik secara umum</span></div>
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<span lang="IN">Penatalaksanaan medik secara umum pada hernia inguinalis
yaitu :</span></div>
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<span lang="IN">a.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN"> Tindakan Non Bedah</span></div>
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<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Tindakan ini dilakukan untuk mengobati
atau mengatakan keluhan
(simptomatik) obat-obatan yang dapat diberikan pada klien hernia
inguinal, biasanya :</span></div>
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<span lang="IN">a).<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Obat anti nyeri ( analgetik )</span></div>
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<span lang="IN">b).<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Obat anti mikrobial ( antibiotik )</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l22 level1 lfo3; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c).<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Obat anti mual ( antiemetik )</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l22 level1 lfo3; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d).<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Vitamin</span></div>
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<span lang="IN">2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Reposisi Bimanual </span></div>
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<span lang="IN">Teknik ini dilakukan dengan cara
memegang isi hernia membentuk corong, sedangkan tangan kanan mendorongnya
kearah cincin hernia dengan tekanan lambat tapi menetap sampai terjadi reposisi</span></div>
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<span lang="IN">3)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Reposisi Spontan</span></div>
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<span lang="IN">Reposisi dilakukan dengan menidurkan
anak dengan pemberian sedatif dan kompres es diatas hernia. Bila resposisi ini
berhasil anak disiapkan untuk operasi pada hari berikutnya. Jika resposisi
tidak berhasil, dalam waktu 6 jam harus dilakukan operasi segera.</span></div>
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<span lang="IN">b.</span><span lang="IN"> Tindakan Bedah</span></div>
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<span lang="IN">Prinsip dasar operasi hernia terdiri
dari :</span></div>
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<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Herniotomi</span></div>
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<span lang="IN">Merupakan suatu
tindakan pembedahan dengan pembebasan kantong hernia sampai kelehernya, kantong
dibuka dan isi hernia dibebaskan kalau ada perlengketan, kemudian direposisi.
Kantong hernia dijahit dan diikat setinggi mungkin lalu dipotong (Sjamsuhidayat, 2004 :
531 )</span></div>
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<span lang="IN">2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Hernioplastik</span></div>
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<span lang="IN">Adalah suatu tindakan memperkecil
annulus inguinalis internus dan memperkuat dinding belakang kanalis inguinalis
(Sjamsuhidayat, 2004 : 531 ).</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 27.0pt; mso-list: l9 level2 lfo1; tab-stops: list 27.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">7.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Dampak Post Herniotomi Terhadap
Sistem Tubuh</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; mso-list: l9 level3 lfo1; tab-stops: list 45.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Gastrointestinal</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; text-align: justify; text-indent: 18.0pt;">
<span lang="IN">Pembedahan traktus gastrointestinal
sering kali mengganggu proses fisiologi normal pencernaan dan penyerapan. Mual,
muntah dan nyeri dapat terjadi selama pmbedahan ketika digunakan anestesia
spinal. Dan penurunan peristaltik usus ini mengakibatkan distensi abdomen dan
gagal untuk mengeluarkan feses dan flatus. motalitas gastrointestinal dapat
mengakibatkan distensi abdomen dan gagal untuk mengeluarkan feses dan flatus (
Brunner & Suddarth 2002 : 484 & 455 ).</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; mso-list: l9 level3 lfo1; tab-stops: list 45.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Neurologi</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Luka pembedahan mengakibatkan spasme
otot dan pembuluh darah sehingga merangsang pelepasan mediator kimia (
seratonin, bradikinin, histamin ). Pross ini merangsang reseptor nyeri kemudian
rangsangan ditransmisikan ke thalamus, kortek cerebri sehingga terasa nyeri.
Nyeri akan merangsang RAS ( Retikular Activating Sistem ) stimulus ini
menyebabkan sikap terjaga dan berkurangnya stimulus untuk mengantuk.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; mso-list: l9 level3 lfo1; tab-stops: list 45.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Pernapasan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Peningkatan frekuensi nafas dapat
terjadi akibat nyeri pada luka operasi,hal ini merangsang sinyal dari sum-sum
tulang belakang yang dihantarkan melalui dua jalur yaitu Spinal Thalamus
Traktus ( STT ) ke Spinal Respiratory Traktus ( SRT ). Dari spinal thalamus
traktus akan dihantarkan ke korteks cerebri sehingga nyeri dipersepsikan,
sedangkan dari spinal respirator, traktus akan dihantarkan ke medula oblongata
sehingga mengakibatkan neural inspiratory yang akan meningkatkan frekuensi
pernapasan. Nyeri pada luka operasi dapat menekan pengembanahan rongga dada dan
pasien dapat memerlukan sangat banyak dorongan untuk beergerak, ambulasi, dan
bernafas dalam ( C.Long, Barbara, 1996 : 251 ).</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; mso-list: l9 level3 lfo1; tab-stops: list 45.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Kardiovaskuler</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada klien post herniotomi biasanya
dapat terjadi peningkatan denyut nadi, hal ini disebabkan dari rasa nyeri
akibat luka operasi sehingga mengakibatkan medula oblongata untuk meningkatkan
frekuensi pernapasan dan merangsang epineprin sehingga menstimulasi jantung
untuk memompa lebih cepat selain itu juga dapat terjadi akibat faktor
metabolik, endokrin dan keadaan yang menghasilkan adrenergik sehingga
dimanifestasikan peningkatan denyut nadi.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; mso-list: l9 level3 lfo1; tab-stops: list 45.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">e.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Integumen</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Luka operasi akan mengakibatkan
kerusakan kontinuitas jaringan dan keterbatasan gerak dapat mengakibatkan
kerusakan kulit pada daerah yang tertekan karena sirkulasi perifer terhambat.
Akibat dari keadaan post operatif seperti peradangan, edema dan perdarahan,
sering terjadi pembekakan skrotum setelah perbaikan hernia inguinal lateral (
C.Long, Barbara, 1996 : 247 ).</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; mso-list: l9 level3 lfo1; tab-stops: list 45.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">f.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Muskuloskeletal</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Nyeri pada luka operasi timbul akibat
terputusnya kontinuitas jaringan serta adanya spasme otot, terjadi penekanan
pada pembuluh darah yang mengakibatkan metabolisme anaerob sehingga
menghasilkan asam laktat, hal ini mengakibatkan terjadinya gangguan pergerakan
( otot persendian ) sehingga aktivitas sehari-hari dapat terganggu. Selain itu
nyeri akibat luka operasi dapat mengakibatkan klien mengalami keterbatasan
gerak.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; mso-list: l9 level3 lfo1; tab-stops: list 45.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">g.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Perkemihan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 45.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Terjadinya retensi urine dapat terjadi
setelah prosedur pembedaha. Retensi terjadi paling sering setelah pembedahan
pada rektum, anus, dan vagina setelah pembedahan pada abdomen bagian bawah,
penyebabnya diduga adalah spasme spinkter kandung kemih ( Brunner &
Suddarth 2002 : 484 ).</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; text-align: justify;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; mso-list: l9 level1 lfo1; tab-stops: list 22.4pt; text-align: justify; text-indent: -11.7pt;">
<b style="mso-bidi-font-weight: normal;"><span lang="IN">B.<span style="font: 7pt "Times New Roman";"> </span></span></b><b style="mso-bidi-font-weight: normal;"><span lang="IN">Proses Keperawatan </span></b></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Proses Keperawatan pada klien dengan
post herniotomi adalah metode sistematik dimana secara langsung perawat bersama
klien secara bersama menentukan masalah keperawatan sehingga membutuhkan asuhan
keperawatan, membuat perencanaan dan rencana implementasi, serta mengevaluasi
hasil asuhan keperawatan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Proses Keperawatan menurut Yura dan
Walsh (1967) yang dikutip oleh Gaffar dalam buku asuhan keperawatan profesional
terdiri dari 5 tahap, yaitu :</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level2 lfo1; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pengkajian</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pengkajian adalah pendekatan sistematis
untuk mengumpulkan data melalui wawancara, observasi, pemeriksaan fisik,
pemeriksaan laboratorium dan diagnostik serta reviu catatan sebelumnya.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify;">
<span lang="IN">Untuk mengkaji klien dengan post herniotomi meliputi :</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level3 lfo1; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pengumpulan Data</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Identitas</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l14 level1 lfo4; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Identitas klien mencakup : nama.
Umur, jenis kelamin, pendidikan, pekerjaan, suku/bangsa, nomor medik, status,
diagnosa medis, tanggal masuk rumah sakit, tanggal pengkajian dan alamat.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l14 level1 lfo4; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Identitas penanggung jawab
meliputi : nama, umur, pekerjaan, agama, hubungan dengan klien dan alamat</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Riwayat Kesehatan Sekarang</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Alasan Masuk Perawatan </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; tab-stops: list 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Disini
menggambarkan tentang hal-hal yang menjadikan pasien di bawa ke rumah sakit dan
dirawat. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Keluhan utama</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-pagination: none; tab-stops: list 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Keluhan utama merupakan keluhan klien yang bersifat subyektif pada saat
dikaji. Biasanya keluhan utama yang dirasakan klien post herniotomi adalah
nyeri daerah luka operasi.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Riwayat Kesehatan sekarang</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; tab-stops: list 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Bagian ini menguraikan
keluhan pertama yang muncul secara kronologis meliputi faktor yang mencetuskan
memperingan gejala, kualitas, lokasi / penyebaran, upaya yang dilakukan serta
waktu dirasakannya keluhan, durasi dan frekuensi. Dengan menggunakan alat bantu
yang mencakup PQRST :</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 81.0pt; text-align: justify; text-indent: -9.0pt;">
<span lang="IN">P : Provokative / palliative</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 9.0pt;">
<span lang="IN">Merupakan hal atau faktor yang pencetus
terjadinya penyakit, hal yang memperberat atau memperingan, nyeri yang
dirasakan biasanya bertambah bila klien berjalan, bersin, batuk atau napas
dalam.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 9.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN">Q : Quality / Quantity</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 126.0pt; text-align: justify;">
<span lang="IN">Qualitas dari suatu keluhan atau penyakit yang dirasakan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN">R : Region / Radition</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 126.0pt; text-align: justify;">
<span lang="IN">Region adalah daerah atau tempat dimana keluhan dirasakan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN">S : SaveQuality / Quantity</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN"> Region / Radition</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN">S : </span></div>
<div class="MsoNormal">
<span lang="IN">ale</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN">rity Scale</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 126.0pt; text-align: justify;">
<span lang="IN">Severity scale adalah keganasan atau intensitas dari
keluhan tersebut.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN">T : Time</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 126.0pt; text-align: justify;">
<span lang="IN">Time adalah waktu dimana keluhan dirasakan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Riwayat kesehatan yang lalu</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; tab-stops: list 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada tahap ini
dikaji mengenai latar belakang kehidupan klien sebelum masuk rumah sakit yang
menjadi faktor predisposisi seperti riwayat bekerja mengangkat benda-benda
berat, tanyakan juga tentang riwayat penyakit menular dan atau penyakit
keturunan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">e)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Riwayat keluarga</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-pagination: none; tab-stops: list 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada tahap ini dikaji tentang riwayat kesehatan keluarga, adakah dalam
keluarga yang mengalami penyakit sama dengan klien saat ini dan atau riwayat
penyakit keturunan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 89.85pt; mso-pagination: none; text-align: justify;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">3)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Data Biologis</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pola nutrisi</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada aspek ini dikaji mengenai kebiasaan
makan klien sebelum dan sesudah masuk rumah sakit. Dikaji mengenai riwayat diet
klien. Bagaimana kebiasaan makan dalam sehari, jenis makan. Apakah dijumpai
perubahan pada makan akibat penyakit, setelah itu dikaji tentang kebiasaan
minum ( jenis, jumlah dalam sehari ) dan kebiasaan minum-minuman beralkohol.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pola eleminasi</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dikaji mengenai frekuensi, konsistensi,
warna dan kelainan eleminasi, kesulitan-kesulitan eliminasi dan keluhan-keluhan
yang dirasakan klien pada saat bab dan bak.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Istirahat dan tidur</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dikaji mengenai kebutuhan istirahat dan
tidur, apakah ada gangguan sebelum dan pada saat tidur, lama tidur dan
kebutuhan istirahat tidur.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Personal hygiene</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dikaji mengenai kebiasaan mandi, gosok
gigi, mencuci rambut dan dikaji apakah memerlukan bantuan orang lain atau
secara mandiri.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">e)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Aktivitas dan latihan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dikaji apakah aktivitas yang
dilakukan klien dirumah dan dirumah
sakit dibantu atau secara mandiri.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">4)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pemeriksaan Fisik</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pemeriksaan fisik dilakukan dengan cara
inspeksi, palpasi, perkusi dan auskultasi. Pemeriksaan fisik dilakukan head to
toe tetapi hasilnya dituliskan persistem tubuh. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -36.0pt;">
<span lang="IN">a)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Keadaan umum</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Keadaan klien dengan hernia biasanya
mengalami kelemahan, dan periksa status gizinya serta tingkat kesadaran compos
mentis.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -36.0pt;">
<span lang="IN">b)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Tanda-tanda vital</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada tahap ini dilakukan pemeriksaan
tanda-tanda vital biasanya pada pasien post herniotomi terjadi penurunan
tekanan darah, peningkatan suhu dan demam, pernapasan cepat dan dangkal.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -36.0pt;">
<span lang="IN">c)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Tinjauan sistem</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem respirasi</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dikaji dengan cara inspeksi, palpasi,
auskultasi, perkusi.Dalam sistem ini perlu dikaji mengenai bentuk hidung,
kebersihannya, adanya sekret, adanya pernafasan cuping hidung, bentuk dada,
pergerakan dada apakah simetris atau tidak, bunyi nafas, adanya ronchi atau
tidak, frekuensi dan irama nafas teratur.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem cardiovaskuler</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dikaji mulai dari warna konjungtiva,
warna bibir, tidak ada peningkatan JVP,
peningkatan frekuensi dan irama denyut nadi, bunyi jantung tidak disertai suara
tambahan, penurunan tekanan darah.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(3)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem pencernaan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Sistem pencernaan dikaji mulai dari
mulut sampai anus, dalam sistem ini perlu dikaji adanya stomatitis, jumlah
gigi, caries, bau mulut, mukosa mulut, ada tidaknya pembesaran tonsil, bentuk
abdomen datar, turgor kulit kembali lagi, fokus pada pemeriksaan dengan kasus
hernia apakah ada distensi abdomen, nyeri tekan dan nyeri lepas. Adakah lesi
pada daerah abdomen adanya massa, pada auskultasi dapat diperiksa peristaltik
usus.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(4)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem perkemihan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-pagination: none; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dikaji ada tidaknya
pembengkakan dan nyeri pada daerah pinggang, observasi dan palpasi pada daerah
abdomen untuk mengkaji adanya retensio urine, ada tidaknya nyeri tekan dan
benjolan serta pengeluaran urine apakah ada nyeri pada waktu miksi atau tidak.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(5)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem neurologis</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Secara umum pada kasus hernia inguinalis
lateral tidak mengalami gangguan, namun gangguan terjadi dengan adanya nyeri
sehigga perlu dikaji tingkat skala ( 0-5) serta perlu dikaji nilai GCS dan
pemeriksaan fungsi syaraf kranial untuk mengidentifikasi kelainan atau
komplikasi.<br style="mso-special-character: line-break;" />
<br style="mso-special-character: line-break;" />
</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(6)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem integumen</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dalam sistem ini perlu dikaji keadaan
kulit (turgor, kebersihan, pigmentasi, tekstur dan lesi), serta perlu dikaji
kuku dan keadaan rambut, sekitar kulit atau ekstremitas adakah oedema atau tidak.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada klien dengan post herniotomi akan
didapatkan kelamaan integumen karena adanya luka insisi pada daerah abdomen,
sehingga perlu dikaji ada atau tidaknya tanda radang didaerah terkena adalah
ada tidaknya tanda lesi dan kemerahan, pengukuran suhu untuk mengetahui adanya
infeksi.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(7)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem penglihatan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada post herniotomi sistem ini tidak mengalami gangguan. Untuk
mengetahui keadaan kesehatan maka harus diperiksa tentang fungsi penglihatan,
kesimetrisan mata kiri dan kanan, oedema atau tidak.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(8)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Endokrin</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Dalam sistem ini perlu dikaji adanya
pembesaran kelenjar tiroid dan kelenjar getah bening.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l1 level1 lfo5; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">(9)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Sistem Muskuloskeletal</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-pagination: none; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pada hernia
inguinalis lateral biasanya post operasi secara umum tidak mengalami
gangguan,tapi perlu dikaji kekuatan otot
ekstremitas atas dan bawah, dengan nilai kekuatan otot (0-5). Diperiksa juga adanya kekuatan
pergerakan, atau keterbatasan gerak.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 108.0pt; mso-pagination: none; text-align: justify; text-indent: 16.6pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">5)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Data psikologis</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Data psikologis yang perlu dikaji adalah
status emosional, konsep diri, mekanisme koping klien dan harapan serta pemahaman
klien tentang kondisi kesehatan sekarang. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Status emosional</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify;">
<span lang="IN">Kemungkinan ditemukan emosi klien jadi gelisah dan labil,
karena proses penyakit yang tidak di ketahui/ tidak pernah diderita sebelumnya.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Konsep diri</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Konsep diri didefinisikan sebagai semua
pikiran, keyakinan dan kepercayaan yang membuat orang mengetahui tentang
dirinya dan mempengaruhi hubungannya dengan orang lain. (Stuart and Sundeen,
1997 : 227). </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Konsep diri terdiri atas
komponen-komponen berikut ini ( keliat, Budi Anna : 2001). </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l18 level1 lfo11; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">1)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Citra tubuh</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Kumpulan dari sikap individu yang
disadari dan tidak disadari terhadap tubuhnya. Termasuk persepsi masa lalu dan
sekarang serta perasaan tentang ukuran, fungsi, penampilan dan potensi. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l18 level1 lfo11; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">2)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Ideal diri</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Persepsi individu tentang bagaimana dia
seharusnya berperilaku berdasarkan standar, aspirasi, tujuan, atau nilai
personal tertentu. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l18 level1 lfo11; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">3)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Harga diri</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-pagination: none; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Penilaian individu
tentang nilai personal yang diperoleh dengan menganalisa seberapa baik prilaku
seseorang sesuai ideal diri.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l18 level1 lfo11; mso-pagination: none; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">4)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Penampilan peran</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-pagination: none; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Serangkaian pola
perilaku yang dihapkan oleh lingkungan sosial berhubungan dengan fungsi
individu diberbagai kelompok sosial</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; mso-list: l18 level1 lfo11; tab-stops: list 90.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">5)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Identitas personal</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 90.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pengorganisasian perinsip dari
kepribadian yang bertanggung jawab terhadap kesatuan, kesinambungan,
konsistensi, dan keunikan individu</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Stressor </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Stressor adalah faktor-faktor yang
menambah beban klien baik dari pelayanan kesehatan ataupun pribadi dan
keluarga.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Seseorang yang mempunyai stressor akan
mempersulit dalam proses suatu penyembuhan penyakit. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Koping Mekanisme </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Koping mekanisme ini merupakan suatu
cara bagaimana seseorang untuk mengurangi atau menghilangkan stress yang
dihadapi </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-list: l9 level5 lfo1; tab-stops: list 72.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">e)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Harapan dan pemahaman klien
tentang kondisi kesehatan yang dihadapi. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Hal ini perlu dikaji agar tim kesehatan
dapat memberikan bantuan dengan efisien. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pengkajian psikososial post herniotomi
meliputi bagaimana status emosi klien, harapan klien tentang penyakit yang
dideritanya, gaya komunikasi, sosialisasi klien dengan keluarga atau
masyarakat, interaksi klien dirumah sakit, gaya hidup klien sehari-hari, serta
kepuasan pelayanan keperawatan yang klien rasakan dirumah sakit.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; text-align: justify; text-indent: 27.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-pagination: none; text-align: justify; text-indent: 27.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; mso-pagination: none; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">6)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Aspek Sosial dan Budaya </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pengkajian ini menyakit pada pola
komunikasi dan interaksi interpersonal, gaya hidup, faktor sosial serta support
sistem yang ada pada klien.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">7)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Data Spiritual</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-pagination: none; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Data spiritual
menyangkut keyakinan terhadap Tuhan Yang Maha Esa, harapan terhadap kesembuhan
serta kegiatan spiritual yang dilakukan saat ini.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">8)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Pemeriksaan Penunjang</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Pemeriksaan laboratorium dan atau
radiologi perlu dilakukan untuk memvalidasi dalam menegakkan diagnosa sebagai
pemeriksaan penunjang.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l9 level4 lfo1; tab-stops: list 54.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">9)<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Data Pengobatan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-pagination: none; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Data ini digunakan
untuk mengetahui jenis obat apa saja yang digunakan pada kasus hernia
inguinalis lateral. Untuk mengetahui keefektifan penyembuhan penyakit. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level3 lfo1; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Analisa Data</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Setelah dilakukan pengkajian secara
lengkap, maka tahap selanjutnya adalah menganalisa data untuk menentukan
diagnosa keperawatan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify; text-indent: 18.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level2 lfo1; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Diagnosa Keperawatan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Diagnosa keperawatan adalah penyataan
yang status atau masalah kesehatan aktual atau potensial. Tujuannya adalah
mengidentifikasi adanya masalah aktual berdasarkan respon klien terhadap
masalah atau penyakit, penyebab adanya masalah, kemampuan klien untuk mencegah
atau menghilangkan masalah (Gaffar, 1999 : 61) </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Diagnosa keperawatan yang mungkin muncul
pada post herniotomi adalah : </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 63.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Nyeri berhubungan dengan
terputusnya kontinuitas jaringan akibat pembedahan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 63.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap komplikasi,
Atelektasis, Ileus Paralitik, Dehisens, Infeksi, Kekurangan cairan dan
biokimia, Tromboflebitis berhubungan dengan pembedahan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 63.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Kurang perawatan diri berhubungan
dengan keterbatasan mobilitas fisik sekunder terhadap pembedahan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 63.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap kerusakan
penatalaksanaan pemeliharaan dirumah berhubungan dengan kurangnya pengetahuan
tentang perawatan diri saat pasien pulang.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 63.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">e<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap infeksi
berhubungan dengan retensi perkemihan akut, insisi pembedahan, dan inflamasi
skrotum sekunder terhadap herniorafi.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 63.0pt; text-align: justify; text-indent: -18.0pt;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level2 lfo1; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Perencanaan Keperawatan </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Setelah merumuskan diagnosa keperawatan
maka perlu dibuat perencanaan intervensi keperawatan dan aktivitas keperawatan.
Tujuan perencanaan adalah untuk mengurangi, menghilangkan dan mencegah masalah
keperawatan klien. Tahapan perencanaan keperawatan adalah penentuan prioritas
diagnosa keperawatan,penetapan sasaran <i style="mso-bidi-font-style: normal;">(goal</i>)
dan tujuan <i style="mso-bidi-font-style: normal;">(objective</i>), penetapan kriteria
evaluasi dan merumuskan intervensi keperawatan ( Gaffar, 1999:63 ) </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 18.0pt; text-align: justify; text-indent: 27.0pt;">
<span lang="IN">Perencanaan keperawatan pada tahap ini
dibahas rencana tindakan keperawatan berikut rasionalnya :</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l0 level1 lfo6; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">a.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Nyeri berhubungan dengan
terputusnya kontinuitas jaringan akibat pembedahan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 27.0pt; tab-stops: list 36.0pt; text-align: justify; text-indent: 9.0pt;">
<span lang="IN">Tujuan : Nyeri
berkurang sampai dengan hilang, dengan kriteria :</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 27.0pt; mso-list: l6 level1 lfo13; tab-stops: list 36.0pt 54.0pt; text-align: justify; text-indent: 9.0pt;">
<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Klien tampak tenang</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 27.0pt; mso-list: l6 level1 lfo13; tab-stops: list 36.0pt 54.0pt; text-align: justify; text-indent: 9.0pt;">
<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Skala nyeri 0 ( 0-5)</span></div>
<div class="MsoNormal" style="line-height: 150%; tab-stops: list 54.0pt; text-align: justify;">
<br /></div>
<div align="center">
<table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse: collapse; border: none; mso-border-alt: solid windowtext .5pt; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-yfti-tbllook: 480;">
<tbody>
<tr style="height: 18.1pt; mso-yfti-firstrow: yes; mso-yfti-irow: 0;">
<td style="border: solid windowtext 1.0pt; height: 18.1pt; mso-border-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.2pt;" valign="top" width="258"><div align="center" class="MsoNormal" style="text-align: center;">
<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt;">INTERVENSI</span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; height: 18.1pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.8pt;" valign="top" width="258"><div align="center" class="MsoNormal" style="text-align: center;">
<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt;">RASIONAL</span></b></div>
</td>
</tr>
<tr style="height: 40.95pt; mso-yfti-irow: 1; mso-yfti-lastrow: yes;">
<td style="border-top: none; border: solid windowtext 1.0pt; height: 40.95pt; mso-border-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.2pt;" valign="top" width="258"><div class="MsoNormal" style="margin-left: 9.5pt; mso-list: l6 level2 lfo13; text-indent: -9.5pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Pantau :</span></div>
<div class="MsoNormal" style="margin-left: 21.7pt; mso-list: l19 level1 lfo15; text-indent: -12.2pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Tekanan
darah, nadi dan pernapasan setiap 4 jam.</span></div>
<div class="MsoNormal" style="margin-left: 21.7pt; mso-list: l19 level1 lfo15; text-indent: -12.2pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Intensitas
nyeri</span></div>
<div class="MsoNormal" style="margin-left: 21.7pt; mso-list: l19 level1 lfo15; text-indent: -12.2pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Tingkat
kesadaran</span></div>
<div class="MsoNormal" style="margin-left: 9.5pt; mso-list: l6 level2 lfo13; text-indent: -9.5pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Informasikan ke dokter jika nyeri diberikan
sampai pemberian obat respon terhadap analgetik yang bertambah buruk atau
tidak ada selanjutnya.</span></div>
<div class="MsoNormal" style="margin-left: 9.5pt; mso-list: l6 level2 lfo13; text-indent: -9.5pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Bantu pasien untuk mengambil posisi yang
nyaman. Tinggikan ekstremitas yang terasa sakit. Tekuk lutut dengan
menggunakan bantal atau penyokong lutut ditempat tidur untuk menurunkan
ketegangan otot-otot perut setelahtindakan bedah atau bila ada nyeri
dipunggung.</span></div>
<div class="MsoNormal" style="margin-left: 9.5pt; mso-list: l6 level2 lfo13; text-indent: -9.5pt;">
<span lang="IN" style="font-size: 10pt;">4.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Ajarkan pasien teknis bernapas berirama untuk nyeri yang
ringan sampai sedang dalam hubungannya deengan nyeri yang lain meringankan
intervensi :</span></div>
<div class="MsoNormal" style="margin-left: 36.0pt; mso-list: l20 level1 lfo14; tab-stops: list 36.0pt; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Instrusikan
pasien untuk memelihara kontak mata pada suatu objek sambil menarik napas
perlahan melalui mulut dan mengeluarkan napas melalui bibir yang dikerutkan.</span></div>
<div class="MsoNormal" style="margin-left: 9.5pt; mso-list: l6 level2 lfo13; text-indent: -9.5pt;">
<span lang="IN" style="font-size: 10pt;">5.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Berikan istirahat sampai nyeri hilang.
Kurangi kebisingan dan sinar yang terang. Jaga kehangatan pasien dengan
selimut ekstra.</span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; height: 40.95pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.8pt;" valign="top" width="258"><div class="MsoNormal" style="margin-left: 14.7pt; mso-list: l6 level3 lfo13; text-indent: -14.7pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Untuk
mengenal indikasi kemajuan atau penyimpangan dari hasil yang diharapkan.</span></div>
<div class="MsoNormal" style="margin-left: 14.7pt; text-indent: -14.7pt;">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; mso-list: l6 level3 lfo13; text-indent: -14.7pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Ini
merupakan indikasi bahwa perlu analgetik yang lebih keras atau mulai ada
komplikasi.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; mso-list: l6 level3 lfo13; text-indent: -14.7pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Tempatkan
tubuh pada posisi yang nyaman untuk mengurangi penekanan dan mencegah
otot-otot tegang membantu menurunkan rasa tidak nyaman.</span></div>
<div class="MsoNormal" style="margin-left: 14.7pt; text-indent: -14.7pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; text-indent: -14.7pt;">
<br /></div>
<div class="MsoNormal" style="margin-right: 68.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; mso-list: l6 level3 lfo13; text-indent: -14.7pt;">
<span lang="IN" style="font-size: 10pt;">4.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Distraksi
mengganggu stimulus nyeri dengan mengurangi rasa nyeri. Distraksi tidak
mengubah intensitas nyeri. Paling baik digunakan untuk periode pendek pada
nyeri ringan sampai sedang.</span></div>
<div class="MsoNormal" style="margin-left: 14.7pt; text-indent: -14.7pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; text-indent: -14.7pt;">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; text-indent: -14.7pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; text-indent: -14.7pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.7pt; mso-list: l6 level3 lfo13; text-indent: -14.7pt;">
<span lang="IN" style="font-size: 10pt;">5.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Istirahat
menurunkan pengeluaran energi. Vasokontriksi perifer terjadi pada nyeri hebat
dan menyebabkan pasien merasa dingin. Biasanya rangsangan lingkungan yang
kuat, memperhebat persepsi nyeri.</span></div>
</td>
</tr>
</tbody></table>
</div>
<div class="MsoNormal" style="text-align: justify;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l0 level1 lfo6; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap komplikasi,
Atelektasis, Ileus Paralitik, Dehisens, Infeksi, Kekurangan cairan dan
biokimia, Tromboflebitis berhubungan dengan pembedahan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; tab-stops: list 36.0pt; text-align: justify; text-indent: -36.0pt;">
<span lang="IN">Tujuan :
Mendemonstrasikan tidak adanya komplikasi.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; tab-stops: list 36.0pt; text-align: justify; text-indent: -36.0pt;">
<br /></div>
<table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse: collapse; border: none; margin-left: 23.4pt; mso-border-alt: solid windowtext .5pt; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-yfti-tbllook: 480;">
<tbody>
<tr style="height: 18.1pt; mso-yfti-firstrow: yes; mso-yfti-irow: 0;">
<td style="border: solid windowtext 1.0pt; height: 18.1pt; mso-border-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.2pt;" valign="top" width="258"><div align="center" class="MsoNormal" style="text-align: center;">
<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt;">INTERVENSI</span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; height: 18.1pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.8pt;" valign="top" width="258"><div align="center" class="MsoNormal" style="text-align: center;">
<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt;">RASIONAL</span></b></div>
</td>
</tr>
<tr style="height: 26.7pt; mso-yfti-irow: 1; mso-yfti-lastrow: yes;">
<td style="border-top: none; border: solid windowtext 1.0pt; height: 26.7pt; mso-border-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.2pt;" valign="top" width="258"><div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">Atelektasis :</span></div>
<div class="MsoNormal" style="margin-left: 12.4pt; mso-list: l4 level1 lfo16; text-indent: -12.4pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Pantau
bunyi paru-paru tiap 4 jam selama 24 jam, kemudian 8 jam sekali terutama pada
orang yang berisiko tinggi ateletaksis pascaoperasi (perokok,lansia,dan
orang-orang yang mempunyai penyakit paru kronis).</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 12.4pt; mso-list: l4 level1 lfo16; text-indent: -12.4pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Ubah
posisi tiap 2 jam. Biarkan pasien melakukannya sesering mungkin. Melakukan
ambulasi sesuai perintah.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 12.4pt; mso-list: l4 level1 lfo16; text-indent: -12.4pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Pastikan
rasa sakit dapat dikontrol</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">Paralitic ileus :</span></div>
<div class="MsoNormal" style="margin-left: 13.1pt; mso-list: l8 level1 lfo17; text-indent: -13.1pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Pantau
:</span></div>
<div class="MsoNormal" style="margin-left: 36.0pt; mso-list: l20 level1 lfo14; tab-stops: list 36.0pt; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Selang
nasogastrik (warna dan jumlah drainase setiap 8 jam).</span></div>
<div class="MsoNormal" style="margin-left: 36.0pt; mso-list: l20 level1 lfo14; tab-stops: list 36.0pt; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Status
abdomen (mengauskultasi bising usus, menanyakan tentang flatus) setiap 8 jam.</span></div>
<div class="MsoNormal" style="margin-left: 13.1pt; mso-list: l8 level1 lfo17; text-indent: -13.1pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Ukur
dan catat besarnya lingkaran perut setiap 8 jam jika diperkirakan terjadi
distensi abdomen.</span></div>
<div class="MsoNormal" style="margin-left: 13.1pt; mso-list: l8 level1 lfo17; text-indent: -13.1pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Berikan
makan melalui mulut jika bising usus telah ada, keluar flatus dan distensi
abdomen berkurang.</span></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">Dehisens :</span></div>
<div class="MsoNormal" style="margin-left: 18.0pt; mso-list: l2 level1 lfo7; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Pantau
keadaan tepi luka ketika mengganti perban.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 18.0pt; mso-list: l2 level1 lfo7; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Agar
pasien menahan insisi abdomen ketika batuk.</span></div>
<div class="MsoNormal" style="margin-left: 18.0pt; mso-list: l2 level1 lfo7; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Berikan
perawatan luka dengan menggunakan teknik
aseptik yang ketat.</span></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">Infeksi :</span></div>
<ol start="1" style="margin-top: 0cm;" type="1">
<li class="MsoNormal" style="mso-list: l21 level1 lfo8; tab-stops: list 36.0pt;"><span lang="IN" style="font-size: 10pt;">Pantau :</span></li>
</ol>
<div class="MsoNormal" style="margin-left: 45.9pt; mso-list: l11 level1 lfo23; text-indent: -9.9pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Suhu
badan setiap 4 jam</span></div>
<div class="MsoNormal" style="margin-left: 45.9pt; mso-list: l11 level1 lfo23; text-indent: -9.9pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Keadaan
luka ketika melakukan perawatan luka</span></div>
<div class="MsoNormal" style="margin-left: 45.9pt; mso-list: l11 level1 lfo23; text-indent: -9.9pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Hasil
laporan JDL terutama jumlah leukosit (terutama SDP ).</span></div>
<ol start="2" style="margin-top: 0cm;" type="1">
<li class="MsoNormal" style="mso-list: l21 level1 lfo8; tab-stops: list 36.0pt;"><span lang="IN" style="font-size: 10pt;">Berikan antibiotik yang
diresepkan.Berikan paling sedikit 2 liter cairan setiap hari ketika
melaksanakan terapi antibiotik</span></li>
<li class="MsoNormal" style="mso-list: l21 level1 lfo8; tab-stops: list 36.0pt;"><span lang="IN" style="font-size: 10pt;">Ganti perban sesuai aturan dengan
menggunakan tekhnik aseptik.</span></li>
</ol>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">Kekurangan cairan
dan biokimia :</span></div>
<ol start="1" style="margin-top: 0cm;" type="1">
<li class="MsoNormal" style="mso-list: l7 level1 lfo9; tab-stops: list 36.0pt;"><span lang="IN" style="font-size: 10pt;">Pantau :</span></li>
</ol>
<div class="MsoNormal" style="margin-left: 54.0pt; mso-list: l17 level1 lfo18; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Masukan
dan haluaran setiap 8 jam.</span></div>
<div class="MsoNormal" style="margin-left: 54.0pt; mso-list: l17 level1 lfo18; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Hasil
elektrolit serum</span></div>
<div class="MsoNormal" style="margin-left: 54.0pt; mso-list: l17 level1 lfo18; text-indent: -18.0pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Status
umum setiap 8 jam</span></div>
<ol start="2" style="margin-top: 0cm;" type="1">
<li class="MsoNormal" style="mso-list: l7 level1 lfo9; tab-stops: list 36.0pt;"><span lang="IN" style="font-size: 10pt;">Lakukan terapi yang ditentukan untuk
mengatasi retensi cairan :</span></li>
</ol>
<div class="MsoNormal" style="margin-left: 47.9pt; mso-list: l10 level1 lfo19; text-indent: -11.9pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Diet
natrium dibatasi</span></div>
<div class="MsoNormal" style="margin-left: 47.9pt; mso-list: l10 level1 lfo19; text-indent: -11.9pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Masukan
cairan dibatasi</span></div>
<div class="MsoNormal" style="margin-left: 47.9pt; mso-list: l10 level1 lfo19; text-indent: -11.9pt;">
<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Terapi
diuretik.</span></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">Evaluasi
keefektifan terapi :resolusi manifestasi kelebihan volume cairan, natrium
serum kembali kerentang normal.</span></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">Tromboflebitis :</span></div>
<div class="MsoNormal" style="margin-left: 37.5pt; mso-list: l3 level1 lfo10; tab-stops: list 37.5pt; text-indent: -19.5pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Bantu
sirkulasi pada anggota badan bawah setiap 8 jam sampai dimulai ambulasi
:denyut nadi telapak kaki, tanda-tanda Homan's, betis nyeri tekan, pengisian
kapiler,warna dan badan.</span></div>
<div class="MsoNormal" style="margin-left: 37.5pt; mso-list: l3 level1 lfo10; tab-stops: list 37.5pt; text-indent: -19.5pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;"> Anjurkan latihan gerak ditempat tidur setiap
2 jam. Ketika ambulasi dimulai,pastikan pasien melakukannya secara progresif
paling sedikit 3 kali sehari.</span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; height: 26.7pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 193.8pt;" valign="top" width="258"><div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; mso-list: l3 level2 lfo10; text-indent: -13.0pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Untuk
mengidentifikasi kemajuan atau penyimpangan dari hasil yang diharapkan.</span></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; mso-list: l3 level2 lfo10; text-indent: -13.0pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Aktivitas
mendorong bernapas dalam.</span></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; mso-list: l3 level2 lfo10; text-indent: -13.0pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Individu
melakukan pernapasan cepat dan dangkal bila mengalami nyeri hebat, yang
membatasi ekspansi penuh dari alveoli.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 10.55pt; mso-list: l3 level3 lfo10; text-indent: -10.55pt;">
<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Untuk mengidentifikasi indikasi kemajuan
atau penyimpangan dari hasil yang diharapkan.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 10.55pt; mso-list: l3 level3 lfo10; text-indent: -10.55pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Untuk memperoleh data yang objektif.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 10.55pt; mso-list: l3 level3 lfo10; text-indent: -10.55pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Keadaan tersebut mengindikasikan adanya
peristaltik dan fungsi usus normal.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<span lang="IN" style="font-size: 10pt;">1. Untuk mengidentifikasi kemajuan atau
penyimpangan dari hasil yang diharapkan.</span></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<span lang="IN" style="font-size: 10pt;">2. Untuk mencegah tegangan pada jahitan.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">3. Infeksi luka
adalah penyebab utama dehisens.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<span lang="IN" style="font-size: 10pt;">1. Untuk mengidentifikasi kemajuan atau
penyimpangan dari hasil yang diharapkan.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 12.3pt; text-indent: -12.3pt;">
<span lang="IN" style="font-size: 10pt;">2. Terapi antibiotik diperlukan untuk
mencegah dan mengatasi infeksi. Cairan membantu menyebarkan obat kejaringan
tubuh.</span></div>
<div class="MsoNormal" style="margin-left: 13.7pt; text-indent: -13.7pt;">
<span lang="IN" style="font-size: 10pt;">3. Perban yang lembab merupakan media kultur
untuk pertumbuhan bakteri. Dengan mengikuti teknik aseptik akan mengurangi
risiko kontaminasi bakteri.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 12.3pt; text-indent: -12.3pt;">
<span lang="IN" style="font-size: 10pt;">1. Untuk mengidentifikasi indikasi kemajuan
atau penyimpangan dari hasil yang diharapkan</span></div>
<div class="MsoNormal" style="margin-left: 11.6pt; text-indent: -11.6pt;">
<span lang="IN" style="font-size: 10pt;">2. Natrium menahan air. Diuretik membantu
membuang kelebihan air tubuh.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.0pt; text-indent: -13.0pt;">
<span lang="IN" style="font-size: 10pt;">1. Untuk mengidentifikasi kemajuan atau
penyimpangan dari hasil yang diharapkan.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">2. Latihan
merangsang sirkulasi.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
</td>
</tr>
</tbody></table>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l0 level1 lfo6; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Kurang perawatan diri berhubungan
dengan keterbatasan mobilitas fisik sekunder terhadap pembedahan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; tab-stops: list 36.0pt; text-align: justify;">
<span lang="IN">Tujuan : Perawatan diri terpenuhi
dengan kriteria :</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l15 level1 lfo20; tab-stops: list 36.0pt 54.0pt; text-align: justify; text-indent: 0cm;">
<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Klien dapat memenuhi kebutuhan
aktifitas</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l15 level1 lfo20; tab-stops: list 36.0pt 54.0pt; text-align: justify; text-indent: 0cm;">
<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Perawatan diri terpenuhi scara
mandiri</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; tab-stops: list 90.0pt; text-align: justify;">
<br /></div>
<table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse: collapse; border: none; mso-border-alt: solid windowtext .5pt; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-yfti-tbllook: 480;">
<tbody>
<tr style="mso-yfti-firstrow: yes; mso-yfti-irow: 0;">
<td style="border: solid windowtext 1.0pt; mso-border-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.2pt;" valign="top" width="283"><div align="center" class="MsoNormal" style="text-align: center;">
<span lang="IN" style="font-size: 10pt;">INTERVENSI</span></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.25pt;" valign="top" width="283"><div align="center" class="MsoNormal" style="text-align: center;">
<span lang="IN" style="font-size: 10pt;">RASIONAL</span></div>
</td>
</tr>
<tr style="mso-yfti-irow: 1; mso-yfti-lastrow: yes;">
<td style="border-top: none; border: solid windowtext 1.0pt; mso-border-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.2pt;" valign="top" width="283"><div class="MsoNormal" style="margin-left: 11.9pt; text-indent: -11.9pt;">
<span lang="IN" style="font-size: 10pt;">1. Tentukan tingkat bantuan yang
diperlukan.Berikan bantuan AKS sesuai keperluan. Membiarkan pasien melakukan
sebanyak mungkin untuk dirinya.</span></div>
<div class="MsoNormal" style="margin-left: 13.3pt; text-indent: -13.3pt;">
<span lang="IN" style="font-size: 10pt;">2. Berikan waktu yang cukup bagi pasien
untuk melaksanakan aktifitas.</span></div>
<div class="MsoNormal" style="margin-left: 11.9pt; text-indent: -11.9pt;">
<span lang="IN" style="font-size: 10pt;">3. Instruksikan pasien adaptasi yang
diperlukan untuk melaksanakan AKS. Dimulai dengan tugas yang mudah dilakukan
dan berlanjut sampai tugas yang sulit. Berikan pujian untuk keberhasilan
tersebut.</span></div>
<div class="MsoNormal" style="margin-left: 13.3pt; text-indent: -13.3pt;">
<span lang="IN" style="font-size: 10pt;">4. Menaruh bel ditempat yang mudah
dijangkau.</span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.25pt;" valign="top" width="283"><div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">1. Untuk mendorong
kemandirian</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 11.8pt; text-indent: -11.8pt;">
<span lang="IN" style="font-size: 10pt;">2. Membebani pasien dengan aktifitas
menyebabkan frustasi.</span></div>
<div class="MsoNormal" style="margin-left: 11.8pt; text-indent: -11.8pt;">
<span lang="IN" style="font-size: 10pt;">3. Untuk mendorong kemandirian. Pujian
memotivasi untuk terus belajar.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">4. Untuk memberikan
rasa aman.</span></div>
</td>
</tr>
</tbody></table>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l0 level1 lfo6; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap kerusakan
penatalaksanaan pemeliharaan dirumah berhubungan dengan kurangnya pengetahuan
tentang perawatan diri saat pasien pulang.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; tab-stops: list 36.0pt;">
<span lang="IN">Tujuan : Kerusakan penatalaksanaan dirumah tidak terjadi dengan
kriteria hasil :</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l5 level1 lfo21; tab-stops: list 36.0pt left 54.0pt; text-indent: 0cm;">
<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Klien dan keluarga mengerti
tentang penatalaksanaan dirumah.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 54.0pt; mso-list: l5 level1 lfo21; tab-stops: list 54.0pt; text-indent: -18.0pt;">
<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Klien dan keluarga mengatakan akan
melaksanakan perawatan, aktifitas yang baik dirumah.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l5 level1 lfo21; tab-stops: list 36.0pt left 54.0pt; text-indent: 0cm;">
<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Mengidentifikasi bagian-bagian
yang memerlukan perawatan.</span></div>
<div class="MsoNormal">
<br /></div>
<table border="1" cellpadding="0" cellspacing="0" class="MsoTableGrid" style="border-collapse: collapse; border: none; mso-border-alt: solid windowtext .5pt; mso-padding-alt: 0cm 5.4pt 0cm 5.4pt; mso-yfti-tbllook: 480;">
<tbody>
<tr style="mso-yfti-firstrow: yes; mso-yfti-irow: 0;">
<td style="border: solid windowtext 1.0pt; mso-border-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.2pt;" valign="top" width="283"><div align="center" class="MsoNormal" style="line-height: 150%; text-align: center;">
<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt; line-height: 150%;">INTERVENSI</span></b></div>
</td>
<td style="border-left: none; border: solid windowtext 1.0pt; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.25pt;" valign="top" width="283"><div align="center" class="MsoNormal" style="line-height: 150%; text-align: center;">
<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt; line-height: 150%;">RASIONAL</span></b></div>
</td>
</tr>
<tr style="mso-yfti-irow: 1; mso-yfti-lastrow: yes;">
<td style="border-top: none; border: solid windowtext 1.0pt; mso-border-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.2pt;" valign="top" width="283"><div class="MsoNormal" style="margin-left: 10.5pt; text-indent: -10.5pt;">
<span lang="IN" style="font-size: 10pt;">1. Pastikan pasien memiliki instruksi
tertulis tentang perawatan diri dan perjanjian tertulis untuk kunjungan
evaluasi.</span></div>
<div class="MsoNormal" style="margin-left: 9.1pt; text-indent: -9.1pt;">
<span lang="IN" style="font-size: 10pt;">2. Ajarkan dan biarkan pasien merawat luka
jika penggantian perban perlu dilakukan dirumah. Tekankan pentingnya mencuci
tangan sebelum dan sesudah merawat luka.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 11.2pt; text-indent: -11.2pt;">
<span lang="IN" style="font-size: 10pt;">3. Evaluasi kebutuhan bantuan perawatan
dirumah dan tersedianya sistem pendukung yang memadai untuk memberikan
bantuan yang diperlukan. Hubungi perencana atau bagian pemulangan pasien untuk mengatur bantuan perawatan
dirumah jika pasien memerlukan bantuan tetapi tidak mempunyai sistem
pendukung dirumah.</span></div>
<div class="MsoNormal" style="margin-left: 11.2pt; text-indent: -11.2pt;">
<span lang="IN" style="font-size: 10pt;">4. Instruksikan pasien untuk memberitahu
dokter jika terjadi infeksi luka : kemerahan, nyeri tekan, drainase, demam.</span></div>
<div class="MsoNormal" style="margin-left: 10.5pt; text-indent: -10.5pt;">
<span lang="IN" style="font-size: 10pt;">5. Pastikan pasien mempunyai persediaan yang
cukup untuk perawatan luka dan resep untuk analgetik.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 9.8pt; text-indent: -9.8pt;">
<span lang="IN" style="font-size: 10pt;">6. Instruksikan agar pasien beristirahat
sepanjang hari, secara bertahap melakukan
aktivitas serta menghindari mengangkat benda-benda berat dan latihan
yang berlebihan.</span></div>
</td>
<td style="border-bottom: solid windowtext 1.0pt; border-left: none; border-right: solid windowtext 1.0pt; border-top: none; mso-border-alt: solid windowtext .5pt; mso-border-left-alt: solid windowtext .5pt; mso-border-top-alt: solid windowtext .5pt; padding: 0cm 5.4pt 0cm 5.4pt; width: 212.25pt;" valign="top" width="283"><div class="MsoNormal" style="margin-left: 11.8pt; text-indent: -11.8pt;">
<span lang="IN" style="font-size: 10pt;">1. Instruksi verbal akan mudah terlupakan.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 9.0pt; text-indent: -9.0pt;">
<span lang="IN" style="font-size: 10pt;">2. Praktik akan membantu pasien mengembangkan keyakinannya dalam perawatan diri. Juga
memungkinkan perawat mengevaluasi kemampuan pasien melaksanakan keterampilan
tersebut sendiri dan menentukan apakah diperlukan bantuan. Tindakan untuk
mencegah infeksi harus dilanjutkan sampai luka benar-benar sembuh.</span></div>
<div class="MsoNormal" style="margin-left: 10.4pt; text-indent: -10.4pt;">
<span lang="IN" style="font-size: 10pt;">3. Layanan sosial atau perencana pemulangan
pasien berfungsi sebagai penghubung yang penting untuk pemindahan pasien
kelingkungan rumah atau fasilitas perawatan luar untuk memastikan kelanjutan
penyembuhan atau rehabilitasi.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 14.6pt; text-indent: -14.6pt;">
<span lang="IN" style="font-size: 10pt;">4. Diperlukan antibiotik untuk mengatasi
infeksi.</span></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 11.1pt; text-indent: -11.1pt;">
<span lang="IN" style="font-size: 10pt;">5. Persediaan penting untuk mengurangi
kecemasan yang pada umumnya berhubungan dengan pemulangan pasien. Analgesik
memberi kenyamanan dan mendorong untuk tidur.</span></div>
<div class="MsoNormal" style="margin-left: 11.1pt; text-indent: -11.1pt;">
<br /></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">6. Pembedahan
adalah stresor.</span></div>
</td>
</tr>
</tbody></table>
<div class="MsoNormal" style="line-height: 150%; text-align: justify;">
<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l0 level1 lfo6; tab-stops: list 36.0pt; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">e.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap infeksi berhubungan
dengan pembedahan </span></div>
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<span lang="IN">Tujuan : Risiko tinggi terhadap
infeksi tidak terjadi dengan kriteria :</span></div>
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<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Suhu tubuh normal 37<sup>0</sup>C</span></div>
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<span lang="IN">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Tanda-tanda infeksi tidak terjadi</span></div>
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<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt;">INTERVENSI</span></b></div>
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<b style="mso-bidi-font-weight: normal;"><span lang="IN" style="font-size: 10pt;">RASIONAL</span></b></div>
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<span lang="IN" style="font-size: 10pt;">1.<span style="font: 7pt "Times New Roman";"> </span></span><span lang="IN" style="font-size: 10pt;">Pantau :</span></div>
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<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Suhu
badan setiap 4 jam</span></div>
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<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Keadaan
luka ketika melakukan perawatan luka</span></div>
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<span lang="IN" style="font-size: 10pt;">-<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Hasil
laporan JDL terutama jumlah leukosit.</span></div>
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<span lang="IN" style="font-size: 10pt;"> 2.
Tetap pada fasilitas kontrol infeksi, sterilisasi dan prosedur/kebijakan
aseptik.</span></div>
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<span lang="IN" style="font-size: 10pt;">3. Identifikasi gangguan pada teknik aseptik
dan atasi dengan segera pada waktu terjadi.</span></div>
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<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">4. Sediakan
pembalut yang steril</span></div>
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<br /></div>
<div class="MsoNormal">
<span lang="IN" style="font-size: 10pt;">5. Berikan
antibiotik sesuai petunjuk</span></div>
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<span lang="IN" style="font-size: 10pt;">1. Untuk mengidentifikasi kemajuan atau
penyimpangan dari hasil yang diharapkan </span></div>
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<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal">
<br /></div>
<div class="MsoNormal" style="margin-left: 13.9pt; mso-list: l13 level1 lfo12; text-indent: -13.9pt;">
<span lang="IN" style="font-size: 10pt;">2.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Tetapkan
mekanisme yang dirancang untuk untuk mencegah infeksi.</span></div>
<div class="MsoNormal" style="margin-left: 13.9pt; mso-list: l13 level1 lfo12; text-indent: -13.9pt;">
<span lang="IN" style="font-size: 10pt;">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Kontaminasi
dengan lungkungan/ kontak personal akan menyebabkan daerah yang steril
menjadi tidak steril sehingga dapat meningkatkan risiko infeksi.</span></div>
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<span lang="IN" style="font-size: 10pt;">4.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Mencegah
kontaminasi lingkungan pada luka yang baru.</span></div>
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<span lang="IN" style="font-size: 10pt;">5.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN" style="font-size: 10pt;">Dapat
diberikan secara profilaksis bila dicurigai terjadinya infeksi atau
kontaminasi.</span></div>
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<br /></div>
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<span lang="IN">3.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Implementasi</span></div>
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<span lang="IN">Implementasi merupakan pelaksanaan
perencanaan keperawatan oleh perawat dan klien. Hal-hal yang harus diperhatihan
ketika melakukan implementasi adalah intervensi dilaksanakan sesuai dengan
rencana setelah dilakukan validasi
(Gaffar, 1999:65 ).</span></div>
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<span lang="IN">Perencanaan tindakan keperawatan akan
dapat dilaksanakan dengan baik, jika klien mempunyai keinginan untuk
berpartisipasi dalam pelaksanaan tindakan keperawatan. Selama tahap
pelaksanaan, perawat terus melakukan pengumpulan data dan memilih tindakan
perawatan yang paling sesuai dengan kebutuhan klien.</span></div>
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<span lang="IN">4.<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Evaluasi</span></div>
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<span lang="IN">Evaluasi adalah
fase akhir dari proses keperawatan. Evaluasi menyediakan nilai informasi
mengenai pengaruh intervensi yang telah direncanakan dan merupakan perbandingan
dari hasil yang diamati dengan kriteria hasil yang telah dibuat pada tahap
perencanaan, disamping itu evaluasi juga digunakan sebagai alat ukur suatu
tujuan yang mempunyai kriteria tertentu yang memberikan tujuan tercapai, tidak
tercapai atau tercapai sebagaian (Hidayat, 2002 : 41) </span></div>
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<span lang="IN">Terdapat 2 tipe
dokumentasi evaluasi yaitu evaluasi formatif yang menyatakan evaluasi yang
dilakukan pada saat memberikan intervensi dengan respon segera dan evaluasi
sumatif yang merupakan rekapitulasi dari hasil observasi dan analisis status
pasien pada waktu tertentu. </span></div>
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<span lang="IN">Evaluasi sumatif
dapat dilakukan dengan menggunakan pendekatan SOAP, sebagai berikut : </span></div>
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<span lang="IN">S : Respon
Subjektif klien terhadap tindakan keperawatan yang telah dilaksanakan</span></div>
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<span lang="IN">O : Respon objektif
klien terhadap tindakan keperawatan yang telah dilaksanakan </span></div>
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<span lang="IN">A : Analisa ulang atas subjektif dan objektif
untuk menyimpukan apakah masalah masih tetap atau muncul. Masalah baru atau
data yang kontradiksi dengan masalah yang ada. </span></div>
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<span lang="IN">P : Perencanaan atau tindak lanjut berdasarkan
hasil analisa pada respon klien </span></div>
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<br /></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 16.75pt; mso-pagination: none; text-align: justify; text-indent: 28.25pt;">
<span lang="IN">Selanjutnya setelah
evaluasi dilakukan pada hari berikutnya dituliskan dalam catatan perkembangan. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 16.75pt; mso-pagination: none; text-align: justify; text-indent: 19.3pt;">
<span lang="IN">Catatan perkembangan merupakan catatan tentang
perkembangan keadaan klien yang didasarkan pada setiap masalah yang ditemui
pada klien (Hidayat, 2002 : 46) </span></div>
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<span lang="IN">S : Data
Subjektif </span></div>
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<span lang="IN">Perkembangan keadaan didasarkan pada
apa yang dirasakan, dikeluhkan, dan dikemukakan klien. </span></div>
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<span lang="IN">O : Data objektif </span></div>
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<span lang="IN">Perkembangan yang bisa diamati dan
diukur oleh perawat atau tim kesehatan lain. </span></div>
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<span lang="IN">A : Analisis </span></div>
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<span lang="IN">Data subjektif dan objektif dinilai dan
dianalisis, apakah berkembang ke arah perbaikan atau kemunduran. Hasil analisis
dapat menguraikan sampai dimana masalah yang ada dapat diatasi atau adakah
perkembangan masalah baru yang menimbulkan diagnosa keperawatan baru. </span></div>
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<span lang="IN">P : Perencanaan </span></div>
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<span lang="IN">Rencana penangan klien dalam hal ini
didasarkan pada hasil analisis diatas yang berisi melanjutkan rencana
selanjutnya apabila keadaan atau masalah belum teratasi dan membuat rencana
baru bila rencana awal tidak efektif. </span></div>
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<span lang="IN">I : Implementasi</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 72.0pt; mso-pagination: none; text-align: justify;">
<span lang="IN">Tindakan yang dilakukan berdasarkan
rencana</span></div>
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<span lang="IN">E : Evaluasi </span></div>
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<span lang="IN"> Evaluasi berisikan penilaian sejauh
mana tindakan dan evaluasi telah dilaksanakan dan sejauh mana masalah bisa
teratasi </span></div>
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<span lang="IN">R : Reassement </span></div>
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<span lang="IN">Bila hasil evaluasi
menunjukkan masalah belum teratasi, pengkajian ulang perlu dilakukan kembali
melalui proses pengumpulan data subjektif, data objektif dan proses
analisisnya. </span></div>
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<span lang="IN">a<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Nyeri berhubungan dengan
terputusnya kontinuitas jaringan akibat pembedahan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify;">
<span lang="IN">Kriteria hasil : Menyatakan tidak nyeri, intensitas nyeri
berkurang, tanda-tanda vital stabil, ekspresi muka dan postur tubuh rileks.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">b<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap komplikasi,
Atelektasis, Ileus Paralitik, Dehisens, Infeksi, Kekurangan cairan dan
biokimia, Tromboflebitis berhubungan dengan pembedahan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify;">
<span lang="IN">Kriteria hasil : tidak ada infeksi, bunyi napas bersih,
penyembuhan luka, dan tidak ada perdarahan.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">c<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Kurang perawatan diri berhubungan
dengan keterbatasan mobilitas fisik sekunder terhadap pembedahan</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify;">
<span lang="IN">Kriteria hasil : Mengidentifikasi area kebutuhan,
mengungkapkan aktifitas terprnuhi.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">d<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap kerusakan
penatalaksanaan pemeliharaan dirumah berhubungan dengan kurangnya pengetahuan
tentang perawatan diri saat pasien pulang.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify;">
<span lang="IN">Kriteria hasil :
Klien dan keluarga mengerti tentang penatalaksaan di rumah. </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; mso-list: l9 level6 lfo1; text-align: justify; text-indent: -18.0pt;">
<span lang="IN">e<span style="font: 7pt "Times New Roman";">
</span></span><span lang="IN">Risiko tinggi terhadap infeksi
berhubungan dengan retensi perkemihan akut, insisi pembedahan, dan inflamasi
skrotum sekunder terhadap herniorafi.</span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify;">
<span lang="IN">Kriteria hasil : Mendemonstrasikan tidak ada tanda-tanda
infeksi </span></div>
<div class="MsoNormal" style="line-height: 150%; margin-left: 36.0pt; text-align: justify; text-indent: -18.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 36.0pt; text-indent: -18.0pt;">
<br /></div>
<div class="MsoNormal" style="margin-left: 36.0pt; text-indent: -18.0pt;">
<br /></div>
Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com83tag:blogger.com,1999:blog-7259934002090567583.post-81681457757812193162011-08-26T09:25:00.001+07:002011-08-26T09:25:50.283+07:00VIROLOGICAL COMPLIANCE<p align="justify">7.1 Introduction</p> <p align="justify">7.2 Health significance of human viruses in drinking-water</p> <p align="justify">7.3 Occurrence of human viruses in source waters</p> <p align="justify">7.4 Risk management</p> <p align="justify">7.4.1 International approaches</p> <p align="justify">7.4.2 Virus removal by current water treatment processes</p> <p align="justify">7.5 Sampling, testing and data interpretation</p> <p align="justify">7.6 C.t values</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000" size="1">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"><b></b></p> <p align="justify"><b>REFERENCES</b></p> <p align="justify"><b>Figures and Tables</b></p> <p align="justify">Table 7.1: UV dose requirements for virus inactivation credit</p> <p align="justify">Table 7.2: C.t values for inactivation of viruses by free chlorine, pH 6 – 9</p> <p align="justify">Table 7.3: C.t values for inactivation of viruses by chloramine</p> <p align="justify">Table 7.4: C.t values for inactivation of viruses by chlorine dioxide, pH 6 – 9</p> <p align="justify">Table 7.5: C.t values for inactivation of viruses by ozone</p> <p align="justify"> </p> <p align="justify"><strong><font size="3">7.1 INTRODUCTION</font></strong></p> <p align="justify"><i>No maximum acceptable values (MAVs) have been set for human viruses in the Drinking-water Standards for New Zealand (2005). It is likely that a MAV or MAVs will be established in a future edition. This chapter foreshadows such developments.</i></p> <p align="justify"><i></i></p> <p align="justify"><i>In the absence of any MAVs for viruses in the current DWSNZ it should be understood that if they are specifically sought, they should not be detected. If detected, advice should be sought from the relevant health authorities.</i></p> <p align="justify">There are more than 140 different types of human enteric viruses that may contaminate potable source waters. These include several important groups: Hepatitis A virus, Hepatitis E virus, norovirus, enterovirus and adenovirus, that have been associated with waterborne illness and are capable of causing severe, and in some cases fatal, infections.</p> <p align="justify">Human viruses are obligate intracellular parasites, which means they cannot grow or multiply outside their host. Viruses consist of a nucleic acid genome surrounded by a protein capsid and, in some cases, a lipoprotein envelope. These viruses are very small, ranging from 20 – 70 nm in diameter.</p> <p align="justify">Human enteric viruses are shed in the gut, respiratory tract and occasionally urine of an infected person, and are discharged with body wastes into wastewater. Infected people do not always show signs of illness (asymptomatic) but they will still produce virus in their wastes. Specific viruses or strains of viruses are not always present in a community at any one time, but representatives of the large groups (e.g. adenovirus or enterovirus) are generally present on most occasions.</p> <p align="justify">Enteric viruses may be found in high numbers in domestic wastewater. Recent New Zealand studies have shown adenovirus and enteroviruses to be present in concentrations greater 10,000 infectious virus units per litre of wastewater (Watercare Services Surrogate Study 2002). The numbers of viruses in wastewater varies with the level of virus infection in the community but, in general, human viruses will always be present in wastewater, even of small communities, (average human virus load 100 – 1000 infectious viruses/L) and will occasionally reach very high levels (>10,000 infectious virus/L) (Lewis et al 1986). Wastewater treatment processes that do not include a disinfection step are often inefficient in removing viruses (<90% removal) and some viruses may reach potable water supplies.</p> <p align="justify">Human enteric viruses cannot multiply in the environment once outside the host. The viruses are characterised by the ability to survive for days, weeks or more, in the environment depending on the type of water, season and other factors (Hunter 1997).</p> <p align="justify">A large proportion of the human viruses present in source drinking-waters will normally be removed or inactivated by well-operated standard drinking-water treatment processes.</p> <p align="justify">Routine monitoring for viruses in treated water and source water is currently impractical in most situations in New Zealand because of the high cost of sampling and analysis, and problems of detection of a full range of the viruses occurring.</p> <p align="justify"> </p> <p align="justify"><font size="3"><strong>7.2 Health significance of human viruses in drinking-water</strong></font></p> <p align="justify">Hepatitis A virus, Hepatitis E virus, norovirus, enterovirus and adenovirus may occur in drinking-water where they are present in the source water and when water treatment does not remove them completely. Very few human enteric viruses (1 – 50 virus particles depending on type) are required to produce an infection in a susceptible person (Hunter 1997). The symptoms generally attributed to enteric viruses are gastroenteritis and diarrhoea but they can also cause respiratory, central nervous system, liver, muscular and heart infections. Some waterborne viruses have also been associated with some forms of diabetes, chronic fatigue syndrome and dementia (Nwachcuku and Gerba 2004). The major groups of viruses contaminating water are discussed below but may not represent all the viruses likely to be transmitted by water. It is reasonable to expect that further important groups of waterborne viruses will be detected in the future and that these will most likely cause atypical waterborne disease (Nwachcuku and Gerba 2004).</p> <p align="justify"><b>Norovirus</b>: this group of caliciviruses includes the Norwalk and Norwalk-like viruses. Members of this group are strongly associated with waterborne outbreaks in many parts of the world. Symptoms of infection are mild and self-limiting and include vomiting, diarrhoea and nausea over 24 – 48 hours. Norovirus is quite prevalent in New Zealand and is responsible for a large proportion of viral gastroenteritis reported to health authorities (ESR 2004). This virus is one of the easiest to link to a common source outbreak as the symptoms occur rapidly after contact with the virus (approximately 24 hours).</p> <p align="justify"><b>Hepatitis A and E:</b> Hepatitis A and E have a relatively low occurrence in New Zealand (ESR 2004) but induce quite significant symptoms including fever, malaise anorexia and jaundice. The disease is generally self-limiting but has a 2 percent mortality rate. The infectious doses for these viruses are relatively low (10 – 100 viruses) and symptoms do not occur until 10 – 50 days after infection. Internationally Hepatitis A and E outbreaks have frequently been associated with water.</p> <p align="justify"><b>Enteroviruses and adenoviruses:</b> these two different groups represent the viruses that are most commonly found in contaminated surface water. These viruses produce a very broad range of symptoms including respiratory, skin and eye, nervous system, liver, heart and muscular involvement. Gastroenteritis with vomiting and diarrhoea is a less common outcome of infection with these viruses and is limited to only a few adenovirus and enterovirus types. Reported waterborne outbreaks of these viruses, other than in swimming pools, are very infrequent. It is not clear whether lack of reporting is because the dominant symptoms produced by these viruses are not those traditionally associated with water or food borne disease, or because such outbreaks are indeed rare (Hunter 1997).</p> <p align="justify">Virus infections resulting from treated water have not been reported in New Zealand (ESR 2004) in recent years although internationally such outbreaks are recognised (Hunter 1997). Human viruses have been reported to occur at very low levels (0.1 – 1/100 L) in conventionally treated drinking-water in many countries (Vivier et al 2004) including New Zealand (Kim 2005).</p> <p align="justify">Estimations of viral disease risk using standard risk assessment techniques with a high infectivity virus predict the surprisingly high annual risk of infection of between 1:3 and 1:25 from conventionally treated drinking-water contaminated by viruses at low levels (~1 virus per 100 litres) (Gerba and Rose 1992).</p> <p align="justify"> </p> <p align="justify"><font size="3"><strong>7.3 Occurrence of Human viruses in Source waters</strong></font></p> <p align="justify">The New Zealand freshwater microbiology study (McBride et al 2002) is the most significant study of human viruses occurrence in surface water in New Zealand to date. This study carried out in collaboration between the Ministries for the Environment, Agriculture and Forestry, and Health tested recreational water locations on 25 rivers and lakes every 2 weeks for 15 months.</p> <p align="justify">Human adenovirus and/or enterovirus were detected, by qualitative molecular methods, in more than 50% of the 275 samples collected. This data suggests that human virus occurs quite frequently in surface waters and in a wide range of source water locations and types.</p> <p align="justify">Subsequent culture based studies of virus occurrence in the Waikato River show that adenovirus and enterovirus levels are generally low, less than 5 per 100 L, but on some occasions may be as high as 10 per 100 L (Watercare Waikato River Monitoring studies 2003 - 2004).</p> <p align="justify">Studies using sensitive qualitative molecular-based virus detection methods suggest that adenovirus occurrence may be 10 times higher than this level on some occasions in the Waikato River (Kim et al 2005) although it is not clear whether all of these viruses are able to produce infections.</p> <p align="justify">International data collated by WHO suggest that typical surface source waters may contain 0 - 10 viruses per litre (WHO 2004).</p> <p align="justify"> </p> <p align="justify"><font size="3"><strong>7.4 Risk management</strong></font></p> <p align="justify">Potential for disease outbreaks associated with human virus contamination of source waters, and the potential for carry over to treated drinking-water is recognised throughout the developed world. Approaches to controlling the risks are largely through protection of source water quality by control of human activity in reservoir catchments, and through adequate treatment and disinfection of drinking-water. It is now well accepted that bacterial indicators such as <i>E. coli</i> are not adequate surrogates of viral occurrence. Human viruses tend to be more resistant to environmental stresses and water treatment mechanisms than are bacterial indicators, so the absence of the indicator may not equate with absence of the virus contaminant.</p> <p align="justify"><strong>7.4.1 International approaches</strong></p> <p align="justify">The paucity of knowledge on the specific occurrence of human viruses in source waters, and the problems of virus detection and regular monitoring, mean that most guideline documents include only the qualitative requirement that, if tested for, human viruses should not be detected in treated drinking-water.</p> <p align="justify">Where virus guidelines or standard requirements are in place these are stated either in terms of virus occurrence, or as water treatment plant virus removal efficiency. Such values are either derived from acceptable levels of health risk or, pragmatically, reflect virus detection capability.</p> <p align="justify">Recent standard and guideline recommendations have moved towards risk-based evaluation of water treatment requirements. The USEPA Surface Water Treatment Rule includes a virus treatment requirement and requires that treatment of both filtered and unfiltered water sources is sufficient to remove or inactivate 99.99% (4 log) of viruses (USEPA 1994). This requirement is principally based on the acceptable (USEPA 1994) level of waterborne illness in a community (1 case per 10,000 consumers) and the likely level of viruses in surface water. Recent US proposals for surface water disinfection (USEPA 2003a) use the adenovirus group as the target virus.</p> <p align="justify">The WHO Guidelines recognise that water treatment requirements will differ for different communities, and propose a risk-based approach for setting performance targets for surface water treatment plants (WHO 2004).</p> <p align="justify">The risk-based approach takes into account a broad range of factors including virus occurrence and infectivity, water type, community health status and treatment characteristics. Such an approach requires a detailed knowledge of the water supply, water treatment performance and community activities and health status.</p> <p align="justify">Approaches to managing viruses in treated water also recognise that the greatest health risk to a community occurs when water treatment conditions are atypical such as when source water condition is unusual, very high levels of virus occur, or through poor performance, or even failure, within the water treatment process.</p> <p align="justify"><strong>7.4.2 Virus removal by current Water treatment processes</strong></p> <p align="justify">Reduction of virus numbers in water as a result of treatment can occur through either virus removal or virus inactivation. Each virus type may react somewhat differently to particular water treatment methods, but the bulk of research to-date suggests that some broad generalisations can be made.</p> <p align="justify"><b>Virus removal</b> can occur by physical association of a virus with other particles. Particle flocs containing viruses are then removed by settlement or filtration. Virus association with particles and flocs may be enhanced by addition of coagulants and certain salts. The extremely small size of viruses means that they are unlikely to be removed if they are not associated with other particles. Water treatment processes such as flocculation, sand filtration, microfiltration and ultrafiltration, and prolonged standing in reservoirs, will result in physical removal of particle-associated viruses. Only reverse osmosis and dialysis membranes have pore sizes small enough to trap virus particles that are not associated with larger particles or flocs.</p> <p align="justify">The effectiveness of virus removal is affected by those factors that act against particle association or floc formation including water condition and pH (LeChevelier and Au 2004).</p> <p align="justify"><b>Virus inactivation</b> occurs through disruption of the external protein coat (capsid), modification of specific surface sites needed for infection (host receptor recognition sites) or major change to the nucleic acid (RNA or DNA). Disinfectants such as chlorine, chlorine dioxide, and ozone will cause disruption of the virus coat and of the exposed nucleic acids (Shin and Sobsey 2003, Tree et al 2003). Ultraviolet light in the range of 200 – 310 nm (antimicrobial range) will disrupt the nucleic acids by causing cross-linking that leaves them unable to replicate.</p> <p align="justify">Viruses can also be inactivated by prolonged holding in reservoirs exposed to sunlight, elevated temperature and extremes of pH (e.g. lime treatment) (Sobsey 1989). Different virus types and strains will show different levels of resistance to chemical or physical inactivation. Adenoviruses are considered to be the most resistant virus group to many disinfection treatments, because of its structure and nucleic acid makeup, and have been used by the USEPA as a model virus for designing UV criteria for surface water treatment (USEPA 2003a).</p> <p align="justify">The potential for virus inactivation by disinfectants is reduced by the presence of other particles or organic matter that will consume disinfectants or of light adsorbing or blocking materials that reduce UV penetration (LeChevelier and Au 2004).</p> <p align="justify">Repair of disinfection damage is unlikely to occur in viruses as they do not have repair mechanisms as such. It has been suggested that some viruses (e.g. adenovirus) may be able to repair their DNA if there is no damage to the virus coat and they are able to infect a human cell (Nwachcuku and Gerba 2004).</p> <p align="justify">Water treatment plants will normally include both virus removal and virus inactivation processes that act as multiple barriers.</p> <p align="justify">Virus removal and inactivation efficiencies for a range of water treatment processes are reviewed in the WHO (2004) Guidelines (chapter 7), and by LeChevallier and Au (2004).</p> <p align="justify"> </p> <p align="justify"><strong><font size="3">7.5 Sampling, testing and data Interpretation</font></strong></p> <p align="justify">The determination of virus removal efficiency within a water treatment plant, or occurrence in treated water, is dependant on the ability to reliably detect and enumerate the viruses. Determination of the health risk that viruses pose to the community using the water further depends on the ability to demonstrate or infer that the viruses detected are capable of causing human infection.</p> <p align="justify"><b><i>Virus detection and enumeration</i></b><b>.</b> No single method allows detection of all virus types and strains. Traditionally viruses have been concentrated from water samples using filtration or adsorption based techniques with subsequent detection by culture in a permissive human or primate cell line. Many of the virus concentration techniques were developed using poliovirus or other enterovirus types and it is not clear how effectively these work for other virus types particularly norovirus.</p> <p align="justify">Virus concentration from large volumes of water is laborious and time consuming and adds significantly to the cost of virus analysis. Not all virus types are culturable in cell lines, again norovirus has not been cultured routinely and is not detectable using traditional methods.</p> <p align="justify">Viruses (culturable and non-culturable) can be detected at very low levels using polymerase chain reaction (PCR) based molecular methods that target novel DNA or RNA sequences in the genetic information of the virus. Virus assay using PCR can target individual viruses or groups of viruses and multiple analyses would be required to investigate all the relevant viruses in a particular sample (Greening et al 2002). Recent advances in real-time PCR have made these methods both rapid and quantitative and potentially quite routine. PCR based methods are around 10-fold less sensitive that culture based methods for virus detection (Lewis et al 2000). Quantitative PCR based molecular methods are also significantly less expensive than traditional culture methods. It is unusual for a virus concentration and detection method to consistently recover more than 50 - 60% of the virus present in a sample (Lee and Jeong 2004).</p> <p align="justify"><b><i>Virus sampling strategies</i></b><i>.</i> Relatively few viruses are needed for an infection to occur in a susceptible person so low numbers of viruses must be quantified in relatively large volumes of finished water. For example, if source waters contain 5000 viruses per 100 L it would be necessary to sample and analyse at very least 200 L of finished water to demonstrate a 4 log reduction in viruses. Typically, source water sample volumes should be 10 – 100 L, partially treated waters 50 – 200 L, and finished, disinfected water sample volumes 100 – 200 L.</p> <p align="justify">The current cost of virus analysis may make regular monitoring beyond the means of many groups responsible for drinking-water treatment.</p> <p align="justify">Specific short-term studies of virus occurrence and inactivation/removal within a plant are feasible but should be designed carefully to allow adequate interpretation of the data.</p> <p align="justify"><b><i>Determination of virus infectivity.</i></b> Molecular methods for virus detection do not specifically show whether viruses are still infectious. Detection of viruses using a cell-culture based technique shows that the viruses are infectious and pose a risk of illness to water consumers. Infectivity of a virus can however be inferred for certain RNA viruses (norovirus, enteroviruses, Hepatitis A and E) from molecular detection data where the viruses are subjected to chemical disinfection, but not UV disinfection (Greening et al 2002). Virus viability is inferred whenever virus nucleic acid is detected because the nucleic acids (single stranded RNA) are extremely susceptible to degradation in the environmental.</p> <p align="justify"><b><i>Interpretation of virus detection and occurrence data.</i></b> Where viruses are detected in finished drinking-water the response to the data should be based, in consultation with relevant health authorities, on a risk evaluation incorporating the type and number of virus detected, the reproducibility of the result, and the health status and vulnerability of the community.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">7.6 C.t VALUES</font></b></p> <p align="justify">Appendix C of the <i>LT1ESWTR Disinfection Profiling and Benchmarking Technical Guidance Manual</i> (USEPA 2003b) includes C.t tables for disinfection of viruses by various disinfectants. These tables are referenced to AWWA (1991) but in the text of USEPA (1991) it would appear that it was a USEPA publication. Because the 2003 publication still uses the 1991 tables it is assumed that the data the 1991 tables were based on is still the latest information!</p> <p align="justify">The USEPA Surface Water Treatment Rule required (<i>inter alia</i>) that treatment of both filtered and unfiltered sources remove or inactivate 4 log (99.99%) of viruses. This requirement was enacted in 1989. Presumably the 1991 tables were developed to assist water suppliers assess the degree of disinfection of viruses.</p> <p align="justify">USEPA’s LT2ESWTR (2003a) includes a table showing the C.t values for disinfecting viruses using UV light. The proposed UV doses for inactivation of viruses were based on the dose-response of adenovirus because, among viruses that have been studied, it appears to be the most UV resistant and is a widespread waterborne pathogen. Health effects of adenovirus are described in Embrey (1999).</p> <p align="justify">It is doubtful that this same approach was used in developing the 1991 tables; viruses are simply referred to collectively, and viruses are not defined in the 1991 information provided. Some viruses require a much higher C.t than others. Nor is it explained whether the data relate to studies in single virions or cell-associated virions – the latter require a higher C.t.</p> <p align="justify">Table 7.1 shows the UV doses that water suppliers must apply to receive credit for up to 4 log inactivation of viruses. This is Table IV – 21 in USEPA (2003a).</p> <p align="justify"> </p> <p align="center"><strong>Table 7.1: UV dose requirements for virus inactivation credit</strong></p> <p align="center"><strong></strong></p> <p align="center"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="207"> <p><b>Log Credit</b></p> </td> <td valign="top" width="199"> <p><b>Virus<sup>1</sup></b></p> <p><b>UV Dose (mJ/cm<sup>2</sup>)</b></p> </td> </tr> <tr> <td valign="top" width="207"> <p>0.5</p> </td> <td valign="top" width="199"> <p>39</p> </td> </tr> <tr> <td valign="top" width="207"> <p>1.0 (90% removal)</p> </td> <td valign="top" width="199"> <p>58</p> </td> </tr> <tr> <td valign="top" width="207"> <p>1.5</p> </td> <td valign="top" width="199"> <p>79</p> </td> </tr> <tr> <td valign="top" width="207"> <p>2.0 (99% removal)</p> </td> <td valign="top" width="199"> <p>100</p> </td> </tr> <tr> <td valign="top" width="207"> <p>2.5</p> </td> <td valign="top" width="199"> <p>121</p> </td> </tr> <tr> <td valign="top" width="207"> <p>3.0 (99.9% removal)</p> </td> <td valign="top" width="199"> <p>143</p> </td> </tr> <tr> <td valign="top" width="207"> <p>3.5</p> </td> <td valign="top" width="199"> <p>163</p> </td> </tr> <tr> <td valign="top" width="207"> <p>4.0 (99.99% removal)</p> </td> <td valign="top" width="199"> <p>186</p> </td> </tr> </tbody></table> </p> <p align="center"><sup>1 </sup>based on adenovirus studies</p> <p align="justify">Tables 7.2, 7.3, 7.4, 7.5 have been taken from Appendix C of USEPA (2003b) and copied from the 1991 publication, i.e. they refer to undefined viruses.</p> <p align="justify">Based on Table 7.2, a free available chlorine content of 0.20 mg/L after 30 minutes retention time is equivalent to a C.t of 6. This would achieve 4 log inactivations at 10°C. At 5°C the minimum retention time should be 40 minutes, or if that cannot be achieved, the residual free chlorine content should be increased to 0.30 mg/L.</p> <p align="justify"> </p> <p align="center"><strong>Table 7.2: C.t values for inactivation of viruses by free chlorine, pH 6 – 9</strong></p> <p align="center"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="95"> <p><b>Log inactivation</b></p> </td> <td valign="top" width="94"> <p><b>1°C</b></p> </td> <td valign="top" width="94"> <p><b>5°C</b></p> </td> <td valign="top" width="94"> <p><b>10°C</b></p> </td> <td valign="top" width="94"> <p><b>15°C</b></p> </td> <td valign="top" width="94"> <p><b>20°C</b></p> </td> <td valign="top" width="94"> <p><b>25°C</b></p> </td> </tr> <tr> <td valign="top" width="95"> <p>2</p> </td> <td valign="top" width="94"> <p>5.8</p> </td> <td valign="top" width="94"> <p>4.0</p> </td> <td valign="top" width="94"> <p>3.0</p> </td> <td valign="top" width="94"> <p>2.0</p> </td> <td valign="top" width="94"> <p>1.0</p> </td> <td valign="top" width="94"> <p>1.0</p> </td> </tr> <tr> <td valign="top" width="95"> <p>3</p> </td> <td valign="top" width="94"> <p>8.7</p> </td> <td valign="top" width="94"> <p>6.0</p> </td> <td valign="top" width="94"> <p>4.0</p> </td> <td valign="top" width="94"> <p>3.0</p> </td> <td valign="top" width="94"> <p>2.0</p> </td> <td valign="top" width="94"> <p>1.0</p> </td> </tr> <tr> <td valign="top" width="95"> <p>4</p> </td> <td valign="top" width="94"> <p>11.6</p> </td> <td valign="top" width="94"> <p>8.0</p> </td> <td valign="top" width="94"> <p>6.0</p> </td> <td valign="top" width="94"> <p>4.0</p> </td> <td valign="top" width="94"> <p>3.0</p> </td> <td valign="top" width="94"> <p>2.0</p> </td> </tr> </tbody></table> </p> <p align="center"><strong></strong></p> <p align="center"><strong>Table 7.3: C.t values for inactivation of viruses by chloramine</strong></p> <p align="center"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="94"> <p><b>Log inactivation</b></p> </td> <td valign="top" width="94"> <p><b>1°C</b></p> </td> <td valign="top" width="94"> <p><b>5°C</b></p> </td> <td valign="top" width="94"> <p><b>10°C</b></p> </td> <td valign="top" width="94"> <p><b>15°C</b></p> </td> <td valign="top" width="94"> <p><b>20°C</b></p> </td> <td valign="top" width="94"> <p><b>25°C</b></p> </td> </tr> <tr> <td valign="top" width="94"> <p>2</p> </td> <td valign="top" width="94"> <p>1243</p> </td> <td valign="top" width="94"> <p>857</p> </td> <td valign="top" width="94"> <p>643</p> </td> <td valign="top" width="94"> <p>428</p> </td> <td valign="top" width="94"> <p>321</p> </td> <td valign="top" width="94"> <p>214</p> </td> </tr> <tr> <td valign="top" width="94"> <p>3</p> </td> <td valign="top" width="94"> <p>2063</p> </td> <td valign="top" width="94"> <p>1423</p> </td> <td valign="top" width="94"> <p>1067</p> </td> <td valign="top" width="94"> <p>712</p> </td> <td valign="top" width="94"> <p>534</p> </td> <td valign="top" width="94"> <p>356</p> </td> </tr> <tr> <td valign="top" width="94"> <p>4</p> </td> <td valign="top" width="94"> <p>2883</p> </td> <td valign="top" width="94"> <p>1988</p> </td> <td valign="top" width="94"> <p>1491</p> </td> <td valign="top" width="94"> <p>994</p> </td> <td valign="top" width="94"> <p>746</p> </td> <td valign="top" width="94"> <p>497</p> </td> </tr> </tbody></table> </p> <p align="center"><strong></strong></p> <p align="center"><strong>Table 7.4: C.t values for inactivation of viruses by chlorine dioxide, pH 6 – 9</strong></p> <p align="center"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="94"> <p><b>Log inactivation</b></p> </td> <td valign="top" width="94"> <p><b>1°C</b></p> </td> <td valign="top" width="94"> <p><b>5°C</b></p> </td> <td valign="top" width="94"> <p><b>10°C</b></p> </td> <td valign="top" width="94"> <p><b>15°C</b></p> </td> <td valign="top" width="94"> <p><b>20°C</b></p> </td> <td valign="top" width="94"> <p><b>25°C</b></p> </td> </tr> <tr> <td valign="top" width="94"> <p>2</p> </td> <td valign="top" width="94"> <p>8.4</p> </td> <td valign="top" width="94"> <p>5.6</p> </td> <td valign="top" width="94"> <p>4.2</p> </td> <td valign="top" width="94"> <p>2.8</p> </td> <td valign="top" width="94"> <p>2.1</p> </td> <td valign="top" width="94"> <p>1.4</p> </td> </tr> <tr> <td valign="top" width="94"> <p>3</p> </td> <td valign="top" width="94"> <p>25.6</p> </td> <td valign="top" width="94"> <p>17.1</p> </td> <td valign="top" width="94"> <p>12.8</p> </td> <td valign="top" width="94"> <p>8.6</p> </td> <td valign="top" width="94"> <p>6.4</p> </td> <td valign="top" width="94"> <p>4.3</p> </td> </tr> <tr> <td valign="top" width="94"> <p>4</p> </td> <td valign="top" width="94"> <p>50.1</p> </td> <td valign="top" width="94"> <p>33.4</p> </td> <td valign="top" width="94"> <p>25.1</p> </td> <td valign="top" width="94"> <p>16.7</p> </td> <td valign="top" width="94"> <p>12.5</p> </td> <td valign="top" width="94"> <p>8.4</p> </td> </tr> </tbody></table> </p> <p align="center"><strong></strong></p> <p align="center"><strong>Table 7.5: C.t values for inactivation of viruses by ozone</strong></p> <p align="center"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="94"> <p><b>Log inactivation</b></p> </td> <td valign="top" width="94"> <p><b>1°C</b></p> </td> <td valign="top" width="94"> <p><b>5°C</b></p> </td> <td valign="top" width="94"> <p><b>10°C</b></p> </td> <td valign="top" width="94"> <p><b>15°C</b></p> </td> <td valign="top" width="94"> <p><b>20°C</b></p> </td> <td valign="top" width="94"> <p><b>25°C</b></p> </td> </tr> <tr> <td valign="top" width="94"> <p>2</p> </td> <td valign="top" width="94"> <p>0.9</p> </td> <td valign="top" width="94"> <p>0.6</p> </td> <td valign="top" width="94"> <p>0.5</p> </td> <td valign="top" width="94"> <p>0.3</p> </td> <td valign="top" width="94"> <p>0.25</p> </td> <td valign="top" width="94"> <p>0.15</p> </td> </tr> <tr> <td valign="top" width="94"> <p>3</p> </td> <td valign="top" width="94"> <p>1.4</p> </td> <td valign="top" width="94"> <p>0.9</p> </td> <td valign="top" width="94"> <p>0.8</p> </td> <td valign="top" width="94"> <p>0.5</p> </td> <td valign="top" width="94"> <p>0.40</p> </td> <td valign="top" width="94"> <p>0.25</p> </td> </tr> <tr> <td valign="top" width="94"> <p>4</p> </td> <td valign="top" width="94"> <p>1.8</p> </td> <td valign="top" width="94"> <p>1.2</p> </td> <td valign="top" width="94"> <p>1.0</p> </td> <td valign="top" width="94"> <p>0.6</p> </td> <td valign="top" width="94"> <p>0.50</p> </td> <td valign="top" width="94"> <p>0.30</p> </td> </tr> </tbody></table> </p> <h3 align="center"> </h3> <h3 align="center"><strong>REFERENCES</strong></h3> <p align="justify">Embrey, M. (1999). Adenovirus in drinking water, literature summary. Final report. Prepared by The George Washington University School of Public Health and Health Services, Department of Environmental and Occupational Health, Washington, DC.</p> <p align="justify">ESR (2004). <i>Annual Summary of Outbreaks in New Zealand: 2003</i>. Report to Ministry of Health ISSN 1176-3485.</p> <p align="justify">Gerba C. P. and J. Rose (1992). <i>Estimating viral risk from drinking-water: in Comparative Environmental Risk Assessment</i>. Ch. 9, pp 117 – 137. CR Conthern Lewis Publishers.</p> <p align="justify">Greening G., J. Hewitt and G. Lewis (2002). Evaluation of integrated cell culture-PCR (C-PCR) for virological analysis of environmental samples. Journal of Applied Microbiology, <b>93</b>, pp 745 – 750.</p> <p align="justify">Hunter, P. (1997). <i>Viral gastroenteritis. in waterborne disease. Epidemiology and Ecology</i>. Chapter 28, pp 222-231. John Wiley and Sons.</p> <p align="justify">Kim J. (2005). Human adenovirus in the Waikato River: Implication for water supply and public health. MSc thesis. University of Auckland Library.</p> <p align="justify">LeChevelier M., K-K. Au (2004). <i>Water treatment and pathogen control: Process efficiency in achieving safe drinking-water</i>. WHO Drinking-Water Quality Series. WHO, Geneva.</p> <p align="justify">Lee, H. K. and Y.S. Jeong (2004). Comparison of total culturable virus assay and multiplex integrated cell culture-PCR for reliablity of waterborne virus detection. Applied & Environmental Microbiology, <b>70</b>, pp 3632-3636.</p> <p align="justify">Lewis, G. D., F. J. Austin, M. W. Loutit and K. Sharples (1986). Enterovirus removal from sewage - the effectiveness of four different treatment plants. Water Research, <b>20</b>, pp 1291 - 1297.</p> <p align="justify">Lewis G., S. L. Molloy, G. E. Greening and J. Dawson (2000). Influence of environmental factors on virus detection by RT-PCR and cell culture. Journal of Applied Microbiology, <b>88</b>, pp 633-640.</p> <p align="justify">McBride, G., D. Till, T. Ryan, A. Ball, G. Lewis, S. Palmer and P. Weinstein (2002). Freshwater Microbiology Research Programme Report: Pathogen Occurrence and Human Health Risk Assessment Analysis. Ministry of Health, Wellington.</p> <p align="justify">Ministry of Health (2005). <i>Drinking-water Standards for New Zealand 2005</i>. Ministry of Health, Wellington.</p> <p align="justify">Nwachcuku N. and C. P. Gerba (2004). Emerging waterborne pathogens: can we kill them all? Current Opinion in Biotechnology, <b>15</b>, pp 175-180.</p> <p align="justify">Shin G-A and M. D. Sobsey (2003). Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by ozone disinfection of water. Appl. Environ. Microbiol., <b>69</b> (7), pp 3975-3978.</p> <p align="justify">Sobsey M. D. (1989). Inactivation of health-related microorganisms in water by disinfection processes. Water Science and Technology, <b>21</b> (3), pp 179-195.</p> <p align="justify">Tree J. A., M. R. Adams, D. N. Lees (2003). Chlorination of indicator bacteria and viruses in primary sewage effluent. Applied & Environmental Microbiology, <b>69</b> (4), pp 2038-43.</p> <p align="justify">USEPA (1994). <i>National Primary Drinking Water Regulations: Enhanced Surface Water Treatment Regulations</i>. 59 FR 38832; July 29.</p> <p align="justify">USEPA (2003a). <i>Long Term 2 Enhanced Surface Water Treatment Rule</i>; Proposed Rule. National Primary Drinking Water Regulations: 40 CFR Parts 141 and 142, August 11, 2003.</p> <p align="justify">USEPA (2003b). <i>LT1ESWTR Disinfection Profiling and Benchmarking Technical Guidance Manual.</i> EPA 816-R-03-004, Office of Water, May 2003. Available at: http://www.epa.gov/safewater/mdbp/pdf/profile/lt1profiling.pdf</p> <p align="justify">Vivier J. C., M. M. Ehlers and W. O. Grabow (2004). Detection of enteroviruses in treated drinking-water. Water Research, <b>38</b> (11), pp 2699-705, 2004.</p> <p align="justify">Watercare Services Ltd (personal communication): Surrogate study: Mangere wastewater treatment plant 2002.</p> <p align="justify">Watercare Services Ltd (personal communication): Adenovirus and enterovirus monitoning data, Waikato River at Mercer, 2003, 2004.</p> <p align="justify">World Health Organisation (2004). <i>Guidelines for drinking-water quality</i>, 3rd edition. Volume 1: recommendations. ISBN 92 4 154638 7. World Health Organisation, Geneva.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000" size="1">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-48523750588428235682011-08-26T09:04:00.001+07:002011-08-26T09:04:38.525+07:00HUMAN IMMUNODEFICIENCY VIRUS: VIROLOGY AND VACCINE DEVELOPMENT<p align="justify"> </p> <p align="justify">Elizabeth A. Cahill</p> <p align="justify"><b>Abstract. </b>Figures from 2004 suggest that as many as 42.3 million people, 1.1 percent of the world’s population, are currently infected with the human immunodeficiency virus (HIV). In Sub-Saharan Africa, the region suffering most from this pandemic, it is estimated that one in four adults will be killed by acquired immune deficiency syndrome (AIDS) (deWaal, 2004). HIV is a Lentivirus that infects T-helper cells, macrophages and monocytes. The host immune system reacts by removing its own infected T-cells, making the patient more susceptible to opportunistic infection. Chemotherapeutic drugs may drastically reduce morbidity and mortality of patients. These are available to less than 2% of persons with advanced AIDS. Despite much research into various types of vaccines, an effective vaccine against HIV has yet to be developed.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <blockquote> <p align="left">15 September 2004</p> <p align="left">Research sponsored by DARPA Grant DAAD19-02-1-0288, P00001</p> <p align="left">1 September 2004</p> <p align="left">Reed College, Portland, OR</p> </blockquote> <blockquote> <p align="justify"><b>DARPA Grant </b></p> </blockquote> <p align="justify"><b></b></p> <b> <p align="justify"> <br /></p> </b> <p align="justify"><b><font size="3">Introduction</font></b></p> <p align="justify">Just under twenty five years have passed since the Human Immunodeficiency Virus (HIV) was first described (Barre-Sinoussi et al., 1983) and already twenty million people have died (UNAIDS global report, 2004), approximately 14 times the population of the island of Manhattan (US Census data 2000). Despite the enormous amount of research that has gone into vaccine and therapy development, no cure has been found and the number of people with HIV continues to grow. At the close of the year 2003, UNAIDS estimated that 42.3 million people were currently infected. Of the twenty million people dead, approximately 2.9 million died last year alone (Steinbrook, 2004). This pandemic is changing the face of the world.</p> <p align="justify">The enormity of these numbers makes them difficult to comprehend. Some more staggering numbers help to make this figure more accessible. Among adults aged 15-49, 1.1 percent are currently infected (Steinbrook, 2004). Each day approximately 14,000 new infections are established; 95% of new infections occur in developing countries (Emini and Koff, 2001). Every country in southern Africa reports HIV infection rates ranging from 20%-35% (deWaal, 2004). In sub-Saharan Africa approximately 1/3 of children (under age 15) have lost one or both of their parents. In some countries in Africa, there are more than one million orphans (Lewis, 2004). Reversing these trends will be an enormous struggle but is tremendously important. The implications of these numbers and the social challenges surrounding this epidemic cannot be discussed in this paper. However, it is important to consider the macroscopic reality of the HIV crisis when considering the virology of the disease. This virus has created a crisis that demands a global effort. For scientists, politicians and individual citizens it must simultaneously be treated as a pressing crisis and a long-term reality. Both types of strategies are essential.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">Basic virology</font></b></p> <p align="justify">Human Immunodeficiency Virus (HIV) is an infection of the immune system. Other immune system infections include Lupus, asthma and Crohn’s disease. To date, no human has been able to overcome an infection with HIV, although some persons have been able to force viral loads to below the level of detection. The most common modes of HIV infection are direct blood exchange (intravenous drug use or blood transfusion), sexual contact and mother to child transmission. Each of these infection routes can be dampened with appropriate protection strategies.</p> <p align="justify">An understanding of the basic virology of HIV is necessary for discussions of vaccine and chemotherapeutic developments and challenges. HIV is a retrovirus. This indicates that it is an enveloped RNA virus that uses the enzyme reverse transcriptase (RT) to convert its viral RNA into a complementary DNA (cDNA). The resultant cDNA, during a successful infection, is inserted into the host chromosomal DNA where it is able to utilize host machinery, and energy, to further replication and infection. One important feature of RT is that it is error-prone. This serves to increase the virus’ genetic variability and the rate of variant evolution. Retroviral infection can cause numerous diseases, malignancies and cancer. HIV belongs to the genus Lentivirus (“slow virus”), a subset of the family <i>Retroviridae</i>. Lentiviruses are generally larger than other retroviruses and, as their name suggests, have long incubation periods. Every lentivirus causes immune deficiencies and nervous system dysfunctions (Flint et al., 2000) and can be responsible for malignancies such as arthritis or autoimmune disorders.</p> <p align="justify">For an infection to be established the virus must adsorb to the host cell surface. Therefore, the availability of surface receptors determines the host and tissue specificity of any viral infection. In HIV the major viral receptor is a cell surface CD4 protein found on T-helper cells, macrophages and monocytes (Prescott et al., 2001). The humoral and cellular host immune systems both respond to the new infection. The humoral immune system is responsible for the production of antibodies again HIV-1. Antibodies bind to the virus, targeting them for destruction. The cellular immune response is activation of cytotoxic T lymphocytes (CTLs) that directly remove cells presenting viral antigens. The combined efforts of these two limbs of the immune system are insufficient to clear the virus. The continued viral replication in the cells of the lymph nodes ultimately leads to the destruction of the host lymph node structure (Prescott et al., 2001).</p> <p align="justify">The fact that the virus targets both regulatory and antigen presenting immune system cells partially explains the virus’ total ability to avoid destruction by the host immune system (Emini and Koff, 2004). A coreceptor is necessary for viral fusion to the host membrane; it is the binding ability of the coreceptor that determines the tropism of the HIV strain (Flint et al., 2000). During the initial stages of HIV infection the corecptor to the moncyte or macrophage CD4 (M-tropic), CCR5, is essential. Persons homozygous for a mutated, and therefore nonfunctional, CCR5 receptor are impervious to HIV infection (Sullivan et al., 2001). Those heterozygous for the CCR5 receptor maintain a lower viral load during the pre-AIDS course of the disease and appear to progress to AIDS more slowly (Prescott et al., 2001). Mutations in the CCR5 receptor are extremely rare in many regions, such as sub-Saharan Africa and Asia, a fact that may alter the face of HIV/AIDS epidemiology in those regions (Sullivan et al., 2001). After the virus has been established in the host system, the T-cell tropic fusin (CXCR-4) protein is the coreceptor that determines the success of the virus (Prescott et al., 2001). Fusion facilitates the formation of syncitia. This is not seen in M-tropic strains.</p> <p align="justify">All retroviruses have three common structural proteins Gag, Pol, and Env, which code for the core proteins and structural components of the virion, the reverse transcriptase and the envelope glycoprotein, respectively (Flint et al., 2004). Once the HIV-1 virus gains access to the host system, the viral gp120 Env protein binds the CD4 plasma membrane receptor on a host T lymphocyte. The gp120 protein is inaccessible to the host antibodies when it is unattached. It is able to bind CD4 only after a structural adjustment that exposes part of the gp120 to a chemokine receptor, such as CCR5 (Emini and Koff, 2004). The exposed part of the gp120 remains protected from antibodies by either steric hinderance or extensive glycosylation (Emini and Koff, 2004). Every fusion event is followed by the release of the virion core and RNA strands into the cytoplasm of the host T lymphocyte cell. The viral RNA is translated by viral RT into a single-stranded DNA (ssDNA). The RNA strand is then degraded; the ssDNA is used as a template to create a double-stranded DNA (dsDNA). At this point the virus either becomes latent in the cell or forces the cell to transcribe viral mRNAs. The formation of new virions within a host cell will ultimately destroy it, facilitating the release of thousands of new virions into the host system.</p> <p align="justify">Because infected T cells are removed by the host’s own immune response, the patient’s immune response is necessarily compromised. As the loss of T cells becomes more advanced, the infected person becomes increasingly vulnerable to opportunistic infections because their immune system is less functional. These opportunistic infections are often the cause of patient mortality. The progression of HIV infection may be more aggressive in developing countries such as those in sub-Saharan Africa because of chronic infections with pathogens and parasites. Growing antibiotic resistance compounds this problem, making the treatment of other infections more challenging. However, the success of CTLs in removing the infected cells is not wholly destructive. The ability of the patient to clear infected cells, and therefore set the level of viremia during the asymptomatic phase, is a determinant of long-term HIV control. Therefore, the CTLs exert a strong selective pressure, which drives the formation of viral escape mutations (Leslie et al., 2004). HIV is also believed to carry oncogenes; cancer is another common demise of HIV patients. The central nervous system (CNS) can also be damaged because the virus is small enough to cross the blood-brain barrier.</p> <p align="justify">There are roughly four stages described for the average course of an HIV infection. During the primary stage of infection the body produces an acute response to the introduction of the virus. Clinically, this period is best described by a general malaise but HIV antibodies can be detected in the body at this point and the patient is now infectious for the remainder of their lives. Normally, the host system manages to gain control over viral replication at this point and the viremia falls. The virus is not cleared but evades the host immune response and establishes a persistent infection (Emini and Koff, 2004). The patient is often asymptomatic during this phase, which lasts for a variable amount of time. Only approximately 10% of HIV positive adults exhibit disease progression in the first two or three years of infection. After ten years, 80% of HIV positive adults have signs of disease progression (Ho, 1997). During this period of clinical latency, destruction of the lymphoid tissues continues as the virus replicates (Fauci, 2003). The length of the asymptomatic phase poses an enormous challenge for disease control and epidemic modeling (Anderson and May, 1990). Eventually, the virus does overcome the host immune system and the symptoms of HIV/AIDS begin to emerge, the number of host T-cells drops and the patient historically will progress to AIDS at this point (T-cell count below 200 units). No survivors of AIDS have yet been recorded. The symptoms of AIDS are variable but can often include: fever, weight loss, skin rashes, diarrhea, dementia, myelopathy, peripheral neuropathy and increased susceptibility to any opportunistic pathogen (Fauci, 2003). More rare symptoms include Kaposi sarcoma, oral hairy leukoplakia and lymphomas (Flint et al., 2004).</p> <p align="justify">Chemotherapeutic drugs have changed the quality and quantity of life that can be expected for HIV-positive persons who can afford to take them. The three general types of palliative drugs are RT inhibitors that are nucleoside analogs, nonnucleoside inhibitors of RT, fusion inhibitors and protease inhibitors (Prescott et al., 2002). The first effective treatment used to combat HIV was azidovudine (AZT), which is a RT inhibitor (McCleod and Hammer, 1992). A combination of these three types of drugs is most effective and results in greatly reduced morbidity and mortality in countries where they are available (Fauci, 2003). However, the virus does remain latent in T-helper cells and treatment must be continued indefinitely to avoid activation of the virus.</p> <p align="justify"> </p> <p align="justify"><b></b></p> <p align="justify"><b><font size="3">Vaccine development</font></b></p> <p align="justify"><b></b></p> <p align="justify">In addition to the continued arms race for improved chemotherapeutic agents, a vaccine will be a necessary step towards slowing down the spread of this virus. Despite great efforts an effective vaccine remains elusive. Vaccines have historically been most effective in blocking diseases that have a period of acute illness such as smallpox, polio or tetanus. The fact that the host immune response has never been effective in clearing this virus indicates the challenge that biologists face in vaccine development. However, with approximately 14,000 new infections each day (Emini and Koff, 2004) it is a task of enormous importance.</p> <p align="justify">An effective vaccine will have to induce, upon introduction of the virus, an immune response that is different, not just stronger or faster, than the naïve immune response. An obvious target in vaccine development is the Env surface protein, which is integral in establishing HIV infection, as discussed above. A vaccine based on the humoral arm of the immune system would produce antibodies to block new infections. A trimeric Env complex, which includes gp120 and gp41, triggers fusion when complexed with the CD4 receptor and an appropriate coreceptor. Neutralizing monoclonal antibodies (mAbs) have been developed for gp120 that interfere with binding to the CD4 receptor. Similarly mAbs designed for gp41 seem to block fusion. Neutralizing antibodies have been developed for many distinct regions in these glycoproteins but have not been effective <i>in vivo </i>(Burton et al., 2004). Gp120 quickly adapts to immune pressure forming variable surface proteins and gp41, although considered to be more stable, is kinetically inaccessible (Burton et al., 2004). Fortunately, there is a simian immunodeficiency virus (SIV) that is almost identical to HIV. Unfortunately, an effective vaccine has not yet been developed to withstand an SIV challenge (Desrosiers, R.C. 2004).</p> <p align="justify">Another notable obstacle in the vaccine development effort is the sequence variability of the HIV-1 viruses to which humans are exposed. Goulder and Walker (2002) described cases where patients already infected with one strain of HIV-1 became infected again when exposed to a different strain. This has serious consequences for treatment as well as vaccination. In cases of superinfection, the same drug regime that is effective for one strain may not be effective for the other. The antiretroviral drugs used for treating HIV have severe side effects for many patients and increasing the drug dosage or number of drugs may not be possible. A fully effective vaccine would have to protect against many different strains of HIV.</p> <p align="justify">A vaccine designed for a cellular immune response would create anti-HIV-1 CD4+ memory and CD8+ CTLs (Emini and Koff, 2004). The humoral response would, theoretically, be more important to block the establishment of an infection. However, a stronger, faster cellular immune response could help to establish a lower viral load. This is important both for the morbidity of individuals and epidemiologically (Emini and Koff, 2004). This should not be undervalued. The fact that teenagers in every country in sub-Saharan Africa have a 50% chance of being infected with HIV (deWaal, 2002) makes it important to use realistic triage tactics in addition to seeking out a totally effective vaccine.</p> <p align="justify">Microbicides are another strategy being pursued. An entirely effective microbicide (non-toxic, contraceptive or non-contraceptive, fully effective) is many years away (Lewis, 2003). Yet even a partially effective microbicide could save millions of lives. Vaccine and microbicide development can be seen as an important strategy for protecting women (Lewis, 2003). In many cultures women have neither the ability to protect themselves from exposure to this virus nor the power to handle the consequences of the illness. Both a vaccine and a microbicide could give a woman the ability to protect herself without the necessary consent of her partner.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">Conclusions</font></b></p> <p align="justify"><b></b>This epidemic is unlike any the human race has encountered before and must be handled as such. In 1981, during the conservative years under President Reagan, when doctors in New York and California began to note this remarkable and horrible illness in young, gay men, the reaction was slow (Shilts, R., 1987). Public fear and indifference to the gay community made the work of the doctors dedicated to uncovering the root of this disease a battle (Shilts, R., 1987). Similarly, in September 2001, President Mbeki of South Africa questioned the link between HIV and AIDS and decided that zidovudine was too toxic to distribute in clinics. This ignored substantial research demonstrating that zidovudine could dramatically reduce the mother to child transmission of HIV (Lallemant et al., 2004). This trend of insistent ignorance and cruelty is seen repeatedly. In Russia, a group of medical school graduates wrote, in 1997, "We are . . . categorically opposed to combating<sup> </sup>the `new disease' AIDS! We intend . . . to impede the search<sup> </sup>. . . to combat this `noble' epidemic. We are certain that .<sup> </sup>. . AIDS will destroy all drug addicts, homosexuals, and prostitutes.<sup> </sup>. . . Long live AIDS!" (Field, 2004). Despite all of this, there is great cause, and need, for optimism. The pandemic has reached such staggering levels that honest discussions and massive changes are beginning. Education programs, condom and needle distribution programs and health care programs are growing. The World Health Organization aims to have 3 million people in treatment by the year 2005. This is a step towards the ultimate goal of total access to antiretroviral medications. HIV introduces a nontraditional security threat in addition to the massive human tragedy. Countries with high incidence of HIV infection are experiencing severe economic crises along with enormous demographic changes. The effects of the epidemic in regions such as sub-Saharan Africa will be felt globally. There is a constant call for the change in the behavior of those in high-risk populations. For health considerations, this is valid. However, an equally substantial change in behavior is needed in the general population to make the necessary funding and support available.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">References</font></b></p> <p align="justify">Barre-Sinoussi, F., Chermann,. J.-C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. Isolation of a T-lymphotropic retrovirus from patient at risk for acquired immune deficiency syndrome (AIDS). <i>Science</i> <b>220, </b>868-871.</p> <p align="justify">Boes, M, Ploegh, H.L. Translating cell biology <i>in vitro </i>to immunity <i>in vivo</i>. <i>Nature</i> <b>430</b>, 264-271 (2004).</p> <p align="justify">Bretscher, M.T., Althaus, C.L., Muller, V., Bonhoeffer, S. Recombination in HIV and the evolution of drug resistance: for better or for worse? <i>BioEssays </i><b>26.2</b>, 180-188 (2004).</p> <p align="justify">Coovadia, H. Antiretrovial agents- how best to protect infants from HIV and save their mothers from AIDS. <i>NEJM</i>, <b>351</b>, 289-292 (2004).</p> <p align="justify">deWaal, A. Fucking Soldiers: Militarisation, Secrecy and the AIDS pandemic in Africa. <i>Justice Africa papers</i> (2002).</p> <p align="justify">Dezso, Z. and Barabasi A-L. Halting viruses in scale-free networks. <i>Physical Review </i><b>65, </b>055103-1-4 (2002).</p> <p align="justify">Fauci, A. HIV and AIDS: 20 years of science. <i>Nature</i> <b>9</b>, 839-842 (2003).</p> <p align="justify">Fawzi, W.W. et al. A randomized trial of multivitamin supplements and HIV disease progression and mortality. <i>NEJM</i>, <b>351, </b>23-32 (2004).</p> <p align="justify">Field, M.G., HIV and AIDS in the former Soviet bloc. <i>NEJM</i>, <b>351</b>,117-120 (2004).</p> <p align="justify">Friedrich, T.C. et al. Reversion of CTL escape-variant immunodeficiency viruses <i>in vivo</i>. <i>Nature Medicine </i><b>10, </b>275-281 (2004).</p> <p align="justify">Goulder, P.J., Walker, B.D. HIV-1 superinfection – a word of caution. <i>NEJM</i>, <b>347</b>, 756-758 (2002).</p> <p align="justify">Lallemant, M. et al. Single-dose perinatal Nevirapine plus standard Zidovudine to prevent mother-to-child transmission of HIV-1 in Thailand. <i>NEJM</i>, <b>351</b>, 217-227 (2004).</p> <p align="justify">Leslie, A.J. et al. HIV evolution: CTL escape mutation and reversion after transmission. <i>Nature Medicine </i><b>10</b>, 282-289 (2004).</p> <p align="justify">Merrell, D.S. and Falkow, S. Frontal and stealth attack strategies in microbial pathogenesis. <i>Nature</i> <b>430</b>, 250-256 (2004).</p> <p align="justify">McCleod, G.X., Hammer, S.M. Zidovudine: Five years later. <i>Ann. Int. Med.</i> <b>117</b>, 487-501 (1992).</p> <p align="justify">Oltvai, Z.N., Barabasi, A-L. Life’s complexity pyramid. <i>Science </i><b>298</b>, 763- 764 (2002).</p> <p align="justify">Sarafianos, S.G., Hughes, S.H., Arnold, E. Designing anti-AIDS drugs targeting the major mechanism of HIV-1 RT resistance to nucleoside analog drugs. <i>IJBCB </i><b>36</b>, 1706-1715 (2004).</p> <p align="justify">Shilts, R. And the Band Played On. Penguin, 1988.</p> <p align="justify">Steinbrook, R. The AIDS epidemic in 2004. <i>NEJM</i>, <b>351</b>, 115-117 (2004).</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-63701673131324740732011-08-26T08:59:00.001+07:002011-08-26T08:59:36.792+07:00Retinoic acid-mediated antibody response<p align="justify"><b><font size="3">Supplementary Information</font></b></p> <p align="justify"><strong><font size="3"></font></strong></p> <p align="justify"><b><font size="3">Materials</font>.</b> Amino acid building blocks, resins and coupling agents were obtained from Novabiochem (Darmstadt, Germany), Anaspec (San Jose, CA) or ChemPep (Miami, FL). Cholesterol, dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Distearoylglycerol (DSG; LP-R4-029) was obtained from Genzyme Pharmaceuticals (Cambridge, MA). Anhydrous solvents of 99.8% or greater purity were obtained from Acros Organics (Geel, Belgium). Monophosphoryl lipid A derived from Escherichia coli (MPL; #L6638), all-<i>trans</i> retinoic acid (ATRA; #R2625) and 13-<i>cis</i> retinoic acid (13-cis RA; #R3255) were obtained from Sigma-Aldrich. Unless otherwise specified, all other reagents were obtained from Sigma-Aldrich.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b><font size="3">Lipopeptide synthesis</font>. </b>N-MPR peptide (NEQELLELDKWASLNGGK) was synthesized on NovaPEG resin in an automated solid phase synthesizer (ABI 433A, Applied Biosystems, Foster City, CA) with standard fluorenylmethyloxycarbonyl/o-benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate/n-hydroxybenzotriazole (FMOC/HBTU/HOBT) protocols. An orthogonally protected lysine (Fmoc-Lys(1-(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl)-OH; Fmoc-Lys(ivDde)-OH) was incorporated at the C terminus for on-resin conjugation of lipids or biotin. The N terminus was Boc-protected. Removal of the ivDde group was accomplished by 3 x 15 minute treatments of the peptidyl resin with 2% hydrazine hydrate in dimethylformamide (DMF; 10 mL per g resin). The resin was washed in DMF (3 x 10 mL) and dichloromethane (DCM; 3 x 10 mL) and dried under vacuum.</p> <p align="justify">Lipid conjugation was accomplished via amidation of a carboxylated lipid and a deprotected lysine e-amine at the C terminus. A carboxyl group was introduced to DSG via reaction of an available alcohol with succinic anhydride. Briefly, 1.8 mmol DSG was dissolved in 5 mL anhydrous DCM and combined with 3.6 mmol succinic anhydride in 10 mL anhydrous pyridine. The mixture was refluxed at 60 <sup>o</sup>C overnight. The reaction was continued to completion as monitored by thin layer chromatography (TLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS; Voyager DE, Applied Biosystems, Foster City, CA) in para-nitroaniline matrix. The product was washed twice with 1M hydrochloric acid (HCl), dried over sodium sulfate and stored dry until use. Carboxylated lipids were obtained in approximately 90-100% yield. The molecular weight and TLC R<sub>F</sub> value were as follows: DSG-Suc, 749.6 (+ Mg<sup>2+</sup>) Da, R<sub>F</sub> 0.08 (DCM/acetone, 20/1). </p> <p align="justify">Lipidation was accomplished by activation of 270 mmol carboxylated lipid with 270 mmol each of HBTU, HOBT and diisopropylethylamine (DIEA) in anhydrous DMF/DCM (DCM as needed for lipid solubilization) for 30 min at room temperature followed by addition of 67.5 mmol resin and continued reaction under argon for 24h at room temperature. Following the reaction, the resin was washed with DMF (4 x 10 mL) and DCM (4 x 10 mL) to remove unreacted lipids and dried under vacuum. The lipopeptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% water, 2.5% ethanedithiol and 1% triisopropylsilane for 4 hours under argon. The lipopeptide was precipitated into cold ethyl ether, pelleted by centrifugation at 3000 rpm (RT6000, Sorvall, Waltham, MA) and washed once with cold ethyl ether. The ether was poured off and the pellet was re-dissolved in methanol (MeOH), transferred to a round bottom flask, dried by rotary evaporation under reduced pressure and further dried under high vacuum. Stock lipopeptide solutions were prepared MeOH/CHCl<sub>3</sub> and stored at -20 <sup>o</sup>C. The molecular weight was as follows: N-MPR-DSG 2963.2 Da.</p> <p align="justify">Biotinylated N-MPR peptide was prepared for use in ELISA by an analogous method. Biotin was attached to the deprotected C terminal amine by activation of 500 mmol D-biotin with 500 mmol HBTU/HOBT/DIEA in 1.65 mL anhydrous 1:1 DMF/dimethylsulfoxide (DMSO) for 30 min followed by addition of resin and continued reaction under argon for 24h at room temperature. Following the reaction, the resin was washed with 1:1 DMF/DMSO (3 x 10 mL), DMF (3 x 10 mL) and DCM (3 x 10 mL) and dried under vacuum. Biotinylated peptides were cleaved and purified as described above. Molecular weight of N-MPR-biotion was 2455.1 Da as determined by MALDI-MS. Biotin content was quantified by 4´-hydroxyazobenzene-2-carboxylic acid dye exclusion (Sigma #H2153) according to the manufacturer’s instructions. </p> <p align="justify"> </p> <p align="justify"><b><font size="3">RAL Synthesis</font>.</b> The synthesis of all-<i>trans</i> retinoic acid phospholipid (RAL) is depicted in <b>Scheme S1</b>. First, 1-O-octadecyl-2-O-benzyl-sn-glycerol was converted to phosphocholine by phosphorylation with phosphorus oxychloride and coupling to the choline tetraphenyl borate salt. Then the benzyl group was removed by catalytic transfer hydrogenation. Finally, all-<i>trans</i> retinoic acid was attached to the 2-hydroxy group with the typical DCC/DMAP method. </p> <p align="justify"><b></b></p> <p align="justify">1-O-octadecyl-2-O-benzyl-sn-glycerol was from BACHEM (Torrance, CA). Other reagents were from Aldrich (Milwaukee, WI). TLC analyses were performed on 0.25-mm silica gel F<sub>254</sub> plates using a variety of developing systems. High performance flash chromatography (HPFC) was carried out on a Biotage (Charlottesville, VA) Horizon™ HPFC™ system with pre-packed silica gel columns (60 Ǻ, 40-63 mm). Unless noted otherwise, the ratios describing the composition of solvent mixtures represent relative volumes. <sup>1</sup>H NMR spectra were acquired on a Varian 400 MHz instrument. Chemical shifts are expressed as parts per million using tetramethylsilane as internal standard. <i>J</i> values are in Hertz. MALDI-TOF mass spectra were obtained at the Mass Spectrometry Facility, University of California San Francisco.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">1-O-octadecyl-2-O-benzyl-sn-glycero-phosphate</font> (1):</b> A solution of 1-O-octadecyl-2-O-benzyl-sn-glycerol (10 g, 23 mmol) and anhydrous pyridine (3.72 mL, 2 equiv.) in anhydrous tetrahydrofuran (40 mL) was added dropwise to the freshly distilled phosphorus oxychloride (2.36 mL, 1.1 equiv.) in tetrahydrofuran (20 mL) with stirring at 0 °C. Stirring was continued for 3 h at 0 °C. Then 10% sodium bicarbonate (60 mL) was added, and the mixture was stirred at 0 °C for 15 min. The solution was then poured on ice water (200 mL), acidified with conc. HCl (pH ca. 2), and extracted with diethyl ether (400 mL × 2). The ether extracts were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure, azeotropically dried with toluene twice, and used directly for next step reaction. Yield: 11.8 g, 100%. TLC: <i>R<sub>f</sub></i> = 0.05 (CHCl<sub>3</sub>/MeOH/NH<sub>4</sub>OH, 65/25/4).</p> <p align="justify"> </p> <p align="justify"><b><font size="3">1-O-octadecyl-2-O-benzyl-sn-glycero-phosphocholine</font> (2):</b> Compound <b>1</b>(5.14 g, 10 mmol), choline tetraphenyl borate (8.46 g, 2 equiv.) and 2,4,6-triisoproylbenzene sulfonyl chloride (6.04 g, 2 equiv.) were dissolved in anhydrous pyridine (100 mL) with heating. The reaction mixture was stirred at 70 °C for 1h, then 3 h at room temperature. After the addition of water (10 mL), the solvents are removed by rotary evaporation. The residue was extracted with diethyl ether (250 mL × 2). The extracts were combined and evaporated. The crude product was purified by HPFC using a solvent gradient of 20% -35% MeOH-H<sub>2</sub>O (25/4) in chloroform. Yield: 5.8 g, 96.7%. TLC: <i>R<sub>f</sub></i> = 0.17 (CHCl<sub>3</sub>/MeOH/H<sub>2</sub>O, 65/25/4). <sup>1</sup>H NMR (CDCl<sub>3</sub>-CD<sub>3</sub>OD), <i>d</i> 0.87 (t, <i>J = </i>7.2, 3H); 1.27 (br, 30H); 1.57 (m, 2H); 3.15 (s, 9H); 3.36-3.60 (m, 7H); 3.83 (m, 1H); 4.04 (m, 1H); 4.16 (m, 2H); 4.68 (m, 2H); 7.31-7.45 (m, 5H). MALDI-MS calculated for C<sub>33</sub>H<sub>63</sub>NO<sub>6</sub>P<sup>+</sup> [M + H]<sup>+</sup> 600.45, found 600.54.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">1-O-octadecyl-2-hydroxy-sn-glycero-phosphocholine (3):</font></b> To as solution of compound <b>2</b>(5.77 g, 9.6 mmol) in 70 mL methanol, were added 10% Palladium on activated carbon (2 g) and ammonium formate (3.03 g, 48 mmol) under nitrogen atmosphere. The mixture was stirred vigorously under nitrogen for 10 h at 60 °C. The mixture was cooled to room temperature, filtered through Celite 545. The filtrate was concentrated, and applied to a flash40+M column, purified with the HPFC system using an elution gradient of 30% -50% MeOH-H<sub>2</sub>O (25/4) in chloroform. Yield: 3.3 g, 67%. TLC: <i>R<sub>f</sub></i> = 0.05 (CHCl<sub>3</sub>/MeOH/H<sub>2</sub>O, 65/25/4). <sup>1</sup>H NMR (CDCl<sub>3</sub>-TFA, 10:1), <i>d</i> 0.88 (t, <i>J = </i>6.9, 3H); 1.27 (br, 30H); 1.63 (m, 2H); 3.25 (s, 9H); 3.64-3.85 (m, 6H); 4.30 (m, 1H); 4.58 (m, 1H); 4.68 (m, 2H); 5.03 (m, 1H). MALDI-MS calculated for C<sub>26</sub>H<sub>57</sub>NO<sub>6</sub>P<sup>+</sup> [M + H]<sup>+</sup> 510.40, found 510.62.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">1-O-octadecyl-2-all-trans-retinoyl-sn-glycero-phosphocholine (4; RAL):</font></b> To a solution of compound 3 (0.6 g, 1.1 mmol) and all-trans retinoic acid (0.73 g, 2 equiv.) in dry ethanol-free chloroform, were added dimethylaminopyridine (0.15 g) and dicyclohexylcarbodiimide (0.5 g, 2.1 equiv.). The reaction mixture was kept in dark with stirring for 36 h. The precipitate was filtered and the filtrate was concentrated, applied to flash 25+M column, purified with the HPFC using a elution gradient of 10%-30% MeOH/28% NH<sub>4</sub>OH (25/4) in chloroform. Yield: 626 mg, 72%. TLC: <i>R<sub>f</sub></i> = 0.52 (CHCl<sub>3</sub>/MeOH/NH<sub>4</sub>OH, 65/25/4). <sup>1</sup>H NMR (CDCl<sub>3</sub>), <i>d</i> 0.89 (t, <i>J = </i>6.9, 3H); 1.04 (s, 6H); 1.26 (br, 30H); 1.45-1.53 (m, 4H); 1.63 (m, 2H); 1.72 (s, 3H); 2.00 (s, 3H); 2.04 (m, 2H); 2.34 (s, 3H); 3.32 (s, 9H); 3.41 (m, 2H); 3.58 (m, 2H); 3.78 (m, 2H); 4.00 (m, 2H); 4.30 (m, 2H); 5.17 (m, 1H); 5.79 (m, 1H); 6.12-18 (m, 2H); 6.28 (m, 2H); 7.01 (m, 1H). MALDI-MS calculated for C<sub>46</sub>H<sub>83</sub>NO<sub>7</sub>P<sup>+</sup> [M + H]<sup>+</sup> 792.60, found 792.84.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">RAL digestion by phospholipase A<sub>2</sub></font>.</b> The cleavage of retinoic acid from RAL by phospholipase A<sub>2</sub> (PLA<sub>2</sub>) was examined <i>in vitro</i>. 10 mmol RAL were dried to a thin film in a borosilicate glass tube by rotary evaporation under reduced pressure and following by further drying under high vacuum overnight. The film was hydrated in 2 mL containing 10 mM HEPES, 25 mM KCl, 10 mM CaCl<sub>2</sub> and 20 mM Triton-X, pH 8.8 and heated at 45 <sup>o</sup>C for 20 min. Fifty mL of a 100 U/mL solution of snake venom PLA<sub>2</sub> (Sigma #P7778) were added to 250 mL of the RAL solution and incubated for 30 min. When analyzed by TLC, incubation with PLA<sub>2</sub> resulted in total loss of the original RAL spot (R<sub>f</sub> = 0.38) and appearance of two new spots, corresponding to free retinoic acid (R<sub>f</sub> = 0.28) and lysolipid (R<sub>f</sub> = 0.18; CHCl<sub>3</sub>/MeOH/NH<sub>4</sub>OH, 65/25/4).</p> <p align="justify"> </p> <p align="justify"><b><font size="3">Liposome preparation</font></b>. Lipopeptides were formulated in liposomes composed of 15:2:3 DMPC:DMPG:Cholesterol with MPL, RA and lipopeptide as indicated. Prior to use, glassware was rinsed with MeOH and CHCl<sub>3</sub> and dried for at least 90 min at 150 <sup>o</sup>C to destroy pyrogens. Lipid solutions were combined in borosilicate glass tubes and dried to a thin film by rotary evaporation under reduced pressure. Films were further dried under high vacuum overnight. Lipids were hydrated in sterile PBS (UCSF Cell Culture Facility) by intermittent vortexing and bath sonication under argon for a brief period (approximately 15 seconds) to disperse the lipids into the buffer. Defined diameter vesicles were formed by extrusion 11 times through 400 nm polycarbonate membranes using a hand-held extruder (Avestin, Ottowa, Canada). To prevent contamination, the extruder was disassembled and thoroughly cleaned with MeOH and sterile PBS between samples. Vesicle size was characterized by dynamic light scattering and zeta potential was determined by electrophoretic mobility (Zetasizer 3000, Malvern, New Bedford, MA). Liposomes were prepared fresh prior to each injection and stored at 4 <sup>o</sup>C under argon until use. </p> <p align="justify"> </p> <p align="justify"><b><font size="3">Liposome association of N-MPR-DSG and RA</font>.</b> Liposomal association of antigen and retinoic acid was measured following sedimentation of liposomes by an ultracentrifugation method. Essentially, liposomes were formulated with antigen, MPL and RA exactly as described in <b>Methods</b> and centrifuged at 150,000g for 45 minutes in a Beckman TL-100 ultracentrifuge. When control liposomes containing fluorescently labeled lipids were centrifuged in this manner, greater than 90% of the fluorescence was consistently recovered in the liposome pellet.<i> </i>The liposome pellet was resuspended by pipetting and an aliquot was disrupted by mixing with 1.5% (v/v) C<sub>12</sub>E<sub>10</sub> detergent. Antigen concentration in the pellet and supernatant was quantified by the absorbance of aromatic side chains at 280 nm and RA concentration was quantified by the absorbance at 350 nm (NanoDrop, Thermo Scientific, Wilmington, DE). The percent of liposome association was defined as (Pellet)/(Pellet + Supernatant) x 100. </p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com3tag:blogger.com,1999:blog-7259934002090567583.post-90880313336439988322011-08-26T08:52:00.001+07:002011-08-26T08:52:19.734+07:00ASSIGNMENT SHEET FOR VIROLOGY TERM PAPER Spring 2010<p align="justify"></p> <p align="justify"> <p align="justify"></p> <p align="justify"></p> Your assignment is to survey the primary literature in a well-focussed area of virology and to write a literature research paper (also known as a "term paper") of approximately 3000-4000 words reviewing the current state of knowledge in the chosen area. The purposes of the assignment are to acquaint you with the current literature in virology, provide you with in-depth knowledge in an area of virology that is interesting to you and to familiarize you with some "cutting edge" research in virology.</p> <p align="justify"></p> <p align="justify">The term paper assignment is separate and distinct from the oral report assignment although there is some overlap in purpose between the two assignments.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"><strong><font size="3">A: Selection of topic </font></strong></p> <p align="justify"></p> <p align="justify">There is no restriction as to topic except that it must involve some aspect of viruses and it must be approved by the instructor. The latter restriction is enforced to keep topics well-defined and manageable and to ensure that no two students select an identical topic.</p> <p align="justify"></p> <p align="justify">The "first come first served" rule is in effect here. Students who make a topic selection early are less likely to run into conflicts with other students. Register your choice with the instructor to receive priority. Email is a good option here.</p> <p align="justify"></p> <p align="justify">In order to keep to a schedule, the term paper topic must be elected fairly early in the semester. This means you will need to carry on some extra reading outside class and consult with the instructor as you define your interests and narrow your choices. The deadline date for topic selection is given in the Term Paper schedule.</p> <p align="justify"></p> <p align="justify">Your term paper topic may be related to but not identical to the topic of your oral report. For graduate students, your term paper topic may be related to but not identical to your thesis topic. I will expect an OK from your major advisor that the term paper topic you have chosen is not too closely related to your thesis. These may be subsequently revised, if necessary.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"></p> <p align="justify"><strong><font size="3">B. Library Research</font></strong></p> <p align="justify"></p> <p align="justify">You will need to survey the primary literature in order to write your paper (and perhaps to select your term paper topic in the first place). Some scientific journals devoted to reporting the results of studies with viruses include (but are not limited to) the following.</p> <p align="justify"></p> <p align="justify"><i>Virology</i>: publishes mechanistic and molecular reports on all types of viruses; perhaps the most prestigious virological journal</p> <p align="justify"></p> <p align="justify"><i>Journal of General Virology</i>: publishes studies on all aspects of virology, including studies considered too biologically oriented to be accepted for Virology; because of the wide range of studies published here, this tends to be the most interesting virological journal</p> <p align="justify"></p> <p align="justify"><i>Journal of Virology</i>: concentrates mainly on medical aspects of virology and on animal virology, but does publish studies on plant and bacterial viruses; papers with molecular, biochemical, structural, biological orientation may appear here</p> <p align="justify"></p> <p align="justify"><i>Intervirology</i>: publishes fewer articles, but does accept a wide range of topics; this is the journal in which matters of taxonomy, nomenclature, classification are usually discussed</p> <p align="justify"></p> <p align="justify"><i>Phytopathology</i>: publishes a few papers each issue pertaining to plant viruses, usually reporting biological studies </p> <p align="justify"></p> <p align="justify"><i>Journal of Bacteriology</i>: publishes papers on various aspects of bacterial viruses</p> <p align="justify"></p> <p align="justify"><i>Journal of Clinical Microbiology</i>: publishes a few articles each issue strictly on medical aspects of virology</p> <p align="justify"></p> <p align="justify"><i>Journal of Medical Virology</i>: devoted to a wide range of aspects of medical virology, including case studies</p> <p align="justify"></p> <p align="justify"><i>Journal of Virological Methods</i>: reports new methods for the study of animal, plant and bacterial viruses</p> <p align="justify"></p> <p align="justify"><i>Cell</i>: the most widely-cited journal in all the biological sciences occasionally publishes an article pertaining to virology, when the studies reported have very great significance</p> <p align="justify"></p> <p align="justify">These journals are by no means the only ones which contain papers on viruses. They are merely the leading journals in the field. Unfortunately, the IUP library receives few of them. Obtaining copies of articles which appear in other prominent journals is thus made somewhat problematic.</p> <p align="justify"></p> <p align="justify">There are several ways to deal with this difficulty. The most effective way is to travel to a university-level library in another community (Pittsburgh, State College, Morgantown, to name a few) and look up the article there. The second way, which is less reliable and ultimately more time-consuming, is to use the inter-library loan service (ILLIAD). The third way, the slowest and least reliable of all, is to use reprint request cards to ask authors directly for a copy of the needed article. Your instructor will demonstrate the use of these cards. Another way that may prove effective is to download from the internet, when possible.</p> <p align="justify"></p> <p align="justify">Your paper <i>MUST</i> be based mainly on primary articles published within the last few years. Primary articles are those which make the initial report of original research and have undergone a process of checking by other scientists that is called peer review. Primary reports contrast with secondary, or review reports, which make use of primary sources to discuss and summarize the current state of information in an area. They bring together many primary articles to present an overall, up-to-date picture of a topic. Secondary articles are often helpful in locating primary articles or defining a topic. Because it will be based upon primary sources, your term paper will ultimately resemble one of these review articles. You should consciously model your term paper on a review article such as those which appear in the “Annual Review” series, available electronically on the IUP campus. </p> <p align="justify"></p> <p align="justify">Tertiary sources, such as textbooks, will be of little help in your work, since they are usually outdated by years at the time of publication</p> <p align="justify"></p> <p align="justify">You will probably find that internet searches of the literature will make your job much easier. You will also probably find that there are many pages and sites that deal with different aspects of virology. You may use these sites for background information but the main thrust of your paper should deal with reports in the primary literature. Unlike web sites, these reports have been reviewed by peers and may be assumed to be of greater value and accuracy.</p> <p align="justify"></p> <p align="justify">Web sites such as Wikipedia are not acceptable sources. Your citations must come from peer-reviewed materials.</p> <p align="justify"></p> <p align="justify">You will be required to keep the instructor up-to-date on the progress of your literature review and you will be graded on how well you do so. See the Term Paper Schedule for details.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"></p> <p align="justify"></p> <p align="justify"></p> <p align="justify">ALL STUDENTS WILL NEED TO DISCUSS THEIR REFERENCES WITH ME AND SHOW ME THE REPRINTS!!!!!</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"></p> <p align="justify"></p> <p align="justify"></p> <p align="justify"></p> <p align="justify"><strong><font size="3">C: Organization of paper</font></strong></p> <p align="justify"></p> <p align="justify">The final paper must organized into the four main sections listed below. To organize your paper further, use subheadings wherever possible within each section.</p> <p align="justify"></p> <p align="justify">(1) Introduction </p> <p align="justify">(2) Body</p> <p align="justify">(3) Conclusion</p> <p align="justify">(4) Bibliography</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"></p> <p align="justify">(1) In the Introduction, briefly review and describe the viruses you will discuss as well as the biological property being reviewed (replication or transmission for example). The general state of knowledge in the area and the pertinence of the topic should also be described. Include in the Introduction a list of the abbreviations you will use in the term paper.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify">(2) In the Body of your paper you summarize the knowledge in the field, discussing the pertinent contributions of each important primary report, one at a time. In essence, you use the Body to present the results of your literature review. In this part of the paper you are getting your message across, explaining the material to your readers. It is your job as writer to organize, reorganize, synthesize, paraphrase, summarize----whatever it takes to package the information in a form which is easy for the reader to understand.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify">(3) The Conclusion does two things. First, it summarizes the results of your literature review. Second, it pulls together the information you have collected to produce a "take-home message". In other words it <u>synthesizes</u> the important information.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify">(4) Bibliography lists the primary, secondary and tertiary literature you have consulted. For literature citations, use the same format as that used by articles in the <i>Journal of Virology</i>. I expect all students, both graduate and undergraduate, to do a professional job of searching the literature and I expect their bibliographies to show evidence of this. Microscopic bibliographies containing many tertiary sources or only a half-dozen or so references will count against your grade. I fully realize the difficulties of attempting to do library work at IUP and I also realize that the only way to surmount them is to start soon and to keep with it. Waiting until the last minute guarantees extra problems and lower grades.</p> <p align="justify"></p> <p align="justify">Papers not conforming to format guidelines will be returned ungraded.</p> <p align="justify"></p> <p align="justify">You will be expected to keep the instructor posted as you progress through the stages of writing your paper. See the Term Paper Schedule for details.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"></p> <p align="justify"><strong><font size="3">D. Style</font></strong></p> <p align="justify"></p> <p align="justify">The paper is to be written in the general style of a regular scientific review article (see the library's electronic collection of "Annual Review" serials for examples). Write the paper in standard scientific English, using jargon where appropriate and with definitions. Use complete sentences with correct punctuation and minimal spelling errors. Assume that your audience consists of a group of upper-division, advanced undergraduate students of at least your own level of education. If there are questions of style, usage, spelling, abbreviations, format, etc. refer to the <i>Journal of Virology</i> for guidance. Papers must be typewritten or neatly handwritten. Papers not conforming to stylistic guidelines will be returned ungraded.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"><strong><font size="3">E. Schedule</font></strong></p> <p align="justify"></p> <p align="justify">The deadlines for submitting various stages of the term paper are given below. These deadlines are in place to ensure that students make regular and steady progress toward completion of the paper and to discourage procrastination. Except in cases of dire emergency (as judged by the instructor on a case-by-case basis), failure to meet a deadline will result in a grade of zero for the appropriate segment of the term paper.</p> <p align="justify"></p> <p align="justify">March 1 (Monday) at 4:30 PM This is the deadline for turning in a preliminary term paper topic in hard copy format and discussing it with me (aka First Progress Report). These may be subsequently revised, if necessary. You have to have a topic and a few references at this point. We will also discuss your literature search.</p> <p align="justify"></p> <p align="justify">April 5 (Monday) at 4:30 PM At this time a brief outline of the paper is due (aka Second Progress Report) in hard copy format. The major subheadings of Introduction, Body, and Conclusion must be in place. The important points in each subheading should be listed. The bibliography should be well advanced, with all of the key references and most of the others listed. Subsequent revisions are permitted. We will have a more in-depth discussion of your literature search at this meeting.</p> <p align="justify"></p> <p align="justify">May 3 (Monday) Finished papers due at 4:30 PM. </p> <p align="justify"></p> <p align="justify">If you have any questions about the format or other requirements for brief outline, preliminary bibliography, and so on, feel free to contact me for clarification.</p> <p align="justify"> </p> <p align="justify"></p> <p align="justify"><strong><font size="3">F. TERM PAPER GRADING</font></strong></p> <p align="justify"></p> <p align="justify">Your paper will be graded on a 100-pt. scale with points awarded according to the following schedule.</p> <p align="justify"></p> <p align="justify">Source Points Due Date</p> <p align="justify"></p> <p align="justify">Brief Outline/</p> <p align="justify">First Progress Report 5 Monday March 1 at 4:30 PM</p> <p align="justify">Final Outline/</p> <p align="justify">Second Progress Report 5 Monday April 5 at 4:30 PM</p> <p align="justify"></p> <p align="justify">Finished Paper 90 Monday May 4 at 4:30 PM</p> <p align="justify"></p> <p align="justify">You will receive full credit for the term paper topic, the brief outline and the final outline as long as you turn them in ON TIME and written according to the guidelines established above.</p> <p align="justify"></p> <p align="justify">Finished papers will be assigned first a letter grade and then a number grade from the range corresponding to the letter. Comments will be written on each individual paper Letter grades and their corresponding number range are given below.</p> <p align="center"> </p> <p align="center"> <table border="0" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="49"> <p align="center"><strong>Letter</strong></p> </td> <td valign="top" width="74"> <p align="center"><strong>%</strong></p> <p align="center"><strong>Range</strong></p> </td> <td valign="top" width="461"> <p align="center"><strong>Comment</strong></p> </td> </tr> <tr> <td valign="top" width="49"> <p align="center"></p> </td> <td valign="top" width="74"> <p align="center"></p> </td> <td valign="top" width="461"> <p align="center"></p> </td> </tr> <tr> <td valign="top" width="49"> <p align="center">A</p> </td> <td valign="top" width="74"> <p align="center">>92-100</p> </td> <td valign="top" width="461"> <p align="justify">Excellent; you conducted a thorough survey of the literature, concisely synthesized the data, communicated the results effectively, summarized and produced a take-home-message, used correct scientific terminology and followed the format</p> </td> </tr> <tr> <td valign="top" width="49"> <p align="center">B</p> </td> <td valign="top" width="74"> <p align="center">>82-92</p> </td> <td valign="top" width="461"> <p align="justify">Above Average; you did many but not all of the above; see paper for more comments</p> </td> </tr> <tr> <td valign="top" width="49"> <p align="center">C</p> </td> <td valign="top" width="74"> <p align="center">>72-82</p> </td> <td valign="top" width="461"> <p align="justify">Average; you did some but not all of the above; see paper for more comments</p> </td> </tr> <tr> <td valign="top" width="49"> <p align="center">D</p> </td> <td valign="top" width="74"> <p align="center">>62-72</p> </td> <td valign="top" width="461"> <p align="justify">Below Average; you did not do most of the above; see paper for more comments</p> </td> </tr> <tr> <td valign="top" width="49"> <p align="center">F</p> </td> <td valign="top" width="74"> <p align="center"><u><</u>62</p> </td> <td valign="top" width="461"> <p align="justify">Far Below Average; you did few or none of the above, did not turn in a paper, or did not meet a deadline</p> </td> </tr> </tbody></table> </p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com1tag:blogger.com,1999:blog-7259934002090567583.post-62499146362882555212011-08-26T08:44:00.001+07:002011-08-26T08:44:22.880+07:00UK POLICY FOR MANAGEMENT OF POTENTIAL ORGAN/TISSUE DONORS WITH CONFIRMED POSITIVE VIROLOGY RESULTS (UPDATE)<h3 align="justify"><strong></strong></h3> <h3 align="justify"><strong><font size="3">1. INTRODUCTION</font></strong></h3> <p align="justify">1.1 This policy provides guidance on the management of potential organ/tissue donors with positive virology results. It will give healthcare professionals guidance on how to proceed<del datetime="2004-01-08T14:23" cite="mailto:red033"> when donation cannot take place as a consequence</del><ins datetime="2004-01-08T14:23" cite="mailto:red033"> in light </ins>of these results, the subsequent care of the potential donor, and their next of kin/significant other. </p> <p align="justify">1.2 Transplantation is now well established, as the treatment of choice for the majority of patients with end stage organ failure. However, the transmission of infection is one of the associated risks. All potential organ/tissue donors have blood taken for a number of virology tests. Please refer to<ins datetime="2004-01-08T14:24" cite="mailto:red033"> the</ins> Microbiological Safety of Blood and Tissues for Transplantation<ins datetime="2004-01-08T14:24" cite="mailto:red033"> (MSBT)</ins> guidelines.<sup><ins datetime="2004-01-08T14:33" cite="mailto:red033">1</ins></sup> In addition, the donor/tissue transplant co-ordinator is required to take a detailed medical and social history.</p> <p align="justify">1.3 When organ donation is discussed with the family of the potential donor, and consent for donation is agreed,<del datetime="2004-01-08T14:25" cite="mailto:red033"> this process also informs the donor’s family that tests for a</del><ins datetime="2004-01-08T14:25" cite="mailto:red033"> the consent will include</ins> <ins datetime="2004-01-08T14:26" cite="mailto:red033">testing for a </ins>number of possible viral infections<ins datetime="2004-01-08T14:26" cite="mailto:red033">.</ins><del datetime="2004-01-08T14:27" cite="mailto:red033"> will be carried out on</del><del datetime="2004-01-08T14:26" cite="mailto:red033"> the blood of the potential donor.</del> Moreover, the consent form also says that in the event of a confirmed positive result becoming available that may have clinical relevance to any family member, then the result and its implications will be discussed with them.</p> <p align="justify">1.4 Confidentiality is one of the most significant concepts in healthcare and is an important factor in client/practitioner relationships as stated in the codes of conduct (General Medical Council (GMC) 1993<sup><ins datetime="2004-01-08T14:27" cite="mailto:red033">2</ins> </sup><del datetime="2004-01-08T14:27" cite="mailto:red033">/</del>Nursing Midwifery Council 2002).<sup><ins datetime="2004-01-08T14:28" cite="mailto:red033">3</ins></sup></p> <p align="justify">1.5 Under The NHS (Venereal Diseases) Regulation 1974<sup><ins datetime="2004-01-08T14:34" cite="mailto:red033">4</ins></sup> and the NHS Trusts (Venereal Diseases) Directions 1991<sup><ins datetime="2004-01-08T14:31" cite="mailto:red033">5</ins> </sup><del datetime="2004-01-08T14:31" cite="mailto:red033"></del>it:<b><i></i></b></p> <p align="justify"><b><i></i></b></p> <p align="justify"><b><i>“prevents the disclosure of any identifying information about a patient examined or treated for a sexually transmitted disease (including HIV & AIDS) other than to a medical practitioner (or to a person employed under the direction of a medical practitioner) in connection with and for the purpose of whether the treatment of the patient and/or the prevention of the spread of the disease.”</i></b></p> <p align="justify">This regulation demonstrates that there are statutory duties to ensure all steps are taken to prevent any information, that is capable of identifying a patient with a sexually transmitted disease being disclosed. However the disclosure of this information could arguably be possible for the purposes of treatment and prevention of the spread of the disease to partners and others who may be affected as a result of their relationship to the potential organ donor.</p> <p align="justify">1.6 This Policy has been devised to provide guidelines, information and support for healthcare professionals where positive virology results have been confirmed on potential organ/tissue donors and where these results have implications for others. It is important that this is dealt with in an appropriate manner pertinent to the merit of each individual case. </p> <p align="justify"> </p> <h6 align="justify"><strong><font size="3">2. REVIEW </font></strong></h6> <p align="justify"><b></b></p> <p align="justify">2.1 This document will be reviewed every two years or as required. </p> <p align="justify"> </p> <h3 align="justify"></h3> <h3 align="justify"><strong><font size="3">3. AIM</font></strong></h3> <p align="justify">3.1 To identify all potential donors with positive virology</p> <p align="justify">3.2 To determine the necessity for disclosure of this information to the next of kin/significant other.</p> <p align="justify">3.3 To obtain expert advice.</p> <p align="justify">3.4 To repeat and confirm virology results.</p> <p align="justify"><b></b></p> <p align="justify">3.5 To determine who discloses this information. </p> <p align="justify">3.6 To disclose the findings with the appropriate healthcare specialists.</p> <p align="justify">3.7 To ensure that any discussions with the next of kin/significant other are appropriately documented in the potential organ/tissue donor’s medical notes. </p> <p align="justify">3.8 To ensure documentation of reasons for disclosure are clear and concise including contact/advise given from appropriate individual healthcare professionals.</p> <p align="justify">3.9 Disclosure to take place within an appropriate time scale.</p> <p align="justify">3.10 To advise the next of kin/significant other<b> </b>of support networks available.</p> <p align="justify"> </p> <h3 align="justify"><strong><font size="3">4. RATIONALE</font></strong></h3> <p align="justify">4.1 It is acknowledged that in the UK there is no current legislation regarding the breach of confidentiality when disclosing positive virology results of potential cadaver donors (especially when a third party health is at risk). In contrast there are many states in America where there is a legal obligation to disclose confidential information if there is a third party who may be affected (Cochran 1999).<sup><ins datetime="2004-01-08T14:34" cite="mailto:red033">6</ins></sup><del datetime="2004-01-08T14:34" cite="mailto:red033"> </del></p> <p align="justify">4.2 The British Medical Association (BMA) Confidentiality and Disclosure of Health Information document (1999)<sup><ins datetime="2004-01-08T14:33" cite="mailto:red033">7</ins></sup> states:</p> <p align="justify"><b><i></i></b></p> <p align="justify"><b><i>‘post mortem test of a cadaver may reveal the presence of previously undiagnosed infectious conditions which people close to the deceased person may need to be aware of in order to protect their own health or that of others’ </i></b></p> <p align="justify">4.3 The ethos of the Department of Health (DOH), BMA and GMC is that the decision to disclose information regarding positive virology results should be made by balancing the benefits with the harm of the public interest. This includes the legitimate concern for the interests of others who may be affected by the person with the virus. The GMC (1993) has concluded:</p> <p align="justify"><b><i>‘there are grounds for such disclosure only where there is a serious and identifiable risk to a specific individual who, if not so informed, would be exposed to the infection………………………but where such consent is withheld the doctor may consider it a duty to seek to ensure that any sexual partner is informed, in order to safeguard such persons from a possible fatal infection’ (GMC cited in Kennedy, Chapter 9, pg 664<sup><ins datetime="2004-01-08T14:35" cite="mailto:red033">8</ins></sup>).</i></b></p> <p align="justify">4.4 The DOH issued guidance on partner notification for HIV in December 1992<sup><ins datetime="2004-01-08T14:36" cite="mailto:red033">9</ins></sup> and stated that the benefits of notification are the following:</p> <p align="justify">q Identified contacts are given the opportunity to consider whether they wish to be tested</p> <p align="justify">q Those who have unknowingly been infected may wish to take steps to prevent transmission to others</p> <p align="justify">q Access to treatment and support programmes so that they may benefit from long term monitoring of their clinical condition and from appropriate therapies.</p> <p align="justify">4.5 Although the above guidance has only stipulated the disclosure for HIV infection, Hepatitis B and C are important viruses and the implications of such infections can be equally as life threatening as HIV. These guidelines therefore may be used to address all positive virology results where there are implications for a third party.</p> <p align="justify">4.6 Informing potentially infected individuals may have various implications. However, advances in medical treatments, therapies and support networks may balance the benefit and outweigh the harm for most individuals. </p> <p align="justify"><b></b></p> <p align="justify">4.7 A nationwide policy will give guidance to the healthcare professionals in dealing with this ethically and morally difficult issue.</p> <p align="justify"> </p> <p align="justify"><strong><font size="3">5. HIV</font></strong></p> <p align="justify">5.1 When a potential organ/tissue donor has been identified as HIV positive this information will be relayed to the duty donor/tissue transplant co-ordinator.</p> <p align="justify">5.2 The donor/tissue transplant co-ordinator will stop the donation process, informing all the relevant parties but maintaining the need for confidentiality.</p> <p align="justify">5.3 The result will be reconfirmed by the laboratory medical staff prior to disclosure.</p> <p align="justify"> </p> <p align="justify"><strong><font size="3">6. HEPATITIS B/C</font></strong></p> <p align="justify">6.1 When a potential organ/tissue donor has been identified as hepatitis positive this information will be relayed to the donor/tissue transplant co-ordinator.</p> <p align="justify">6.2 The donor/tissue transplant co-ordinator will inform UKT and the relevant retrieval teams of the result.</p> <p align="justify">6.3 If the retrieval teams decline the offer the donor transplant co-ordinator will inform UKT. The organs will continue to be offered in accordance with national guidance.</p> <p align="justify">6.4 This result will be reconfirmed by the laboratory medical staff prior to disclosure </p> <p align="justify"> </p> <p align="justify"><font size="3"><b>7. </b><b>SYPHILIS</b></font></p> <p align="justify">7.1 On obtaining a positive<b> </b>syphilis result the donor/tissue transplant co-ordinator will inform the transplant team and UKT, as appropriate.</p> <p align="justify">7.2 This result will be reconfirmed by the laboratory medical staff prior to disclosure. </p> <p align="justify"> </p> <p align="justify"><b><font size="3">8. NOTIFICATION OF NEXT OF KIN/SIGNIFICANT OTHERS </font></b></p> <p align="justify"><b></b></p> <p align="justify">8.1 The donor/tissue<b> </b>transplant co-ordinator will discuss with the clinician in charge of the potential donor an action plan on what information is to be disclosed to the next of kin/significant other at this early stage and how this information will be disclosed and by whom. <b></b></p> <p align="justify">8.2 An appointment will be arranged to discuss results with the next of kin/significant<b> </b>other, however<b>,</b> this should take place after the confirmatory results have been received and expert advice has been obtained. </p> <p align="justify">8.3 The clinician in charge of the care of the potential donor should be involved in this meeting and it may be appropriate to include an expert practitioner within this field. This meeting should include no less than two appropriate healthcare professionals. Advice may be obtained from the hospital legal department and the medical director of UKT before any meeting takes place if necessary.</p> <p align="justify">8.4 The information to be discussed at this meeting should include:</p> <ul> <li> <div align="justify">A detailed discussion of the positive result and the implications to the next of kin/significant other </div> </li> <li> <div align="justify">An offer to screen all those who may be at risk </div> </li> <li> <div align="justify">The need for precautions to be taken until their results are confirmed. If they decline to be tested then advice should be given on prevention of transmission.</div> </li> <li> <div align="justify">Approval should be sought and encouraged for permission to divulge these findings to their General Practitioner.</div> </li> <li> <div align="justify">Contact numbers for local clinics and support groups/counsellors should be given to the next of kin/significant other. </div> </li> </ul> <p align="justify"> </p> <p align="justify">8.5 Detailed records of all conversations should be entered into the notes of the potential donor/ donor medical records.</p> <p align="justify">8.6 Letter should be sent to the General Practitioner of the potential donor/donor.</p> <p align="justify"> </p> <p align="justify"><strong>REFERENCES</strong></p> <p align="justify"><ins datetime="2004-01-08T14:39" cite="mailto:red033">1</ins> <ins datetime="2004-01-08T14:39" cite="mailto:red033">Department of Health, Advisory Committee on the Microbiological Safety of Blood and Tissues for Transplantation</ins>.<i><ins datetime="2004-01-08T14:39" cite="mailto:red033">Guidance on the Microbiological Safety of Human Organs, Tissues and Cells used in Transplantation</ins> (August 2000).</i><ins datetime="2004-01-08T14:39" cite="mailto:red033"></ins></p> <p align="justify"><ins datetime="2004-01-08T14:39" cite="mailto:red033"></ins></p> <p align="justify"><ins datetime="2004-01-08T14:39" cite="mailto:red033">2</ins> <ins datetime="2004-01-08T14:39" cite="mailto:red033">General Medical Council (1993) Confidentiality: Protecting and Providing Information</ins></p> <p align="justify"><ins datetime="2004-01-08T14:39" cite="mailto:red033"></ins></p> <p align="justify"><ins datetime="2004-01-08T14:40" cite="mailto:red033">3</ins> <ins datetime="2004-01-08T14:40" cite="mailto:red033">Nursing and Midwifery Council (2002) Code of Professional Conduct</ins></p> <p align="justify"><ins datetime="2004-01-08T14:40" cite="mailto:red033"></ins></p> <p align="justify"><ins datetime="2004-01-08T14:40" cite="mailto:red033">4</ins> <ins datetime="2004-01-08T14:40" cite="mailto:red033">The National Health Service (Venereal Diseases) Regulation 1974, No 29 </ins></p> <p align="justify"><ins datetime="2004-01-08T14:40" cite="mailto:red033"></ins></p> <p align="justify"><ins datetime="2004-01-08T14:40" cite="mailto:red033">5</ins> <ins datetime="2004-01-08T14:40" cite="mailto:red033">The National Health Service Trusts (Venereal Diseases) Directions 1991</ins></p> <p align="justify"><ins datetime="2004-01-08T14:40" cite="mailto:red033"></ins></p> <p align="justify"><ins datetime="2004-01-08T14:41" cite="mailto:red033">6</ins> <ins datetime="2004-01-08T14:41" cite="mailto:red033">Cochran M (1999) The real Meaning of Patient-Nurse Confidentiality, Critical Care Nurse Quarterly 22 (1) pp 42-51</ins></p> <p align="justify"><ins datetime="2004-01-08T14:37" cite="mailto:red033">7</ins> British Medical Association (1999) Confidentiality and Disclosure of Health Information<ins datetime="2004-01-08T14:41" cite="mailto:red033"></ins></p> <p align="justify"><ins datetime="2004-01-08T14:41" cite="mailto:red033">8</ins> <ins datetime="2004-01-08T14:41" cite="mailto:red033">Kennedy I, Grubb A (1994) Medical Law, Chapter 9, pg 664, 2<sup>nd</sup> Edition, Butterworth & Co Ltd, London</ins></p> <p align="justify"><del datetime="2004-01-08T14:39" cite="mailto:red033"></del></p> <p align="justify"><ins datetime="2004-01-08T14:37" cite="mailto:red033">9</ins> Department of Health (1992) PLICO (92) 5 Guidance on Partner Notification for HIV Infection</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com1tag:blogger.com,1999:blog-7259934002090567583.post-21418613507811436792011-08-26T00:46:00.001+07:002011-08-26T00:46:22.296+07:00Appendix XXI – Specimens, Cell Lines and Stain Table<p><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhximxtOq2lYHZxSBw4XdHReNHyN78O5OCZZE8L9PUHUTFm9rn5190LoXRj28J_fnsuFvyotSyGe7_ECZqtHj-SzK4AAHjP5fyfbkPYzxN4y172MyMH3gdijOyQ0mCvNrrfwNqPTewjcmE/s1600-h/image%25255B5%25255D.png"><img style="border-bottom: 0px; border-left: 0px; display: inline; border-top: 0px; border-right: 0px" title="image" border="0" alt="image" src="http://lh3.ggpht.com/-ftcTdXtvJZo/TlaKaprWDmI/AAAAAAAABLs/mMDNBtfbHlI/image_thumb%25255B6%25255D.png?imgmax=800" width="516" height="612" /></a> </p> <p align="justify">* DFA= SimulFluor DFA ,Flu season is from the 1st of November to the end of April. Do DFA for Baby’s Auger Suction/Nasopharyngeal Swab year round.</p> <p align="justify">** RD June to Nov.</p> <p align="justify">Treat sputum with sputolysin if is purulent. <b>Working solution</b>: 1 ml of conc.+ 10 ml of DH<sub>2</sub>O ,<b>only stable</b> <b>for 48 hours at 2-8</b><b><sup>0</sup></b><b>C</b>. </p> <p align="justify"><sup>1 </sup>Use 6 drops of untreated specimen.</p> <p align="justify"><sup>2 </sup>Use Simulfluor Para1,2,3/Adeno,report as ‘Negative or Positive for Adenovirus’.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-59746109742630463122011-08-26T00:42:00.001+07:002011-08-26T00:42:53.642+07:00CYTOSPIN PREPARATION (Appendix XX)<p align="justify"><b>I. </b><b><u>Introduction</u></b></p> <p align="justify">A cytospin preparation is a concentration of cells taken directly from specimens or from scraped cell cultures.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b>II. </b><b><u>Reagents and Materials</u></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">Virus-specific or pooled antibody</p> <h3 align="justify"><font size="2">Phosphate buffered saline (PBS)</font></h3> <p align="justify">Cold acetone (4<sup>o</sup>C)</p> <p align="justify">Distilled water</p> <p align="justify">Mounting fluid</p> <p align="justify">Non-immune antibody</p> <p align="justify">vortex</p> <p align="justify">sterile pipettes</p> <p align="justify">cytospin and accessories</p> <p align="justify">humidified chamber</p> <p align="justify">coplin jars</p> <p align="justify">fluorescence microscope</p> <p align="justify"> </p> <p align="justify"><b>III. </b><b><u>Procedure</u></b></p> <p align="justify"><strong><u></u></strong></p> <p align="justify"><b><u></u></b></p> <p align="justify"><b>1. Shell Vial</b></p> <p align="justify"><b></b></p> <p align="justify"><b></b>i. Remove all except 1 ml maintenance media from shell vial using a sterile pipette.</p> <p align="justify">i. Scrape cells from top of coverslip using a sterile pipette. Break up cell clumps by pipetting the cells up and down several times.</p> <p align="justify">ii. Pipette 200 ul (4 drops) of scraped cells into funnel for each well.</p> <p align="justify">iii. Cytospin at 2000 rpm (700g) for 5 minutes.</p> <p align="justify">iv. Remove slide and air dry.</p> <p align="justify">v. Fix in cold acetone for 10 minuets in a coplin jar. Remove slide and air dry.</p> <p align="justify">Proceed to staining. Refer to Appendix IV for Indirect fluorescent antibody staining techniques or Appendix V for Direct fluorescent antibody staining techniques.</p> <p align="justify"><b>or</b></p> <p align="justify">Refer to Appendices IV and V for immunofluorescent staining techniques for shell vials.</p> <p align="justify"> </p> <p align="justify"><b>1. </b><b>Tube culture (or Shell Vials for CPE)</b></p> <p align="justify"><b></b></p> <p align="justify">i. Remove all except 1 ml maintenance media from the culture tube using a sterile pipette.</p> <p align="justify">ii. Scrape cells from side of tube using a sterile pipette. Break up cell clumps by pipetting the cells up and down several times.</p> <p align="justify">iii. Pipette 200 ul (4 drops) of scraped cells into funnel for each well.</p> <p align="justify">iv. Cytospin at 2000 rpm (700 x g) for 5 minutes.</p> <p align="justify">v. Remove slide and air dry.</p> <p align="justify">vi. Fix in cold acetone for 10 minutes in a coplin jar. Remove slide and air dry.</p> <p align="justify">vii. Proceed to staining. Refer to Appendix IV for Indirect fluorescent antibody stains or Appendix V for Direct fluorescent antibody stains.</p> <p align="justify"> </p> <p align="justify"><b>or</b></p> <p align="justify">Refer to Appendices IV and V for immunofluorescent staining techniques for shell vials.</p> <p align="justify"> </p> <p align="justify"><b>3. Direct from specimen</b></p> <p align="justify"><strong></strong></p> <p align="justify"><b>IV. </b><b><u>Reference</u></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">Thermo Shandon, cytospin. Manufacturer's manual. Refer to Appendix VI for procedure.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-13713787152237086672011-08-26T00:41:00.001+07:002011-08-26T00:41:01.229+07:00PNEUMOCYSTIS CARINII DFA TEST (Appendix XIX)<p align="justify"> <p align="justify"><b></b></p> <p align="justify"><b></b></p> <b>I. <u>Introduction</u></b><u></u></p> <p align="justify"><b><i></i></b></p> <p align="justify">The Merifluor-Pneumocystis DFA test is an in vitro test for the direct detection of Pneumocystis carini cysts and trophozoites in bronchoalveolar lavage (BAL), bronchial wash (BW); sputum or biopsy specimen.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b>II. <u>Collection and Transport</u></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">BAL, wash and sputum should be collected using standard procedures. Biopsy specimens e.g. transbronchial, open lung or others must not be fixed and are transported to the lab on a saline moistened piece of gauze in a sterile container. Tissue should not be allowed to dry or become immersed in saline. All specimens should be transported as soon as possible to the laboratory. PCP testing can be done on the day after receipt <u>except</u> specimens received Friday or the day before a holiday must be stained and read that day.</p> <p align="justify"> </p> <p align="justify"><b>III</b><b>. <u>Procedure</u></b></p> <p align="justify"><strong><u></u></strong></p> <p align="justify"><b><u></u></b></p> <p align="justify"><b>Reagents</b></p> <p align="justify"><b></b></p> <p align="justify"><b></b>FITC- <i>P. carinii</i> conjugate</p> <p align="justify">Control slides</p> <p align="justify">Distilled water</p> <p align="justify">FA mounting fluid</p> <p align="justify">Sputolysin: diluted 1:10 (i.e. 300 Ul sputolysin 3.0 mL distilled water)</p> <p align="justify"> </p> <p align="justify"><b>Materials</b></p> <p align="justify"><b></b></p> <p align="justify"><b></b>Vortex</p> <p align="justify">Sterile pipettes</p> <p align="justify">10 - 100 uL Eppendorf pipette</p> <p align="justify">Humidified chamber</p> <p align="justify">Coplin jars</p> <p align="justify">Fluorescent microscope</p> <p align="justify"> </p> <p align="justify"><b>Preparation of Slides</b></p> <p align="justify"><b></b></p> <p align="justify"><b></b>BAL and BW:</p> <p align="justify">1. Centrifuge the BAL or BW for 10 minutes at 1800 x g.</p> <p align="justify">2. Remove and discard all but 0.5 mL of the supernatant. Thoroughly resuspend the pellet in the remaining 0.5 mL of fluid.</p> <p align="justify">3. Make a thin smear twice the size of a cytospin spot and allow to air dry.</p> <p align="justify">4. Fix in acetone for 5 minutes in a coplin jar, then air dry.</p> <p align="justify">5. Slide must be stained within 8 hours or freeze at -20<sup>0</sup>C.</p> <p align="justify"> </p> <p align="justify"><b>Sputum .. See Sputolysin Procedures AppXXII</b></p> <p align="justify"><b></b></p> <p align="justify">1. Combine equal volumes (3 mL each) of sputum and diluted sputolysin. Vortex mixture.</p> <p align="justify">2. Incubate for 3 minutes at 35<sup>0</sup>C.</p> <p align="justify">3. Vortex the mixture briefly and add an equal volume of PBS and entrifuge at 1300 x g for 5 minutes.</p> <p align="justify"><i>4. </i>Remove the supernatant, leaving 0.5 mL to resuspend the pellet<i>.</i></p> <p align="justify">5. Make a smear twice the size of a cytospin spot. Allow to air dry.</p> <p align="justify">6. Fix in acetone for 5 minutes in a coplin jar, then air dry.</p> <p align="justify">7. Slide must be stained within 8 hours or freeze at -20<sup>0</sup>C.</p> <p align="justify"> </p> <p align="justify"><b>Biopsy Specimen</b></p> <p align="justify"><b></b></p> <p align="justify">1. Prepare a freshly cut surface on a fragment of tissue.</p> <p align="justify">2. Touch the cut surface to a FA slide. Make several non-overlapping imprints within the well, avoiding smearing using several cuts.</p> <p align="justify">3. While imprints are still moist on the slide, fix by adding 1 - 2 drops of acetone and allow to air dry.</p> <p align="justify">4. Slide must be stained within 8 hours or freeze at -20<sup>0</sup>C.</p> <p align="justify"> </p> <p align="justify"><b>Staining - DFA</b></p> <p align="justify"><b></b></p> <p align="justify">1. Cover the smear with 30 uL of <i>P. carinii</i> FITC-conjugate antibody. </p> <p align="justify">2. Incubate in a humidified chamber for 30 minutes at 36<sup>0</sup>C.</p> <p align="justify">3. Wash slide twice with distilled water for 2 minutes in a coplin jar.</p> <p align="justify">4. Allow the slide to dry.</p> <p align="justify">5. Mount using coverslip and mounting fluid.</p> <p align="justify">6. Read with fluorescence microscope with the FITC / Evans Blue filter and 40x objective.</p> <p align="justify"> </p> <p align="justify"><b>Interpretation of Results</b></p> <p align="justify"><b></b></p> <p align="justify">POSITIVE: Any specimen which contains two typical cysts exhibiting apple-green fluorescence of characteristic morphology. Generally cysts, 5 - 8 um diameter, are found together with trophozoites in clusters. Clusters can be variable in size and may appear with or without "honeycomb" like structure. Some cysts fluoresce evenly throughout their structure whereas other cysts may fluoresce mainly on their periphery and produce a "honeycomb" appearance within the clusters.</p> <p align="justify">NEGATIVE: Red fluorescence <u>without</u> any characteristic apple-green fluorescence as described above.</p> <p align="justify"> </p> <p align="justify"><b>IV. </b><b><u>Reporting</u></b></p> <p align="justify">POSITIVE: "<i>Pneumocystis jiroveci (previously known as P. carinii)</i> positive by immunofluorescence".</p> <p align="justify">NEGATIVE: "<i>Pneumocystis (previously known as P. carinii)</i> negative by immunofluorescence".</p> <p align="justify">Telephone all positive results and document. </p> <p align="justify"> </p> <p align="justify"><b>V. </b><b><u>Quality Controls</u></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">Positive and negative control slides should be stained each time the staining procedure is performed. Refer to a senior technologist if controls do not work or for any other problems with staining, reading or reporting results.</p> <p align="justify">External QC (slides from a source other than the reagent supplier or the daily QC) should be done on new reagent lots and if the batch (daily) QC fails.</p> <p align="justify">Check Calcoflour stain result in the LIS for concordance and notify the Mycology section as well as Senior/Charge if their result is different. Appropriate actions should be taken to reconcile the difference.</p> <p align="justify"> </p> <p align="justify"><b>VI. </b><b><u>Reference</u></b></p> <p align="justify">Merifluor Pneumocystis, Meridian Diagnostics, Inc. 3471 River Hills Drive, Cincinnati, Ohio, 45244. Tel. 513-271-3700</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-24915928302697251002011-08-26T00:38:00.001+07:002011-08-26T00:38:48.039+07:00Appendix XVIII (Cont'd) QUALITY CONTROL OF MONOCLONAL ANTIBODIES<p align="justify"> <p align="justify"><b></b></p> <p align="justify"><b></b></p> <p align="justify"><b></b></p> <b>Reagent quality controls</b>:</p> <p align="justify">These must be perform prior to patient testing to ensure each component of the reagent performs as expected.</p> <p align="justify">a. Check expiratory date then perform DFA, SimulFlour DFA or IFA accordingly.</p> <p align="justify">b. External QC slides (different manufacturer, unless not available) of the same batch are used to test both current and the new reagents in parallel</p> <p align="justify">c. Results must fall within range of expected results before reagents are released for use (eg. all 7 viruses must be positive and negative wells are negative for the Bion 14-well Respiratory Panel).</p> <p align="justify">d. Record reagent expiry date and QC results in Reagent Log and/or LIS.</p> <p align="justify">e. Report abnormal QC results to Charge/Senior technologist.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify">Expected reagent QC results:</p> <p align="center"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="132"> <p><strong>External (commercial) QC slide</strong></p> </td> <td valign="top" width="126"> <p><strong>Current Reagent</strong></p> </td> <td valign="top" width="108"> <p><strong>New Reagent</strong></p> </td> </tr> <tr> <td valign="top" width="132"> <p>Positive well</p> <p>(for each virus)</p> </td> <td valign="top" width="126"> <p>+</p> </td> <td valign="top" width="108"> <p>+</p> <p>(no weaker than current reagent)</p> </td> </tr> <tr> <td valign="top" width="132"> <p>Negative well</p> </td> <td valign="top" width="126"> <p>-</p> </td> <td valign="top" width="108"> <p>-</p> </td> </tr> </tbody></table> </p> <p align="justify">Failed reagent QC results:</p> <p align="justify">i. Inform charge/senior technologist to investigate cause of failed QC.</p> <p align="justify">ii. Record in Reagent Log Chart. (Instrument Maintenance Log if microscope/incubator is involved in the failure and Incident Report if necessary).</p> <p align="justify">iii. May need to re-run failed control materials in parallel to fresh controls to evaluate the QC material itself.</p> <p align="justify">iv. If the re-run shows the old QC material still fails and fresh QC is satisfactory, the error may be attributed to the old QC material itself and the reagent is satisfactory.</p> <p align="justify">v. If the re-run shows both the old and fresh QC material fail (or other QC not satisfactory), the error may be attributed to the reagent then the reagent cannot be released for use. Supplier of the reagent should be contacted and the appropriate incident report should be filled.</p> <p align="justify"> </p> <p align="justify"><u></u></p> <p align="justify"><b>Daily QCs</b>: </p> <p align="justify">These are performed within each batch of patient samples to monitor assay performance and techniques within the batch.</p> <p align="justify">a. Check reagent expiratory date and verify that Reagent QC is satisfactory for the reagent lot/kit being used.</p> <p align="justify">b. Appropriate positive and negative control slides (eg. ATCC 4-well slide with RSV/Para3 for SimulF RS stain) should be stained with each batch. These slides should be placed in various random positions within the batch.</p> <p align="justify">c. Examine the negative control well first to establish the dull red colour (Evans blue counterstained) and to determine if there is any nonspecific staining.</p> <p align="justify">The positive control must be clearly distinguishable from the negative control or the test is invalid.</p> <p align="justify"><i></i></p> <p align="justify">d. Record QC results in LIS and/or wosksheet.<i></i></p> <p align="justify"><i></i></p> <p align="justify">Failed Daily QC:</p> <p align="justify">i. Do not release patient results pending resolution of QC error.</p> <p align="justify">ii. Inform charge/senior technologist.</p> <p align="justify">iii. Record in Reagent Log Chart (and Instrument Maintenance Log if microscope/incubator is involved in the failure).</p> <p align="justify">iv. Re-run failed controls in parallel to fresh controls (and/or external QC) to evaluate the QC material itself.</p> <p align="justify">v. If the re-run shows the old QC material still fails, fresh QC passes and nothing else is wrong with the batch (only the old QC material failed, patient results valid) patient results may be released.</p> <p align="justify"> </p> <p align="justify">Marked decrease/absence in fluorescence can be due to:</p> <p align="justify">a. Reagent deterioration/skipping (did not apply primary/secondary stain)</p> <p align="justify">b. Microscope (filter, bulb, alignment)</p> <p align="justify">c. Other equipment, reagents or technique</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-64895371612946110742011-08-26T00:36:00.001+07:002011-08-26T00:36:13.565+07:00Appendix XVII VIROLOGY TRAINING GUIDE (optional) & Appendix XVIII QUALITY CONTROL OF MONOCLONAL ANTIBODIES<p> </p> <p align="center"><b><font size="3"></font></b></p> <p> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="145"> <p align="center">WEEK</p> </td> <td valign="top" width="240"> <p align="center">ACTIVITY</p> </td> <td valign="top" width="288"> <p align="center">MATERIALS</p> <p><b></b></p> </td> </tr> <tr> <td valign="top" width="145"> <p>1</p> </td> <td valign="top" width="240"> <p>SHELL VIAL UNKNOWNS</p> </td> <td valign="top" width="288"> <p>CMV / HHF</p> <p>HSV / HHF</p> </td> </tr> <tr> <td valign="top" width="145"> <p>1 - 2</p> </td> <td valign="top" width="240"> <p>TUBE CULTURE UNKNOWNS</p> </td> <td valign="top" width="288"> <p>CMV / HHF</p> <p>HSV / HHF</p> <p>ECHOVIRUS / HHF, RMK, HEP-2</p> <p>COXSACKIE B / HHF, RMK, HEP-2</p> <p>ADENOVIRUS / HFF, RMK, HEP-2</p> <p>*INFLUENZA A / HHF, RMK, HEP-2</p> <p>*PARAINFLUENZA 3 / HHF, RMK, HEP-2</p> <p>*RSV / HHF, RMK, HEP-2</p> </td> </tr> <tr> <td valign="top" width="145"> <p>START 1<sup>ST</sup> WEEK</p> </td> <td valign="top" width="240"> <p>SPECIMEN PLANTING</p> </td> <td valign="top" width="288"> <p>PATIENT SAMPLES</p> </td> </tr> <tr> <td valign="top" width="145"> <p>START 2<sup>ND</sup> WEEK</p> </td> <td valign="top" width="240"> <p>SHELL VIAL STAINING</p> </td> <td valign="top" width="288"> <p>PATIENT SAMPLES</p> </td> </tr> <tr> <td valign="top" width="145"> <p>START 3<sup>RD</sup> WEEK</p> </td> <td valign="top" width="240"> <p>TUBE CULTURE READING</p> </td> <td valign="top" width="288"> <p>PATIENT SAMPLES</p> </td> </tr> <tr> <td valign="top" width="145"> <p>1 - 8 </p> </td> <td valign="top" width="240"> <p>READ LAP - 1, 2, 3</p> </td> <td valign="top" width="288"> <p>CACMILE SELF-STUDY</p> <p>VIROLOGY COURSE</p> </td> </tr> <tr> <td valign="top" width="145"> <p>8 (END OF ROTATION)</p> </td> <td valign="top" width="240"> <p>WRITTEN EXCERCISES / </p> <p>SELF-EXAM</p> </td> <td valign="top" width="288"> <p>CACMILE SELF-STUDY</p> <p>VIROLOGY COURSE</p> </td> </tr> </tbody></table> </p> <p>*November to April training periods only.</p> <p> </p> <p align="center"><b><font size="3">Appendix XVIII</font></b></p> <p align="center"><b><font size="3"></font></b></p> <p align="center"><b><font size="3">QUALITY CONTROL OF MONOCLONAL ANTIBODIES</font></b></p> <p> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="271"> <p><b>Monoclonal Antibodies</b></p> <p><b></b></p> </td> <td valign="top" width="126"> <p><b>Method</b></p> </td> <td valign="top" width="241"> <p><b>Expected Use</b></p> </td> </tr> <tr> <td valign="top" width="271"> <p>Respiratory Viral Screen/RSV panel</p> <p>FluA/B panel</p> <p>RSV/para3 panel</p> <p>Para123/Adeno panel</p> <p>Specific Parainfluenza 1</p> <p>Specific Parainfluenza 2</p> </td> <td valign="top" width="126"> <p>SimulFluor DFA</p> <p>SimulFluor DFA</p> <p>SimulFluor DFA</p> <p>SimulFluor DFA</p> <p>DFA</p> <p>DFA</p> </td> <td valign="top" width="241"> <p>R-Mix Shell Vials / direct specimen</p> </td> </tr> <tr> <td valign="top" width="271"> <p>Coxsackie A9</p> <p>Coxsackie B</p> <p>Echovirus</p> <p>Poliovirus</p> <p>Enterovirus 70 / 71</p> <p>Mumps (not in routine use)</p> </td> <td valign="top" width="126"> <p>IFA</p> </td> <td valign="top" width="241"> <p>E-Mix /MRC-5 Shell Vials</p> </td> </tr> <tr> <td valign="top" width="271"> <p>CMV pp65</p> <p>CMV Immediate Early</p> </td> <td valign="top" width="126"> <p>IFA</p> <p>IFA</p> </td> <td valign="top" width="241"> <p>Direct polymorph, leukocytes</p> <p>MRC-5 Shell Vial</p> </td> </tr> <tr> <td valign="top" width="271"> <p>Specific Herpes simplex 1</p> <p>Specific Herpes simplex 2</p> <p>Specific Varicella zoster virus</p> <p>CMV early & late</p> <p>Herpes simplex bivalent</p> </td> <td valign="top" width="126"> <p>DFA</p> </td> <td valign="top" width="241"> <p>MRC-5 Shell Vial /direct specimen</p> <p>MRC-5 Shell Vial /direct specimen</p> <p>MRC-5 Shell Vial /direct specimen</p> <p>MRC-5 Shell Vial</p> <p>MRC-5 Shell Vial /direct specimen</p> </td> </tr> </tbody></table> </p> <p><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-31584640978178549742011-08-26T00:33:00.001+07:002011-08-26T00:33:34.458+07:00Appendix XVI WEEKLY WORK SCHEDULE<p align="justify"> <p align="justify"><b></b></p> 1. Specimen Management Bench - Monday to Friday :</p> <p align="justify">a) Accessioning and processing all Virology specimens.</p> <p align="justify">b) For PCR testing: mix well and transfer CSF/ EDTA blood / Amniotic fluid to Blue-topped vial (e.g. HSV, EBV, CMV, Parvovirus B19, Polyomavirus, Enterovirus.)</p> <p align="justify">b) Preparing and staining of Direct Smears, PCP slides.</p> <p align="justify">c) Send out stool or Urine for EM.<a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify">2. Shell Vial Bench – Monday to Friday :</p> <p align="justify">a) Staining and reading of all shell vials.</p> <p align="justify">b) Reading of all Direct Smears and PCP slides.</p> <p align="justify">c) Read Super E-mix Shell vials daily.</p> <p align="justify">d) Enter all shell vial related (e.g. new reagent lots, daily staining, weekly viral propagation and MRC-5 growth) QC results either in LIS and/or Data Sheet.</p> <p align="justify">e) Tabulate and fax weekly viral isolates data (on/after Friday).</p> <p align="justify">f) Maintain stocks of viral isolates as well as preparation of QC slide.</p> <p align="justify"> </p> <p align="justify">3. CMV Antigenemia Bench :</p> <p align="justify">a) Monday to Friday: Record temperatures and humidity on freezers, fridges and room every morning. </p> <p align="justify">b) Monday to Friday: Process, stain and read all CMV antigenemia specimens.</p> <p align="justify">c) Perform RPR on Wednesday to Friday.</p> <p align="justify">d) Perform VZ Ab testing using VIDAS.</p> <p align="justify">e) Perform Monospot daily.</p> <p align="justify"> </p> <p align="justify">4. Molecular Bench:</p> <p align="justify">a) Perform Tagman HCV RNA PCR using Ampliprep when there</p> <p align="justify">are enough of specimens for a full run.</p> <p align="justify">b) Perform Tagman HBV DNA Assay using Ampliprep when there are enough specimens for a full run.</p> <p align="justify">c) Assist PCR bench with Chlamydia/GC testing.</p> <p align="justify">d) Perform molecular bench inventories.</p> <p align="justify"> </p> <p align="justify">5. AXSYM Bench:</p> <p align="justify">a) Perform all stat Hepatitis markers, HIV1&2, CMV IgG and Rubella </p> <p align="justify">IgG and HIV assays and QC’s using AxSYM.</p> <p align="justify">b) Perform weekly maintenance on Thursday or Friday.</p> <p align="justify">c) Perform monthly maintenance at beginning of each month.</p> <p align="justify">d) Perform RPR (syphilis) on Monday and Tuesday.</p> <p align="justify">e) Do inventory on Wednesday/Thursday.</p> <p align="justify"> </p> <p align="justify">6. Eye Bank bench: Using NEXGEN:</p> <p align="justify">a) Perform Eye Bank -BioRad EIA HBsAg &HIV and Ortho EIA HBcAb & HCV Ab daily.</p> <p align="justify">b) Perform C. difficile Toxin A/B testing daily.</p> <p align="justify">c) Perform EBV VCA IgG once a week.</p> <p align="justify"> </p> <p align="justify">7. PCR Bench :</p> <p align="justify">a) Perform Chlamydia/GC using ProbTec.</p> <p align="justify">b) Perform WNV RT-PCR for TGLN and CSF specimens; freeze samples and enter into LIS store.</p> <p align="justify">d) Perform RNA and DNA extractions from CSF/whole blood.</p> <p align="justify">e) Perform HSV PCR Tuesday and Friday.</p> <p align="justify">f) Perform EBV PCR,CMV PCR and Parvo B19 PCR once a week.</p> <p align="justify">g) Perform Enterovirus PCR when received.</p> <p align="justify"> </p> <p align="justify">8. P-Lab Bench :</p> <p align="justify">a) Perform Ortho HTLV 1&2 Ab</p> <p align="justify">b) Perform Immucor CMV Ab</p> <p align="justify">c) Perform WNV IgM Ab</p> <p align="justify">d) Freeze all transplant patients sera in freezer program under ‘Donor’, Inception Biosciences sera under ‘CTSCP’ and Cord Blood of Canada sera under ‘CBB’. </p> <p align="justify"> </p> <p align="justify">9. Evening Technologist: </p> <p align="justify">a) Perform stat TGLN / stat recipient assays if requested.</p> <p align="justify">b) Perform WNV IgM assay</p> <p align="justify">c) Perform AxSYM assays, QC’s and maintenance</p> <p align="justify">d) Process time-sensitive specimens (eg.molecular tests).</p> <p align="justify"> </p> <p align="justify">10. Night Technologist:</p> <p align="justify">a) Perform stat TGLN / stat recipient assays if requested.</p> <p align="justify">b) Continue WNV IgM assay if set up late.</p> <p align="justify">c) Perform AxSYM assays, QC’s and maintenance.</p> <p align="justify">d) Process time-sensitive specimens (eg.molecular tests).</p> <p align="justify"> </p> <p align="justify">11. Technician 1- Monday to Friday :</p> <p align="justify">a) Disinfect, check centrifuges and document on sheet.</p> <p align="justify">b) Spin and accession all Serology specimens.</p> <p align="justify">c) Send out all PHL specimens and printing out manifest.</p> <p align="justify">d) Process Viral load specimens.</p> <p align="justify">e) Document pressure reading in LIS for Serology Accession Hood.</p> <p align="justify"> </p> <p align="justify">12. Technician 2- Monday to Friday :</p> <p align="justify">a) Disinfect, check centrifuges and document on sheet if not yet done by Technician 1.</p> <p align="justify">b) Process Viral load specimens.</p> <p align="justify">c) Enter patients’ demographic in Viral Load program.</p> <p align="justify">d) Spin, accession and freeze sera for HBV DNA and HCV RNA.</p> <p align="justify">Help technician 2 to spin and accession all serology specimens</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-20750307983312299842011-08-26T00:27:00.001+07:002011-08-26T00:27:17.461+07:00VIRUS ISOLATION AND IDENTIFICATION CHARACTERISTICS (<p align="center"><b>Appendix XV</b></p> <p align="center"><a href="http://lh6.ggpht.com/-Wki5FiheHsM/TlaFr30QRJI/AAAAAAAABLQ/qKgXrBzsd7A/s1600-h/image%25255B10%25255D.png"><img style="border-bottom: 0px; border-left: 0px; display: inline; border-top: 0px; border-right: 0px" title="image" border="0" alt="image" src="http://lh3.ggpht.com/-PpUTT0XHw2g/TlaFu4CJ_QI/AAAAAAAABLU/HD6U3d43-sE/image_thumb%25255B8%25255D.png?imgmax=800" width="549" height="510" /></a> </p> <p align="left"> </p> <p><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgF9I6qgWtBj9Hlp8626HIpFN4ZO2yb8gDmEpk4dJEKDDiYcVhaLqHnUnkGiV99icZOmpNSD6_m_jpg1LMAW8iFOcFoVbnbq1i_SXM069WIaJkZYaZXbfDd_N_PWmnjLnc-s9t5SWNjSho/s1600-h/image%25255B15%25255D.png"><img style="border-bottom: 0px; border-left: 0px; display: block; float: none; margin-left: auto; border-top: 0px; margin-right: auto; border-right: 0px" title="image" border="0" alt="image" src="http://lh6.ggpht.com/-FnRu5SYath0/TlaF4PsL0tI/AAAAAAAABLc/jRYQm9eH-MY/image_thumb%25255B15%25255D.png?imgmax=800" width="547" height="604" /></a> </p> <p></p> <a href="http://lh4.ggpht.com/-9MrAag8YrtY/TlaF6akoLlI/AAAAAAAABLg/NATelXR2AsQ/s1600-h/image%25255B20%25255D.png"><img style="border-bottom: 0px; border-left: 0px; display: inline; border-top: 0px; border-right: 0px" title="image" border="0" alt="image" src="http://lh4.ggpht.com/-u-wLZZJPU3I/TlaF8vQvYQI/AAAAAAAABLk/_eSVlrEoD74/image_thumb%25255B24%25255D.png?imgmax=800" width="549" height="426" /></a> <p><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-44941365859297881822011-08-26T00:20:00.001+07:002011-08-26T00:20:00.754+07:00Appendix XIII PRESERVATION OF CELL CULTURE MONOLAYERS & Appendix XIV QUALITY CONTROL OF CELL CULTURES USED FOR ROUTINE VIRUS ISOLATES<p align="justify"><b></b></p> <p align="justify"><b></b></p> <p align="justify">1. Aspirate the medium from the cell culture tubes to be preserved.</p> <p align="justify">2. Add 8 mL buffered formaldehyde preservative medium to each tube.</p> <p align="justify">3. Record the following information on the tube:</p> <p align="justify">- virus</p> <p align="justify">- number of days incubation</p> <p align="justify">- lab number</p> <p align="justify">4. Store preserved culture at room temperature.</p> <p align="justify"><u>Formaldehyde Preservative</u></p> <p align="justify"><u></u></p> <p align="justify">100 mL Formaldehyde Solution (37 - 40%)</p> <p align="justify">900 mL Distilled Water</p> <p align="justify">20 mL Phenol Red (0.5%)</p> <p align="justify">4.0 g NaH<sub>2</sub>PO<sub>4</sub> - H<sub>2</sub>O</p> <p align="justify">6.5 g Na<sub>2</sub>HPO<sub>4</sub></p> <p align="justify"><sub></sub></p> <p align="justify"><b>Materials</b></p> <p align="justify"><b></b></p> <p align="justify">Vortex</p> <p align="justify">Sterile pipettes</p> <p align="justify">10 - 100 uL Eppendorf pipette</p> <p align="justify">Humidified chamber</p> <p align="justify">Coplin jars</p> <p align="justify">Fluorescent microscope</p> <p align="justify"> </p> <p align="justify"><b></b></p> <p align="justify"><b>Appendix XIV</b></p> <p align="justify"><b></b></p> <p align="justify"><b>QUALITY CONTROL OF CELL CULTURES USED FOR ROUTINE VIRUS ISOLATES </b></p> <p align="justify"><strong></strong></p> <p align="justify"><b></b></p> <p align="justify"><b><u>Tube Culture </u></b><b>(Not in routine use)<u></u></b></p> <p align="justify"><u></u></p> <p align="justify">Upon receipt of cell culture tubes, the record of the date received, vendor, lot number, and passage number is kept in the QC binder for cell lines. The monolayer is checked microscopically for sterility and appearance of an acceptable confluent monolayer.</p> <p align="justify"> </p> <p align="justify"><b>A. <u>Uninoculated Negative</u> <u>Controls:</u></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">Reserve 4 tubes of each lot cells for use as controls and label as follows:</p> <p align="justify">N1, date;</p> <p align="justify">N2, date;</p> <p align="justify">N3 date;</p> <p align="justify">C, date;</p> <p align="justify">V, date</p> <p align="justify"> </p> <p align="justify">a. <u>Negative Controls with refeed:</u> (3 tubes, N1; N2; N3)</p> <p align="justify">Select one tube each on Wednesday Friday and Monday to set up along with inoculated specimens. Incubate, observe and refeed these tubes in parallel with patient inoculated cultures to monitor monolayer quality, toxicity and sterility. They can also be used to provide a baseline for comparison of inoculated cultures when reading for CPE and immunostaining. CMK and HFF tubes are kept for 3 weeks, HEp-2 and RD for 2 weeks</p> <p align="justify">b. <u>Unopened Negative Controls</u> <u>without refeed: </u>(2 tubes, C; V)</p> <p align="justify">These tubes are left unopened and observed to identify toxicity and contamination originating with the vendor. All tubes, CMK, HEp-2 , HFF and RD tubes are kept for only one week. One tube (C) is kept in the clean room and one (V) is placed on the Virology drum.</p> <p align="justify"> </p> <p align="justify">Record sterility and cell appearance for these tubes into the LIS Manual (REGISTRATION OF TUBE CULTURE MEDIA)</p> <p align="justify"><b>B. <u>Positive Controls:</u></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">Each week scrape from an HFF tubes containing the following QC strains of HSV-1 HSV-2 and CMV to propagate the QC strains in the new lot of HFF tubes, Use CMK tubes for RSV and nfluenza A. Examine microscopically for CPE and record the results into the LIS as growth control. At the same time also inoculate MRC-5 and R-Mix shell vials to perform quality control for MRC-5 shell vials (see II. <b><u>Shell Vial Cell Line<s>s </s>(MRC-5 cell suspension below)</u></b></p> <p align="justify">HSV-1 (ATCC VR-5539)</p> <p align="justify">HSV-2 (ATCC VR-540)</p> <p align="justify">CMV (ATCC VR-807)</p> <p align="justify">RSV (ATCC VR-284)</p> <p align="justify">Influenza A (ATCC VR-544)</p> <p align="justify">The MRC-5 shell vials are then stained with HSV-1, HSV-2 and CMV IEA monoclonal antibodies at 24 hours, R-Mix shell vials with RS. Record the results into the LIS as shell vial growth control as well as stain controls.</p> <p align="justify">Each month (and whenever necessary), remove from the liquid nitrogen storage, cryovials of above strains to propagate the QC strains in HFF tubes.</p> <p align="justify">Additional positive controls may be set up with the following strains and for the following reasons:</p> <p align="justify">Coxsackie B1 (ATCC VR-28)</p> <p align="justify">Parainflunza 3 (ATCC )</p> <p align="justify">Varicella-zoster (ATCC VR-1367)</p> <p align="justify">i) Low isolation rates</p> <p align="justify">ii) Comparison of cell lines</p> <p align="justify">iii) Vendor changes</p> <p align="justify">iv) Proficiency test failures</p> <p align="justify">v) Training purposes</p> <p align="justify">vi) Consistent problems with negative controls</p> <p align="justify">vii) Preparation of QC material (i.e. Positive control slides)</p> <p align="justify">Consult a senior technologist to determine the cell lines and viruses to be set up.</p> <p align="justify"><b></b></p> <p align="justify"><b></b></p> <p align="justify"><b>II. <u>Shell Vial Cell Line </u>(Suspension seeding is not in routine use)<u></u></b></p> <p align="justify">Upon receipt of a shipment of cells, initial and date the record sheet accompanying the shipment. The record should contain vendor, lot number, passage number and QC data. File in the QC binder for cell lines.</p> <p align="justify">Before seeding the shell vials, aspirate about 30 mL of MRC-5 cell suspension into a 125 cm<sup>2</sup> tissue culture flask, label on the side of flask with “MRC-5, date and ‘<b>Pre</b>’”.</p> <p align="justify">After seeding shell vials, aspirate about 30 mL of MRC-5 cell suspension into another 125 cm<sup>2</sup> tissue culture flask, label on the side of flask with “MRC-5, date and ‘<b>Post</b>’”.</p> <p align="justify">Reserve 6 shell vials for use as negative and positive controls as follows:</p> <p align="justify"> </p> <p align="justify">A. <u>Negative Controls for this week, also become Positive Old Lot for following week (3 Vials)</u></p> <p align="justify">These are incubated at 36<sup>o</sup>C and observed daily for one week or more to identify toxicity and contamination originating with the vendor. Results are recorded on the QC chart.</p> <p align="justify">Three shell vials are reserved to QC the next shipment in parallel after the cells are added. </p> <p align="justify"> </p> <p align="justify">B. <u>Positive Controls</u> (3 Vials)</p> <p align="justify">Each week, usually 2-3 days after seeding the shell vials, HFF tubes containing HSV-1 (ATCC VR-5539), HSV-2 (ATCC VR-540) and CMV (ATCC VR-807) are scraped from and used to inoculate 6 MRC-5 shell vials, 3 from the current lot and 3 from the previous lot (the same 3 vials were used as Negative Controls for a week). The shell vials are then stained with HSV-1, HSV-2 and CMV IEA monoclonal antibodies after 1 day incubation. Record the results into the LIS daily QC under the following codes:</p> <p align="justify">a. VHSV1D HSV1 daily SLIDE SV QC</p> <p align="justify">b. VHSV1D HSV1 daily SLIDE SV QC</p> <p align="justify">c. VCMV-D CMV-IE daily SLIDE SV QC</p> <p align="justify">d. VQCSV Shell Vial MRC-5 Quality Control</p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-91853920363283094862011-08-26T00:17:00.001+07:002011-08-26T00:17:51.171+07:00CRYOPRESERVATION OF VIRUS ISOLATES (Appendix XII)<p align="justify">1. Record in the computer Freezer Program details of the isolate stored.</p> <p align="justify">a. Select "A. Clinical Records".</p> <p align="justify">b. "Add new entries?": enter "Yes".</p> <p align="justify">c. Enter lab number, ensure patient data is correct, enter date frozen, enter study (ie "VIR"), ensure specimen type is correct.</p> <p align="justify">d. Enter name of isolate to be frozen once.</p> <p align="justify">e. Note the freezer locations in which the frozen isolates are to be placed.</p> <p align="justify">- Freezer "G" & "J" refer to Liquid Nitrogen freezers.</p> <p align="justify">- Shelf no. refers to the canister number.</p> <p align="justify">- Box no. refers to the cane number. Each cane holds 5 freezer vials.</p> <p align="justify"> </p> <p align="justify">2. Discard maintenance medium on monolayer to be frozen.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify">3. Add 1.0 mL viral freezing medium (see Appendix VII) to positive cells. Scrape monolayer and mix well with freezing medium.</p> <p align="justify"> </p> <p align="justify">4. Distribute into freezing vial labelled with LIS label.</p> <p align="justify"> </p> <p align="justify">5. Store the vial immediately in liquid nitrogen freezer in the location specified by the freezer program.</p> <p align="justify"> </p> <p align="justify"><strong><u>FREEZING CAP SPECIMENS AND ISOLATES</u>:</strong></p> <p align="justify">1. After planting CAP specimens, add 1 mL of viral freezing medium to the sample and transfer to a freezing vial. Enter into the freezer program as described above using "VQC" for study. Enter "CAPSAM" as isolate #1.</p> <p align="justify"> </p> <p align="justify">2. Freeze one vial of CAP viral isolates after identification using steps 2 - 5 described above. The frozen isolate will be isolate #2 and is entered using the code for the name of the virus. (The original sample and frozen isolate may be on different canes, this is acceptable).</p> <p align="justify"> </p> <p align="justify"><b><u></u></b></p> <p align="justify"><b><u>Reference</u></b></p> <p align="justify">Laboratory Diagnosis of Infectious Diseases. 1988 Volume II. Pg. 268.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-64701327003082169252011-08-26T00:16:00.001+07:002011-08-26T00:16:20.942+07:00RECOVERY OF CRYOPRESERVED CELLS (Appendix XI)<p>Generally research use only:</p> <p>1. Obtain the frozen cells from liquid nitrogen storage and immediately place the vial in a 37<sup>o</sup>C water bath <b>WITH A PROTECTIVE COVER. ** </b>Allow to thaw for 2 minutes (no more than 3 minutes).</p> <p>2. Wipe the outside of the vial with 95% alcohol.</p> <p>3. Transfer the contents of the vial to a new 125 cm<sup>2</sup> flask using a sterile transfer pipette.</p> <p>4. Gradually add 30 mL growth medium to the cells (slowly over 2 minutes) to dilute the cells.</p> <p>5. Incubate at 37<sup>o</sup>C and observe at 24 hours for cell adherence and growth.</p> <p>6. Discard old medium and refeed cells with 30 mL of growth medium at 24 hours to remove all traces of DMSO and reincubate the cells.</p> <p>7. Replace growth medium with maintenance medium when the monolayer is confluent (usually after 2-4 days).</p> <p>8. Replace maintenance medium with fresh maintenance medium once a week.</p> <p><b><u>Reference</u></b></p> <p>Isenberg, HD: Clinical Microbiology Procedures Handbook. American Society of Microbiology, 1992. Pg. 8.20.7.</p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-90732737877180179422011-08-26T00:14:00.001+07:002011-08-26T00:14:24.748+07:00CRYOPRESERVATION OF CELL CULTURES (Appendix X)<p align="justify">Generally research use only:</p> <p align="justify">1. When a new bottle of cells to be frozen arrives, transfer 30 mL aliquots of cells into 125 cm<sup>2</sup> flasks (about 3 flasks).</p> <p align="justify">2. Incubate at 36<sup>o</sup>C until a complete monolayer is obtained (approximately 4 - 5 days).</p> <p align="justify">3. Follow steps 1 - 5 in Appendix IX (Trypsinization and maintenance of monolayer cell cultures).</p> <p align="justify">4. Transfer the trypsinized monolayer to a sterile 15 mL centrifuge tube.</p> <p align="justify">5. Centrifuge at 2000 rpm (700 x <i>g</i>) for 10 minutes.</p> <p align="justify">6. Discard the supernate and resuspend the cells in 10 mL cell freezing medium.</p> <p align="justify">7. Distribute 1.0 mL volume into freezing vials labelled with cell line information.</p> <p align="justify">8. Place the vials into a "Mr. Frosty" freezing container and hold in -70<sup>o</sup>C freezer for 2 - 3 hours.</p> <p align="justify">9. Record in the computer Freezer Program details of the cell lines stored. Use study "CEL".</p> <p align="justify">10. Transfer the vials to liquid nitrogen freezer.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b><u>Reference</u></b></p> <p align="justify">Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infection. A.P.H.A. 1989. Sixth Edition. Pg. 72.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-45677267007150154162011-08-26T00:12:00.001+07:002011-08-26T00:12:48.460+07:00TRYPSINIZATION AND MAINTENANCE OF MONOLAYER CELL CULTURES (Appendix IX)<p align="justify">Monolayer cell cultures may be kept active and available for seeding of tubes, vials, dishes and plates. A constant supply of cell cultures can be maintained by routine subpassage of cell lines to new flasks. (Generally research use only.)</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify">1. Discard medium from the cell culture flask (125 cm<sup>2</sup>). Rinse monolayer with 15 mL of Hank's Balanced Salt Solution and discard.</p> <p align="justify">2. Add 5 mL of trypsin EDTA mixture (pre-warmed to 36<sup>o</sup>C) to flask.</p> <p align="justify">3. Incubate the culture flask for 3 minutes (no more than 5 minutes) at 36<sup>o</sup>C. Observe after 3 minutes to see whether the cell sheet is breaking loose from the flask surface. Tap flask sharply against palm of hand to aid in loosening tissue.</p> <p align="justify">4. When tissue has loosened completely, add 15 mL of pre-warmed growth medium into the flask.</p> <p align="justify">5. Mix cells by drawing cells and fluid up and down in a pipette. </p> <p align="justify">6. Adjust the volume to 90 mL with growth medium.</p> <p align="justify">7. Aliquot 30 mL of suspended cell mixture into at least one new 125 cm<sup>2</sup> culture flask. Aliquot the remaining to shell vials at 1.5 mL each or tube culture (16 x 125 mm) at 2 mL each.</p> <p align="justify">8. Incubate the culture flasks, shell vials or tube cultures at 36<sup>o</sup>C.</p> <p align="justify">9. Observe daily for growth of cells (3 - 5 days) and for change in pH of medium. If the medium becomes acidic or basic, replace it with fresh growth medium.</p> <p align="justify">10. Replace growth medium with maintenance medium when a confluent monolayer is obtained (usually after 2 - 3 days).</p> <p align="justify">11. Replace with fresh maintenance medium once a week.</p> <p align="justify"><b></b></p> <p align="justify"><b>Note:</b> Freeze first passage of cell culture whenever a new shipment is received. (See Appendix VIII).</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-36069823394340140412011-08-26T00:11:00.001+07:002011-08-26T00:11:34.116+07:00Appendix VIII MEDIA<p> <p><b></b><b></b></p> <p><b></b></p> <b><u>GROWTH MEDIUM</u></b> (Not in routine use)</p> <p> </p> <p>500 mL Eagles' MEM with Hanks' salt/without glutamine</p> <p>10 mL Vitamins</p> <p>5 mL L-glutamine (200 mM)</p> <p>50 mL Inactivated fetal calf serum</p> <p>Store at 4<sup>o</sup>C. Stable for 2 weeks.</p> <p><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p> </p> <p><b><u>MAINTENANCE MEDIUM</u></b> (Made in-house, not in routine use)</p> <p>500 mL Eagles' MEM with Hanks' salt/without glutamine (stored at 4<sup>o</sup>C)</p> <p>5 mL L-glutamine (200 mM, stored frozen at -20<sup> o</sup>C in clean room freezer)</p> <p>10 mL Inactivated fetal calf serum (stored frozen at -20<sup> o</sup>C in clean room freezer)</p> <p>5 mL Fungizone (250 μg/mL, stored frozen at -20<sup> o</sup>C in clean room freezer)</p> <p>*5 mL Gentamicin (1 mg/mL, stored frozen at -20<sup> o</sup>C in clean room freezer)</p> <p>**5 mL Vancomycin (10 mg/mL, stored frozen at -20<sup> o</sup>C in clean room freezer)</p> <p>Store at 4<sup>o</sup>C. Stable for 2 weeks.</p> <p> </p> <p>*<b> Gentamicin Solution (1 mg/mL):</b></p> <p>Dilute entire contents (10 mL) of 10 mg/mL Gentamicin Sulphate vial into 90 mL of d. H<sub>2</sub>O to achieve 1 mg/mL. Filter sterilize, then dispense into 5 mL aliquots (stored frozen at -20<sup> o</sup>C in clean room freezer).</p> <p> </p> <p>**<b> Vancomycin Solution (10 mg/mL):</b></p> <p>Add contents of 1 gram vial of Vancomycin to 100 mL of distilled H<sub>2</sub>O. Filter sterilize, then dispense into 5 mL aliquots (stored frozen at -20<sup> o</sup>C in clean room freezer.</p> <p> </p> <p><b><u>MAINTENANCE MEDIUM</u></b><b> (Diagnostic Hybrids Inc., ready to use)</b></p> <p><strong></strong></p> <p><u></u></p> <p><b>Media Quality Control:</b></p> <p><b></b></p> <p><b><i></i></b>a. A 1.5 mL aliquot each of the corresponding maintenance media is to be placed into each of MRC-5, R-Mix and E-Mix shell vials for a check on sterility/toxicity before the media are used on patient samples.</p> <p>b. MRC-5, R-Mix and E-Mix Maintenance Media are to be registered into the LIS (micqc) as “on receipt” with lot numbers and expiry dates.</p> <p>c. Maintenance Media QC results are all entered into the LIS daily (R-Mix for 2 days, MRC-5 for 4 days and E-Mix for 5 days).</p> <p>d. Activate and inactivate the media lots in the LIS as appropriate.</p> <p>Failed Media QC: </p> <p>a. Any media/cell lines showing microbial contamination or toxicity is considered abnormal.</p> <p>b. Do not release media lot for use pending resolution of the QC failure.</p> <p>c. Inform charge/senior technologist to discuss further actions including the following.</p> <p>d. Record in Incident Report (if necessary).</p> <p>e. Repeat QC/re-make media to evaluate if it is the cell media, the supplement(s) or other materials causing the problem.</p> <p> </p> <p><b><u>CELL</u></b><b><u> WASHING MEDIUM</u></b> (Purchased)</p> <p>Hanks' Balanced Salt solution without CaCL<sub>2</sub>, MgCL<sub>2</sub> and MgSO<sub>4</sub>.7H<sub>2</sub>O</p> <p> </p> <p><b><u>CELL</u></b><b><u> FREEZING MEDIUM</u></b> (SIGMA C6164) (Purchased)</p> <p>MEM supplemented with a mixture of fetal bovine serum and calf serum containing DMSO.</p> <p> </p> <p><b><u>VIRAL FREEZING MEDIUM</u></b> (SIGMA C6039) (Purchased)</p> <p>MEM supplemented with a mixture of fetal bovine serum and calf serum containing 10% glycerol.</p> <p> </p> <p><b><u>TRANSPORT MEDIA</u></b>:</p> <p> </p> <p><b><u>BARTEL'S VIRAL TRANSPORT MEDIUM</u></b> (B1029-36A) (Purchased)</p> <p>Eagles' MEM with nonessential amino-acid and L-glutamine in HBSS.</p> <p>2% FBS</p> <p>10 μg/mL Gentamicin</p> <p>50 μg/mL Streptomycin</p> <p>50 μg/mL Penicillin</p> <p>4 μg/mL Amphotericin B</p> <p>15 mM HEPES</p> <p>9.5 mM Sodium bicarbonate</p> <p> </p> <p><b></b><b><u>CHLAMYDIA TRANSPORT MEDIUM</u></b> (Not in routine use)</p> <p>500 mL Eagles' MEM with Hank's salt/without glutamine.</p> <p>50 mL Fetal Calf Serum (normal)</p> <p>5 mL 3M Glucose</p> <p>5 mL Gentamicin (1000 μg/mL)</p> <p>5 mL Vancomycin (1000 μg/mL)</p> <p>5 mL Fungizone (250 μg/mL)</p> <p>Combine all components aseptically. Dispense 1.5 mL transport medium into sterile screw-cap vials containing 3 glass beads. Store at -20<sup>o</sup>C.</p> <p><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-46667466411997182802011-08-26T00:10:00.001+07:002011-08-26T00:10:01.850+07:00HEMADSORPTION OF TUBE CULTURE MONOLAYERS (NOT ROUTINELY DONE) Appendix VII<p align="justify">The hemadsorption (HAD) technique is used primarily to detect viruses that produce little or no cytopathic effect (CPE) in tube culture monolayers. Using guinea pig RBC, it is used to screen inoculated cell cultures for the presence of influenza, parainfluenza, mumps and Newcastle disease viruses.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b><font size="3">I. <u>Procedure</u></font></b></p> <p align="justify"><strong><u><font size="3"></font></u></strong></p> <p align="justify"><b>Reagents</b></p> <p align="justify">guinea pig RBC in Alsever's solution</p> <p align="justify">sterile phosphate buffered saline(PBS)</p> <p align="justify"> </p> <p align="justify"><b>Materials</b></p> <p align="justify">sterile pipettes</p> <p align="justify">precision pipettes</p> <p align="justify">inverted microscope</p> <p align="justify">centrifuge</p> <p align="justify">15 mL sterile centrifuge tube</p> <p align="justify"> </p> <p align="justify"><b>Preparation of 10% stock guinea pig RBC suspension</b></p> <p align="justify">The stock suspension should be prepared every Monday and stored at 4<sup>o</sup>C and used within 7 days of preparation.</p> <p align="justify">1. Transfer 5 mL of blood to 15 mL tube and add equal volume of PBS.</p> <p align="justify">2. Centrifuge at 3000 rpm (700 x <i>g</i>) for 5 minutes at room temperature.</p> <p align="justify">3. Discard the supernate and add 10 mL of PBS.</p> <p align="justify">4. Centrifuge and wash the cells until the supernatant is clear. (approx. 2-3 washings). </p> <p align="justify">5. Determine the packed-cell volume and add PBS equal to 9 times the packed-cell volume to yield a 10% suspension.</p> <p align="justify"> </p> <p align="justify"><b>Preparation of 0.4% working guinea pig RBC suspension</b></p> <p align="justify">The working suspension should be prepared from 10% suspension on the day of testing.</p> <p align="justify">1. Add 0.4 mL of the 10% suspension to 9.6 mL of PBS.</p> <p align="justify"> </p> <p align="justify"><b>HAD Test</b></p> <p align="justify">The HAD test is performed on day 5 and day 10 for respiratory specimens with no evidence of CPE.</p> <p align="justify">1. Select one RMK tube to be used for HAD. Transfer the medium in the tube to another sterile, labelled capped tube. Place medium at 4<sup>o</sup>C pending HAD results.</p> <p align="justify">2. Add 0.2 mL of the 0.4% RBC suspension to each culture tube, to be tested.</p> <p align="justify">3. Incubate the tubes horizontally at 4<sup>o</sup>C for 30 minutes. Make sure the RBC suspension is distributed over the monolayer.</p> <p align="justify">4. Gently rotate or tap the tubes to resuspend nonadsorbed cells. Immediately examine the tubes with inverted microscope with the 40X objective. Do not handle the tubes in such a way that the monolayers will become warm. </p> <p align="justify"> </p> <p align="justify"><b>Interpretation of Results</b></p> <p align="justify">1. Positive HAD test should show RBCs firmly attached to the monolayer. Hemagglutinated cells (clumped RBCs) are also seen in the fluid overlaying the monolayer.</p> <p align="justify">2. Negative HAD test should show no or minimal RBCs attached to the monolayers, with almost all cells floating above the monolayers.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">II. <u>Quality Control</u></font></b></p> <p align="justify">Positive and negative controls should be set up for HAD test prior to the expected "flu" season.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">III. Positive HAD Test</font></b></p> <p align="justify">1. Place the tube in a 36<sup>o</sup>C water bath for 15 minutes.</p> <p align="justify">2. Wash the eluted monolayers twice with PBS.</p> <p align="justify">3. Perform indirect immunofluorescence test for influenzae and parainfluenzae viruses. See Appendix III.</p> <p align="justify">4. The culture medium harvested prior to the HAD test may be used for subpassage, storage or identification by hemagglutination inhibition.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">IV. Negative HAD Test</font></b></p> <p align="justify">1. Discard tube that is HAD negative at 10 days after inoculation.</p> <p align="justify">2. Tube inoculated after 5 days should be washed 3 times with 5 mL Hank's Balanced Salt Solution to remove all RBCs.</p> <p align="justify">3. Refeed tubes with 2 mL of maintenance medium and reincubate the cultures for another 5 days.</p> <p align="justify">4. Discard the culture fluid harvested from negative cultures unless subpassage is to be performed.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">V. <u>Reference</u></font></b></p> <p align="justify">Isenberg, H.D. 1992. Clinical Microbiology Procedures Handbook. Vol. 2. ASM.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-42587129066420530552011-08-26T00:03:00.001+07:002011-08-26T00:03:22.142+07:00DIRECT ANTIGEN DETECTION FROM SPECIMENS (Appendix VI)<p align="justify"><font size="3"><b><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi4hxr0Pq-Fk_f1n7_8MHy2sw9IV-lXMlHKVmyzVV4g986luU46euvTAHW3xdg25LIK4UIhjPeC6AxROWGIdjf7cFdnHjkJaDjMeMTwWsy7bAHOZPVBNqwfuObqZDS5rgSEWU086mLWc8I/s1600-h/elisa_principle2.jpg"><img style="border-right-width: 0px; display: inline; border-top-width: 0px; border-bottom-width: 0px; margin-left: 0px; border-left-width: 0px; margin-right: 0px" title="elisa_principle" border="0" alt="elisa_principle" align="left" src="http://lh6.ggpht.com/-IMDYPKFBJ-E/TlaAECi7tII/AAAAAAAABK8/JiVg_OWWDNg/elisa_principle_thumb.jpg?imgmax=800" width="244" height="132" /></a> I. </b><b><u>Introduction</u></b></font></p> <p align="justify"><b></b></p> <p align="justify">Immunofluorescent staining may be used to rapidly detect viral antigen directly in a clinical specimen. Smears prepared at the patient's bedside may be used, or a smear may be prepared in the laboratory from cellular material in a specimen. Specimens for which direct antigen detection may be requested include bronchoscopy or nasopharyngeal specimens for respiratory virus detection or vesicular lesion scraping for HSV/VZV antigen detection. Cells are stained using pooled or specific monoclonal antibody stains and examined under the fluorescence microscope looking for specific fluorescence of cell cytoplasm or nucleus.</p> <p><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000" size="2">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"><font size="3"><b>II.</b> <b><u>Reagents and Materials</u> </b></font></p> <p align="justify">Virus-specific or pooled FITC and Rodamine conjugated antibody stains (with Evans blue counter stain):</p> <p align="justify">Respiratory Viral Screen/RSV panel</p> <p align="justify">FluA/B panel</p> <p align="justify">RSV/para3 panel</p> <p align="justify">Para1,2,3/Adeno panel</p> <p align="justify">Specific Parainfluenza 1</p> <p align="justify">Specific Parainfluenza 2</p> <p align="justify">Herpes simplex 1</p> <p align="justify">Herpes simplex 2</p> <p align="justify">Herpes simplex bivalent</p> <p align="justify">Varicella zoster virus</p> <h3 align="justify">Phosphate buffered saline (PBS)</h3> <p align="justify">Cold acetone (4<sup>o</sup>C)</p> <p align="justify">Distilled water</p> <p align="justify">FA mounting fluid</p> <p align="justify">Vortex</p> <p align="justify">Sterile pipettes</p> <p align="justify">Cytospin and accessories</p> <p align="justify">Humidified chamber</p> <p align="justify">Coplin jars</p> <p align="justify">Fuorescence microscope with FITC/Rodamine/Evans blue filters</p> <h4 align="justify"> </h4> <h4 align="justify"><strong>II. <u>Procedure</u></strong></h4> <p align="justify"> </p> <p align="justify"><b>A. Preparation of slide</b></p> <p align="justify">a) For swabs:</p> <p align="justify">i. Vortex patient sample in transport medium for 30 seconds.</p> <p align="justify">Remove excess fluid from the swab and discard the swab.</p> <p align="justify">ii. Transfer 0.5 - l.0 ml of this specimen to a microcentrifuge tube. Centrifuge 1 minute at 14,000 rpm.</p> <p align="justify">iii. Remove supernatant (can be placed back with original specimen to be processed further) down to 400 ul (8 drops). Vortex 5-10 seconds.</p> <p align="justify">iv. Pipette 200 ul (4 drops) of this sediment into funnel for each well. Prepare the appropriate number of cytospin wells according to table below.</p> <p align="justify">v. Cytospin at 2000 rpm (700g) for 5 minutes.</p> <p align="justify">vi. Remove slide and air dry.</p> <p align="justify">vii. Fix in cold acetone for 10 minutes in a coplin jar.</p> <p align="justify">viii. Remove slide and air dry.</p> <p align="justify">ix. Proceed to staining. Refer to Appendix V (DFA) for staining procedures.</p> <p align="justify"> </p> <p align="justify">b) For Bronchoscopy Specimens (BAL,Washings):</p> <p align="justify">i. Mix specimen gently and examine for cellular turbidity. Specimens which are more turbid than a 0.5 McFarland standard are diluted to that approximate turbidity using Hank's Balanced Salt Solution (Gibco BRL).</p> <p align="justify">ii. Pipette 200 ul (4 drops) of specimen into funnel for each well. Prepare the appropriate number of cytospin wells according to table below.</p> <p align="justify">iii. Cytospin 200 uL at 2000 rpm (700 x g) for 5 minutes. Prepare the appropriate number of cytospin preparations according to the table below.</p> <p align="justify">iv. Remove slide and air dry.</p> <p align="justify">v. Fix in cold acetone 10 minutes in coplin jar.</p> <p align="justify">vi. Remove and air dry.</p> <p align="justify">vii. Proceed to staining. Refer to Appendix V (DFA) for staining procedures.</p> <p align="justify"> </p> <p align="justify">c) For Smears Prepared Outside of the Laboratory</p> <p align="justify">For smears which have been prepared outside of the laboratory, examine macroscopically for evidence of material on slide. Specimens for which only one slide is received may be stained with <u>ONE</u> monoclonal antibody <u>only</u> according to the physician's request. It is not possible to stain a negative control for such specimens. <u>A swab for viral culture</u> should be requested if not received along with the smear.</p> <p align="justify">Draw a circle around material on slide with a diamond pencil prior to staining.</p> <p align="justify">i. Fix in cold acetone 10 minutes in coplin jar.</p> <p align="justify">ii. Remove and air dry</p> <p align="justify">iii. Proceed to staining. Refer to Appendix V (DFA) for staining procedures. </p> <p align="justify"> </p> <p align="justify"><b>1. </b><b>Staining Protocol</b></p> <p align="justify"><b></b></p> <p align="justify">Prepare appropriate number of cytospin wells and stain as per chart. Refer to Appendix V (DFA) for staining procedures.</p> <p align="justify"> <table border="0" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="200"> <p align="center"><strong>Specimen</strong></p> </td> <td valign="top" width="258"> <p align="center"><strong>Monoclonal Antibody</strong></p> </td> <td valign="top" width="188"> </td> </tr> <tr> <td valign="top" width="200"> <p align="left">Nasopharyngeal/</p> <p align="left">Bronchoscopy/</p> <p align="left">Throat</p> </td> <td valign="top" width="258"> <p align="left">Respiratory screen <sup>a</sup></p> <p align="left">(November to April)</p> </td> <td valign="top" width="188"> <p align="left">SimulFluor, Light Diagnostics</p> </td> </tr> <tr> <td valign="top" width="200"> <p align="left">(prepare 2 cytospin wells)</p> </td> <td valign="top" width="258"> <p align="left">Flu A/ Flu B </p> </td> <td valign="top" width="188"> <p align="left">SimulFluor, Light Diagnostics</p> </td> </tr> <tr> <td valign="top" width="200"> <p align="left">Vesicular Lesion Swab</p> </td> <td valign="top" width="258"> <p align="left">HSV-bivalent</p> </td> <td valign="top" width="188"> <p align="left">Bartels</p> </td> </tr> <tr> <td valign="top" width="200"> <p align="left">(prepare 2 cytospin wells)</p> </td> <td valign="top" width="258"> <p align="left"></p> </td> <td valign="top" width="188"> <p align="left"></p> </td> </tr> <tr> <td valign="top" width="200"> <p align="left"></p> </td> <td valign="top" width="258"> <p align="left">VZV</p> </td> <td valign="top" width="188"> <p align="left">Meridan Bioscience Inc.</p> </td> </tr> </tbody></table> </p> <p align="justify"><sup>a</sup> If respiratory screen or Flu A/B is positive, prepare more cytospin preparations and stain according to interpretation chart that follows.</p> <p align="justify"><b><u></u></b></p> <p align="justify"><b><u>SimulFluor Respiratory Screen Staining Protocol and Interpretations – Scheme 1</u></b></p> <p align="justify"><b><u></u></b></p> <p align="left">Using filter for FITC/Evans Blue (Filter no. 3 in fluorescence microscope no.2, Leica I)</p> <p>First double-well cytospin slide </p> <p align="justify"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgg1ciWiZOLj5trJgOq6BiFcexCqJYSmu9H1ojocoHlXasB2AmDFSbIIlGcfakcjw50PF5Vc-BAr0CAjQqolmYSxGPYO3t4n1DrsrRMpdNbSGbyCkSbzf3mSOHATrXjeHUqNHKan8uzCqI/s1600-h/image.png"><img style="border-bottom: 0px; border-left: 0px; display: block; float: none; margin-left: auto; border-top: 0px; margin-right: auto; border-right: 0px" title="image" border="0" alt="image" src="http://lh6.ggpht.com/-SGRpFfrpUmg/TlaAMq3NocI/AAAAAAAABLE/F4jeEyYn1kI/image_thumb.png?imgmax=800" width="580" height="529" /></a> </p> <p><b><u>SimulFluor Respiratory Screen Staining Protocol and Interpretations- Scheme 2</u></b></p> <p><b><u></u></b></p> <p>Using filter for FITC/Evans Blue (Filter no. 3 in fluorescence microscope no.2, Leica I)</p> <p>First double cytospin slide </p> <p align="justify"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjsbPXOuYaXByPDgNUXp9FqfoPXT1-0UzI_BlfSsB6qFH4lFK-7gmqR09V7NnDpDFySnXV542AaqKFv8PYprpfqOacHbB33QP8ceV2PYEo-5VmbL9HCSGhMnMe0o-QnyA3j5so39ypFY-0/s1600-h/image%25255B1%25255D.png"><img style="border-bottom: 0px; border-left: 0px; display: block; float: none; margin-left: auto; border-top: 0px; margin-right: auto; border-right: 0px" title="image" border="0" alt="image" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhz5v7_rteo6xMOWHIkxd7xFRfY3KW8_qpEhfQcOFOiOr10-WASB_tQfPRnSFgYGzjyo-AEtDT31NIuA-YZhEX7rxH-6KmIzjMEPXWd0uoWYQlbZQNnU9mkJVCjZ-ex5NvJ11_xGml14L4/?imgmax=800" width="580" height="536" /></a> </p> <p align="justify"><font size="3"><b>III</b><b>. Interpretation of Results</b></font></p> <p align="justify">Examine smear for adequate numbers and types of cells. Respiratory specimens should consist of columnar (ciliated or goblet) epithelial cells. Scrapings from lesions should contain basal epithelial cells. (See reference 2, page 69). <b><u>Adequate interpretation of results requires a minimum of approximately 20-50 cells per smear.</u></b> Samples with inadequate numbers of cells should be shown to a charge/senior technologist and reported as having insufficient cellular material.</p> <p align="justify">Positive: Specific, apple-green fluorescence in the cytoplasm and/or nucleus of the exfoliated cells. This specific fluorescence must be absent in the negative control and/or smears stained with other antibodies.</p> <p align="justify">Negative: Dull-red stained cells with no viral specific apple-green fluorescence.</p> <p align="justify">OR</p> <p align="justify">Dull-red stained cells with pinpoint non-specific nuclear staining. </p> <p align="justify">Insufficient cells: Fewer than 20 epithelial cells per smear</p> <p align="justify"> </p> <p align="justify"><b><font size="3">IV. <u>Quality Control</u></font></b></p> <p align="justify"><strong><u><font size="3"></font></u></strong></p> <p align="justify"><strong><u>Reagent</u><u> QC</u>:</strong></p> <p align="justify">a. Check expiratory date. </p> <p align="justify">b. Verify that Reagent QC is satisfactory for the reagent lot/kit being used (recorded in Reagent Log and/or on the kit). </p> <p align="justify">c. If necessary, perform the Reagent QC procedure ( external QC slide with all components eg. Bion 14-well Respiratory Panel). </p> <p align="justify"> </p> <p align="justify"><u><strong>Failed Reagent QC:</strong></u></p> <p align="justify">Test is invalid without satisfactory Reagent QC results.</p> <p align="justify">a. Do not release reagent lot for use pending resolution of QC error.</p> <p align="justify">b. Inform charge/senior technologist.</p> <p align="justify">c. Record in Reagent Log Chart, Instrument Maintenance Log if microscope/incubator is involved in the failure (and Incident Report if necessary).</p> <p align="justify">d. Re-run failed control materials in parallel to fresh controls to evaluate the QC material itself.</p> <p align="justify">a. If the re-run shows the old QC material still fails and fresh QC is satisfactory, the error may be attributed to the old QC material itself and the reagent is satisfactory.</p> <p align="justify">b. If the re-run shows both the old and fresh QC material fail (or other QC not satisfactory), the error may be attributed to the reagent then the reagent cannot be released for use.</p> <p align="justify"> </p> <p align="justify"><strong><u>Daily QC</u>:</strong></p> <p align="justify">a. Appropriate positive and negative control slides (eg. ATCC 4-well slide with RSV/Para3 for SimulF RS stain) should be stained with each batch.</p> <p align="justify">b. Examine the negative control well first to establish the dull red colour (Evans blue counterstained) and to determine if there is any nonspecific staining.</p> <p align="justify">The positive control must be clearly distinguishable from the negative control or the test is invalid.</p> <p align="justify"> </p> <p align="justify"><strong><u>Failed Daily QC</u>:</strong></p> <p align="justify">a. Do not release patient results pending resolution of QC error.</p> <p align="justify">b. Inform charge/senior technologist.</p> <p align="justify">c. Record in Reagent Log Chart (and Instrument Maintenance Log if microscope/incubator is causing the failure).</p> <p align="justify">d. Re-run failed controls in parallel to fresh controls (and/or external QC) to evaluate the QC material itself.</p> <p align="justify">e. If the re-run shows the old QC material still fails and fresh QC passes and nothing else is wrong with the batch (only the old QC material failed, patient results valid) patient results may be released.</p> <p align="justify"> </p> <p align="justify">Marked decrease/absence in fluorescence can be due to:</p> <p align="justify">a. Reagent deterioration/skipping (did not apply the correct stain)</p> <p align="justify">b. Microscope (filter, bulb, alignment)</p> <p align="justify">c. Other equipment, reagents or technique</p> <p align="justify"><b><font size="3"></font></b></p> <p align="justify"><b><font size="3">V. <u>Reporting</u></font></b></p> <p align="justify">See individual specimen protocols.</p> <p align="justify"><b></b></p> <p align="justify"><b><font size="3">VI. <u>Reference</u></font></b></p> <p align="justify">1. Wiedbrauk, D.L. 1993, Raven Press. Manual of Clinical Virology.</p> <p align="justify">2. Rossier, E., Miller, H., Phipps, P. 1989, University of Ottawa Press. Rapid Viral </p> <p align="justify">Diagnosis by Immunofluorescence: An Atlas & Practical Guide.</p> <p align="justify">3. SimulFluor Product Insert for cat. no. 3296, June 2002, Revision C: 40729</p> <p align="justify">Light Diagnostics Chemicon International Temecula, CA 92590</p> <p align="justify"></p> <p></p> <p></p> <p><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000" size="2">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com1tag:blogger.com,1999:blog-7259934002090567583.post-44903753939825892412011-08-25T23:37:00.001+07:002011-08-25T23:37:03.784+07:00DIRECT IMMUNOFLUORESCENT ANTIBODY (DFA) STAINING (Appendix V )<p align="justify"> <p align="justify"><b></b></p> <p align="justify"><b></b></p> <b><font size="3"><a href="http://lh4.ggpht.com/-ogNYdUJL8w4/TlZ6Im1_fXI/AAAAAAAABKg/yiBt26TFSc0/s1600-h/apendix%2525205%25255B2%25255D.jpg"><img style="border-bottom: 0px; border-left: 0px; display: inline; margin-left: 0px; border-top: 0px; margin-right: 0px; border-right: 0px" title="apendix 5" border="0" alt="apendix 5" align="left" src="http://lh4.ggpht.com/-Rg7wMmVaVmY/TlZ6LJytuCI/AAAAAAAABKk/HS_eW-CJ3rA/apendix%2525205_thumb.jpg?imgmax=800" width="244" height="164" /></a> I. <u>Introduction</u></font></b></p> <p align="justify">The DFA staining technique is used to detect viruses either directly in patient specimens or which have been isolated in shell vial or tube cultures. The method consists of a single staining step using a virus-specific antibody which is conjugated with a fluorochrome. Viruses which we currently identify by DFA staining include HSV-1, HSV-2, VZV, CMV (late antigen) and respiratory viruses (SimulFluor stains for respiratory syncytial virus, parainfluenza, influenza, adenovirus).</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b><font size="3">II. <u>Reagents and Materials</u> </font></b></p> <p align="justify">FITC-conjugated virus-specific antibody</p> <p align="justify">FITC/Rodamine-conjugated virus-specific antibody (SimulFluor)</p> <p align="justify">Phosphate Buffered Saline (PBS)</p> <p align="justify">dH<sub>2</sub>O</p> <p align="justify">cold acetone (4<sup>o</sup>C)</p> <p align="justify">mounting fluid</p> <p align="justify">sterile pipettes</p> <p align="justify">cytospin and accessories (for tube culture)</p> <p align="justify">humidified chamber</p> <p align="justify">glass slides</p> <p align="justify">coverslips</p> <p align="justify">paper towels for blotting</p> <p align="justify"> </p> <h4 align="justify"><strong>III. <u>Procedure</u></strong></h4> <p align="justify"> </p> <p align="justify"><b></b></p> <p align="justify"><b>1. </b><b>Shell Vial</b></p> <p align="justify"><b></b></p> <p align="justify">This procedure is for staining of cells directly in shell vial. If staining a cytospin slide or slide made directly from a patient specimen, follow the tube culture procedure below.</p> <p align="justify">i. Discard cap. Remove maintenance medium from the shell vial using sterile pipette.</p> <p align="justify">ii. Add 1 mL of cold acetone. Cover with tray lid and let sit for 10 minutes.</p> <p align="justify">iii. Decant acetone and blot shell vial on paper towel.</p> <p align="justify">iv. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS. </p> <p align="justify">v. Add 75ml (2 drops from bottle) of appropriate FITC or Rodamine-conjugated virus-specific antibody. Cover with tray lid.</p> <p align="justify">vi. Incubate at 36<sup>o</sup>C for 30 minutes.</p> <p align="justify">vii. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS. Repeat.</p> <p align="justify">viii. Remove the coverslip from each shell vial and place cell side down onto a drop of mounting fluid on a glass slide.</p> <p align="justify">ix. For HSV 1, HSV 2, VZ and CMV, read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.</p> <p align="justify">x. For respiratory viruses, read using fluorescence microscope with the FITC/Evans Blue Rodamine bi-filter and the 40x objective.</p> <p align="justify"> </p> <p align="justify"><b>2. Tube Culture</b></p> <p align="justify">i. Prepare cytospin slide from cell culture tube as outlined in Appendix XX.</p> <p align="justify">ii. Fix slide in cold acetone for 10 minutes in a coplin jar. Remove slide and air dry.</p> <p align="justify">iii. Add 20ml of appropriate FITC or Rodamine -conjugated antibody onto the fixed cytospin slide.</p> <p align="justify">iii. Incubate in a humidified chamber at 36<sup>o</sup>C for 30 minutes.</p> <p align="justify">iv. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.</p> <p align="justify">v. Wash with distilled water for 1 minute in a coplin jar.</p> <p align="justify">vi. Wipe excess water from the slide without touching the cytospin preparation.</p> <p align="justify">vii. Mount using coverslip and mounting fluid.</p> <p align="justify">xi. For HSV 1, HSV 2, VZ and CMV, read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.</p> <p align="justify">viii. For respiratory viruses, read using fluorescence microscope with the FITC/Evans Blue Rodamine bi-filter. </p> <p align="justify"> </p> <p align="justify"><b>Interpretation of Results</b></p> <p align="justify">Positive: Bartel CMV monoclonal antibody: Bright apple green fluorescence of cytoplasmic inclusion (late antigen) and homogenous early nuclear antigen in CMV-CPE cells.</p> <p align="justify">Chemicon SimulFluor Respiratory Screen:</p> <p align="justify">All respiratory viruses except RSV show bright apple green fluorescence of the cytoplasm and/or nucleus of the infected cell.</p> <p align="justify">RSV shows bright gold fluorescence of the cytoplasm and/or nucleus of the infected cell.</p> <p align="justify">Chemicon SimulFluor Flu A/Flu B:</p> <p align="justify">Influenzae A virus shows bright apple green fluorescence.</p> <p align="justify">Influenzae B virus shows bright gold fluorescence.</p> <p align="justify">Chemicon SimulFluor RSV/Para 3:</p> <p align="justify">RSV virus shows bright apple green fluorescence.</p> <p align="justify">Parainfluenzae 3 shows bright gold fluorescence.</p> <p align="justify">Chemicon SimulFluor Para 123/Adeno:</p> <p align="justify">Parainfluenza 1,2,3 viruses show bright apple green fluorescence.</p> <p align="justify">Adenovirus shows bright gold fluorescence.</p> <p align="justify">Chemicon individual monoclonal antibodies:</p> <p align="justify">Parainfluenzae 1 and 2, and adenovirus show bright apple green fluorescence.</p> <p align="justify">Negative: Red Cells with no apple-green fluorescence.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">IV. <u>Quality Control</u></font></b></p> <p align="justify">Appropriate positive and negative control slides should be stained with each batch.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">VI. <u>Reference</u></font></b></p> <p align="justify">Isenberg, H.D., 1992, ASM. Clinical Microbiology Procedures Handbook Vol. 2.</p> <p><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-90292813684306289662011-08-25T23:30:00.001+07:002011-08-25T23:30:57.453+07:00INDIRECT IMMUNOFLUORESCENT ANTIBODY (IFA) STAINING (Appendix IV)<p align="justify"> </p> <p align="justify"><b></b></p> <p align="justify"><strong>FOR VIRAL CULTURE CONFIRMATION</strong></p> <p align="justify"><strong></strong></p> <p align="justify"><b></b></p> <p align="justify"><b><font size="3"><a href="http://lh3.ggpht.com/-uYvbJHodP9g/TlZ4mLPYUeI/AAAAAAAABKY/jV1XOokF6ws/s1600-h/imuniflourence%25255B5%25255D.jpg"><img style="border-bottom: 0px; border-left: 0px; display: inline; margin-left: 0px; border-top: 0px; margin-right: 0px; border-right: 0px" title="imuniflourence" border="0" alt="imuniflourence" align="left" src="http://lh4.ggpht.com/-8FeJMdHkGIc/TlZ4uR3o7DI/AAAAAAAABKc/mhP3OMycYsI/imuniflourence_thumb%25255B1%25255D.jpg?imgmax=800" width="244" height="180" /></a> I. <u>Introduction</u></font></b></p> <p align="justify"><b></b></p> <p align="justify">The IFA technique is used to identify viral isolates in the cells obtained from shell vials and tube cultures. The indirect method consists of two steps. In the first step, primary antibodies are allowed to react with viral antigens in the cells. These specific complexes are detected in a second step using a species-specific antibody conjugated with a fluorochrome. Viruses which we currently identify by IFA staining include cytomegalovirus immediate early antigen (CMV-IE) and enteroviruses.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b><font size="3">II. <u>Reagents and Materials</u> </font></b></p> <p align="justify">Virus-specific antibody </p> <p align="justify">FITC-conjugated antimouse antibody</p> <p align="justify">Phosphate buffered saline (PBS) </p> <p align="justify">Distilled water</p> <p align="justify">Cold acetone (4<sup>o</sup>C)</p> <p align="justify">Mounting fluid</p> <p align="justify">Sterile pipettes</p> <p align="justify">Cytospin and accessories (for tube cultures)</p> <p align="justify">Humidified chamber</p> <p align="justify">Sterile freezer vial</p> <p align="justify">Glass slides</p> <p align="justify">Coverslips</p> <p align="justify">Paper towels for blotting</p> <p align="justify">Humidified chamber (for tube culture)</p> <p align="justify"> </p> <p align="justify"><font size="3"><b>III. </b><b><u>Procedure</u></b></font></p> <p align="justify"><strong><u><font size="3"></font></u></strong></p> <p align="justify"><b>1. Shell Vial</b></p> <p align="justify">Follow outline in Appendix II to determine if staining should be done in the shell vial itself or if a cytospin needs to be prepared. If the staining is to be done in the shell vial itself, proceed to step i) below.</p> <p align="justify">i. Discard cap. Remove maintenance medium from the shell vial using a clean sterile pipette.</p> <p align="justify">a) Add 1 mL of cold acetone. Cover and fix for 10 minutes.</p> <p align="justify">b) Decant acetone and blot on paper towel.</p> <p align="justify">c) Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS.</p> <p align="justify">d) Add 75 ml (2 drops from bottle) of appropriate antibody. Cover.</p> <p align="justify">vi. Incubate at 36<sup>o</sup>C for 30 minutes.</p> <p align="justify">vii. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS. Repeat.</p> <p align="justify">viii. Add 75ml (2 drops from bottle) of appropriate FITC -conjugated antibodies, cover and repeat steps vi and vii.</p> <p align="justify">x. Remove the coverslip and place cell side down onto a drop of mounting fluid on a glass slide.</p> <p align="justify">xi. Read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective. </p> <p align="justify"> </p> <p align="justify"><b>2. Tube Culture (or Shell Vials for CPE)</b></p> <p align="justify">i. Prepare cytospin preparation from cell culture as outlined in Appendix XX.</p> <p align="justify">ii. Add 20 ml of appropriate antibodies onto the fixed cytospin slide.</p> <p align="justify">iii. Incubate in a humidified chamber for 30 minutes at 36<sup>o</sup>C.</p> <p align="justify"> </p> <p align="justify">iv. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.</p> <p align="justify">v. Wipe excess PBS from the slide without touching the cell spot.</p> <p align="justify">vi. Add 20 ml of appropriate FITC-conjugated antibodies.</p> <p align="justify">vii. Incubate in a humidified chamber at 36<sup>o</sup>C for 30 minutes.</p> <p align="justify">viii. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.</p> <p align="justify">ix. Wash with distilled water for 1 minute in a coplin jar.</p> <p align="justify">x. Wipe excess water from the slide without touching the cell spot.</p> <p align="justify">xi. Mount using coverslip and mounting fluid.</p> <p align="justify">xii. Read with fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.</p> <p align="justify"> </p> <p align="justify"><b>Interpretation of Results</b></p> <p align="justify">Positive: Enterovirus:</p> <p align="justify">An green fluorescence.</p> <p align="justify">Negative: Red cells with no apple-green fluorescence.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">IV. <u>Quality Control</u></font></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">Appropriate positive and negative control slides should be stained with each batch.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">V. <u>Reporting</u></font></b></p> <p align="justify">See individual specimen protocols.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">VI. <u>Reference</u></font></b></p> <p align="justify">Isenberg, H.D., 1992. Clinical Microbiology Procedures Handbook Vol. 2. ASM Press.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-86257674878383702452011-08-25T23:23:00.001+07:002011-08-25T23:23:17.633+07:00TUBE CULTURE (not in routine use) or SHELL VIALS for CPE PROCEDURE (Appendix III)<p align="justify"><b><font size="3"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhw8WkcJmKDH2WAsgiVmy5ZIV8_MPvwd3hcqQE7r5yaj2RgokF9tBAT_5SHZeQ581d6uhK3Z3X41XeR7kS47iPLMHWBzVEm9pNzNG_v_M4DPLthhvnffSmoAlfHgTeip7WoB8hDYrPoHr8/s1600-h/69763578%25255B2%25255D.png"><img style="border-bottom: 0px; border-left: 0px; display: inline; margin-left: 0px; border-top: 0px; margin-right: 0px; border-right: 0px" title="69763578" border="0" alt="69763578" align="left" src="http://lh4.ggpht.com/-R6XNjhFEAjE/TlZ28FhxJII/AAAAAAAABKU/VkxYuoofn9Q/69763578_thumb.png?imgmax=800" width="104" height="104" /></a> I. <u>Introduction</u></font></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">Tube culture is the conventional method used by diagnostic virology laboratories for virus isolation. Since there is no universal cell line for recovery of all clinically significant viruses, a combination of cell types is used routinely depending on the symptoms, clinical specimen type and specific viruses being sought. Shell Vials (E-Mix ) can also be adapted to extend their normal incubation times and continue as tube cultures.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> <p align="justify"> </p> <p align="justify"><b><font size="3">II. <u>Reagents and Materials</u> </font></b></p> <p align="justify">Fluorescence microscope Leica DBRB with #2 filter for Rodamine/FITC Evans blue and #4 filter for FITC Evans blue or </p> <p align="justify">Fluorescence microscope Leica DC300F with #3 filter for Rodamine/FITC Evans blue and #1 filter for FITC Evans blue</p> <p align="justify">Inverted microscope</p> <p align="justify">Control slides</p> <p align="justify">Virus-specific antibody (eg. Enterovirus D3 stain, DHI)</p> <p align="justify">FITC-conjugated antimouse antibody</p> <p align="justify">Phosphate buffered saline (PBS) </p> <p align="justify">Distilled water</p> <p align="justify">Cold acetone (4<sup>o</sup>C)</p> <p align="justify">Mounting fluid</p> <p align="justify">Sterile pipettes</p> <p align="justify">Cytospin and accessories</p> <p align="justify">Vortex </p> <p align="justify">Sterile freezer vial</p> <p align="justify">Glass slides</p> <p align="justify">Coverslips</p> <p align="justify">Paper towels for blotting</p> <p align="justify">Humidified chamber </p> <p align="justify"> </p> <p align="justify"><font size="3"><b>III</b> <b><u>Procedure</u></b></font></p> <p align="justify">1. Registration</p> <p align="justify">i) Upon receipt in the lab, register cell culture received from the supplier in the lab information system (LIS). Refer to virology LIS manual for procedure.</p> <p align="justify">ii) File the vendor QC sheet (received with the shipment) in the QC binder.</p> <p align="justify">iii) Randomly, select two tubes from each lot and check the monolayer microscopically for confluent growth and quality of cells. Use these two tubes as the “unopened controls” outlined under the Quality Control section below.</p> <p align="justify">iv) MRC-5, R-Mix and E-MIx cell lines are stored for 18-24 hours at 36<sup>o </sup>C in O<sub>2 </sub>(or until cell lines reach >50% confluency) and then are kept at room temperature until expiry.</p> <p align="justify">2. Inoculation of cell culture shell vials</p> <p align="justify">i) Aliquot 50 mL maintenance medium and allow to come to room temperature before using.</p> <p align="justify">ii) Refer to the protocol for each specimen type to determine the number of tubes and types of cell lines to be inoculated. Also refer to Appendix XV if needed.</p> <p align="justify">iii) Prior to inoculation, check the cell culture tubes for acceptable confluent monolayer formation and sterility.</p> <p align="justify">iv) Decant the medium from the tube. </p> <p align="justify">v) Using a clean, sterile pipette for each tube, add 1.5 mL of the aliquotted maintenance medium to each tube and re-cap. After set up is complete, discard any remaining maintenance medium.</p> <p align="justify">vi) Inoculate 0.2 mL (4 drops) of processed specimen into each tube, recapping immediately afterward.</p> <p align="justify">vii) Incubate the tubes in the roller drum at 36<sup>o</sup>C. Refer to the appropriate specimen protocol for the incubation time for each tube. </p> <p align="justify">viii) Refeed MRC-5, R-Mix and E-Mix minimally once per 5 days. Shell vials showing signs of chemical toxicity (red media / sloughing cells), bacterial / fungal contamination (yellow / turbid media) or aging should be refed within the day.</p> <p align="justify"> </p> <p align="justify"><font size="3"><b>III</b><b>. <u>Reading of Cultures (Shell Vials for CPE)</u></b></font></p> <p align="justify"><b><u></u></b></p> <p align="justify">i) <b>Cytopathic effect (CPE)</b>: E-Mix or other cell lines should be examined daily for CPE. Any culture demonstrating <u>></u> 2+ CPE should be confirmed by staining. The cells should be scraped, a cytospin slide prepared and appropriate monoclonal antibody staining performed. If no CPE is present, refeed with corresponding maintenance medium and Reincubate.</p> <p align="justify"> </p> <p align="justify">ii) <b>Enteroviruses (D3 enterovirus, DHI)</b>: Perform<b> </b>enterovirus stain when CPE is observed:</p> <p align="justify"><b></b></p> <p align="justify">a. Prepare cytospin preparation from cell culture as outlined below:</p> <p align="justify">b. Remove about 0.5 mL (leaving about 1 mL) maintenance media from the culture using a sterile pipette.</p> <p align="justify">c. Scrape cells using a sterile pipette. Break up cell clumps by pipetting up and down several times.</p> <p align="justify">d. Pipette 4 x 200 uL (4 x 4 drops) of scraped cells into 4 funnels on 2 double slides.</p> <p align="justify">e. Cytospin at 2000 rpm (700 x g) for 5 minutes.</p> <p align="justify">f. Remove slide and air dry.</p> <p align="justify">g. Fix in cold acetone for 10 minutes in a coplin jar. Remove slide and air dry.</p> <p align="justify">h. An enterovirus QC control slide should be stained in parallel with the patient as follows.</p> <p align="justify">i. Stain the 4 wells by adding 20 mL each of Enterovirus D3, ECHO, Coxsackie B and Polio stains onto the fixed cell spots.</p> <p align="justify">j. Incubate in a humidified chamber for 30 minutes at 36<sup>o</sup>C.</p> <p align="justify">k. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.</p> <p align="justify">l. Wipe excess PBS from the slide without touching the cell spot.</p> <p align="justify">m. Add 20 mL of FITC-conjugated antibodies.</p> <p align="justify">n. Incubate in a humidified chamber at 36<sup>o</sup>C for 30 minutes.</p> <p align="justify">o. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.</p> <p align="justify">p. Wash with distilled water for 1 minute in a coplin jar.</p> <p align="justify">q. Wipe excess water from the slide without touching the cell spot.</p> <p align="justify">r. Mount using coverslip and mounting fluid.</p> <p align="justify">s. Read with fluorescence microscope Leica DC300F with #3 filter for Rodamine/FITC Evans blue and the 40x objective (<b>warning:#1 filter is for FITC Evans blue only</b>).</p> <p align="justify"><b>Interpretation of Results</b></p> <p align="justify">Positive for enterovirus: Green fluorescence</p> <p align="justify">Negative: Dull-red counterstained cells with no apple-green fluorescence.</p> <p align="justify">Invalid: If no counterstain is visible, repeat staining </p> <p align="justify">QC slide failed, report to senior/charge</p> <p align="justify">If positive, record in freezer program and freeze cells and supernate. Refer to Appendix X and XII for procedure.</p> <p align="justify"> </p> <p align="justify">iii) <b>Confirmation by PHL</b>: Any culture demonstrating CPE for which a virus cannot be detected using monoclonal antibodies or other in-house methods and for which toxicity has been ruled out (see below) should be referred to the Public Health Laboratory (PHL) for further work-up. Pass cells to a new culture before sending. Scrape and add 0.2 ml (4 drops) of scraped cells to a fresh culture containing 2 mL of fresh maintenance media (1:10 dilution). Consult the charge/senior technologist or medical microbiologist before referring the specimen to PHL.</p> <p align="justify"> </p> <p align="justify"><b></b></p> <p align="justify">iv) <b>Culture Toxicity:</b> If chemical toxicity is suspected in a culture (rounding of cells, sloughing of cells, granular cytoplasm of cells or unusual CPE, consult senior/charge technologist if unsure), proceed as follows:</p> <p align="justify"> </p> <p align="justify">v) Pass cells by scraping and adding 0.2 ml (4 drops) of these scraped cells to a fresh culture containing 2 mL of fresh maintenance media (1:10 dilution). Proceed with tube culture method as outlined above.</p> <p align="justify"> </p> <p align="justify">vi) The effects of chemical toxicity would be reduced by dilution whereas the effects of CPE (caused by viral replication) would be the same, if not accelerated on passage. If CPE is suspected, identify virus by antibody stains. If chemical toxicity is suspected, continue to incubate (may need further refeeding to reduce toxicity). If unsure of cell toxicity or CPE , refer to the charge/senior technologist for review. </p> <p align="justify"> </p> <p align="justify">vii) <b>Contaminated Culture:</b> If the culture appears visibly contaminated (eg. cloudy and/or yellow medium) and thus uninterpretable, proceed as follows: </p> <p align="justify">a. On 1<sup>st</sup> or 2<sup>nd</sup> reading - change the maintenance medium, and reincubate.</p> <p align="justify">b. On 3<sup>rd</sup> or later reading or recurrence - issue a final report stating:</p> <p align="justify">“Virology culture: Specimen is heavily contaminated with bacteria and/or fungus. Unable to interpret virology culture.”</p> <p align="justify">c. Replant if specimen is from a sterile site or contamination is attributed to the lab. If multiple specimens are contaminated, report to senior/charge.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">IV. <u>Quality Control</u> (Tube Cultures are not in routine use)</font></b></p> <p align="justify"><b></b></p> <p align="justify">Record all results of QC in LIS (or log). Refer to virology LIS manual for procedure. Report any abnormal results to charge/senior technologist.</p> <p align="justify">Five tubes are reserved from each lot of cell cultures received, and used as controls as follows:</p> <p align="justify">i) <u>Negative controls</u>: (tubes labelled N1, N2, N3)</p> <p align="justify">On Wednesday, Friday and Monday, an uninoculated tube from each cell line used that day is placed in the roller drum with the inoculated specimens. These tubes are incubated, read and refed with the patient inoculated cultures to monitor the monolayer quality, medium toxicity/contamination. They can also be used </p> <p align="justify">to provide a baseline for comparison for inoculated cultures when reading for CPE. HFF, CMK, HEp-2 and RD tubes are kept for 5, 2, 2 and 1 weeks respectively.</p> <p align="justify">ii) <u>Unopened Controls</u>: (2 tubes labelled C and V respectively)</p> <p align="justify">These tubes are not opened. One tube is kept at 36<sup>o</sup> C in O<sub>2</sub> in the clean room (C) </p> <p align="justify">and one is placed on the roller drum (V) at 36<sup>o</sup> C in O<sub>2</sub>. These tubes are observed </p> <p align="justify">for 1 week to identify toxicity and contamination originating with the vendor. </p> <p align="justify">iii) <u>Positive Controls</u>:</p> <p align="justify"><u></u></p> <p align="justify">Each week HSV-1 ATCC strain # VR- 539 is scraped from the previous week’s positive control tube and used to inoculate a fresh HFF tube. If the control fails to propagate, a new vial can be retrieved from liquid N<sub>2</sub> tank MINS shelf 6.</p> <p align="justify">Additional positive controls may be set up for the following reasons:</p> <ul> <li> <div align="justify">· Low isolation rates</div> </li> <li> <div align="justify">· Comparison of cell lines</div> </li> <li> <div align="justify">· Vendor changes</div> </li> <li> <div align="justify">· Proficiency test failures</div> </li> <li> <div align="justify">· Training</div> </li> <li> <div align="justify">· Continuing problems with negative controls</div> </li> <li> <div align="justify">· Preparation of QC material (i.e. positive control slides)</div> </li> <li> <div align="justify">Consult a charge/senior technologist to determine the cell lines and viruses to be set up.</div> </li> </ul> <p align="justify"> </p> <p align="justify"><b><font size="3">V. <u>Reference</u></font></b></p> <p align="justify">1) Isenberg, H.D. 1992. Clinical Microbiology Procedures Handbook. Vol. 2. ASM</p> <p align="justify">Press.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0tag:blogger.com,1999:blog-7259934002090567583.post-21830354155706286062011-08-25T23:13:00.001+07:002011-08-25T23:13:42.578+07:00SHELL VIAL PROCEDURE (Appendix II)<p align="justify"><b><font size="3">I. <u>Introduction</u></font></b></p> <p align="justify"><b></b></p> <p align="justify">The shell vial method employs centrifugation of the patient specimen onto a cell monolayer contained in a vial. In general, the centrifugation step shortens the time to a positive culture result. Virus may be detected by direct fluorescent antibody (DFA) or indirect fluorescent antibody (IFA) staining within hours or days of inoculation. Currently, the MRC-5 (Human Fibroblast cells, Diagnostic Hybrid Inc. [DHI]) shell vial is used for the detection of CMV, HSV and VZ, the R-Mix (DHI) is used for the detection of respiratory viruses and the E-Mix (DHI) is use for the detection of enteroviruses.</p> <p align="justify"> </p> <p align="justify"><b><font size="3">II. <u>Reagents and Materials</u></font></b></p> <p align="justify"><b><u></u></b></p> <h3 align="justify"><font size="2">Fluorescence microscope with filter for FITC/Evans blue</font></h3> <p align="justify">Inverted microscope</p> <p align="justify">FITC-conjugated virus-specific antibody stains (HSV1,2; VZV; CMV-IE; D3 enterovirus)</p> <p align="justify">SimulFluor DFA Respiratory Viral Screen/RSV panel-(Chemicon)*</p> <p align="justify">Phosphate buffered saline (PBS) </p> <p align="justify">Distilled water</p> <p align="justify">Cold acetone (4<sup>o</sup>C)</p> <p align="justify">Mounting fluid</p> <p align="justify">Sterile pipettes</p> <p align="justify">Cytospin and accessories </p> <p align="justify">Humidified chamber</p> <p align="justify">Sterile freezer vial</p> <p align="justify">Sterile shell vials with round coverslips and caps</p> <p align="justify">Needle with hooked end attached to syringe</p> <h3 align="justify">Maintenance media</h3> <p align="justify">Glass slides</p> <p align="justify">Coverslips</p> <p align="justify">Paper towels for blotting</p> <p align="justify"><b></b></p> <p align="justify"><b></b></p> <p align="justify"><font size="3"><b>III</b><b>. <u>Procedure</u></b></font></p> <p align="justify"><b></b></p> <p align="justify">1. Registration</p> <p align="justify">i) Upon receipt of a shipment of cells, initial and date the record sheet accompanying the shipment. The record should contain vendor, lot number, passage number and QC data. File in the Cell Culture QC binder.</p> <p align="justify"><b></b></p> <p align="justify">ii) Register the shell vial lots in the lab information system (LIS), this is necessary to generate the Shell Vial MRC-5, E-Mix or R-Mix QC procedures. Print labels. Refer to virology LIS manual for procedure.</p> <p align="justify"> </p> <p align="justify">2. <b>Seeding (not in routine use</b>) of MRC-5shell vials </p> <p align="justify">i) Before seeding the shell vials, aspirate about 15 mL of MRC-5 cell suspension into a 125 cm<sup>2</sup> tissue culture flask, place label <b>on the side</b> of flask and/or write “MRC-5, date and ‘<b>Pre</b>’”.</p> <p align="justify">ii) After seeding shell vials, aspirate about 15 mL of MRC-5 cell suspension into another 125 cm<sup>2</sup> tissue culture flask, label on the side of flask with “MRC-5, date and ‘<b>Post</b>’”.</p> <p align="justify">iii) Aliquot the MRC-5 cells in 1 to 2 mL volumes into a sterile shell vials containing round cover-slips.</p> <p align="justify">iv) Each shell vial is capped tightly (CO<sub>2</sub> produced by growing cells is needed to maintain proper pH for optimal cell growth) and incubated at 36ºC for 2-3 days to form a relatively confluent monolayer before use.</p> <p align="justify"> </p> <p align="justify">3. Incubation</p> <p align="justify">i. For seeded (not in routine use) MRC-5 shell vials, incubate at 36ºC and use them on days 3-10.</p> <p align="justify">ii. Store DHI (MRC-5, E-Mix and R-Mix) shell vials at room temperature in the dark. The DHI shell vials can be placed in service after pre-incubating for 2 to 4 hours. </p> <p align="justify">Only sufficient shell vials for the day should be pre-incubated and the unused ones can be pre-incubated again the next day up to the allowed 24 hr limit..</p> <p align="justify"> </p> <p align="justify">4. Refeed</p> <p align="justify">Shipping media in the shell vials must be replaced with the corresponding refeed media prior to inoculation. This is done by decanting or aspirating with a pipette and adding 1.5 mL of the appropriate aliquot maintenance media (each of MRC-5, E-Mix and R-Mix has its own refeed medium from DHI).</p> <p align="justify"> </p> <p align="justify">5. Inoculation of shell vials</p> <p align="justify">i) Refer to specimen protocol or Appendix XXI Specimen Cell Line Stain Table for the number of shell vials to be inoculated. </p> <p align="justify">ii) Prior to inoculation, check for confluent monolayer formation, sterility and for presence of a coverslip. Ensure that the shipping media have already been replaced with appropriate fresh maintenance media. Record results daily under Shell Vial MRC5, Emix or Rmix quality control procedures in the LIS.</p> <p align="justify">iii) Apply a specimen label (LIS barcode) to the shell vial(s) and a corresponding plane glass slide. Label slides for HSV1; HSV2; VZ; CMV on MRC-5 shell vials and RS on R-Mix shell vials accordingly. E-Mix will be read for CPE as with tube culture and does not require a pre-labeled slide.</p> <p align="justify">iv) Inoculate 0.2 mL for MRC-5, E-Mix and 0.3 mL for R-Mix of processed specimen into the shell vials. Inoculate one specimen at a time, recapping immediately afterward.</p> <p align="justify">v) Centrifuge at room temperature for 15 minutes at 4300 rpm (3500 x <i>g</i>).</p> <p align="justify">vi) Use a new, sterile pipette for each vial. Process one specimen at a time, recapping immediately afterwards. After set up is complete, discard any remaining aliquotted maintenance medium. For specimens that have excess blood or mucous, remove excessive sediment by refeeding shell vials after about 2 hours of incubation. </p> <p align="justify">viii) Incubate the shell vials at 36ºC, lined up in rows of HSV1 and HSV2. CMV, R-Mix and E-Mix should be lined up in different cluster plates (CMV-IE requires an extra step in IFA staining and 2 days of incubation; R-Mix and VZ are DFA staining but require 2 and 4 days of incubation respectively; E-Mix is incubated and read for CPE):</p> <p align="center"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="78"> <p><b>Cell Line</b></p> </td> <td valign="top" width="228"> <p><b>Virus/Stain/Read for CPE*</b></p> </td> <td valign="top" width="84"> <p><b># of Vials</b></p> </td> <td valign="top" width="138"> <p><b>Incubation Time</b></p> </td> </tr> <tr> <td valign="top" width="78"> <p>MRC-5</p> <p>MRC-5</p> <p>MRC-5</p> <p>MRC-5</p> <p>R-Mix</p> <p>E-Mix*</p> </td> <td valign="top" width="228"> <p>HSV 1, 2</p> <p>HSV bivalent</p> <p>VZV</p> <p>CMV</p> <p>Resp virus Screen (RS)</p> <p>Enteroviruses* (read for CPE)</p> </td> <td valign="top" width="84"> <p>2</p> <p>1</p> <p>1</p> <p>1</p> <p>1</p> <p>1</p> </td> <td valign="top" width="138"> <p>1 day</p> <p>1 day</p> <p>4 days</p> <p>2 days</p> <p>2 days</p> <p>5 days*</p> </td> </tr> </tbody></table> </p> <p align="justify"> </p> <p align="justify">4. Staining of shell vials</p> <p align="justify">i) Prior to staining, examine the shell vial monolayer using the inverted microscope:</p> <p align="justify">ii) If there is <75% CPE, perform IFA or DFA staining on the shell vial monolayer using the required antibody conjugate. For CMV, see shell vial staining under Appendix IV (IFA) and for HSV 1&2, VZV and RS, see shell vial staining under Appendix V (DFA). </p> <p align="justify">iii) If >75% of the monoloayer has lifted from the coverslip, check the colour of the maintenance media and proceed as follows:</p> <p align="justify">iv) If the maintenance media is bright pink (suggesting alkaline pH), yellow or cloudy, check with charge/senior technologist before proceeding further.</p> <p align="justify">iii) If the maintenance media is appropriately coloured (salmon pink), perform IFA or DFA staining using cytospin preparations of scraped shell vial cells. Follow the staining procedure for prepared cytospin slides as outlined in the tube culture section in Appendix IV (IFA) and Appendix V (DFA).</p> <p align="justify">iv) Discard cap. Remove maintenance medium from the shell vials, using a different sterile pipette for shell vials of the same specimen number.</p> <p align="justify">v) Add 1 mL of cold acetone to each shell vial. Cover and fix for 10 minutes.</p> <p align="justify">vi) Decant acetone and blot on paper towel.</p> <p align="justify">vii) Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Make sure the stream is gentle enough not to flip the cover-slip. Decant PBS. </p> <p align="justify">viii) Add 75 ml (2 drops from bottle) of HSV1, HSV2 and VZ to the appropriate row of shell vials in the DFA cluster plate (including QC shell vials, if done on that day). Cover. </p> <p align="justify">ix) Add 75 ml (2 drops from bottle) of CMV-IE to the appropriate row of shell vials in the IFA (CMV, 2 day) cluster plate (including QC shell vials, if done on that day). Cover.</p> <p align="justify">x) Incubate both DFA and IFA shell vials at 36<sup>o</sup>C for 30 minutes.</p> <p align="justify">xi) Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Make sure the stream is gentle enough not to flip the cover-slip. Decant PBS. Repeat.</p> <p align="justify">xii) For the DFA shell vials (HSV1, HSV2, VZV) remove the coverslip and place cell side down onto a drop of mounting fluid on the pre-labelled glass slide.</p> <p align="justify">xiii) For the IFA (CMV 2 day) shell vials, add 75ml (2 drops from bottle) of appropriate FITC-conjugated anti-mouse antibodies, cover and repeat the incubation and wash steps (i and j).</p> <p align="justify">xiv) Remove the coverslip and place cell side down onto a drop of mounting fluid on a glass slide.</p> <p align="justify">xv) Read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective. </p> <p align="justify"> </p> <p align="justify"><font size="3"><b>III</b><b>. <u>Reading of Stained Shell Vials</u></b></font></p> <p align="justify"><strong><u><font size="3"></font></u></strong></p> <p align="justify"><b></b></p> <p align="justify">i)<b> CMV – Immediate Early Antigen (CMV-IE)</b></p> <p align="justify"><b></b></p> <p align="justify">Using the fluorescence microscope with the FITC/Evans Blue filter, scan the entire field using the 25x objective. Use the 40x objective to investigate any fluorescing cells.</p> <p align="justify">POSITIVE: An even matte apple-green fluorescence covering the entire kidney bean shaped/oval nucleus. May include specks of brighter fluorescence.</p> <p align="justify">NEGATIVE: No typical cells with apple-green fluorescence</p> <p align="justify">INVALID: If no counterstain is visible or only the edge of cover slip is stained, inform senior/charge technologist. The cover-slip may have flipped before being stained</p> <p align="justify"> </p> <p align="justify">ii)<b> HSV/VZV</b></p> <p align="justify"><b></b></p> <p align="justify">Using the fluorescence microscope with the FITC/Evans Blue filter, scan the entire field using the 10x or 25x objective. Use the 40x objective to investigate any fluorescing cells.</p> <p align="justify">POSITIVE: Distinct apple-green fluorescence of the cytoplasma and /or nucleus of the infected cells. Dull red Evans blue counter stain should be visible for stained nonfluorescent cells.</p> <p align="justify">NEGATIVE: No typical cells with apple-green fluorescence. </p> <p align="justify">Dull red Evans blue counter stain should be visible for negative cells. </p> <p align="justify">INVALID: If no counterstain is visible or only the edge of cover slip is stained, inform senior/charge technologist. The cover-slip may have flipped before being stained.</p> <p align="justify"> </p> <p align="justify">iii) <b>RS- (Respiratory virus Screen)</b></p> <p align="justify"><b></b></p> <p align="justify">Using the fluorescence microscope with the FITC/Evans Blue and Rodamine bi-filter, scan the entire field using the 10x, and 25x objectives. Use the 40x objective to investigate any fluorescing cells.</p> <p align="justify">POSITIVE for respiratory virus: i) green: Cells with apple-green fluorescence fluorescence.</p> <p align="justify">POSITIVE for RSV: i) gold: Cells with gold fluorescence.</p> <p align="justify">NEGATIVE: No typical cells with apple-green or gold fluorescence</p> <p align="justify">INVALID: If no counterstain is visible or only the edge of cover slip is stained, inform senior/charge technologist. The cover-slip may have flipped before being stained.</p> <p align="justify"> </p> <p align="justify"><font size="2">IV. Shell Vial for CPE (E-Mix) can be referred to Appendix III (Tube Culture/Shell Vial for CPE)</font></p> <p align="justify"><strong></strong></p> <p align="justify"><b><font size="3">IV. <u>Quality Control</u></font></b></p> <p align="justify"><b>A. Shell Vial MRC-5 Quality Control: (unopened shell vial)<u></u></b></p> <p align="justify">This is done weekly when cell shipments are received to monitor cell growth. Record daily in LIS.</p> <p align="justify"> <table border="1" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="212"> <p align="center"><strong>Examine daily (for 7 days) for:</strong></p> </td> <td valign="top" width="172"> <p align="center"><strong>Expected results:</strong></p> </td> <td valign="top" width="206"> <p align="center"><strong>Shell Vial MRC5 QC- expected results</strong> <strong>(LIS entry):</strong></p> </td> </tr> <tr> <td valign="top" width="212"> <p align="justify">Absence of contamination</p> </td> <td valign="top" width="172"> <p align="justify">Visual inspection: (1) medium colour not yellow (2) medium not cloudy</p> </td> <td valign="top" width="206"> <p align="justify">OK*</p> </td> </tr> <tr> <td valign="top" width="212"> <p align="justify">Healthy cell growth</p> </td> <td valign="top" width="172"> <p align="justify">Under inverted microscopy: (1) confluent monolayer (2) medium colour pink</p> </td> <td valign="top" width="206"> <p align="justify">OK*</p> </td> </tr> <tr> <td valign="top" width="212"> <p align="justify">Cover slip</p> </td> <td valign="top" width="172"> <p align="justify">Under inverted microscopy: cover slip present</p> </td> <td valign="top" width="206"> <p align="justify">OK*</p> </td> </tr> </tbody></table> </p> <p align="justify">At the end of 7 days, one unopened shell vial in good condition is used as “Previous lot MRC-5” for the following week.</p> <p align="justify"> </p> <p align="justify"><b>B. Shell Vial Inoculation QC procedure (6 shell vials + 1 previous lot):<u></u></b></p> <p align="justify"><b><u></u></b></p> <p align="justify">This QC procedure is performed once a week utilizing HSV-1 (ATCC 539) to:</p> <p align="justify">1. Show that each MRC-5 lot supports the propagation of the intended viruses.</p> <ol start="start"> <li> <div align="justify">Monitor entire shell vial procedures from inoculation to reading including incubation, staining and reading (HSV1 & 2 are DFA, CMV-IE is IFA).</div> </li> <li> <div align="justify">The inoculation part is done by Tube Culture bench, the Shell Vial bench completes the procedure including reporting in the LIS.</div> </li> </ol> <p align="justify"> </p> <p align="justify"> </p> <p align="justify">Gr* = stained positive for the intended virus</p> <p align="justify">Pos*= stained positive with the specified stain</p> <p align="justify">Neg*= stained negative with the specified stain </p> <p align="justify"> </p> <p align="justify"><b>2. Daily Slide Shell Vial QC procedure:</b></p> <p align="justify"><b></b></p> <p align="justify">Done and recorded each work day to monitor the staining of each batch (except the day when Inoculated Shell Vial QC procedure is done).</p> <p align="justify"> <table border="0" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="76"> <p>4-well HSV daily QC slide</p> </td> <td valign="top" width="81"> <p>2-well CMV daily QC slide</p> </td> <td valign="top" width="156"> <p>Well containing:</p> </td> <td valign="top" width="120"> <p>Stain with:</p> </td> <td valign="top" width="120"> <p>HSV1 daily SLIDE SV QC- expected results on <b>Staining Reaction</b> (LIS entry):</p> </td> </tr> <tr> <td valign="top" width="76"> <p>1</p> </td> <td valign="top" width="81"> </td> <td valign="top" width="156"> <p>HSV-1 (ATCC 539)</p> </td> <td valign="top" width="120"> <p>HSV-1</p> </td> <td valign="top" width="120"> <p>Pos*</p> </td> </tr> <tr> <td valign="top" width="76"> <p>2</p> </td> <td valign="top" width="81"> </td> <td valign="top" width="156"> <p>Uninoculated MRC-5 cells</p> </td> <td valign="top" width="120"> <p>HSV-1</p> </td> <td valign="top" width="120"> <p>Neg*</p> </td> </tr> <tr> <td valign="top" width="76"> <p>3</p> </td> <td valign="top" width="81"> </td> <td valign="top" width="156"> <p>HSV-2 </p> </td> <td valign="top" width="120"> <p>HSV-2</p> </td> <td valign="top" width="120"> <p>Pos*</p> </td> </tr> <tr> <td valign="top" width="76"> <p>4</p> </td> <td valign="top" width="81"> </td> <td valign="top" width="156"> <p>Uninoculated MRC-5 cells</p> </td> <td valign="top" width="120"> <p>HSV-2</p> </td> <td valign="top" width="120"> <p>Neg*</p> </td> </tr> <tr> <td valign="top" width="76"> </td> <td valign="top" width="81"> <p>1</p> </td> <td valign="top" width="156"> <p>CMV (ATCC 807)</p> </td> <td valign="top" width="120"> <p>CMV-IE</p> </td> <td valign="top" width="120"> <p>Pos*</p> </td> </tr> <tr> <td valign="top" width="76"> </td> <td valign="top" width="81"> <p>2</p> </td> <td valign="top" width="156"> <p>Uninoculated MRC-5 cells </p> </td> <td valign="top" width="120"> <p>CMV-IE</p> </td> <td valign="top" width="120"> <p>Neg*</p> </td> </tr> </tbody></table> </p> <p align="justify">Gr* = stained positive for the intended virus</p> <p align="justify">Pos*= stained positive with the specified stain</p> <p align="justify">Neg*= stained negative with the specified stain </p> <p align="justify"> <table border="0" cellspacing="0" cellpadding="0"><tbody> <tr> <td valign="top" width="76"> <p>4-well R-Mix daily QC slide</p> </td> <td valign="top" width="156"> <p>Well containing:</p> </td> <td valign="top" width="120"> <p>Stain with:</p> </td> <td valign="top" width="120"> <p>R-Mix daily SLIDE SV QC- expected results on <b>Staining Reaction</b></p> </td> <td width="182"></td> </tr> <tr> <td valign="top" width="76"> <p>1</p> </td> <td valign="top" width="156"> <p>Flu A (ATCC)</p> </td> <td valign="top" width="120"> <p>RS</p> </td> <td valign="top" width="120"> <p>Pos*</p> </td> <td width="182"></td> </tr> <tr> <td valign="top" width="76"> <p>2</p> </td> <td valign="top" width="156"> <p>Uninoculated cells</p> </td> <td valign="top" width="120"></td> <td valign="top" width="120"> <p>Neg*</p> </td> <td width="182"></td> </tr> <tr> <td valign="top" width="76"> <p>3</p> </td> <td valign="top" width="156"> <p>RSV (ATCC)</p> </td> <td valign="top" width="120"> <p>HSV-2</p> </td> <td valign="top" width="120"> <p>Pos*</p> </td> <td width="182"></td> </tr> <tr> <td valign="top" width="76"> <p>4</p> </td> <td valign="top" width="156"> <p>Uninoculated MRC-5 cells</p> </td> <td valign="top" width="120"> <p>HSV-2</p> </td> <td valign="top" width="120"> <p>Neg*</p> </td> <td width="182"></td> </tr> <tr> <td width="7"></td> <td valign="top" width="324"> <h3>UHN/MSH Microbiology Department </h3> <p>Policy & Procedure Manual</p> </td> <td valign="top" width="210"> <p><b>Policy # MI/VIR/16/02/v03</b></p> </td> <td valign="top" width="114"> <p>Page 10 of 10</p> </td> </tr> <tr> <td width="7"></td> <td valign="top" width="324"> <h3><b>Virology Manual</b></h3> </td> </tr> <tr> <td width="7"></td> <td width="68"></td> <td width="156"></td> <td width="99"></td> <td width="21"></td> <td width="120"></td> <td width="68"></td> <td width="114"></td> </tr> </tbody></table> </p> <p align="justify"><b>D. Reagent QC (HSV1, HSV2, HSV bivalent, CMV-IE and VZ stains): </b></p> <p align="justify">a. Performed prior to patient testing and must pass before reagents are released for use.</p> <p align="justify">a. Done on external QC slides.</p> <p align="justify">b. Record QC results in Reagent Log and LIS.</p> <p align="justify">Failed QCs:</p> <p align="justify">a. Do not release patient results pending resolution of QC failure.</p> <p align="justify">b. Inform charge/senior technologist.</p> <p align="justify">c. Record in Reagent Log Chart, Instrument Maintenance Log (if eg. microscope/incubator is involved in the failure) and file incident report if necessary.</p> <p align="justify">d. Re-run failed controls in parallel to fresh controls (and/or external QC) to evaluate the QC material itself (already done routinely for MRC5 cells).</p> <p align="justify">e. If the re-run shows the old QC material still fails, fresh QC passes and nothing else is wrong with the batch (only the old QC material failed, patient results valid) patient results may be released.</p> <p align="justify">Marked decrease/absence in fluorescence can be due to:</p> <p align="justify">a. Reagent deterioration/skipping (did not apply primary/secondary stain)</p> <p align="justify">a. Microscope (filter, bulb, alignment)</p> <p align="justify">c. Other equipment, reagents or technique</p> <p align="justify"><i></i></p> <p align="justify"><b>V. <u>Reference</u></b></p> <p align="justify">1. Isenberg, H.D. 1992. Clinical Microbiology Procedure Handbook Vol. 2. ASM Press.</p> <p align="justify">2. Gleaves, Curt A. et al, J Clin Micro., Feb. 1985. Comparison of Standard Tube and Shell Vial Cell Culture Techniques for the Detection of Cytomegalovirus in Clinical Specimens.</p> <p align="justify">3. Engler, Howard D., Selepak, Sally T., J Clin Micro., June 1994. Effect of Centrifuging Shell Vials at 3,500 x <i>g</i> on Detection of Viruses in Clinical Specimens.</p> <p align="justify"><a title="http://bahankuliahkesehatan.blogspot.com/" href="http://bahankuliahkesehatan.blogspot.com/"><font color="#000000">http://bahankuliahkesehatan.blogspot.com/</font></a></p> Rafless bencoolenhttp://www.blogger.com/profile/03049098776512473429noreply@blogger.com0