Diagnosa Lab 2

8/9/2019 Diagnosa Lab 2

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Virological Tests

An Overview


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DIAGNOSIS LABORATORIUM PENYAKIT VIRUS

PEMERIKSAAN MIKROSKOPIK

ISOLASI VIRUS Pemilihan, Pengambilan, Pengiriman, Penyimpanan

bahan pemeriksaan Pemilihan Penggunaan Sis im!"#spes Lab#ra #riumyang baik!sesuai

PEMERIKSAAN SEROLO$IK Reaksi Pengika an K#mplemen Reaksi "amba an "emaglu inasi Reaksi Aglu inasi!Presipi asi!%l#kulasi Reaksi Ne ralisasi Reaksi Imun#&lu#resensi

PER'O(AAN "E)AN


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Diagnostic Methods in

Virology1. Direct Examination

2. Indirect Examination (VirusIsolation)

3. Serology


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Direct Examination1. Antigen Detection immunofluorescence, ELISA etc.

2. Electron Microscopy morphology of virus particlesimmune electron microscopy

3. Light Microscopy histological appearance inclusion bodies

4. Viral Genome Detection hybridization with specificnucleic acid probes

polymerase chain reaction !"#$


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Indirect Examination1. Cell Culture cytopathic effect "!E$

haemabsorption

immunofluorescence

2. Eggs poc%s on "A& haemagglutination

inclusion bodies

3. Animals disease or death


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Serology'etection of rising titres of antibody between acute and convalescentstages of infection, or the detection of Ig& in primary infection.

Classical Techni ues !e"er Techni ues

(. "omplement fi)ation tests "*+$ (. #adioimmunoassay #IA$. -aemagglutination inhibition tests . Enzyme lin%ed immunosorbent assay EIA$. Immunofluorescence techni/ues I*$ . !article agglutination

0. 1eutralization tests 0. 2estern 3lot 23$4. "ounter5immunoelectrophoresis 4. #I3A, Line immunoassay


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REPRODUKSI PEMBIAKAN VIRUS

SE'ARA IN VI*RO+alam biakan sel aringan -*issue 'ell 'ul ure.

(iakan Primer -Primary 'ul ure. (iakan Sel S abil *erus Menerus!Les ari

-S able '#n inu#us 'ell 'ul ure. / "ella 'ell, %L 'ell, ("K 'ell, Ae0es 'ell

SE'ARA IN OVO+alam elur ayam!bebek berembri#

SE'ARA IN VIVO+alam he1an yang lengkap!u uh -in a2 .

Men2i , *ikus, Kelin2i, "ams er, Kera Nyamuk Ae0es alb#pi2 us!Ae0es aegyp i


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Virus Isolation"ell "ultures are most widely used for virus isolation, there are types of cell cultures6

(. !rimary cells 5 &on%ey 7idney. Semi5continuous cells 5 -uman embryonic %idney and s%infibroblasts

. "ontinuous cells 5 -eLa, 8ero, -ep , LL"5&7 , &'"7

!rimary cell culture are widely ac%nowledged as the best cell culturesystems available since they support the widest range of viruses.-owever, they are very e)pensive and it is often difficult to obtain areliable supply. "ontinuous cells are the most easy to handle but the rangeof viruses supported is often limited.


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ell ultures9rowing virus may produce

(. "ytopathic Effect "!E$ 5 such as the ballooning of cells orsyncytia formation, may be specific or non5specific.

. -aemadsorption 5 cells ac/uire the ability to stic% tomammalian red blood cells.

"onfirmation of the identity of the virus may be carried out usingneutralization, haemadsorption5inhibition or immunofluorescencetests.


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yto!athic E"ect (1)

"ytopathic effect of enterovirus :( and -S8 in cell culture6 note the ballooning of cells .8irology Laboratory, ;ale51ew -aven -ospital, Linda Stannard,


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yto!athic E"ect (2)

Syncytium formation in cellculture caused by #S8 top$, andmeasles virus bottom$.

courtesy of Linda Stannard,


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#aemadsor!tion

Syncytial formation caused by mumps virus andhaemadsorption of erythrocytes onto the surface of the cell

sheet.courtesy of Linda Stannard,


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PEMBIAKKAN VIRUS (IN OVO)


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$ro%lems &ith cell cultureLong period up to 0 wee%s$ re/uired for result.

Often very poor sensitivity, sensitivity depends on alarge e)tent on the condition of the specimen.

Susceptible to bacterial contamination.

Susceptible to to)ic substances which may be present in

the specimen.&any viruses will not grow in cell culture e.g. -epatitis3, 'iarrhoeal viruses, parvovirus, papillomavirus.


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'a!id ulture Techni ues#apid culture techni/ues are available whereby viral antigensare detected to 0 days after inoculation. +he "&8 'EA**test is the best e)ample, whereby

+he cell sheet is grown on individual cover slips in a plastic bottle.

*ollowing inoculation, the bottle then is spun at a low speed

for one hour to speed up the adsorption of the virus$ andthen incubated for to 0 days.

+he cover slip is then ta%en out and e)amined for the presence of "&8 early antigens by immunofluorescence.


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DE ** test +or MV

8irology Laboratory, ;ale51ew -aven -ospital$


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Viruses Isolated %y ell

ultureViruses rea#ily isolate# $y cell culture Less %re uently isolate# &iruses

-erpes Simple) 8aricella5=oster

"ytomegalovirus &easles

Adenoviruses #ubella

!olioviruses #hinoviruses

"o)sac%ie 3 viruses "o)sac%ie A viruses

Echoviruses

Influenza

!arainfluenza

&umps

#espiratory Syncytial 8irus


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Electron Microsco!y(> ? virus particles per ml re/uired for visualization, 4>,>>> 5 ?>,>>>magnification normally used. 8iruses may be detected in the followingspecimens.

'aeces #otavirus, Adenovirus 1orwal% li%e virusesAstrovirus, "alicivirus

Vesicle 'lui# -S88=8

()in scrapings papillomavirus, orf molluscum contagiosum


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Electronmicrogra!hs

Adenovirus #otavirus

courtesy of Linda Stannard,


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Immune ElectronMicrosco!y

The sensiti,ity and s!eci-city o+ EM may %e enhanced%y immune electron microsco!y. There are t&o,ariants /

"lassical Immune electron microscopy IE&$ 5 the sample istreated with specific anti5sera before being put up for E&. 8iral

particles present will be agglutinated and thus congregate

together by the antibody.Solid phase immune electron microscopy S!IE&$ 5 the grid iscoated with specific anti5sera. 8irus particles present in thesample will be absorbed onto the grid by the antibody.


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$ro%lems &ith Electron

Microsco!yEx!ensi,e e ui!ment

Ex!ensi,e maintenance'e uire ex!erienced o%ser,er

Sensiti,ity o+ten lo&


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Serology"riteria for diagnosing !rimary Infection

0 fold or more increase in titre of Ig9 or total antibody between

acute and convalescent sera!resence of Ig&SeroconversionA single high titre of Ig9 or total antibody$ 5 very unreliable

"riteria for diagnosing #einfection

fold or more increase in titre of Ig9 or total antibody between acuteand convalescent sera

Absence or slight increase in Ig&


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Ty!ical Serological $ro-le +ter cuteIn+ection

1ote that during reinfection, Ig& may be absent or present at a low level transiently


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om!lement *ixation Test

"omplement *i)ation +est in &icrotiter !late. #ows ( and e)hibit complementfi)ation obtained with acute and convalescent phase serum specimens,respectively. 5fold serum dilutions were used$ +he observed 05fold increase issignificant and indicates recent infection.


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E0IS +or #IV anti%ody

&icroplate ELISA for -I8 antibody6 coloured wells indicate reactivity


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estern lot-I85( 2estern 3lot

Lane(6 !ositive "ontrolLane 6 1egative "ontrolSample A6 1egativeSample 36 IndeterminateSample "6 !ositive


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se+ulness o+ Serological

'esults-ow useful a serological result is depends on the individual virus.

*or e)ample, for viruses such as rubella and hepatitis A, the onset ofclinical symptoms coincide with the development of antibodies. +hedetection of Ig& or rising titres of Ig9 in the serum of the patient wouldindicate active disease.

-owever, many viruses often produce clinical disease before theappearance of antibodies such as respiratory and diarrhoeal viruses. So inthis case, any serological diagnosis would be retrospective and thereforewill not be that useful.

+here are also viruses which produce clinical disease months or yearsafter seroconversion e.g. -I8 and rabies. In the case of these viruses, themere presence of antibody is sufficient to ma%e a definitive diagnosis.


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$ro%lems &ith SerologyLong period of time re/uired for diagnosis for paired acute and convalescentsera.

&ild local infections such as -S8 genitalis may not produce a detectable

humoral immune response.E)tensive antigenic cross5reactivity between related viruses e.g. -S8 and8=8, @apanese 3 encephalitis and 'engue, may lead to false positive results.

immunocompromised patients often give a reduced or absent humoralimmune response.

!atients with infectious mononucleosis and those with connective tissuediseases such as SLE may react non5specifically giving a false positiveresult.

!atients given blood or blood products may give a false positive result due to

the transfer of antibody.


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S* anti%odies>

'iagnosis depends on the presence of an intact blood5brain barrier


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'a!id Diagnosis ased onthe Detection o+ Viral

ntigens 1asopharyngeal Aspirate #S8Influenza A and 3!arainfluenzaAdenovirus

*aeces #otavirusesAdenovirusesAstrovirus

S%in -S88=8

3lood "&8 pp?4 antigenaemia test$


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MV !!56 antigenaemia

test

8irology Laboratory, ;ale51ew -aven -ospital$


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d,antages andDisad,antages

Advantages

#esult available /uic%ly, usually within a few hours.

!otential !roblems

Often very much reduced sensitivity compared to cell culture,can be as low as >C. Specificity often poor as well.

#e/uires good specimens.+he procedures involved are often tedious and time5consuming and thus e)pensive in terms of laboratory time.


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S!ecimens +or 'outine

TestsClinical Category *loo# Throat

s"a$'aeces C(' +ther

(. &eningitis D D D D. Encephalitis D D D D 3rain biopsy. !aralytic disease D D D D

0. #espiratory illness D D 1asopharyngeal aspirate4. -epatitis D?. 9astroenteritis D:. "ongenital diseases D . &yocarditis D !ericardial fluid

((. &yositis D D( . 9landular fever D( . !ost &ortem D Autopsy

After use, swabs should be bro%en into a small bottle containing ml of virus transport medium.Swabs should be sent to the laboratory as soon as possible without freezing. *aeces, "S*, biopsy or

autopsy specimens should be put into a dry sterile container.


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Molecular Methods&ethods based on the detection of viral genome are alsocommonly %nown as molecular methods. It is often said thatmolecular methods is the future direction of viral diagnosis.

-owever in practice, although the use of these methods isindeed increasing, the role played by molecular methods in aroutine diagnostic virus laboratory is still small compared toconventional methods.

It is certain though that the role of molecular methods willincrease rapidly in the near future.


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lassical Molecular

Techni ues'ot5blot, Southern blot, in5situ hydridization are e)amples ofclassical techni/ues. +hey depend on the use of specific

'1AG#1A probes for hybridization.+he specificity of the reaction depends on the conditions usedfor hybridization. -owever, the sensitivity of these techni/uesis not better than conventional viral diagnostic methods.

-owever, since they are usually more tedious and e)pensivethan conventional techni/ues, they never found widespreadacceptance.


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$olymerase hain 'eaction

(1)!"# allows the in vitro amplification of specific target '1A se/uences by afactor of (> ? and is thus an e)tremely sensitive techni/ue.

It is based on an enzymatic reaction involving the use of synthetic

oligonucleotides flan%ing the target nucleic se/uence of interest.+hese oligonucleotides act as primers for the thermostable +a/ polymerase.#epeated cycles usually 4 to 0>$ of denaturation of the template '1A atF0o"$, annealing of primers to their complementary se/uences 4> o"$, and

primer e)tension : o"$ result in the e)ponential production of the specifictarget fragment.

*urther sensitivity and specificity may be obtained by the nested !"#.

'etection and identification of the !"# product is usually carried out byagarose gel electrophoresis, hybridization with a specific oligonucleotide

probe, restriction enzyme analysis, or '1A se/uencing.


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$olymerase hain 'eaction

(2)Advantages of !"#6

E)tremely high sensitivity, may detect down to one viral genome per sample volume

Easy to set up

*ast turnaround time

'isadvantages of !"# E)tremely liable to contamination

-igh degree of operator s%ill re/uired

1ot easy to set up a /uantitative assay.

A positive result may be difficult to interpret, especially with latent viruses such as "&8,

where any seropositive person will have virus present in their blood irrespective whetherthey have disease or not.

+hese problems are being addressed by the arrival of commercial closed systems such as the#oche "obas Amplicor which re/uires minimum handling. +he use of synthetic internalcompetitive targets in these commercial assays has facilitated the accurate /uantification of

results. -owever, these assays are very e)pensive.


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Schematic of !"#

Each cycle doubles the copy number of the target


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7ther 8e&er Molecular

Techni ues3ranched '1A is essentially a sensitive hydridization techni/ue which involveslinear amplification. 2hereas e)ponential amplification occurs in !"#.

+herefore, the sensitivity of b'1A lies between classical amplificationtechni/ues and !"#. Other 1ewer molecular techni/ues depend on some form ofamplification.

"ommercial proprietary techni/ues such as L"#, 1AS3A, +&A depend one)ponential amplification of the signal or the target.

+herefore, these techni/ues are as susceptible to contamination as !"# and sharethe same advantages and disadvantages.

!"# and related techni/ues are bound to play an increasingly important role inthe diagnosis of viral infections.

'1A chip is another promising technology where it would be possible to detect alarge number of viruses, their pathogenic potential, and their drug sensitivity atthe same time.


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Metho#TargetAmpli%ication

(ignalAmpli%ication Thermocycling (ensiti&ity

CommercialE,amples

-C. E)ponential 1o ;es -igh #oche Amplicor

LC. 1o E)ponential ;es -igh Abbot L"H

!A(*A E)ponential 1o 1o -igh Organon +e%ni%a

TMA E)ponential 1o 1o -igh 9enprobe

/01.eplicase 1o E)ponential 1o -igh 1one

*ranche# D!A 1o Linear 1o &edium "hiron uantiple)

"omparison between !"# and other nucleicacid Amplification +echni/ues


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PENGOBATAN INFEKSI VIRUS

+ASAR POLA RASIONAL AN*IVIRAL O(A* KEMO*ERAPE*IK Peman&aa an perbe0aan bi#kimia1i an ara

Sel h#spes Sel yang 0iin&eksi 3irus Virus

Virus Parasi #bliga in raseluler Siklus per umbuhan*ergan ung / perlengkapan s ruk ural 0an en4ima ik 0ari

sel h#spes

Virus mengan0ung m#lekul &ungsi#nal khas yang 5Virus 2#0e06 +apa mene apkan k#mp#sisi 0an s ruk ur asam nuklea ,

pr# ein kapsi0 0an en4im khas 3irus


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Sasaran terapi antivir s Setiap ta!ap "a#a$ rep#i%asi vir s

Perubahan m#lekul yang khas 3irus #leh 4a kimia1i 0apamenghamba sa u!lebih langkah 0alam siklus replikasi 3irusyang n#rmal, seper i /

Pr#ses a0s#rpsi -penempelan. Pene rasi 5Un2#a ing6 Sin esis gen#m 3irus *ranslasi M#rgenesis -peraki an. Ma urasi

Kesuli an 0apa bersi&a #ksik bagi h#spes 7i0#3u0in "IV 0epresi sumsum ulang +i0an#sin pankrea i is sp#ra0ik Vi0arabin mual 0an ra0ang ura


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Oba An i3iral ber0asarkan mekanisme ker anya /

A0s#rpsi 0ihamba #leh P#liani#n heparin -muk#p#lisakari0a asam. "erpes3irus

Pene rasi 0ihamba #leh Alpha8aman a0in My9#3irus -In&luen4a g#l A0an '., Paramy9#3irus -Virus Sen0ai., *#ga3irus -Rubella.,

"erpes3irus

5Un2#a ing6 0ihamba #leh 'hl#r#:uin -inhibi #r plasm#0ium. N+V

Sin esis gen#m 0ihamba #leh Anal#g nukle#si0a -an i um#r, an i 3iral. I+U -I#0#0e#ksiuri0in., Vi0arabin -Arabin#&uran#sil8

a0enin., 'y arabin -Arabin#&uran#sil82y #sin. "erpesSimple9, Vari2ella87##s er, 'y #mega8l#3irus, SV;


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*A"AP SIN*ESA $ENOM VIRUS / Sin esa 4a pen0ahuluan -Pre2urs#r syn he i2. %ungsi 2e akan -*empla e &un2 i#n. %ungsi en4im -En4yme &un2 i#n.

*ranslasi 0ihamba #leh *hi#semi2arba4#ne I(* -Isa in8(e a8*hi#semi2arba4#ne., Me hia4#ne!Marb#rah, N8Me hyl8I(* Vari#lla,Va22inia $angren#sa, Va22inia 5+issemina e06

M#rgenesis 0ihamba #leh =80e#9y8+8$lu2#se Virus In&luen4a,"erpes3irus -menghamba sin esa glik#pr# ein 0an glik#lipi0.

Ma urasi 0ihamba #leh Ri&ampisin p#93irus, A0en#3irus

An i 3irus "erpes Simple9 A2y2l#3ir -A2y2l#guan#sine. %#s8rilasi #ba #leh Kinase *imi0in 3irus Menghamba p#limerase+NA 3irus


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SENYA&A ANTI VIRAL

O'at Ana#

N %#e si"aMe%anis$e Ker*a Ma+a$ Vir s

A+,+# vir Ya Pen !a$'at p #i$erase vir s -erpesvir s

A$anta"in Ti"a% Men !a$'at pa"a vir s ti"a%'erse# ' n

In.# en/a A

Di"an sin (""#) Ya Pen !a$'at trans%riptase 'a#i% -IV012 -IV03 1

F s%arnet Ti"a% Pen !a$'at p #i$erase vir s 4,t $e a# vir s2 -erpes

Si$p#e52 Vari+e##a06 ster2-IV01

Gan+,+# vir Ya Pen !a$'at p #i$erase vir s 4,t $e a# vir s

I" %s ri"in Ya Pen !a$'at era%an ti$i"in vir s -erpesvir s Keratitis

Ri'avirin Ya Pen !a$'at $RNA vir s Vir s Sinsitia# pernapasan2In.# en/a A "an B2 "e$a$Lassa

Tri.# ri"in Ya 7 -erpesvir s Keratitis

Vi"ara'in Ya Pen !a$'at p #i$erase vir s -erpes Si$p#e52 Vari+e##a06 ster

6a#sita'in (""+) Ya Pen !a$'at trans%riptase 'a#i% -IV012 -IV03

6i" v "in (A6T) Ya Pen !a$'at trans%riptase 'a#i% -IV012 -IV03


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INTERFERON Pr# ein 0isan0ikan inang +ihasilkan #leh he1an a au biakan sel resp#n in&eksi 3irus a au

pengin0uksi lain Menga ur imuni as seluler 0an hum#ral

Per ahanan per ama erha0ap in&eksi 3irus

NOMEN'LA*URE "UMAN IN*ER%ERON I%N8 I%N Leu2#2y e, ype >, S abil pa0a p" =

I%N8 I%N %ibr#blas , ype >, S abil pa0a p" = I%N8 I%N Immune, ype =, Labil pa0a p" =, Ag8in0u2e0,Mi #gen8in0u2e0 -phy #hemaglu inin.


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PENEMUAN INTERFERON

(PER4OBAAN ISAA4S DAN LINDERMANN2 189:)Vir s In.# en/a("i$ati%an)

Vir s In.# en/a-i" p vir #en

IFN

In.e%si pri$er (pre in.e+ti n)

S per in.e+ti n(in.e%si se% n"er ))

Rep#i%asi"i!a$'at

"i' an

In% 'asi 1 $a#a$ In% 'asi 1 $a#a$ In% 'asi1 $a#a$

IFN

IFN ; Inter.er n

Se#ap t K! ri A#ant is


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Va%sin vir s !i" p "i#e$a!%an Bia%an


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FAKTOR YANG MEMPENGARU-I STABILITAS VIRUS

SU"U Pemanasan -"ea ing. Pen0inginan -Re&rigera i#n. Pembekuan -%ree4ing. Peleburan - ha1ing.

RA+IASI Sinar Ul ra 3i#le Sinar ? Sinar

7A* KIMIA Asam Oksigen -Aera i#n.(asa An ibi# ika!An i3iral$aram An i serum spesi&ik"al#gen 7a 1arna -Ph# #8Serum n#rmal 0ynami2 ina2 i3a i#n.

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Diagnosa Lab 2