Basic Serological

Tests
Out line

3.1 Introduction to serology

3.2 Serology of syphilis

3.3 Serological test for febrile disease

3.4 Pregnancy Test

3.5 HIV Tests

3.1 Introduction to serology
Definitions
 Serology is the scientific study of blood serum.

 In practice, the term usually refers to the diagnostic

identification of antibodies in the serum.
+ Such antibodies are typically formed in response to
an infection (against a given microorganism)

 against other foreign proteins (in response, for

example, to a mismatched blood transfusion), or to
one's own proteins (in instances of autoimmune
disease).
Introduction…

 Serological tests may be performed for

diagnostic purposes when an infection is
suspected, in rheumatic illnesses, and in many
other situations, such as checking an individual's
blood type.
 Serology blood tests help to diagnose patients
with certain immune deficiencies associated with
the lack of antibodies.
In such cases, tests for antibodies will be
consistently negative.
Introduction…

 Antibody molecules combine reversibly with

antigens to form immune complexes.

Ag+Ab Ag.Ab complex

 The detection and measurements of these

reactions form the basis of serology a sub
discipline of immunology.
Introduction…....cont

 Therefore;
 Serology - is the science of measuring
antibody or antigen in body fluids.

 Widely used in the serological diagnosis of

 bacterial,

 viral,

 fungal, and

 parasitic diseases.

 They are usually sensitive and give

reproducible results.
 3.2 Syphilis Serology

 Syphilis is a chronic systemic disease, which

leads to lesions on the body.

 It is derived from a Greek word "syphilos"

meaning crippled, maimed (heart victim).

Etiology and Transmission
 Is a systematic infection caused by the spirochate
Treponema pallidium

 Transmitted by:
 Mainly Sexual contact (Venereal syphilis)
 Less commonly via the placenta (congenital
syphilis) OR
 By accidental inoculation from infectious
material
e.g. fresh blood transfusion
Morphology
 T. pallidum is seen
microscopically as a closed
coiled, thin, delicate
regular spiral organism
varying in length from 6 to
15m and consisting of 8 to
24 coils.
 The only known natural host
for T. pallidum is the
human.
 Serologic tests for syphilis

 Generally grouped into TWO, based upon the

type of Ag used and Ab detected

A. Standard non-treponemal tests: - which

use either cardioipin or lipoid extracts as
antigens and detect reagin. (E.g.,VDRL, RPR)
B. Standard treponemal tests: - which use
treponemal antigens and peak up specific
antibodies. ( E.g., FTA-ABS, and TPI)
SEROLOGICAL TECHNIQUE
RPR (Rapid reagin card test for syphilis)
Principle
 Destructive syphilitic lesions cause tissue damage.
Circulating antibodies called reagin are produced
against some of the tissue components.
 The rapid reagin card test uses a modified form
of the VDRL (Vernal Disease Research Laboratory)
antigen called cardiolipin in suspension with
carbon particles.
 When cardiolipin antigen reacts with reagin
antibody in patient’s serum the carbon particles in
the suspension clump together.
Materials

The following are provided in the test kits:

 Reagin antigen suspension

 Reagin positive control serum

 Reagin negative control serum

 Reagin test card

 Dispensing bottle and needle

 Dropper tubes

 Mixing sticks and etc

Qualitative test method
Procedure (RPR)
 Let the reagents and specimens warm up to room
temperature.
 Dispense one drop of negative control serum on to
one circle on the test card using a disposable
dropper tube.
 Repeat step two with the positive control serum
using a clean dropper tube.
 Dispense on drop of each sample serum or plasma
on to one circle on the card using a clean dropper
tube for each specimen.
Procedure (RPR) ….
 Spread all the drops to cover the whole area of the
circles using the mixing sticks.
 Mix with reagain antigen suspension in the
dispensing bottle. Hold the bottle vertically and
dispense one drop on to each test sample. Do not
mix again.
 Place the card on the rotor for 8 minutes at
100rpm.
Reading the results

 Negative result:

The carbon particles remain in an even

suspension = Non reactive

 Positive result:

The carbon particles clump together

= Reactive
For the test to be valid the negative control must be non-
reactive and the positive control must be reactive
Quantitative test method
1. Dispense 1 drop of 0.85% saline on to circles 1 to
5 on the test card
2. Dispense 50µl of patient serum or plasma on to
the first circle and mix by filling and discharging
the pipette at least 6 times (do not make
bubbles). You now have a 1 in 2 dilution in the
first circle.
3. Transfer 50µl from the first circle to the second
circle and repeat the mixing procedure.
4. Continue the dilution procedure to circles 3, 4
and 5 and discard the last 50µl.
 You know have the following dilutions:

Circle 1 2 3 4 5

Dilution ½ ¼ 1/8 1/16 1/32

5. Starting at circler number 5 uses a mixing stick to
spread all the drops to cover the whole area of the
circles.

6. Mix the reagin antigen suspension in the

dispensing bottle. Hold the bottle vertically and
dispense one drop on to each test sample. Do not
mix again.

7. Place the card on the rotor for 8 minutes at

100rpm.
Reporting the results of a quantitative test

 The titer reported in a quantitative test is the

highest sample dilution to show a reactive
(positive) result.
 In the example below the result would be
reported as:
 Reactive to 1/8
If the highest dilution (1 in 32) is reactive proceed
as follows:
8. Prepare a 1in 16 dilution of the sample by adding
0.1ml of serum to 1.5ml of 0.85% saline and mix
well.
9. Dispense 1 drop of saline on to circles 6 to 10 on
the test card using a dropper tube.
10.Dispense 1 drop of the 1 in 16 dilution on to
circle number 6 and proceed as before (step 3
and 4) making doubling dilutions up to well
number 10
You know have the following dilutions:

Circle 6 7 8 9 10
Dilution 1/32 1/64 1/128 1/256 1/512

11. starting at well number 10 spread all the drops as

before
12. Mix the reagin antigen suspension in the dispensing
bottle. Hold the bottle vertically and dispense one drop
on to each circle. Do not mix again
13. place the card on the rotor for 8 minute at
100rpm

14. report the titer as the highest dilution to show a

positive result
 VDRL TEST

Slide Qualitative Test

Sample: serum
Principle
 During the period of infection with syphilis,
reagin, a substance with the properties of an
antibody, appears in the serum affected patients.
 Reagin has the ability to combine with a colloidal
suspension extracted from animal tissue and
clump together to form visible masses, a process
known as flocculation.
Procedure:
1. Pipette 0.05ml or 1drop of inactivated serum into
one ring of the ringed glass slide.
2. Add one-drop (1/60ml) antigen suspension onto
each serum.
3. Rotate slide for 4 minutes. (If rotated by hand on
a flat surface, this movement should roughly
circumscribed a 2 inch/5mm diameter circle).
4. Tests are read immediately after rotation
microscopically with a 10x ocular and a 10x
objective.
Reading and reporting of results
 Tests are read microscopically with low power
objective at 10x magnification, which appears
short rod forms. Aggregation of these particles
into large or small clumps is interpreted in
degrees of reactivity.

Reporting system

No clumping or very slight roughness : Non-reactive (NR)

Small clumps : Weakly reactive (WR)
Medium and large clumps : Reactive (R)
3.3. serological test for Febrile
diseases
3.3.1 Salmonella

 Serologic diagnosis

3.3.2 Rickettisia

 Serologic diagnosis
 Salmonella

 Are often pathogenic for humans or animals

 transmitted from animal and animal product to

humans

 Three main diseases are

 Enterocolitis (enteritis)

 Septicemia (systemic infection)

 Enteric fever (typhoid fever)

 Morphology
 Microscopic
+ Vary in length

+ Gram –Ve

+ Rod shape bacilli

+ Most isolates are motile

+ has peritrichous flagella

 Classification

 Four serotypes can cause enteric fever

+ S. paratyphi A (Serogroup A)

+ S. paratyphi B (serogroup B)

+ S. choleraesusis (serogroup C1)

+ S. typhi (serogroup D)
Antigenic variation

 Salmonella has
- O-antigen………somatic antigen

- H-antigen……… flagellar antigen

- Vi- antigen ……capsular antigen

+ Organism may lose H antigens and become

non-motile
The enteric fever (Typhoid fever)

 Produced by only S. typhi

 The ingested salmonella reach the small intestine

From which they enter the lymphatics Then
go to blood stream From this to many organs
including intestine The organism multiply in
intestinal lymphoid tissue And excreta stool
 Laboratory Diagnosis

 Serological method

 Widal Test

 Widal test is a serological test widely used for diagnosis of

enteric fever.

 It is suspension of killed S. typhi as antigen to detect anti S.

typhi antibody

 Widal test for typhoid and paratyphoid fever is an

agglutination test.
 Laboratory Diagnosis…

 Widal test measures titres of serum agglutinins against

somatic (O) and flagellar (H) antigens which usually
begin to appear during the 2nd week.

 In the absence of recent immunization, a high titer of

antibody to O antigen > 1:640 is suggestive but not
specific.
 Laboratory Diagnosis….
 The typhoid bacillus causes two types of
agglutinins to be produced. The agglutinins are
called:

 Flagella (H) agglutinins

 Somatic (O) agglutinins.

 The patient serum is tested for those O and H

antibodies against the antigen suspensions.
 Methods of widal tests

+ Tube method

+ Slide method
 Procedures
Slide method
 Modification of tube test method by Welch and
Mickle at 1936
Specimen: serum/plasma

 Take clean slide

 Add a drop of serum, which is obtained, from non-
hemolyzed blood.
 Add a drop of antigen suspension, which is non-
expired,
 Mix well antigen suspension and serum.
 Look for agglutination.
Result interpretation

Positive Result : Reactive

 Aggultination reaction indicate positive for
thyphoid fever
Negative result: Non reactive
 No agglutination reaction--- Negative for typhoid
fever
Tube method
 Used to confirm slide test method

Sample: serum/plasma
Procedure
1. For each antigen arrange 10 small test tubes in
a rack.
2. Place 0.9ml of saline in the 1st tube and 0.5ml
in the remaining 9 tubes.
3. Add 0.1ml of fresh cell-free serum to the 1st
tube.
4. Mix and transfer 0.5ml to tube
2,3,4,5,6,7,8,and tube 9. From tube 9 discard
0.5ml.
+ Tube 10 will contain only saline and will
serve as a negative control (antigen
control)
5. Mix antigens well and add 0.5ml to each tube.
Mix by gently shaking the tubes.
6. The final dilutions are 1:20, 1:40, 1:80,
1:160, etc.
7. Incubate the tubes at 37oC for 2-4 hrs
8. Read the negative control at the end of the
incubation period.
+ It should have no agglutination.
9. Read the test one row of antigen at a time. For
reading a white light shining vertically above
the tube is best and using a black background.
10. Shake the tubes gently.
The H type of agglutination is easily broken
up and may be missed. The agglutination is
more granular and not so fragile.
11. Report the highest dilution with definite clumps.
Tube dilution
0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

0.5
0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

initial
Tube 1 2 3 4 5 6 7 8 9 10

Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120

Dilution
factor 10 20 40 80 160 320 640 1280 2560 5120
Reporting of Widal Reactions

 The Widal test is reported by giving the titer

for both O and H antibodies.

 The antibody titer is taken as the highest

dilution of serum in which agglutination
occurs.
 If no agglutination occurs the test should be
reported as:
 S. typhi O titer less than in 20 (O 1:20)

 S. typhi H titer less than in 20 (H 1:20)

Interpretation of the Widal Reaction

 A Negative test does not necessarily mean the

patient is not infected. Reaction occurs in infected
patients about

 50% during the 1st week

 80% in the 2nd week,

 90-95% in the 3rd or 4th week

Serologic diagnosis of rikettsiaceae

 The most reliable and useful serological technique

for diagnosing rickettsial infections is the
indirect fluorescent antibody (IFA) test. It is
of value not only in diagnosing acute infections
but also in serological epidemiological studies.
Another important test is CFT test, which is not
sensitive as IFA test.
Weil-Felix reaction test

 Weil-Felix is an agglutination test for various

rickettsial infections using particular strains of
bacteria of the genus Proteus that have antigens
in common with the rickettsiae to be
 The Weil-Felix reaction test, developed by Weil
and Felix (1916), is based on the fact that certain
strains of Proteus vulgaris and P. mirabilis share
antigens with several of the rickettsia species that
produce febrile disease, such as typhus.
 Three strains of Proteus species have been found
to be useful in diagnosing rickettsial diseases;
these have been labeled OX-2, OX-19 and OX-K.
A test for diagnosis of typhus and certain other
rickettsial diseases.

The blood serum of a patient with suspected

rickettsial disease is tested against certain strains
of (OX-2, OX-19, OX-K).

The agglutination reactions, based on antigens

common to both organisms, determine the
presence and type of rickettsial infection.
 Weil-Felix reaction is an agglutination test based
on the cross-reactions, which occur between
antibodies, produced in acute rickettsial
infections and the OX-19 and X-2 strains of
Proteus vulgaris and the OX-K strains of Proteus
mirabilis.
 In a Weil-Felix reaction a strong agglutination
test with strain OX-19 may indicate epidemic or
endemic typhus, and a weak agglutination may
indicate Rocky Mountain spotted fever,
Mediterranean fever and South African tick fever,
while agglutination with Proteus strain OX-K
indicates scrub typhus.
 The Weil-Felix reactions are non- specific and
cannot be fully relied on to diagnose acute
rickettsial infections.

 False positive Weil-Felix reactions are may occurs

in Proteus infections, R. tsutsugamushi infections,
relapsing fever, brucellosis, rat bite fever,
infectious, mononucleosis, and other acute febrile
illness.
3.4 Pregnancy tests
3.4 Pregnancy tests
Outline

 Introduction

 Serology of hCG in Urine

 Urine pregnancy tests

 Factors that affects urine pregnanacy test

 Urine specimen collection

 Method of determining hCG

Introduction

 Pregnancy is the period during which a woman

carries a baby with in her body before giving
birth.

 Pregnancy begins with conception-that is, the

fertilization of an egg by a sperm.

 The fertilized egg is called zygote.

Production of hCG

 Human chorionic gonadotrophin (hCG) is

synthesized in large amounts by the placenta,
and it appears in urine, blood, amniotic fluid and
serum relatively soon after implantation of the
developing embryo.

 The presence of this hormone serves as basis for

pregnancy testing.
Introduction…

 During pregnancy, the chorionic membrane of the

placenta produces a hormone called human
chorionic gonadotrophin (hCG), which
stimulates the secretion of progesterone, by the
ovary.

 Progesterone maintains the uterus during the

pregnancy and prevents any further release of
eggs from the ovary.
Production of hCG…

 Production of hCG increases steadily during the

first trimester, peaking around the 10th week of
gestation.

 Levels then fall to less than 10% of the first

trimester levels during the reminder of the
pregnancy.
 The urine and serum of pregnant women contain
high concentrations of human chorionic
gonadotrophin (hCG) produced by trophoblast,
which provide the basis of tests for the diagnosis of
pregnancy.
 Specific and sensitive analytical methods for the -
chain sub unit of hCG permit the detection of
pregnancy as early as 8 days after ovulation (1 day
after implantation).
 hCG concentrations climb early in pregnancy,
reaching a maximum by 8 to 10 weeks of
gestation.
 Application of pregnancy tests

 Pregnancy tests are important in investigation of

suspected:

 To detect early pregnancy.

 To determine the adequacy of hormone

production in high risk pregnancies (for
example, habitual abortion)
Application of ……cont

 Ectiopic disease: the implantation of the

fertilized egg in the extra uterine space.
Example in the fallopian tube

 In threatened abortion: to conform the

abortion is complete or not. In this case the
quantity of hGC is decreases gradually.
Detection of hCG in urine

 A number of serologic tests have been used in

pregnancy testing; each designed to detect
minute amounts of hCG when it appears in the
urine during the first few weeks of pregnancy.

 The methods most commonly used now are

based on the agglutination inhibition test

 developed by Noto and Miale (1964)

 Laboratory pregnancy tests are based on

detection of rapidly rising levels of hCG in urine.
Specimens
Urine specimen

 An early morning specimen is preferable

because this is the most concentrated and will
therefore contain the highest level of hCG
 The urine must be collected in
 a clean container, which is free from the all
traces of detergent.
 If the specimen cannot be tested immediately it
should be refrigerated at 4c, but for not longer
than 48 hr’s.
Types of urine testing kits

1. Rapid latex slide test of the inhibition

+ Direct agglutination test

2. Strip Immunochromatographic technique

I. Direct slide agglutination test

Principle

In the direct slide test, the latex reagent consists

of particles coated with anti hCG antibodies. This
reagent is mixed directly with the urine there will
be agglutination, which is visible to the naked
eye if the urine contains the hormone hCG. If not
there is no visible agglutination.
Procedure
 Prepare clean, dry, detergent free slides.
 Take one drop of early morning urine in Pasteur
pipettes and drop on slide.
 Take 1 drop of reagent (anti-hCG antibody).
NB. Don’t use an expired reagent.
 Mix gently; and see for agglutination.

Now a day people use random urine for pregnancy test.

Because they consider as the hCG hormone may present at
any time in the urine. But the morning urine is more

preferable due to content of high concentrated hCG.

 If hCG is present in the urine, it will combine with
the antibodies and causes agglutination of the
latex particles.
Urine latex reagent Result
(hCG antibody agglutination
Coated particles)

hCG Antigen hCG antigen

is present combines with hCG
antibody on latex
particles

Positive result
 If no hCG is present in the urine, there will be no
agglutination of the latex particles.
Urine latex reagent Result
(hCG antibody No agglutination
Coated particles)

No hCG No hCG antigen

Antigen combines with hCG
antibody on latex
particles

Positive result
 In the direct slide test, therefore,

 agglutination ndicates positive tests and

 no agglutination indicates a negative test.

Factors that affect pregnancy tests

 The time in the pregnancy when the test is

carried out.
 The presence of excessive amounts of protein or
blood in the urine may cause false positive
results.
 Detergent contaminated urine
 Turbidity of the specimens
 Drugs, which may cause false-positive results,
include
Performing HIV
Rapid Tests
Definition of terms

 HIV is a human immunodeficiency virus which

causes chronic diseases with other infectious
agent.
 HIV Serology

 Several laboratory methods are available to screen

blood, diagnose infection, and monitor disease
progression in individuals infected by HIV.

 HIV tests can be classified into:

+ detect antibody

+ identify antigen

+ detect or monitor viral nucleic acids, and

+ estimate of T-lymphocyte numbers (cell

phenotyping).
HIV Antibody Tests
 Based on a multi test algorithm for detecting
antibodies to HIV by using screening and
confirmatory tests.
Testing Algorithm Describes the
Sequence of Tests to be Performed

 An HIV Positive Status should be based

upon the outcome of 2 or more different
tests
 When the two test results disagree (one is
reactive, the other non-reactive), the
finding is called “discordant .” In this case,
a third test must be performed.
Testing Algorithm (Currently used)
Blood Sample

Test 1 (KHB)

Non-reactive Reactive

Report Negative Test 2 (Stat Pak)

Non Reactive Reactive

Report Positive

Test 3 (Unigold)

Reactive Result Non-reactive Result

Report Positive Report Negative

Supplies & Materials Checklist

 HIV Rapid testing kits  Gloves

 Alcohol  Aprons or
laboratory coats
 Cotton Gauze / Wool  Timer, clock, or
 Sterile Lancets wrist watch with
 Sharps bin or minute hand
Disinfectant jar for
lancets
 Pen for marking or
labeling
 EDTA Capillary Tube
KHB HIV ½
Make ready the device and
label with code number

123

Control Line

Sample port
Test line

78
Specimen collection
Add 40 l of whole

123

Add 1 drop
of running
buffer
123
Test interpretation (within 30 minutes)
Reactive Result Non reactive Result

123 123

Invalid result

123
The whole procedure (30’)
STAT PACK

83
STAT PAK : Getting Ready

84
STAT PAK: Collecting
Specimen

85
STAT PAK: Adding Specimen
and Reagent to Test Device

86
STAT PAK: Getting Results
(10 minute)

87
STAT PAK: Test interpretation
Reactive Non-reactive Invalid
Uni-Gold

89
Uni-Gold: Getting Ready

2. Remove device from

1. Collect test items
package and label
and other necessary
device with client
lab supplies
identification number

90
Uni-Gold: Collecting
Specimen

3. Collect specimen using the disposable pipette

91
 Uni-Gold: Adding Specimen
and Reagent to Test Device

4. Add 2 drops (approx. 5. Add 2 drops (approx.

60µl) of specimen to 60µl) of the appropriate
the sample port in the wash reagent to sample
device port
Uni-Gold: Getting Results

6. Wait for 10
7. Read and record
minutes (no longer
the results and
than 20 min.)
other pertinent info
before reading the
on the worksheet
results
93
Uni-Gold: Test Interpretation

Reactive Non-reactive Invalid

94
 National Algorithm: Ethiopia
Blood Sample

Test 1 (KHB)

Non-reactive Reactive

Report Negative Test 2 (Stat Pak)

Non Reactive Reactive

Report Positive

Test 3 (Unigold)

Reactive Result Non-reactive Result

Report Positive Report Negative

95
 Possible Outcomes in a serial
Algorithm

KHB Stat Pak Unigold HIV Status

Non-reactive Non-reactive Negative
Reactive Reactive Positive
Reactive Non-reactive Non-reactive Negative
Reactive Non-reactive Reactive Positive

96
Exercise: Identifying appropriate
Testing Algorithm
KHB Stat Pak Unigold Put your
remarks
RDH-001 R NR NR

RDH-002 NR NR NR

RDH-003 R R R

RDH-004 R NR R
Remarks on the excercise

 The two procedures/testing (RDH-002

and RDH-003) were not performed based
on the serial algorithm
THANK YOU