Bagian Patologi Klinik

FK UNHAS

Pengamatan Pada :
- Eritrosit, Lekosit, Trombosit
- Manifestasi : Anemia, lekositosis /lekopeni dan DIC
Lekositosis
- Umumnya Netrofil
, bentuk muda
- Netrofilia lanjut infeksi kronik
- Netrofilia menghebat + sel muda reaksi leukemoid
* Non-Ganas > 25-30 x 106/l
* Inflamasi, Stress, Trauma.

Lekopeni
* Netropenia, mis Demam Tifoid, Brucellosis
* Infeksi hebat netropenia hebat prognosis
buruk
Perubahan morfologik pada sepsis
* Dohle Bodies
* Granulasi Toksik
* Vakuolisasi
Eosinofilia :
* Non-bakterial, biasanya alergi/infeksi parasit

Anemia
* Bisa timbul sekalipun cadangan besi cukup
* Anemia akut : perdarahan / destruksi eritrosit
(misalnya : cold aglutinin sehubungan dengan
Mycoplasma pneumoniae)
* Anemia kronik, dengan :
- Cadangan besi yang normal atau meninggi
di sistem retikuloendotelial
- Penurunan besi dalam plasma
- Penurunan TIBC

Infeksi serius + bakteremia

* Gram negatif DIC (gram positif kurang)
- Trombus
- PT memanjang
- FDP
- Fibrinogen
Trombositopenia bisa juga menjadi tanda sepsis
bakterial dan bisa bermanfaat dalam
mengobservasi respon pasien terhadap terapi.

Laboratory findings
* Hematology
- Leukopenia
- Trombocytopenia
- serum aminotransferase (AST, ALT) elevations
* The diagnosis is made by lab test seroloimmunology
1. Hemaglutination tests
2. Complement Fixation test
3. Neutralization Test
- IgM ELISA or
- paired serology during recovery or
- by antigen-detection ELISA or
- RT-PCR during the acute phase
* Virus is readily isolated from blood in the acute phase if mosquito
inoculation or mosquito cell culture

Diagnosis Demam Dengue/Demam Berdarah

Dengue

Suspicion of dengue infection

Delay after onset of fever

Unknown or 5 days
NS1 Antigen
+

Confirmed early
infection
Acute infection
+

>5 hari

Antisipated Diagnosis
IgM/IgG Serologis
Improve patient Management
+

IgM serology

Late acute or

Dengue

past-infection

is
unlikely

Presumably early early acute infection

Acute infection
is unlikely
A second sample isrequested for confirmation
EARLY DIAGNOSIS
LATE DIAGNOSIS

In dengue
Present

by the 2nd day of fever

By the 4th or 5th day, the wbc count
2000 to 4000/ml,
20 to 40 % granulocytes
Moderate albuminuria and a few casts may be found
- Dengue may be confused with Colorado tick fever,
typhus, yellow fever, or other hemorhagic fevers.
Serologic diagnosis may be made by
- Hemagglutination inhibiting and complement fixation
test using paired sera
- but is complicated by cross-reactions with other
flavivirus antibodies

In dengue hemorrhagic fever

Hct >50 %: ipresent during shock
WBC count
in 1/3 of patients
Coagulaive abnormalities
- Trombocytopenia (<100.000/ml)
- positive tourniquet test
- prolonged PT
- Minimal proteinuria may be present.
- AST levels may be moderately
- Serologic test usually show high complement
fixation antibodies titers againts flavivirus,
suggestive of a secondary immune response

WHO clinical criteria for diagnosis of dengue

hemorrhagic fever :

Acute onset of high, continous fever lasts for 2 to 7

days
Hemorrhagic manifestations, including at least a
positive tourniquet tes and petechiae, purpura,
ecchymoses, bleeding gums, hematemesis or melena
Hepatomegaly
Trombocytopenia (<100.000/ml);or hemoconcentration
(Hct increased by >20%)
Those with dengue shock syndrome also have a rapid
weak pulse with narrowing of the pulse pressure (<20
mmHg) or hypotension with cold, clammy skin and
restlessness.

Laboratory tests are generally not necessary

(viral cultures and Tzanck smear will confirm
diagnosis in patient with atypical presentation)
Antibody to appropriate serotype :
- Seroconversion
- Increase
- Direct immunofluorescent antibodies slide test
(rapid diagnosis)
Tzanck preparation :
- Base of lesion
- Multinucleate giant cells

Laboratory test are generally not

necessary (viral cultures and
Tzanck smear will confirm
diagnosis in patient with atypical
presentation)
The Tzanck preparation shows
multinucleate giant cells for both
varicella-zoster virus and HSV

Darah
- lekopeni
- serum amilase
dalam 10 hari
- Serologi :
* cold agglutinin
* IgM
, max 2 minggu, menetap 6-9 bulan;
kadar serum konvalesens 4x dr pd serum akut
* tes fixasi komplement thd atb positif minggu
pertama
Biakan
- Virus dari ludah 1-5 hari

Komplikasi :
- Inflamasi testis / ovarium : lekositosis, LED
- Pankreatitis : lekositosis, amilase , hiperglikemia
- Meningitis : sel LCS < 500/l, mononuklear; glukosa
normal, protein agak
(20-125 mg/dl)

Temuan Laboratorium
- Darah :
* Lekosit terutama limfosit dan segmen
* Serologi : EIA
- IgM : Fase akut ( 1-2 hari)
- IgG : >10 hari
- Sekret :
* Apusan + pulasan imunofluorosen
* Pulasan Tzanck : Multinucleated Giant Cells
- Biakan :
* Bahan : sekret resp dan urin
* Identifikasi : jaringan

Tes Lab yang bisa dilakukan:

- Sediaan Apus :
* Bahan : Kerokan dasar vesikel
* Pulasan : Tzanck Multinucleated Giant Cells
* Sensitivitas 60 %
- Darah :
* Serologi :
- Titer atb serum konvalesent 4x dpd
serum akut.
- Hemaglutinasi
- ELISA
- FAMA
* PCR : deteksi DNA Virus

Giemsa stained
* Show inclusion bodies within many large cells
or extracellularly

DNA typing : Circular doubel stranded ,

8000 bp
* Crosshybridization :
- > 50% : type seperation
- < 50% : subtype seperation

Generally not necessary

Gram stain and C&S to confirm the diagnosis
when the clinical presentation is unclear
Sedimentation rate parallel to activity of the
disease
Anti-DNAse B and anty hyaluronidase
Urinalysis : hematuria with erytrocyte casts and
proteinuria in patients with acute nephritis

LABORATORY DIAGNOSIS (ANTIBODY TEST)

1. Enzyme-linked Immunosorbent Assay (ELISA)

Most commonly used because of its relatively simple

methodology, high sensitivity and suitability for
testing large numbers of samples, particularly in
blood
testing centres.
Simultaneously detection of antibodies to HIV-1 and
HIV-2 in human serum or plasma.
Detect antibodies directed against major group of HIV-1
and HIV-2 proteins:
HIV-1: envelope protein of gp41
core structural protein of p24
HIV-2: envelope protein of gp36
Methods: indirect, competitive, antigen sandwich

ELISA for HIV antibody

Microplate ELISA for HIV antibody: coloured wells

indicate reactivity

LABORATORY DIAGNOSIS (ANTIBODY TEST: ELISA)

ELISA Test Kit

ELISA Machine

LABORATORY DIAGNOSIS (ANTIBODY TEST: ELISA)

Detection of Ag & Ab in a single test

Utility in primary infection:

pre-seroconversion (window period)

Blood bank

Automated platforms available

2. Simple Test

Requires greater than 30 minutes but has procedures that

can be performed easily without instrumentation.
Within this class of tests are agglutination assays in which
antigen-coated particles (red blood cells [RBC], latex
particles or gelatin particles) are allow to react with serum
antibodies to form visible clumping (agglutination).
If RBC are used, the technique is termed passive
haemagglutination (PHA); latex particles is known as latex
agglutination (LA); gelatin particle is known as particle
agglutination (PA).

Good sensitivity, low cost and ease of performance.

It incorporates a quality system to detect nonspecific
antibodies directed toward the gelatin particles themselves;
sample need to be
treated by adsorption and retested.
False-negative reaction due to prozone (inhibition of
agglutination when excess of antibody present, events
optimal combination) - perform dilution.
Results can be obtained within 2 hours.

LABORATORY DIAGNOSIS (ANTIBODY TEST: SIMPLE TEST)

Particle Agglutination Test

(B) Supplementary/Confirmation Tests

1. Western Blot (WB)

The most widely accepted gold standard for validation of

HIV results.
Based on electrophoretic technique to separate HIV antigens
derived from viral lysate grown in culture and the separate
antigens are transferred (blotted) onto nitrocellulose
membrane.
Commercial WB are supplied with individual strips of
nitrocellulose containing the blotted, separated HIV antigens.
Application of ELISA technique in detection of specific
antibodies to each viral antigens.
Modified WB has the ability to identify and differentiate
infections by HIV-1 and HIV-2.

Western Blot

LABORATORY DIAGNOSIS (ANTIBODY TEST: SUPPLEMENTARY/CONFIRMATORY)

2. Line Immunoassay (LIA)

Another alternative to the classic Western blot.
Recombinant or synthetic peptide antigens are applied onto
nylon strip rather than electrophoresed as in the Western
blot.
Use of artificial antigens decreases the presence of
contaminating substances derived from cell culture that can
cause interference and sometimes false reactions.
A number of reports have verified that the accuracy is
equivalent to the Western blot.

LABORATORY DIAGNOSIS (ANTIBODY TEST: SUPPLEMENTARY/CONFIRMATORY)

Line Immunoassay

Interpretation criteria for Immunoblot tests

Organization
reactive pattern

Minimum band requirements for

American Red Cross

At least one band from each gene

product group: gag AND pol AND env

Centres for Disease

Control (CDC)

Any two of p24, gp41 or gp120/160

World Health Organization

(WHO)

Two env bands (gp160, gp120 or gp41)

+/- pol bands, +/- gag bands

Du Pont

p24 AND p31 AND gp41 or gp120/160

gp160
gp120

p68
p55
p53

Spectrum
of anti-HIV
testing

gp41-45
p40
p34
p24
p18
p12

early

recent / established

advanced

DNA PCR
RNA PCR
p24 Ag
3rd gen ELISA
1st gen ELISA

1wk

2wk

3wk

2mo 6mo

1yr 2yr 3yr

+8yr

(C) Other Antibody Test: Rapid Test

Tests that can be performed in less than 30 minutes, easy to

perform.
Qualitative, intended for use as point-of-care
Technical errors are common with these assays and must be
performed carefully by experienced personnel.
Application includes: emergency rooms, physician's offices,
autopsy rooms. Not recommended for use in a blood
transfusion centre or home-use.
Example:
(i)
"dot blot" or "immunoblot (incorporate a built-in control
that indicates that the test was performed
correctly)
(ii) dipsticks (antigen is attached on the "teeth" of comb-

LABORATORY DIAGNOSIS (ANTIBODY TEST: RAPID TEST)

Several of these rapid tests

differentiate HIV-1 and HIV-2.

have

the

ability

Disadvantages:
subjective interpretation
more expensive
sensitivity may not match the ELISA
Result obtained, must always be confirmed
Types of samples: blood, serum, plasma, saliva and urine

to

HIV RAPID TEST KIT

LABORATORY DIAGNOSIS (ANTIBODY TEST: RAPID TEST)

LABORATORY DIAGNOSIS (ANTIBODY TEST: RAPID TEST)

A woman demonstrates the use of the HIV rapid test

LABORATORY DIAGNOSIS (ANTIBODY TEST: RAPID TEST)

LABORATORY DIAGNOSIS (ANTIBODY TEST: RAPID TEST)

LABORATORY DIAGNOSIS (ANTIBODY TEST)

HIV Testing Strategies

WHO recommends three testing strategies to:

-Maximize accuracy
-Minimize cost
Choice of strategy depends upon:
-objective of test
-prevalence of HIV in population

LABORATORY DIAGNOSIS (ANTIBODY TEST: TESTING STRATEGIES)

WHO Testing Strategies

Objective of testing
Transfusion/donation
safety
Surveillance

Prevalence
of Infection
All

Testing
Strategy
I

>10%

<10%

II

all

II

>10%

II

<10%

III

Clinical signs/
Diagnosis

symptoms of HIV
infection/AIDS
Asymptomatic

LABORATORY DIAGNOSIS (ANTIBODY TEST: TESTING STRATEGIES)

Strategy I:
All samples are tested with one ELISA or rapid/simple assay.
Samples that is reactive is considered HIV Ab positive.
Strategy II:
All samples are first tested with one test.
Any reactive samples are subjected to second test based on
different principle and/or a different antigen preparation.
Strategy III:
All samples are first tested with one test.
Any reactive samples are subjected to second test based on
different principle and/or a different antigen preparation.
Requires a third test if samples are found reactive on the second
test.

LABORATORY DIAGNOSIS (ANTIBODY TEST)

POSITIVE
High Risk Group (WHO 2-tests strategy)
ELISA - Reactive
PA - Detected
Low Risk Group (WHO 3-tests strategy)
ELISA - Reactive
PA - Detected
Immunoblot - Positive
NEGATIVE
Both screening tests: EIA - non-reactive and
PA - not-detected

LABORATORY DIAGNOSIS (ANTIBODY TEST: INTERPRETING TEST RESULTS )

FALSE POSITIVE
The ELISA test may produce false positive results on some
blood from uninfected individuals.
Employs at least 2 different tests.
May due to nonspecific reaction, autoimmune diseases
(Systemic Lupus Erythematosus), etc

LABORATORY DIAGNOSIS (ANTIBODY TEST: INTERPRETING TEST RESULTS )

FALSE NEGATIVE
Test concludes HIV is not present, when in fact the person is
infected
This occurs when the blood is taken during the window
period.
If there has been exposure to risk activities, it is advisable
to repeat the test 3-6 months later.

Sediaan darah malaria

Kegunaan sediaan darah malaria :
Menentukan ada tdknya parasit malaria
Menentukan spesies & stadium plasmodium
Dapat melacak 10 100 parasit/L darah
Menentukan kepadatan parasit

50

Sediaan darah tebal

Cara terbaik menemukan parasit malaria
Mudah dibuat
Diperiksa paling sedikit 100 lapangan pandang

Sediaan darah tipis

Digunakan utk identifikasi jenis Plasmodium bila
dgn sediaan darah tebal sulit ditentukan
Diperiksa paling sedikit 200 lapangan pandang

51

Kelebihan sediaan darah tebal

Lebih banyak sel darah yg diperiksa
Parasit lebih mudah ditemukan

Kekurangan sediaan darah tebal

Tdk dpt membandingkan ukuran Plasmodium dgn
ukuran eritrosit
Spesies Plasmodium sukar ditentukan

52

Kelebihan sediaan darah tipis

Morfologi eritrosit jelas
Spesies Plasmodium bisa ditentukan
Perbandingan ukuran Plasmodium terhdp ukuran
eritrosit bisa dilihat
% parasitemia bisa dihitung

Kekurangan sediaan darah tipis

Jumlah parasit dlm lap. pandang sangat sedikit.

53

Bentuk Tropozoit
Cincin kecil ( eritrosit normal)
Sitoplasma biru
Kromatin inti merah

Bentuk Skizon
Jarang ada dlm sirkulasi darah tepi
Jk ditemukan dlm darah tepi
tanda
malaria berat

54

Bentuk Gametosit
Sgt khas yaitu elips (crescent)
Berpigmen warna hitam
Sitoplasma kuning

55

Teknik Quantitative Buffy Coat (QBC)

Prinsip : Tes Fluoresensi
Eritrosit yg terinfeksi Plasmodium akan terlihat
berfluoresensi di bwh mikroskop fluoresensi

Cepat namun peralatannya mahal

Tdk dpt membedakan spesies
Plasmodium & tdk dpt digunakan utk
hitung parasit.

56

Mendeteksi Ab/Ag spesifik terhadap parasit

malaria atau eritrosit yg terinfeksi Plasmodium
Tes imunoserologis yg melacak Ab tdk dipakai
utk keperluan diagnosis
Tes imunoserologis malaria :
Radioimmunoassay (RIA)
Enzyme Linked Immunoassay (ELISA)
Immunochromatographi (ICT)
Indirect Fluorescent Antibody Test (IFAT)

57

Radioisotop sebagai label

Kadar Ag atau Ab pada sampel dpt ditentukan scr
kuantitatif
Low detection limit 50 parasit/L darah
Sensitif
Kurang praktis & berbahaya

58

Lebih praktis dibanding RIA

Enzyme direaksikan dgn substrat kromogen
intensitas warna sebanding dgn kadar bahan
Mendeteksi Ag & Ab spesifik terhadap Plasmodium

59

Rapid Diagnostic Test

Melacak Ag parasit malaria melalui pengikatan
Ag oleh Ab monoklonal
Ada 3 jenis Ag utama yg sering dijadikan
target ICT utk mendiagnosis malaria yaitu :
Histidine-rich protein (HRP)
Parasite specific lactate dehydrogenase (pLDH)
Plasmodium Aldolase

60

Keunggulan tes ICT

Praktis
Tdk membutuhkan alat pembantu lain
Tdk memerlukan tenaga terampil

Kelemahan tes ICT

Hanya dpt melacak parasit > 100 parasit/L darah
Membutuhkan biaya pemeriksaan yg relatif sdg
Tdk dpt memberi informasi derajat parasitemia

61

Mendeteksi Ab spesifik terhdp Plasmodium

Keadaan dimana parasit sangat minimal
Tdk utk menentukan infeksi baru
Mendeteksi keempat spesies Plasmodium
Manfaat utk penelitian epidemiologi

62

Mendeteksi DNA spesifik terhadap parasit

Plasmodium dlm darah penderita malaria
Teknik Polymerase chain reaction (PCR)
Dpt melacak sampai 5 parasit/L drh
Dpt mengidentifikasi spesies Plasmodium
Waktu pemeriksaan cepat
Sensitivitas & spesifisitasnya tinggi
Mahal

63

Jenis tes laboratorium untuk tuberkulosis

terdiri dari:

Tes Mikrobiologi, terdiri dari:

Tes seluler:

Tes BTA Sputum

Tes Biakan dan Identifikasi M.tuberculosis
Tes Kepekaan Antibiotika

Tes molekuler: PCR

Tes Serologis, terdiri dari:

Semirapid: TB-Dot, ELISA, Tb-kompleks

Rapid: Mycodot, ICT-TB

Anamnesis
Pemeriksaan Fisis
Pemeriksaan Radiologis
Tes Laboratorium
Tes Mikrobiologi
Tes seluler: Tes Apusan BTA
Tes Biakan & Identifikasi
Tes Kepekaan Antibiotika

Tes molekuler: PCR

Tes Serologis
Semirapid: TB-Dot, ELISA, Tb-kompleks
Rapid: Mycodot, ICT-TB seperti Mycotec TB
Ujian Akhir Magister/Ujian Lokal

26 Maret 07

65

Mikroskopik

Kultur (pembiakan)

Ziehl Neelsen
Dekontaminasi
medium Lowenstein-Jensen

PCR

Isolasi DNA: Metode Boom

Proses : denaturasi, annealing, elongasi

IS6110

Ujian Akhir Magister/Ujian Lokal

26 Maret 07

66

Tes Imunoserologi

Deteksi Ab terhadap Ag mikobakterial spesifik

atau gabungan beberapa antigen

Ag60: ELISA
Ag16 : 16 kDa,
spesies-spesific epitop imunodominan
stadium awal infeksi M.tbc & TB primer
38kDa
spesies-spesific epitop imunodominan
ESAT-6
kontak baru terjadi, konversi & meningkatnya risiko
penyakit

Ujian Akhir Magister/Ujian Lokal

26 Maret 07

67

Kultur :
* Darah : positif dalam 10 hari pertama
* Tinja & urin positif dalam minggu 3-5
* Sumsum tulang
Serologi :
* Tes Widal : Serum sembuh 4x
drpada sakit
Darah rutin : lekopeni

Felix-Widal test

- measures levels of agglutinating antibodies against O and H

antigens
- usually appear after 5-8 days of infection
- limited clinical utility in acutely ill patients because positive results
may represent previous infection in endemic areas
- paired titration should be preformed
- In the absence of recent immunization, a high titre of antibody to O
antigen > 1:640 is suggestive but not specific.
- false positive (cross reactivity)

An ELISA for antibodies to the capsular polysaccharide Vi antigen is

useful for detection of carriers but not for the diagnosis of acute
illness

Typhidot test that detects presence of IgM and IgG in one hour
(sensitivity>95%, Specificity 75%)

Typhidot-M, that detects IgM only (sensitivity 90% and

specificity 93%)

Typhidot rapid (sensitivity 85% and Specificity 99%) is a

rapid 15 minute immunochromatographic test to detect IgM.

IgM dipstick test