SEROLOGY

SEROLOGY:
Serology is the scientific study or diagnostic examination of blood serum,
especially about the response of the immune system to pathogens or
Introduced substances.
Serologic tests are blood tests that look for antibodies in your blood. They
can involve several laboratory techniques. Different types of serologic tests can
diagnose various disease conditions. Serologic tests have one thing in
common. They all focus on proteins made by your immune system. This vital
body system helps keep you healthy by destroying foreign invaders that can
make you ill The process for having the test is the same regardless of which
technique the laboratory uses during serologic testing.
To understand serologic tests and why they're useful, it's helpful to know a
little about the immune system and why we get sick.
Antigens are substances that provoke a response from the immune system.
They are most often too small to see with the naked eye. They can enter the
human body through the mouth, through broken skin, or through the nasal
passages. Antigens that commonly affect people include the following:
 Bacteria
 Fungi
 Viruses
 Parasites
The immune system defends against antigens by producing antibodies.
These antibodies are particles that attach to the antigens and deactivate
them. When your doctor tests your blood, they can identify the type of
antibodies and antigens that are in your blood sample and identify the type
of infection you have.
Sometimes the body mistakes its own healthy tissue for outside invaders
and produces unnecessary antibodies. This is known as an autoimmune
disorder. Serologic testing can detect these antibodies to help your doctor
diagnose an autoimmune disorder.
What Are the Types of Serologic Tests?
Because antibodies are so diverse, various tests are useful for detecting the
presence of different types:
 An agglutination assay shows whether antibodies exposed to certain
antigens will cause particle clumping
 A precipitation test shows whether the antigens are similar by
measuring for the presence of antibody in body fluids.
 The Western blot test identifies the presence of antimicrobial antibodies
in your blood by their reaction with target antigens.
What Do the Results Mean?

Normal Test Results

Your body produces antibodies in response to antigens. If testing
shows no antibodies, it indicates you don't have a current or past
infection. Results that show there are no antibodies in the blood sample
are normal.

Abnormal Test Results

Antibodies in the blood sample often mean you've had an immune
system response to a specific antigen from either a current or a past
exposure to a disease or foreign protein.
The testing may also diagnose an autoimmune disorder. In that case,
antibodies to normal or non-foreign proteins or antigens would be
present in the blood.
The presence of certain types of antibodies can also mean that you're
immune to one or more antigen. This means that future exposure to the
antigen or antigens won't result in illness.

Serologic testing can diagnose multiple illnesses, including:

 brucellosis, which is caused by bacteria
 amebiasis, which is caused by a parasite
 measles, which is caused by a virus
 rubella, which is caused by a virus
 HIV
 Syphilis
 fungal infections
 Class of Immunoglobulins:
• There are five classes of antibody-IgG, IgA, IgM, IgD and IgE.. Each B
cell can produce only one specific antibody to an antigen, each
antibody is highly specific and will bind to only one antigen.
1. IgG- This class of antibody is the most important class of
immunoglobulin in secondary immune responses. IgG crosses the
placenta, conferring protection to the new born and is able to activate
the complement system through the classical pathway.
2. IgM is the predominant antibody in the primary immune responses. It
can also activate the classical pathway complement.
3. IgA is found primarily in secretions such as breast milk, tears, saliva
and mucosal membranes.
4. IgE-evolved to provide protection against certain parasitic infections
however in developed countries it is more commonly associated with
allergic diseases such as asthma and hay fever.
5. IgD-there is little known about this antibody.
HIV:

HIV stands for Human Immunodeficiency Virus

HIV belongs to the retrovirus family, and the genus lentivirus, this is how
HIV copies its genetic information, and the slow progression of the disease
(lento = slow). HIV requires a host cell in order to copy its genetic
information and make new virus particles. For HIV to be able to enter a cell,
the cell must have the CD4 receptor. This is found on immune cells, such as
helper T cells that help activate the immune system to fight infections.
HIV TYPE

HIV-1 is the more prevalent and harmful of the two main types of HIV. It also
contains the most number of subtypes which are organized into groups based
on their prevalence. The three groups of HIV subtypes are M, N and O.

HIV type 2

The second type of HIV is HIV type 2. The RNA sequence that makes up HIV-2 is
completely different from the sequence in HIV-1.

HIV TRANSMISSION:

Sexual transmission -- it can happen when there is contact with infected sexual
fluids (rectal, genital, or oral mucous membranes). This can happen. while
having sex without a condom, including vaginal, oral, and anal sex.
Perinatal transmission -- a mother can transmit HIV to her child during
childbirth, pregnancy, and also through breastfeeding

Blood transmission -- the risk of transmitting HIV through blood transfusion is at

high risk, through meticulous screening and precautions, this can be prevented.
However, among people who inject drugs, sharing and reusing syringes
contaminated with HIV-infected blood is extremely hazardous.

SYMPTOMS:

Symptoms of HIV infection appear 2 to 12 weeks after exposure. At this point

the virus begins rapidly taking over immune cells in the blood. The symptoms of
this phase are flu-like. Without treatment, HIV advances in stages. The three
stages of HIV infection are: (1) acute HIV infection, (2) clinical latency, and (3)
AIDS (acquired immunodeficiency syndrome).
The symptoms of early HIV infection may include:
1. Fever
2. Chills
3. Joint pain
4. Muscle aches
5. Sore throat
6. Enlarged glands
7. A red rash
8. Tiredness
9. Weakness
10. Thrush
2. Clinical Latency Stage:
In many cases, after the initial symptoms disappear, there will not be any
further symptoms for many years.
During this time, the virus carries on developing and damaging the
immune system and organs. Without medication that stops HIV replicating,
this process of slow immune depletion can continue, typically for an average
of 10 years. The person living with HIV often experiences no symptoms, feels
well, and appears healthy.
For people who are taking antiretrovirals and are rigidly compliant, this
phase can be interrupted, with complete viral suppression. Effective
antiretrovirals arrest on-going damage to the immune system.
3. Stage 3-Symptomatic

If left untreated, HIV weakens the ability to fight infection. The person
becomes vulnerable to serious illness. Symptoms may include:
1. blurred vision
2. diarrhoea, which is usually persistent or chronic
3. dry cough
4. fever of above 100 °F (37 °C) lasting for weeks
5. night sweats
6. permanent tiredness
7. shortness of breath (dyspnea)
8. swollen glands lasting for weeks
9. unintentional weight loss
10. white spots on the tongue or mouth oral thrush
HIV and AIDS myths and facts

There are many misconceptions about HIV and AIDS. The virus CANNOT be
transmitted from:
1. shaking hands
2. Hugging
3. casual kissing
4. Sneezing
5. touching unbroken skin
6. using the same toilet
7. sharing towels
8. sharing cutlery
9. mouth-to-mouth resuscitation
10. or other forms of "casual contact“
What is AIDS?

AIDS stands for acquired immune deficiency syndrome.

AIDS is defined as having HIV and a specific type of infection

("opportunistic" infection). These infections can be bacterial, fungal,
viral, or parasitic. people with AIDS will usually need to take medications
(such as antibiotics) to prevent opportunistic infections. People who have
AIDS and receive no treatment are also more likely to develop cancer,
especially cancers of the immune system (lymphomas). Another cancer
common for people with AIDS is Kaposi's sarcoma, a type of cancer that
causes bluish red nodules on the legs and that spreads to the lymph
system. Most people with AIDS die from the diseases that AIDS makes
them more susceptible to. The virus occasionally infects the brain,
causing dementia that gets worse over time.
PREVENTION:

Anyone can become infected with HIV. Fortunately, it can be prevented. The
main ways to prevent HIV infection are:
• Use condoms during sex (including vaginal, oral, and anal sex).
• Have fewer sexual partners.
• Don't share needles or other equipment to take drugs.

TREATMENT:

There is currently no cure for HIV or AIDS. Treatments can stap the
progression of the condition and allow most people living with HIV the
opportunity to live a long and relatively healthy life.
• CD4 tests that measure your CD4 cell count.
• Viral load tests that measure the number of viruses in your
bloodstream, and
• Drug resistance tests that find out whether or not the HIV you are
infected with is resistant to any of the anti-HIV medicines that are
available.
Antivirals used are:

1. Nucleoside reverse transcriptase inhibitors, e.g. AZT

2. Nonnucleoside reverse transcriptase inhibitors, e.g. delavirdine
3. Protease inhibitors, e.g. indinavir
4. Fusion inhibitors e.g. enfuvirtide
LABORATORY DIAGNOSIS:

HIV Tri dot test:

Principle:

HIV antigens are immobilized on a porous immunofiltration membrane.

Sample and reagents pass through the membrane and are absorbed into
the underlying absorbent. As the patients sample passes through the
membrane, HIV antibodies, if present, bind to the immobilized antigens.
Conjugate binds to the Fc portion of the HIV antibodies to give distinct
pinkish purple dot (s) against a white background.
Procedure:
1. Buffer solution - 2 drops
2. Serum-2 drops
3. Buffer solution - 2 drops
4. Protein A Conjugate - 2 drops
5. Buffer solution - 2 drops
Several tests can find antibodies to or genetic material (RNA) of the HIV
virus.
These tests include:
 Enzyme-linked immunosorbent assay (ELISA). This test is usually the
first one used to detect infection with HIV. If antibodies to HIV are
present (positive), the test is usually repeated to confirm the diagnosis.
If ELISA is negative, other tests are not usually needed. This test has a
low chance of having a false result after the first few weeks that a
person is infected.
 Western blot. This test is more difficult than the ELISA to perform, but it
is done to confirm the results of two positive ELISA tests.
 Polymerase chain reaction (PCR). This test finds either the RNA of the
HIV virus or the HIV DNA in white blood cells infected with the virus.
PCR testing is not done as frequently as antibody testing, because it
requires technical skill and expensive equipment. This test may be done
in the days or weeks after exposure to the virus. Genetic material may
be found even if other tests are negative for the virus. The PCR test is
very useful to find a very recent infection, determine if an HIV infection
is present when antibody test results were uncertain, and screen blood
or organs for HIV before donation.
 Indirect fluorescent antibody (IFA). This test detects HIV antibodies
using a special fluorescent dye and a microscope. This test may be used
to confirm the results of an ELISA test.
Blood tests for HIV

HIV is diagnosed when antibodies to HIV are found in the blood. The two
main blood tests are:
 ELISA
 Western blot, used to confirm the results of a positive ELISA test.
 HIV is diagnosed when a positive ELISA test is confirmed by a positive
Western blot assay or another test.
 Rapid antibody tests are available that give results right away. One rapid
blood test can detect both HIV antibodies and antigens, which allows an
HIV infection to be found earlier than was possible in the past. Positive
results of a rapid test may need to be confirmed by the ELISA orWestern
blot test.
Testing positive for HIV

 He or she may order several lab tests to check your overall health,
including:
 A complete blood count (CBC), to identify the numbers and types of
cells in your blood.
 A chemistry screen, to measure the blood levels of certain substances
(such as electrolytes and glucose) and to see how well your liver and
kidneys are working.
 Other tests may be done to check for current or past infections that may
become worse because of HIV. You may be tested for.
 Syphilis.
 Hepatitis A, hepatitis B, and hepatitis C.
 Tuberculosis (TB).
Ongoing tests

When you have HIV, two tests are done regularly to see how much of the
virus is in your blood (viral load) and how the virus is affecting your
immune system:
 CD4+ cell counts provide information about the health of your immune
system.
 Viral load measures the amount of HIV in your blood.
 The results of these tests may help you make decisions about starting
treatment or switching to new medicines if the ones you are taking
aren't helping.
HBsAg (HEPATITIS B SURFACE ANTIGEN)/AUSTRALIA ANTIGEN

Introduction:
Hepatitis B virus (HBV) is a double stranded DNA spherical particle with a
double shell. The double shelled particle was discovered by Dane and
colleagues in 1970 and hence often referred to as the Dane particle.
Antigens which have so far been associated with HBV are as follows:

Hepatitis B surface antigen (HBsAg):- This was first noticed in the blood of
an Australian aborigine hence earlier was termed as Australia antigen.
HBsAg has four types, adw, adr, ayw and agr

Hepatitis B core antigen (HBcAg)

Hepatitis B e antigen (HBeAg):- It contains three components

The main uses for HBV tests include:
 To determine whether acute signs and symptoms are due to HBV
infection; two tests, hepatitis B surface Ag and hepatitis B core antibody
IgM, may be performed as part of an acute viral hepatitis panel along
with tests for hepatitis A (HAV) and hepatitis C (HCV) to determine
which virus may be causing the infection.
 To diagnose chronic HBV hepatitis
 To monitor chronic hepatitis B infection and its treatment
 To detect previous exposure to hepatitis B, in a person who is immune
compromised (when the virus can become reactivated)
Some of the secondary reasons to perform testing include: to screen for
hepatitis B infection in at-risk populations or in blood donors, to determine
if someone is a carrier, to detect previous infection (with subsequent
immunity), and to determine if immunity has developed due to
vaccination.
While the tests described above are specific for HBV, other liver tests such as
AST, ALT, and gamma-glutamyl transferase (GGT) may be used to monitor the
progress of the disease. In some cases, a liver biopsy may be performed to
evaluate how much damage has occurred to the liver.

When is it ordered
Hepatitis B tests may be ordered when someone has signs and symptoms
associated with acute hepatitis to determine if they are due to infection with
HBV. Some of these include:
 Fever
 Fatigue
 Loss of appetite
 Nausea, vomiting
 Abdominal pain
 Dark urine
 Pale stools
 Joint pain
 Jaundice
Clinical Significance:
Both HAV (Hepatitis A virus) and HBV infections primarily affect the liver.
Jaundice is usually preceded by anorexia, malaise, nausea, abdominal
discomfort, fever, chilliness and diarrhea. This pre - icteric phase may last
from 2 days to 3 weeks. The icteric stage of HAV usually has an abrupt onset
with a sharp rise in temperature whereas that of HBV appears more
insidiously with no fever (or less fever). Hepatitis caused by HBV is often
severe and leads to chronic hepatitis or post hepatic cirrhosis. HBV can
cause primary liver cancer or produce changes in liver cells which favor the
development of hepatoma.
 Expected normal values in serum:
 IgG: 800-1800 mg/dl
 IgA:-90-350 mg/dl
 IgM:-Men: 60-250 mg/dl
 Women: 70-280 mg/dl
 IgD:-0-14 mg/dl
Method for Diagnosis:
 Spot Method
 Elisa Method
HEPATITIS C VIRUS:

This infection of the liver is caused by the hepatitis C virus.

Symptoms
Many people with Hepatitis have no symptoms. But you could notice these:
• Jaundice (a condition that causes yellow eyes and skin, as well as dark
urine)
• Stomach pain
• Loss of appetite
• Nausea
• Fatigue

How it is spread
The virus spreads through the blood or body fluids of an infected person.
You can catch it from:
 Sharing drugs and needles
 Having sex, especially if you have an STD, an HIV infection, several
partners, or have rough sex
 Being stuck by infected needles
 Birth-a mother can pass it to a child
Hepatitis C isn't spread through food, water, or by casual contact
About 75% to 85% of people who have it develop a long-term infection
called chronic hepatitis C. It can lead to conditions like liver cancer and
cirrhosis, or scarring of the liver. This is one of the top reasons people get
liver transplants.

How Is It Diagnosed
You can get a blood test to see if you have the hepatitis C virus.

Method Used in Lab for Diagnosis:

 Spot Method
 Elisa Method
Widal:

Widal Test is an agglutination test which detects the presence of serum

agglutinins (H and O) in patient's serum with typhoid and paratyphoid fever.
When facilities for culturing are not available, the Widal test is the reliable
and can be of value in the diagnosis of typhoid fevers in endemic areas.It
was developed by Georges Ferdinand Widal in 1896.
The patient's serum is tested for O and H antibodies (agglutinins) against
the following antigen suspensions (usually stained suspensions):
 Salmonella antibody starts appearing in serum at the end of first week
and rise sharply during the 3rd week of endemic fever. In acute typhoid
fever, O agglutinins can usually be detected 6-8 days after the onset of
lever and H agglutinins after 10-12 days.
 It is preferable to test two specimens of sera at an interval of 7 to 10
days to demonstrate a rising antibody titre.
 Salmonella antigen suspensions can be used as slide and tube
techniques.
Procedure of Widal Test
SLIDE TEST
 Place one drop of positive control on one reaction circles of the slide.
 Pipette one drop of Isotonic saline on the next reaction circle. (-ve
Control).
 Pipette one drop of the patient serum to be tested onto the remaining
four reaction circles.
 Add one drop of Widal TEST antigen suspension 'H' to the first two
reaction circles. (PC & NC).
 Add one drop each of 'O', 'H', 'AH' and 'BH' antigens to the remaining
four reaction circles.
 Mix contents of each circle uniformly over the entire circle with
separate mixing sticks.
 Rock the slide, gently back and forth and observe for agglutination
macroscopically within one minute.
 Agglutination is a positive test result and if the positive reaction is
observed with 20 ul of test sample, it indicates presence of clinically
significant levels of the corresponding antibody in the patient serum.
 No agglutination is a negative test result and indicates absence of
clinically significant levels of the corresponding antibody in the patient
serum.
VDRL (Venerable Disease Research in Laboratory) / RPR (Rapid
PlasmaReagin):
Syphilis is sexually transmitted (venereal) disease caused by spirochete
Treponema pallidum
Serological procedures for syphilis include the following:
1. Treponemal tests: detect the antibodies to Treponema pallidum,
eg. Fluorescent Treponema pallidum antibody absorption (FTA-ABS)
and microhemagglutination Treponema pallidum MHA-TP).
2. Non-treponemal tests: detect the antibodies produced in response
tolipoidal material released from the damaged host cell. These
antibodies are traditionally referred to as REAGINS. eg: Venereal
Disease research laboratory (VDRL) and the rapid plasma reagin
(RPR) tests.
Venereal Disease Research Laboratory (VDRL)
The Venereal disease research laboratory (VDRL) test is a non-treponemal
microflocculation test which is used for screening of syphilis. It detects
the IgM and IgG antibodies to lipoidal material released from the
damaged host cells, as well as to lipoprotein-like material and possibly
cardiolipin released from the treponemes.
Procedure
1. Bring the VDRL antigen suspension, controls and samples to room
temperature.
2. Pipette one drop (50 µl) of the test specimen, positive and negative
controls onto separate reaction circles of the disposable slide.
3. Add one drop of well-mixed VDRL antigen next to the test specimen,
positive control and negative control.
4. Using a mixing stick mix the test specimen and the VDRL reagent
thoroughly spreading uniformly over the entire reaction circle.
5. Rotate the slide gently and continuously either manually or on a
mechanical rotor at 180 r.p.m.
6. Observe for flocculation microscopically at 8 minutes.
Observation:
Reactive: Indicated by large or small aggregates in the centre or the
periphery of the test circle.
Non-reactive: Indicated by a smooth, even light gray appearance with no
aggregates visible.
ASO (Anti Streptolysin O):
Antistreptolysin O (ASO) titer is a blood test to measure antibodies
against streptolysin O, a substance produced by group A streptococcus
bacteria.Antibodies are proteins our bodies produce when they detect
harmful substances, such as bacteria.

Why the Test is performed

You will need the test if you have symptoms of a previous infection by
group A streptococcus. Some illnesses caused by this bacterium are:
 Bacterial endocarditis, an infection of the inner lining of your heart
 A kidney problem called glomerulonephritis
 Rheumatic fever, which can affect the heart, joints, or bones
 Scarlet fever
 Strep throat

The ASO antibody may be found in the blood weeks or months after the
strep infection has gone away.
Normal Results
A negative test result means that you do not have strep infection. Your
health care provider may do the test again in 2 to 4 weeks. At times, a
test that was negative the first time may be positive (meaning it finds
ASO antibodies) when done again.
Normal value ranges may vary slightly. Talk to your provider about the
meaning of your test results.

Abnormal Results
An abnormal or positive test result means you recently had a strep
infection, even if you had no symptoms.

Method
 Quantitative
 Qualitative