Transfusion Transmitted Diseases TTDs

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TRANSFUSION TRANSMITTED DISEASES/INFECTIONS (TTDs/TTIs)
With increasing knowledge about mode of spread amongst
population, the HIV infection has reduced. However, In the
year 2015, India is estimated to have around 86000 new HIV
cases, showing 66% decline in new infections compared to the
year 2000 and 32% decline from 2007; the total number of people
living with HIV in India is estimated to be 21.17 lakhs.4
As per the rules of Indian Government and National Aids
Prevention and Control policy, testing of every unit of blood is
mandatory and the blood collected is screened using highest
quality screening tests for 5 TTIs/TTDs, namely:
• HIV 1 and 2
• Hepatitis C virus (HCV)
• Hepatitis B virus (HBV)
• Malaria
• Syphilis
FDA in USA requires infectious disease tests to be tested for
HIV 1 and 2, HBV, HCV, human T cell lymphoma leukemia
(HTLV) I and II, syphilis and West Nile Virus (WNV) and
nucleic acid testing (NAT) for HIV, HBV, HCV and WNV (as
per AABB standards).
Due to prevalence of AIDS virus, the Ministry of Health and
Family Welfare (Government of India) issued a notification in
the year 1989 under the Drugs and Cosmetics Rules and made
the test HIV 1 and 2 antibodies of whole human blood as
mandatory requirement before transfusion.
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Diseases (TTDs)
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Essentials of Blood Banking and Transfusion Medicine
In the year 1990, blood and blood products were regulated
by the Government of India and 3 laboratories, viz. NICD Delhi,
NIV Pune and CMC Vellore, were notified to function as labora-
tories under 3A of Drugs and Cosmetics Rules to test HIV
antibodies.
As trained technicians were not available in the blood banks
to carry out the test for HIV 1 and 2 antibodies, the Ministry of
Health and Family Welfare notified 112 Surveillance Centers
to act as testing laboratories for the blood banks for carrying
out the above tests.
Questionnaire for donors is provided and this is:
• To find out if the donor is having any transmissible diseases.
• TTDs/TTIs are deferrals; some are permanent and some
temporary.
• The aim of blood transfusion is to do good to the patient.
• Thus, screening of blood for infectious diseases is essential.
Signs and symptoms of AIDS and high risk activities, foreign
travel, recent vaccination, drug history, window periods of
infections, etc. are to be looked for.
Donors must document accurate information. The donors
should understand the donation process, blood testing and that
the blood donation is a voluntary informed decision.
Do not donate blood to be tested for a HIV/AIDS, hepatitis
or any other infectious disease.
Counseling for “reactive” donors: If a blood sample tests
positive for any TTI, then
• Tests are repeated in duplicate.
• Complete confidentiality is maintained about the test results.
• Only the donor is informed, counselled and encouraged for
further testing.
Confidentiality/Counselling
The data will be kept confidential, except where required by
law. If the blood tests are positive to any of the administered
standard tests, patient will receive confidential notification. The
blood bank maintains strict confidentiality of all blood donor
records.

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MALARIA
Malaria is one of the major public health problems of the country.
Around 1.5 million confirmed cases are reported annually by
the National Vector Borne Disease Control Programme
(NVBDCP), of which 40–50% are due to Plasmodium falciparum.5
Malaria is transmitted easily through infected RBCs. Many
donors are unaware that they have malaria, which may be latent
and is transmissible by blood donation.
Prospective donors must be asked about malaria or whether
they had been to a region (endemic area) where it is pre-
valent.
Donors who have had a diagnosis of malaria or who are
immigrants, refugees, or citizens from countries in which
malaria is considered endemic are deferred for 3 years; travelers
to endemic countries are deferred for 1 year.
Malaria is curable if effective treatment is started early. Delay
in treatment may lead to serious consequences including
death.
Prompt and effective treatment is also important for controll-
ing the transmission of malaria.
A revised National Drug Policy on malaria has been
adopted by the Ministry of Health and Family Welfare in
2008 and these guidelines have therefore been prepared for
clinicians to treat malaria.
Microscopy of stained thick and thin blood smears remains
the gold standard for confirmation of diagnosis of malaria.
The advantages of microscopy are:
The sensitivity is high. It is possible to detect malarial parasites
at low densities. It also helps to quantify the parasite load.
It is possible to distinguish the various species of malaria
parasite and their different stages.
Malaria: Rapid Diagnostic Tests (RDT)
Rapid diagnostic tests are based on the detection of circulating
parasite antigens. Several types of RDTs are available. Some of
them can only detect P. falciparum, while others can detect
P. vivax and other species as well.

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Essentials of Blood Banking and Transfusion Medicine
RDTs are produced by different companies, so there may be
differences in the contents and in the manner in which the test
is done.
The user’s manual should always be read properly and instruc-
tions followed meticulously. The results should be read at
specified time. The kits should be within expiry date and should
be transported and stored under recommended conditions.
Histidine-rich protein 2 (HRP2-P. falciparum), and p-lactate
dehydrogenase (pLDH-P. vivax) are usually tested.
The kits are expensive and temperature sensitive. Presently,
NVBDCP supplies RDT kits for detection of P. falciparum at
locations where microscopy results are not obtainable within
24 hours of sample collection.
Failure to observe these criteria can lead to false negative
results. It should be noted that Pf HRP2-based kits may show
positive result up to three weeks of successful treatment.
ELISA for Malaria
ELISA detects malaria genus specific pLDH released by
parasitized blood cells.
The microwells are coated with monoclonal anti-pLDH
antibodies. Another anti-pLDH antibody is biotinylated.
Samples along with positive and negative controls are added
in the microwells and incubated with biotinylated antibody.
The wells are washed to remove unbound components.
Procedure of ELISA
100 µl sample diluent containing antibody.
25 µl control/whole blood.
Shake gently.
Incubate for 30 min at 37°C. Wash with buffer.
Add 100 µl diluted conjugate. Incubate for 30 min at room
temperature.
Wash 6 times.
100 µl of substrate and incubate for 30 min at room
temperature.
100 µl of stop solution.

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Read the absorbance at 450 nm with 600–700 nm as reference
within 30 min after stop solution.
Positive or negative reading is given according to the cut-
off value which is calculated as per the manufacture’s manual.
PCR using a modification of the technique originally described
by Snounou et. al. with primers targeting the Plasmodium spp.
18S rRNA genes can be used.6
Nested PCR is more sensitive compared to microscopy,
allowing the detection of Plasmodium in cases with low para-
sitemia, as well as mixed infections of malaria. In all instances,
specimens that are PCR positive and microscopy negative
depict symptomatic patients with a history of travel to malaria
areas of endemicity.
Nested PCR can detect mixed infection with P. falciparum
and P. ovale that was missed by microscopy.
The nested PCR can detect all four species of Plasmodium.
Nested PCR is valuable as a confirmatory test and implementa-
tion should be considered by reference laboratories and
worldwide with adequate laboratory infrastructure to perform
molecular procedures.
Molecular techniques are costly procedures, including the
cost of labor and reagents, compared to the examination of
blood smears. This represents a true impairment for its imple-
mentation in reference laboratories located in poor regions of
the world where malaria is endemic.
HIV INFECTION
HIV infection was recognized only in 1981. AIDS has rapidly
established itself throughout the world.
In the year 2001, number of adults/children living with AIDS
worldwide was 40 millions. In 2012, it was 35.3 millions.
Dying with AIDS worldwide was 3 millions in 2001 and in 2012
it was 1.6 millions.7
• In developed nations, number is decreasing due to combined
anti-retroviral therapy.
• In India, infection is seen in the 2nd to 3rd decade with
increasing number of males being affected with subsequent
infection to wife and children.

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Essentials of Blood Banking and Transfusion Medicine
• Blood is the source of infection for HIV/AIDS. Risk of HIV
from contaminated blood transfusion is 95%. Contaminated
blood, transfusion of whole blood, platelets, factors VIII and
IX (derived from plasma) are the sources for HIV/AIDS.
• Drug addicts have high risk of HIV due to sharing of needles.
• Skin piercing (including injections, ear piercing, tattooing,
acupuncture) are at high risk to this disease.
Laboratory Diagnosis
A.Detection of antibody to HIV:
• ELISA (to detect antibody) is a sensitive test.
• Western Blot (to confirm HIV) is a confirmation test.
B. Virus isolation from cultured lymphocytes non-specific
findings of HIV/AIDS:
• Anaemia
• Leucopenia (especially lymphocytopenia)
• Thrombocytopenia
• Polyclonal hypergammaglobulinemia
Most widely used marker is absolute CD4 lymphocyte count.
As the count decreases, risk of opportunistic infection increases.
Control of AIDS: Prevention of blood borne HIV transmission:
Advise people with high risk groups to avoid donating blood,
body organs, sperm or other tissues.
All blood should be screened for HIV 1 and 2 before blood
transfusion.
Transmission to hemophiliacs can be decreased by intro-
ducing heat treatment of factors VIII and IX.
Strict sterilization practices ensured in hospitals and clinics.
Pre-sterilized disposable syringes and needles should be
used as much as possible. Avoid injections unless absolutely
necessary.
Counselling of HIV positive patients
• Consult a clinician experienced in treating HIV/AIDS.
• Protect ex-partner(s) from HIV by following safe-sex guide-
lines.
• Inform sex partner(s) who may also be infected.

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• Do not share needles.
• Get information and social and legal support from AIDS
service organizations.
• Don’t share HIV status with people who do not need to
know. Only tell people you can count on for support. Think
about whom do you want to share your HIV status with.
• Get psychological support from a counselor and/or join a
support group for people with HIV.
• Maintain a strong immune system with a healthy lifestyle
and regular medical examinations.
• Consider using anti-retroviral therapies that may slow the
progress of the infection in consultation with a qualified
physician.
HEPATITIS B AND HEPATITIS C VIRUS INFECTION
ELISA FOR HBsAg
• It is an in-vitro enzyme immunoassay.
• Specific monoclonal anti-HBs antibodies are used.
• It is direct, non-competitive, solid phase enzyme immuno-
assay with horse radish peroxidase as the marker enzyme.
• HBsAg when present combines with mouse monoclonal anti-
HBsAbs coated on the polystrene surface of the wells and
simultaneously combines with horse radish peroxidase
conjugated sheep poly and mouse monoclonal anti-HBs
antibodies.
• After incubation washed colourless substrate and chromogen
added.
• The enzyme action produces coloured end product.
• Reaction is terminated by stop solution (1.6N H2SO4).
Procedure ELISA for HBsAg Detection
Mix conjugate and conjugate diluent in the ratio of 1:26
• Sample 1 100 µl
• Sample 2 100 µl
• Sample 3 100 µl
Run positive and negative controls.

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Essentials of Blood Banking and Transfusion Medicine
25 µl conjugate to each well and mix, incubate for 60–90 min
at 37°C. (According to Manual)
5–10 min before, mix substrate solution and substrate diluent
1:101. Wash the wells.
100 µl of substrate into all wells, incubate for 30 min at room
temperature.
Add stop solution. Read the absorbance as per the instruc-
tions given by users manual. Positive or negative reading is
given according to the cut-off value which is calculated as per
the manufacture’s manual.
NUCLEIC ACID TESTING (NAT)
• Nucleic acid testing (NAT) is used for the screening of human
immunodeficiency virus, hepatitis B and hepatitis C virus
in donated blood. This test is the first simultaneous, single
tube NAT solution for HIV, HCV and HBV. It is a direct test
where it actually detects the viral nucleic acid (RNA/DNA).
Being a direct test it reduces the window period for detection
for all these three viruses from the current available sero-
logical (ELISA) tests.8
• NAT combines the advantages of direct detection of the
organism with sensitivity several orders of magnitude higher
than that of traditional methods. The screening of blood for
infectious markers (anti-HIV 1 and 2, anti-HCV and HBsAg)
is done using government approved test kits (ELISA or rapid
kits).
• Despite these efforts, residual risk of transfusion-transmitted
infections remains because of donors in the pre-seroconversion
(window period), viral variants, non-seroconverting
(immunosilent) or delayed seroconverting carriers (atypical
seroconversion).
• Nucleic acid testing (NAT) along with serological testing can
reduce this residual risk to a great extent because it involves
highly specific detection of an infectious agent (the virus
itself) with much higher sensitivity.
• NAT is a recently developed technology that allows detection
of very small amounts of genetic material (DNA or RNA) by
a process of massive copying (amplification) of a gene fragment.

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• Currently, donors of blood and plasma are tested for
hepatitis B surface antigen, antibodies to HCV, antibodies
to HIV and HIV antigens, which are the virus’ own proteins.
• NAT has reduced the “window period” for detection of HBV
to 10.34 days, HCV to 1.34 days and HIV to 2.93 days.8
• Given the high rate of sero-positivity of HIV, HCV and HBV
in India and keeping in mind the high percentage of first
time and replacement donors, it is likely that adding NAT
to the current screening tests will have a very significant
reduction in transfusion transmitted infections making
transfusion of blood safer.
VDRL (SYPHILIS)
Blood is tested for syphilis by the following methods:
• Venereal diseases research laboratory (VDRL).
• Treponema pallidum haemagglutination assay (TPHA).
• Fluorescent treponemal antibody-absorbed test (FTA-abs).
• Enzyme immunoassay (EIA) tests.