Blood Transfusion Transmitted Diseases

Introduction

Florence Nightingale, more than 100 years ago said “No stronger condemnation of any hospital or ward could be pronounced than the single fact that zymotic (infectious) disease has originated in it, or that such a disease has attacked other patients than those brought in with them”.

It should, therefore, be obligatory on those who are involved in transfusion of blood to a patient for saving his life, that the blood transfusion does, no harm to the patient. Nothing could be worst than the fact that in an attempt to save life, blood & blood products having transmissible infectious agents have been given. Many of these infectious agents may cause death or prolonged illness. Hence it is necessary to understand the organisms which could be transmitted through blood transfusion and means by which this could be prevented.

Infectious agents

There are four main groups of micro-organisms known to cause infections namely viruses, bacteria, protozoa and fungi  Only first three groups of microbes - viruses, bacteria and protozoa - have been reported to be transmitted by blood transfusion. Individuals with fungal infections are usually too sick to be accepted as blood donors. Viruses are most commonly transmitted by transfusion.

Recently, a new form of infectious agent - the prion - has been identified. At this time, there is no evidence to suggest that they could be transmitted by blood transfusion.

Viruses

Viruses are the simplest forms of life. They infect all forms of life, they lack certain components needed to live and their growth hence depend on the host cell that they infect to provide these missing components .

Following are some of the viruses which are known to be transmitted through blood:

1.          Human immunodeficiency virus (HIV)

2.          Hepatitis B virus

3.          Hepatitis C virus

4.          Hepatitis A virus

5.          Hepatitis G virus

6.          Non - A, Non - B Hepatitis

7.          Epstein Barr Virus

8.          Cytomegalo virus (CMV)

9.          Human T Lymphocytic virus (HTLV - 1 & HTLV - 2)

Some viruses have the property of latency. This is the ability of a virus to join its own nucleic acid with the nucleic acid of the host cell without taking complete control of the host cell as a virus would normally do. Latency usually occurs after an active infection when the individual has recovered and immunity is building up. The viral nucleic acid exists in an inactive form that does not seem to harm the host cell. When the host cell divides, the cell nucleic acid is copied, together with the viral nucleic acid. In this way, the viral nucleic acid becomes part of the cell nucleic acid and is copied every time the cell divides.

Latency is usually indefinite and without any harmful effects on the host cell. However, at any time, the latent nucleic acid could become active and take over the cell functions, resulting in an active infection. This is called a reactivation of infection (recrudescence), which is caused by the reactivation of virus already present in the individual.

TRANSMISSION OF INFECTIOUS AGENTS BY BLOOD TRANSFUSION.

In order to be transmitted by blood transfusion, an infectious agent must be present in the donated blood. Each blood transfusion service or blood bank or laboratory should, therefore, screen for evidence of the microbes that are known to cause infections with this route of transmission .

There are three basic conditions that will determine whether an infectious agent is likely to be transmitted by transfusion:

1.           The agent must be capable of using the blood stream as a route of entry in host.

2.           The infected donor must be essentially free of any noticeable signs and symptoms of disease, otherwise, they would have been identified during donor screening and the donor would have been excluded or deferred.

3.           The agent must exist naturally for a period of time, either free in the plasma or present in a cellular component in the blood stream of an infected donor.

      Any infectious agent meeting all these conditions can be transmitted by blood transfusion. However, whether transmission actually occurs or not            depends on a number of other factors, particularly on the immune status of the patient and the amount of infectious agent transfused.

         It is known that the transmission of certain infectious agents through blood transfusion can, and do occur, and it can be an important route of infection, however, the key point to remember here is that there are a number of ways by which risk can be reduced.

Strategies to reduce the risk of transfusion- transmitted infections.

            The careful selection of donors to ensure that, as far as possible, blood is not collected from people who are likely to be carriers of infectious agents. Safe blood donation depends on, building a panel of regular, voluntary, non - remunerated donors as the first step in ensuring a safe and adequate supply of blood. In our country where most of the blood is collected from family/replacement donors, the risk of transfusion transmissible infection is higher.

            The direct screening of the blood for evidence of the presence of infectious agents or markers produced by them..

            The removal of specific components of blood thought to harbor infectious agents, for example, by the filtration of blood to remove white blood cells.

            The physical/ chemical inactivation of any contaminating agent that may be present: for example, heat treatment of 5 % albumin during production.

        Not all infectious agents can be detected directly in donated blood. Blood is often screened for evidence of previous infection by looking for the presence of specific antibodies raised against the infectious agent. Clearly, it is only by understanding what markers of infection are produced by the infectious agent that screening for the correct marker can be introduced.

INFECTIOUS AGENTS TRANSMISSIBLE THROUGH BLOOD:

Any viral agents can be transmitted by blood, however, important amongst them are HIV, HB V, HCV, CMV, and HTLV-I & HTLV-2 (Table 12 -1)

TRANSFUSION MEDIATED INFECTIONS (VIRAL) (Table 12-1.)

Infection Agents                                                  Illness

 

HIV-1

AIDS

HIV-2

Illness similar to AIDS

HBV

Viral Hepatitis B

HCV

Cirrhosis Liver - Late

 

Carcinoma Liver - Sequelace

Cytomegalovirus

Hepatitis, Lymphadenopathy, rashes,

 

Intrauterine infection of newborns.

HTLV-1, HTLV-II

Adult Cell Leukemia

HUMAN IMMUNODEFICIENCY VIRUS (HIV)

HIV causes AIDS. This syndrome was recognized in 1981, well before the discovery of the causative virus. Wider implications of the immune disorder were noted when, in 1982, AIDS was reported in three hemophiliacs and in a 17-month-old infant whose multiple transfusions at birth included a unit of platelets from a donor who subsequently developed AIDS. Within a few years, over 50% of hemophiliacs receiving clotting factor developed HIV-1 infection.

HIV was first isolated from the cells of an infected patient in 1983 (HIV-1). The virus was subsequently identified as the causative agent of AIDS. In 1986 a second type of HIV, HIV-2, was identified in certain areas of West Africa. HIV-2 appears to cause the same diseases as HIV-1, but may be less pathogenic. It is morphologically similar to HIV-1. The two types can be distinguished by the presence of proteins and glyco-proteins specific to them. Although cross-reactivity occurs between the core protein of both viruses, the envelope proteins are different.

Components of the HIV Virus (Table 12-2.)

Bands Observed

 

Gene

HIV1

HIV2

Proteins

Gag

pl8,p24,pl5

pl6,p26,p55

Core

Pol

p31

 

Endonuclease

 

p55, p65

p68

Reverse transcriptase

Env

gp41

gp36

Transmembrane proteins

 

gpl20,gpl60

gpl40,gpl25

Envelope

p= Protein, gp=Glyco protein,
Number indicates molecules

Cross-reactivity

Cross-reactivity occurs when an antibody recognizes not only its own antigen but also other antigens that have certain similarities. In the case of HIV, this means that an individual infected with HTV-1 would produce antibodies that recognize both core and envelope proteins of HIV-1 and core proteins of HIV-2. Similarly, an individual infected with HIV-2 would produce antibodies that recognize both core and envelope proteins of HIV-2 and core proteins of HIV-1.

THE STRUCTURE OF HIV

There are two types of nucleic acid :

            ribonucleic acid (RNA)

            deoxyribonucleic acid (DNA)

DNA is usually double-stranded. It is the genetic material passed to daughter cells when a cell divides. It is DNA that is responsible for the transmission of hereditary characteristics from parents to children.

The nucleic acid in the HIV virus is RNA. There is no DNA present. Instead, the virus uses the machinery of the human cells that it enters to convert its RNA to DNA so that the virus can replicate or integrate itself in the cell’s DNA.The way that viral and other such proteins are described is based on their molecular weight (measured in daltons) and on whether they are proteins or glycoproteins. The two proteins associated with the RNA of HIV are 7000 daltons (7kDa) and 9000 daltons (9kDa). These are abbreviated to p7 and p9 respectively. The reverse transcriptase enzyme is a 66kDa protein, which is abbreviated to p66. Glycoproteins are similarly abbreviated to “gp”.
The core is totally enclosed in a cone-shaped shell of p24 protein. This is called the major core protein and appears to be the same in both HIV-1 and HIV-2. The whole unit is called the viral capsid. (Fig 12.1)

 


The capsid itself is covered by two layers. The first of these is a shell of pl7 matrix protein to which proteins that project from the surface of the virus particle are attached. This is covered by a lipid bilayer. Projecting through the lipid are many transmembrane proteins. These proteins, gp41, are attached to the p17 matrix and themselves attach the gpl20 envelope proteins. These appear as small projections on the surface of the virus particle. It is the structure of these small projection and their attaching proteins that appears to be the major difference between HIV-1 and HIV-2. The corresponding HFV-2 proteins are gpl 10/130 and gp 36 respectively. Antibodies to these two specific sets of proteins do not cross-react.The entire virus particle is called the virion. This is the infectious particle that is secreted and transmitted between individuals. The complete virion is 100 -120 pm in diameter.
All retrovineses contain three main structrual genes the gag gene which codes for the core proteins, pol (polymerase) and env (envelope) gene which codes for the envelope glycoproteins.Fig. 12.2

 

 

The viral RNA is condensed in a cylindrical core together with two closely-associated structural proteins and an important enzyme called RNA dependent DNA polymerase. This is more commonly known as reverse transcriptase. This enzyme is found in all retroviruses as it is needed to copy the viral RNA into DNA.


Genetic Structure of HIV includes the nine genes, but their are three main genes - gag, pol and env and they are antigenic, others genes serve to regulate the expression of these virion genes.
Gene products marked with asterisk are not usuallay visible on a Western Blot.

ENTRY OF HIV INTO CELLS

HIV enters susceptible cells by binding to a receptor - a protein called CD4 - on the cell surface. CD4 is found on the surface of a number of different cells within the immune system:

            the T cells that help to stimulate the immune response

            the T helper cells (Th cells)

            the macrophages which engulf virus particles in many parts of the body.

Following fusion of the virion to the cell membrane, the uncovered capsid passes into the cytoplasm of the cell. Within the cytoplasm, the RNA is copied to double-stranded DNA by the reverse transcriptase enzyme present in the capsid using raw materials from within the cytoplasm.
The DNA then passes into the nucleus of the cell and integrates into the cellular DNA. Once in the cell, the DNA remains latent.

The final stage of infection occurs when the virus starts to replicate. Large quantities of infectious virus (virions) are produced. As the virus emerges (buds) from the cell, it is packaged in the cell membrane to produce the complete virus particles. These virions are then released and can infect other cells.

THE CLINICAL PRESENTATION OF HIV INFECTION AND AIDS

Initially it was thought that infection with HIV only produces AIDS. It then became clear that HIV infection could lead to a number of different conditions of varying severity, although it usually (and finally) results in AIDS.

HIV have a predilection for infection and destruction of CD or helper T cells, thereby producing severe immundeficiency and associated diseases.

In individuals suffering from AIDS, the main cause of illness is the occurrence of secondary infectious diseases, the opportunistic infections. These opportunistic infections are caused by infectious agents which do not produce disease in normal individual  Following are common infections associated with HIV/AIDS :

          Pneumonia caused by Pneumocystis carinnii

          Tuberculosis produced by Mycobacterium tuberculosis

          Mycobacteriosis caused by Mycobacterium avium/intracellulais

          Chronic cryptosporidiosis

          Toxoplasmosis

          Viral infection, such as cytomegalovirus

Secondary cancers such as Kaposi’s sarcoma and non-Hodgkins’ lymphoma are other conditions sometimes found in AIDS patients. These cancers are usually aggressive and do not respond very well to standard chemotherapy. Kaposi’s sarcoma, as originally described, was a benign malignancy found in elderly men which has no adverse affect on the individual.However, the Kaposi’s sarcoma found mainly in AIDS patients in Africa is a fast-growing, and usually fatal malignancy.

In many parts of the world, patients with AIDS Related Complex (ARC) or AIDS present simply with severe diarrhoea. The presence of opportunistic infections or secondary cancers is only determined following clinical and laboratory investigation.

LABORATORY DIAGNOSIS OF HIV INFECTION

Shortly after exposure, the core protein, p24, has been found in some individuals. Within few weeks antibodies to both envelope (gp41) and core(p24) proteins appear in almost all infected individuals. During the early phase of infection a non-specific acute “viral illness” may occur.Once antibodies appears, they increase in titer even though the host is asymptomatic. During this phase of infection viral cultures of isolated lymphocytes demonstrate the presence of virus. As infection progress, changes in the ratio of T-lymphocytes with specific surface markers, CD4 (helper) to CD8 (suppressor) cell, are observed. The ratio of CD4 to CD8 in healthy and immune competent individual is about 2:1. In acquired immune deficiency syndrome (AIDS), the virus destroys CD4 cells and their number in the body decline and there is decrease in CD4:CD8 ratio.HIV-positive persons with fewer than 200 CD4+ T cells per u.1 are considered as having AIDS in the absence of symptoms and /or opportunistic infection.

Before HIV was identified, AIDS was diagnosed by its clinical appearance. Any laboratory tests that were used were surrogate tests. All of these initial tests measured the results of HIV infection rather than specifically detecting either viral antigen or antibody against the virus.

The production of specific tests for HTV helped to understand both HIV infection and AIDS.
        The finding of anti-HIV antibodies in patients with previously unexplained immunodeficiency is diagnostic of AIDS.

The presence of anti-HIV in an asymptomatic individual means that the individual has been exposed to the virus. It is accepted that, in almost all cases, the virus will be present in the individual. Seroconversion in sequential samples means that the infection has been recent.

Some research papers report that antibodies are present as early as 14 days after infection, while others indicate that they may not be present until 28 days or more after infection. The antibodies produced are directed against both core and envelope proteins. The most important antibodies are specifically anti-p24 (core) and anti-gp41 (envelope). Although antibodies to other virion proteins are produced, the presence of these two antibodies  best confirmation of infection. It has also been found to be the best means of monitoring the progress of infection.

Following infection and prior to the production of antibodies, there is ‘window period’ of varying length during which the infection establishes. During this period when no antibodies is detected, viral antigen (p24, gp41) could be detected. The length of time that antigen can be detected is very short, often no more than 1-2 weeks. Although detection of HIV antigen would theoretically provide evidence of infection at an earlier stage, there are very few commercial antigen assays and they are also not very sensitive. However, in some instance where antigen assays have been used, HIV antigen has been detected at a stage when anti-HIV antibodies had not appeared or just appeared. At present, therefore, it appears that HIV antigen assays may have limited value in blood transfusion service. However, it is possible that their value may grow with an increasing prevalence of HIV in the donor population.

 


       Antibody emerges corresponding with a decrease in free antigen. The levels of antibodies to p-24 and gp-41 peak and remain constant throughout the stages of persistent asymptomatic infection.
       It is not clear whether p24 antigen production stops completely. Using detergent treatment of serum samples, p24 antigen complexed to anti-p24 antobody has been detected in some patients previously considered to possess only anti-p24 antibody. It is possible that p24 antigen production never ceases completely, but that the circulating antigen - antibody complexes are formed, hence the antigen could not be detected.

As ARC develops, level of anti-p24 antibodies falls and detectable p24 antigen appears. At this stage, anti-gp41 antibodies and p24 antigen are detectable. This situation is the prelude to the development of full-blown AIDS.

It may be noted that viremia is maximum during window period and after ARC and AIDS develops.

TRANSMISSION OF HIV INFECTION

The modes of transmission of HIV infection are now well-established. Whilst virus can be isolated from many body secretions, infection is transmitted in only a limited number of ways

There are three principal modes of transmission of HIV infection :

1.           Unprotected penetrative sexual contact with an infected person, either between men or between man and woman.

2.           Inoculation of infected blood, either by blood transfusion or as the result of the use of contaminated needles, syringes or knives used, for example, in injecting drug, ritual scarification or tattooing.

3.          From an infected mother to her child, in the uterus, during birth or by breast feeding.

Blood transfusion can be a significant route of infection. The efficiency of the transmission of HIV through blood transfusion is estimated to be more than 90%. WHO reports that the viral dose in HIV transmission through blood is so large that one HIV-positive transfusion leads to death, on an average, after two years in children and after three to five years in adults. Nevertheless, the extent to which blood transfusion is an actual route of transmission depends on the prevalence of infected individuals in the population and on the effectiveness of the screening program used.
In a population with a low prevalence of infected individuals and a good screening program, transmission by blood transfusion may be extremely rare. In a population with a high prevalence of infected individuals and with a poor or non-existent screening program, transmission by blood transfusion is likely to be relatively common and would be an important route of infection in the population.

Blood transfusion, therefore, can spread HIV infection very widely if blood is not systematically screened.

Transmission of infection by blood transfusion

Transmission of infection by blood transfusion, or the infusion of blood products, can also be avoided. The first approach to the prevention of transmission by transfusion is the selection of donors who are at low risk for transfusion-transmissible infections. Remember that a safe donor makes a safer donation. The following are important points to remember while selecting a donor:

             identifying low-risk donor groups

             avoiding unsuitable blood donors

             recruiting voluntary non-remunerated blood donors

             promotion of self-exclusion by individuals at risk through an effective donor education program

             predonation counselling, including an assessment of risk factors and an opportunity for self-exclusion or confidential unit exclusion

             a brief medical history, including possible signs and symptoms related to transfusion- transmissible infections

             a basic health check, including a brief examination of the arm for needle marks

             promoting regular voluntary non-remunerated blood donation

Self-exclusion is probably the most effective approach in preventing transmission, but is dependent on the, education of potential donors about risk behaviour. It is particularly important to encourage self-exclusion by people such as prostitutes, homosexual and bisexual men, injecting drug users, those who have any unprotected sexual contact other than with a regular partner, and the sexual contacts of any of these people.

Finally, screening tests for HIV infection are needed to enable infected donors to be excluded and the donated blood to be discarded.

Testing for HIV Viral Markers

The detection of anti-HIV is the most suitable approach for identifying HIV-infected blood donations. The three main kinds of screening assays to detect anti-HIV available are:

             Enzyme Linked ImmunoSorbent Assays (ELISA/EIA)

             Particle agglutination assays

             Specialized rapid assays

While selecting assays for testing anti-HIV following features may be taken into account:

             High specificity

             High sensitivity

             Simplicity of test

             Incubation time

             Cost

Enzyme Linked ImmunoSorbent Assay (ELISA)

It is the most common used assay and is based on the use of immobilized viral antigen which captures anti-HIV antibodies present in the test sample.

Manufacturers often use the terms first generation, second generation and third generation assay.

  • First generation assays are purified or unpurified native virus or virus infected cells lysates prepared from cell culture.

·          Second generation assays use recombinant antigen produced by cloning fragments of viral nucleic acid into yeast, growing large amount of the engineered yeast in bulk culture and purifying the viral proteins produced.

·          Third generation assays are synthetic viral polypeptides artificially produced by chemical synthesis.

        Assays are based on the same principles but differ in the way the viral antigen is immobilized:

             On the sides of wells of a polystyrene microplate

             On small polystyrene beads. This method needs specialized equipment.

There are three types of ELISA:

(1)     Antiglobulin type ELISA:

(2)     Viral antibody present in test sample is bound to immobilized viral antigen and is detected by enzyme labeled anti-human antibody.

(3)     Competitive ELISA:

(4)     It is widely used assay, in which antibody present in test sample competes with enzyme- linked specific antibody for binding sites on immobilized antigen.

(5)     Sandwich ELISA:

(6)     This is highly specific type of ELISA in which viral antibody in test sample is bound to immobilized antigen and then detected by free enzyme-labeled viral antigen.

Antiglobulin Type ELISA Method (or EIA)

It is solid phase enzyme immunoassay utilizing polystyrene wells of microplates or beads coated with HIV specific proteins representing HIV core and envelope antigens. See figure 12-4.


(1)           Serum or diluted serum is added to the wells coated with HIV specific proteins (p24 & gp 41). Positive and negative controls are added to a number of wells on each plate run.

(2)           They are incubated for the defined period of time and at the correct temperature.

(3)           During the incubation, any specific antibody present in the test serum binds to the viral antigen.

(4)           At the end of incubation, the wells are washed at least three times with washing fluid to remove unbound serum and to prepare them for the next stage, (manual washing is done using multi-channel washer to fill and then empty the wells with wash fluid or by mechanical  washing  using  an automated plate washer).

(5)           After final wash, the wash fluid is removed. It is very important that well are as much dry as possible. The plate can be turned upside down and gently
tapped dry on some absorbent tissue if the wells are still wet.

(6)           Conjugate solution is added to all wells and they are incubated at the defined period of time and at correct temperature.

Conjugate solution contains anti- human globulin antibody which has been chemically linked to an enzyme usually horse radish peroxidase or alkaline phosphate. Conjugate binds to only human antibodies that are bound to the antigen immobilized on the wells. Conjugate does not bound in those wells that did not contain anti- HIV sera bound to antigen.

(7)      At the end of incubation, the wells are again washed three times to remove excess, unbound conjugate and are prepared for the next stage of the assay as described earlier in step 4&5.


Conjugate

Enzyme labeled human globulin antibody

Fig 12-4.
Antiglobulin Type ELISA Metho


(8)Substrate solution is added immediately to all wells and incubated in dark for the defined period of time at correct temperature

When the substrate solution is added, color develops in the wells containing bound conjugate due the activation of substrate by enzyme. Wells having no bound conjugate do not change the color of the substrate. Thus reactive wells having anti-HIV positive sera are colored, and the wells containing no anti-HIV sera are colorless. The controls show appropriate color changes.

(9)          At the end of incubation, diluted acid (1N H2SO4) solution is added to all wells to stop reaction. The acid inactivates the enzyme and fixes the color. The intensity of color change is directly proportional to the antibody concentrate present in the samples/controls.

The color change can be read visually.

(10)       The optical densities (OD values) of the solutions in the microwells are measured by ELISA reader at the specific wave length after determining the cut-off value and the results are determined.
 

Fig 12-5.
Competitive ELISA Method

Competitive ELISA Method

The principles of the competitive Elisa are the same as that of antiglobulin assay but it differs slightly in the way in which anti-HIV is detected.

             The conjugate and the test sera are added at the same time in wells and incubated together.

             The conjugate is enzyme-labeled anti-HIV antibody in competitive ELISA, rather, than labeled non-specific antibody as in antiglobulin-type ELISA.

             The conjugated anti-HIV competes with anti-HIV in test sera for the antigen binding site. A test smaple containing anti-HIV will block the binding of conjugate to antigen while sample not containing anti-HIV will allow binding of the conjugate to antigen.

Method: (See figure 12-5)

1.           Undiluted sera and conjugate are added in wells at the same time. Positive and negative controls are also put.

        2.           The wells are incubated for the defined period and at correct temperature. During this

 

Period Anti-virus present in  the test serum competes with the conjugated anti-HIV for the binding sites on the viral antigen.

3.          At end of the incubation period, the wells are washed with washing fluid to     remove  excess   sera  and conjugate and prepared for the next step as described in the antiglobulin- type assay.

4.          Substrate solution is immediately added to all wells and incubated in dark for the defined period and at correct temperature.

5.          At the end of the incubation period, dilute acid (1 N H2SO4) is added to all wells to stop the reaction.

6.          An intense color signifies a non- reactive sample, while lack of color signifies a reactive specimen.

7.          The optical densities (OD values) of the solution in the microwells are read by the ELISA reader after determining the cut-off value, and the results are determined.

 Sandwich ELISA Method

sandwich ELISA Method:

The basic principle of the sandwich ELISA is again the same as that of antiglobulin-type ELISA, but it differs in the way in which the anti-HIV is detected.

             Antigen, usually synthetic peptides are attached to the surface of wells in microplates.

             The conjugate is enzyme-labeled synthetic antigen in sandwich ELISA, rather than enzyme- linked anti-human immunglobulin in the antiglobulin-type assay.

             During the incubation period, the conjugated antigen binds anti-HIV antibody bound to the antigen immobilized on the microwells.

             A sandwich is built of antigen-antibody-antigen.

             At the end of the incubation period, dilute acid (1 H2SO4) is added to all wells to stop the reaction.

             An intense color signifies a reactive sample having HIV, while lack of color signifies a non-reactive specimen having no HIV.

             The optical densities (OD values) of the solution in the microwells are read by the ELISA reader after determining the cut-off value, and the results are determined.

Method:   (See figure 12-6)

  1. Sera or diluted sera are added to antigen bound wells. The positive and negative controls are also put. The wells are incubated for the defined period of time and at the correct temperature. During this period anti-HIV present in the sera bind to the antigen.

2.          At the end of incubation period, the wells are washed with washing solution to remove the excess sera and the wells are prepared for the next step as described earlier.

3.          The conjugate is added to the wells. The wells are incubated for the defined period of time and at the correct temperature. During this period, the conjugate binds to antibody bound to the immobilized antigen. A sandwich is built up of antigen-antibody-antigen.

4.          At the end of incubation period, the wells are washed with washing fluid to remove unbound conjugate and the well are prepared for the next step, as described earlier.

5.          Substrate solution is immediately added to all wells and they are incubated at the defined period of time and at correct temperature.

6.          At the end of incubation period, diluted acid (1 N H2SO4) is added to all wells to stop the reaction.

7.          Color develops in the wells having sera containing anti-HIV and no color will develops in the wells having sera with no anti-HIV.

8.          The optical densities (OD values) of the solutions are read by the ELISA reader after calculating cut-off value, and the results are recorded.

 

Precautions in ELISA

            Hemolysed, lipemic or contaminated sample sera should not be used.

            Correct volumes of test samples, conjugate and substrate are added.

            Instructions given by the manufacturers of kits should be strictly followed.

Particle Agglutination Assays

Particle agglutination assay detects the presence of anti-HIV by the agglutination of particles coated with HIV antigens. Particles are made of gelatin or latex. The assay is performed in microplates.

Method:

1.          HIV antigen is immobilized on particles made out of gelatin or latex.

2.           Test samples and controls are diluted in microwells with test diluent which is provided with assays.

                                   

        3.           The HIV coated particles are added to the diluted samples and controls. Then they are incubated, usually at room temperature (20-24°C) for the defined period. During incubation, the particles are agglutinated by anti-HIV present in the serum.

4.           At the end of the incubation period, the result of tests can be read with naked eyes. If gelatin particles are used, they appear in bluish color. If latex particles are used, they appear white in color which can be seen against a black background.

5.           A reactive result appears as an even mat of agglutinated particles across the bottom of wells. A non-reactive result appears as a button or ring of non-agglutinated particles, that settle in the center of well. See figure 12-7.

Advantages of particle agglutination assay:

             Expensive equipment are not needed.

             Do not have different stages of reactions.

             Do not need washing equipment.

             Results can be read visually

Disadvantages of particle agglutination assay:

             Subjective error in weak reaction

             Test serum having non-specific agglutinins, may agglutinate both sensitized and nonsensitized particles.

Specialized Rapid Spot Test

It detects anti-HTV. It is rapid and simple and based on the ELISA/EIA technique. The HIV antigen, usually recombinant or synthetic peptide, is immobilized on either porous or semi-porous membrane usually set in a well, in a plastic cassette containing absorbent pad. Most of the specialized rapid assays are in the form of a kit having every thing required for the test.

Method;

1.           The test sample and buffer solution (provided with the kit) are put on the porous membrane and allowed to soak in. Pre-dilution of the test sample may be required. This is achieved by the addition of drops of diluent and sample in a suitable vial. The diluted sample is then directly put on the porous membrane.

2.           It is incubated for 10-15 minutes. During this period the sample passes through the membrane and if it contain anti-HIV antibodies, it will bind to the HIV antigen on the membrane.

3.           The membrane is rinsed with the buffer solution to remove unbound antibodies.

4.           Then conjugate is added. The composition of the conjugate varies between assays. Some assays use an enzyme conjugated anti-human immunoglobulin as in antiglobulin-type ELISA. When such conjugate is used, a further wash step and addition of chromogen is required to visualize the results. Some assays use protein A labeled with colloidal gold as the conjugate. It will bind to anti-HIV present and gives a red /purple color.

5.           The final result is read visually and compared with the expected results described by the manufacturer.

6.           Although control is not required, it is good practice to set up a negative control and a weak positive control.

Note: The instructions provided by the manufacturers of screening kits should be followed strictly for reliable results.

Merits of Specialized Rapid Test

             Very sensitive

             Has the advantage of speed and sensitivity

             Useful in small blood banks.

             Useful in emergency

             Results can be read visually

             No calculation is required

Demerits of Specialized Rapid Method

             Costly

             False positive results due to particles in the test samples (Ghost Dot)

             Results can not be preserved.

Confirmatory/Supplementary Assay

Western Blot is highly specific confirmatory/supplementary test and at one time it was considered as 'gold standard'. The technique is useful in determining with which viral components the antibodies react. It is relatively specialized and expensive test and is not appropriate for screening blood donations. It is no longer recommended as a routine confirmatory test.

Western Blot or Immunoblot

This is enzyme-linked immunoelectro-transfer blot (immunoblot) technique and is used to detect human anti-HIV. It is the confirmatory test of choice. Detergent disrupted purified HIV virions are separated into various proteins (antigens) according to their relative molecular weight by electrophoresis on a polyacrylamide slab gel in the presence of sodium dodecylsulfate (SDS).
The separated HIV proteins are transferred to nitrocellulose membrane. They retain their relative positions achieved on separation. The antigen impregnated nitrocellulose membrane is then cut into strips, each strip having the full range of viral proteins which were separated in the gel (figure 12.8)

Method:

             Individual nitrocellulose strips are incubated with serum or plasma specimens in wells of incubation tray. The non-reactive and weakly reactive controls are included with each run, regardless of the number of test specimens.

             The strong reactive control is used to establish criteria of reactivity of bands and is  included with the first run for each kit and is not included in subsequent run unless the strip is misplaced. Figure 12.9 shows HlV-specific bands for the strongly reactive control.
             During incubation, if HIV antibodies are present in the specimen, they will bind with viral antigens bound to the nitrocellulose strip.

             At the end of incubation the wells of plastic tray are aspirated and the strips are washed to remove unbound material.

             Anti-HIV bound to HIV antigens on strips are detected by antihuman immunoglobulin antibody to which biotin has been attached. The binding of this tracer antibody to the human immunoglobulin is detected by the addition of an enzyme-avidin conjugate followed by the application of substrate. This substrate changes color in the presence of enzyme and permanently stains the strips.

 

Interpretation of results

The presence or absence of anti-HIV in a specimen is determined by comparison of each nitrocellulose strip of test specimen with the strips used for non-reactive and weakly reactive controls tested with the run, and the strip used for strongly reactive control tested once with the kit. Figure 12-9, indicates the bands on the strip used with the strongly positive control.

             A donor is regarded positive for anti-HIV if bands to the gag protein p24; pol protein p 31 and env glycoprotein gp 41 and gpl 60/120 are present.

             Recently less stringent but equally specific criterion are emerging, requiring the presence of antibodies to at least one of the gag proteins (p 17, p 24, and p 55), one of the pol proteins (p 31, p 51, p 66) and one of the env glcoproteins (gp 41, gp 120, and gp 160).

             Other bands that do not meet the criteria for positive results are evaluated as "indetermined" and are reinvestigated at a later date after 3 to 6 months.

             If there is diagnostic information suggestive of HIV infection, the blot pattern may be interpreted more liberally, and bands representing only two structural genes are required for a positive interpretation.

             A blot pattern without lines is interpreted as "negative"

             One of the difficult patterns to interpret is the finding of the antibodies reactive to one of the gag proteins. This pattern is found during seroconversion in persons exposed to HIV infection and develop a full immune response later. This pattern may also be found among individuals who have no risk for HIV infection and they do not develop full immune response to other HIV gene products afterwards.

Recommendations for HIV 1 & 2 testing

Sera of all blood donations are tested for anti-HIV 1&2 by ELISA/EIA. Sera non-reactive on the first screening are considered as HIV negative and are recommended for transfusion while a serum reactive on the first screening is considered HIV positive and is not used for transfusion and is discarded.

Confirmatory tests are important to determine how to counsel a healthy blood donor and whether or not may be notified.

The general scheme is given below:

ELISA Screening

            Reactive

If both negative,
HIV negative

Repeat ELISA in duplicate If one or both postive

Non-reactive,

donation is safe for transfusion 

Western Blot is done

 


Positive

Counsel or Notify
(if required by law)


Negative

Revaluate after 3 months
(notify-optional)



In the selection of HIV antibody tests for use, the first ELISA kit should have the highest sensitivity, where as for repeat tests ELISA kits should have higher specificity than the first.
WHO has recomended anti-HIV 1 & 2 test with ELISA or Rapid spot assay.

HIV Antigen Detection Assays

These are sensitive and specific tests targeting viral antigens or nucleic acids and their availability has led to proposals for using these assays in screening of donated blood to detect HIV infected blood earlier than with current antibody assays.

The detection of HIV antigen has very limited value in blood transfusion service, however, it may be useful in the following conditions:

             Detection of HIV infection during window period before seroconversion.

             Confirmation of HIV infection in infants born to HIV infected mothers.

             May help to resolve the indeterminate Western Blot test results.

             To monitor HIV infected patients on antiviral therapy.

Methods for HIV antigen testing.

             ELISA for testing HIV p24 antigen

             Polymerase Chain Reaction (PCR.)

             'Viral isolation

Screening for HIV Antigen (ELISA):

Screening for HIV Antigen (ELISA):

Screening for HIV antigen (usually p 24) is performed using sandwich ELISA. The difference between HIV-antigen and anti-HIV antibody screening is that HIV-antigen test uses a sandwich of antibody-antigen- antibody, unlike HIV antibody screening which comprises a sandwich of antigen- antibody-antigen.


•Antibody (usually mono-clonal) is bound  to  the  surface  of the micro wells.

             Antibody (usually mono-clonal) is bound  to  the  surface  of the micro wells.

             Test serum and positive and negative controls are added to the microwells and incubated. At the end of incubation period, the excess serum is washed off.

             Conjugate is added and incubated. The conjugate is an enzyme-labeled specific     antibody     (usually monoclonal).

Fig 12.10 HIV Antigen ELISA

•         During the incubation, the conjugate binds HIV-antigen bound to the anti-

HIV immobilized on the microwell. A sandwich is formed of antibody-antigen-antibody.

·          The excess conjugate is washed away and substrate (chromogen) is added in the same way as in the antiglobulin assay and incubated.

             At the end of the incubation period, diluted acid (1 H2SO4) is added to all wells to stop the reaction.

             An intense color signifies a reactive sample having HIV, while lack of color signifies a non reactive specimen having no HIV.

             The optical densites (OD values) of the solution in the microwells are read by the ELISA reader after determining the cut-off value, and the results are determined (Fig. 12.10).

 

Polymerase Chain Reaction (PCR) for HIV

PCR is the most sensitive assay for the detection of HIV infection. PCR detects HIV infection before tests for antigen or antibody by other methods and thus further shortens window period (time between infection and sero conversion). This test depends on the amplification of HIV integrated in the DNA of infected cells. Polymerase chain reaction systems require several cycles with a thermostable polymerase at carefully controlled temperatures and need specific primers present at templates for the amplified DNA. The detection of the amplification product is by a labeled probe in an immunoblot assay. This method requires very careful control to ensure that positive reactions are specific.

The routine PCR tests may have only a small impact on blood safety with regard to HIV, however the potential for closure of the window period for HIV infection is significant. The implementation of routine PCR testing of donors' blood will require development of automated systems that will eliminate the time consuming steps necessary to extract DNA and to prepare samples.

 

TRANSFUSION ASSOCIATED HEPATITIS

At least four viruses have been associated post-transfusion hepatitis:

1.          Hepatitis A virus (HAV)

2.          Hepatitis B virus (HBV)

3.          Hepatitis C virus (HCV)

4.          Hepatitis D virus (HDV)

Several other viruses causing hepatitis have been reported. Hepatitis E virus has been reported to cause epidemic hepatitis associated with contaminated water. The majority of transfusion associated hepatitis are due to HBV or HCV.

Hepatitis A Virus

Infection is usually a result of viral spread by contaminated food or water due to fecal-oral mode of transmission. HAV is present in blood for a short period and there is no chronic carrier state in humans. Incubation period is 15-40 days.

Transfusion-transmitted HAV infection has occurred, but it is very rare. An outbreak of HAV infection has been reported in recipients of solvent detergent-treated coagulation factor.

Infection by transfusion require that donor has viremia and the recipient be susceptible to virus. Viremia, if it occurs, is usually berief and at the time of illness, when no one will donate blood.
Antibody to HAV regularly appears after infection and thus large number of the population has immunity. The rarity of HAV infection after transfusion does not want routine testing of donors.

Hepatitis B Virus

Hepatitis B virus is in the family of hepadnaviridae. In 1945, it was fond in humans by Blumberg and coworkers in the serum of an Australian aborigine, which they called Australia antigen.



In human the virus is 42 nm in diameter consisting of a 28 nm core with double stranded circular DNA and DNA polymerase. The core is surrounded by a coat protein HBsAg, which also occurs free in the serum as 22 nm spheres and 22 nm to 200 nm filaments. The virion contains further two proteins; hepatitis Be antigen (HBeAg) and core antigen (HBcAg). Both these antigens (proteins) are associated with the capsid core of the virion (Fig. 12-11).

 


 

soluble
HBc antigen

 

 
 


Figure 12.11 Diagramatic representation of HBV

 

Transmission of HBV infection:

The transmission of HBV is mainly by parental route which involves direct contact with body
fluid. The most common route of infection are:

             Contact with infected blood, either by exposure of wounds to infected blood or to
contaminated needles and syringes used in injecting drugs, tattooing, ear piercing, or acupuncture.

             Sexual contact

             Neonatal or perinatal transmission

             Transfusion of infected blood or blood products.

Serological Findings  (Figure   12-12)

The incubation period of HBV infection is about 30 to 150 days during which the patient has no sign and symptom but the virus may be detected.

             When an individual is infected by HBV several of antigens and antibodies can be detected by serologic tests. Usually the first marker of HBV to appear is HBsAg. It remains detectable from a few days to several months. This marker is also found in some (5% - 10%) infected persons who become chronic carriers of HBV.

             Anti-HBs becomes detectable after HBsAg disappear which indicates recovery from acute illness. Sometimes appearance of anti-HBs is delayed for weeks and months after HBsAg becomes undetectable. During this period, called 'core antibody window', anti-HBc may be the only detectable marker of recent HBV infection.

             Shortly after infection and before clinical signs and symptoms or biochemical changes in liver functions occur, two other markers - HBeAg and anti-HBc are detectable in the serum of infected person.

             Initially the anti-HBc is IgM, however as the infection progress IgG anti-HBc appears and the later persists in persons who recover from the infection.

             HBeAg usually disappears when the patient enters the convalescent phase.

             In others with chronic infection (persistence of HBs Ag for longer than 6 months), HBeAg is cleared, and anti-HBe is found.

             In persons who do not develop immunity to HBV, HBsAg, HBeAg, and anti-HBc can be present.

             Individuals, who recover from infection, will have anti-HBs and/or anti-HBc in their sera.

Serologic markers of Hepatitis B infection and their diagnostic significance.

Marker                                                      Significance

HBsAg                                                     Active infection, acute or chronic

Anti-HBs                                                  Clinical recovery, infection resolved, immunity develop

Anti HBc (IgM)                                       Early acute infection

Anti- HBc (IgG)                                       Active or past infection (carrier state)

HBeAg                                                     Acute or serious chronic infection

Anti- HBe                                                 Resolution of acute infection, may signal late sequelae

Screening Test for HBV

A variety of serological markers appear following the infection with HBV, and one of these is HBsAg. This antigen appears before biological evidence of liver disease or jaundice. This persists throughout the acute phase of the disease and declines during convalescence.

Procedures for the detection of HBsAg have evolved from the relative insensitive agar gel diffusion method to the sensitive and reliable techniques of radioimmunoassay and enzyme
immunoassay. Both are third generation tests and are equally sensitive. ELISA procedure has wide application in the detection of HBsAg and anti-HBs antibodies.

 

Enzyme Immunoassay (ELISA/EIA)

It is based on a one step 'sandwich' principle. It involves the use of solid support (wells of microplate or beads) coated with unlabeled anti-HBs antobody. Number of commercial kits are available and the recommended procedure by the manufacturers should be followed. The general steps of the technique of the test in microplate are given below. See figure 12-13.

1.           Put appropriate volume of the test sample and equal volume of positive and negative controls in the wells of microplate coated with anti-HBs.

2.           Incubate for the correct period of time at defined temperature.

3.           At the end of incubation period, the wells are washed to remove excess of serum and dry the wells as much as possible before the next stage of the assay.

4.           Then add appropriate volume of conjugate (enzyme-linked anti-HBs anti-body) to each well. Enzyme label is usually horseradish peroxidase.

5.           Incubate for the correct period of time at defined temperature.

6.           After incubation, the wells are washed and are prepared for the next step.

7.           Add appropriate volume of chromogen substrate into wells.

8.           Incubate the plate in dark for correct period of time, development of colour suggests the presence of HBsAg, and no or low color suggests absence of HBsAg.

9.           The results can be read visually or by ELISA reader.

Radio Immuno Assay (RIA)

The RIA method is similar to ELISA. It is also solid phase sandwich test. Plastic beads, tubes or microtiter wells are coated with anti-HBs antibody. The general principles and the steps of technique are described below: (Fig. 12-14.)

1.           In the first stage HBsAg in the test serum is bound to the solid phase antibody. After incubation unbound extraneous proteins and tluid in the test is removed by aspiration and washing.

2.           In the second stage radio-active iodine (1 l25)-linked anti-HBs antibody is added. After incubation, the unbound iodine-linked antibody is removed by aspiration and washing. 

  1. Measure the amount of bound iodine-linked anti-HBs with gamma counter. Calculate the mean of the negative controls.

A positive result is usually any value more than 2 to 2.5 times of the mean count of negative control.

False positive reactions for HBsAg may occur with EIA or RIA. The results may be confirmed by repeat or confirmatory neutralization test.

 

Safety measures for personnel working in the laboratory.

             Eating, drinking and smoking are prohibited in the laboratory.

             Mouth pipetting is forbidden.

             Personnel should wear disposable gloves when handling specimen.

             Hands should be washed before leaving laboratory.

             Laboratory staff should bear laboratory coats to protect their clothes.

             HBsAg positive material and disposable used in the test should be put in leak proof containers. They should be labeled as infectious and autoclaved before disposing them or incinerated.

             Working table should be cleaned after test. Remove spills with swabs soaked in 1.0 per cent hypochlorite solution immediately.

             In case of accidental innoculation of HBsAg positive blood or secretion in the individuals, prophylactic injection of HBIG should be given.

The types HCV, HDV, HEV and HGV have been identified, from amongst the Non - A, Non-B (NANB) hepatitis virus group, however, there are still some types of Hepatitis which do not show antibodies to any of these known types and hence the term Non-A, Non-B (NANB) is still in use Search for yet more and new hepatotropic viruses is continuing.

HEPATITIS C (HCV)

Hepatitis C Virus (HCV) is the most common cause of post transfusion Non-A, Non-B hepatitis all over the world. The prevalence of HCV antibodies in blood donors in   ever in Egypt it is as high as 14 %. The prevalence of HCV in Indian blood donors has not been studies extensively but as per some available reports the prevalence of anti HCV varies form 0.6% to 5.2% in voluntary/ replacement donors.75% - 80% of HCV infection is reported to progress to chronic infection of which 10% - 20% may progress to cirrhosis and hepatocellular carcinoma.

The average incubation period is 6 - 7 weeks, it may be as less as 2 weeks or as much as 26 weeks, the acute illness (jaundice) is mild.

HCV shares relationship with flaviviruses. Structure of Hepatitis C virus (HCV) has been recently identified by cloning techniques. The virus is an enveloped single standed RNA virus, 50-60 mm in diameter with a genome containing approximately 9500 bases coding for approximately 3000 aminoacids. The genome consists of a highly conserved 5' noncoding region (NCR), generally used for PCR amplification and have regulatory functions. Downsteam to the noncoding region are the regions coding for the structural elements, including the core or nucleocapsid (C) and the envelope (El, E2/NSI), it is presently unclear whether NS1 is apart of the envelope region or the first nonstructural gene. The 5' end of E2/NSI contains a hypervariable region (HV) that mutates rapidly and probably plays a key role in the virus ability to escape neutralization. Next follows a series of non-structural genes (NS2-NS5) with enzymatic on membrane-binding functions (Figure 12.15).

The initial clone discovered was from the NS 4 region, the derived protein was designated 5-1-1. This was expanded to form the cl00-3 antigen, the basis of the first generation anti-HCV EIA assay.



 

Second generation EIA assay added the c22 core antigen and c33c antigen from the NS3 region.
These antigens increased the sensitivity of the second generation assay by about 20% over the  first generation assay.

Figure 12.15: Proposed genome of HCV: functional equivalents and major antigens used in antibody detection assay. Third generation EIA assay adds an NS5 protein and reconifigures some of the earlier antigens. Now it has synthetic peptides which offer the advantage of minimizing the incidence of non- specific reactions. The third generation EIA have only incremental benefits in the disease prevention.

Instructions given for the methods by the manufacturers of the kits should be strictly followed.

Because of the problem of non-specificity, it is important to confirm EIA reactivity with a supplemental assay, a recombinant immunoblot assay (RIBA) that displays the key epitopes in a linear form on a nitrocellulose strip. Now in RIBA the recombinant proteins c22-3 and cl00-3 are replaced with synthetic peptides representing only a portion of the respective coding regions and replaces 5-1-1 antigen with recombinant NS5.

Polymerase Chain Reaction (PCR) for HCV

Detection of HCV infection during early phase of infection is possible by the detection of HCV- RNA by various molecular biological techniques-nucleic acid amplification techniques.

PCR amplification can detect low level of HCV RNA in serum. Testing of HCV RNA is a reliable way of demonstrating that hepatitis C infection is present and is the most spefific test.Testing of  HCV RNA by PCR is useful in:

            In imunodepressed patient.

            In patient who have organ transplant recently.

            In indetermined recombinant immunoblot assay.

            In acute infection as HCV viremia occurs well before the development of anti-HCV.

Certain rapid antibody detection techniques such as immunochromatic test (ITC) are also available.Prior to the development of specific markers certain surrogate markers i.e. determination of serum alanine aminotransferase (ALT) and antibodies to hepatitis core antigen (anti-HBc) havebeen used to identify the high risk donors for transmission of NANB hepatitis (HCV). Although the surrogate markers played an important role in the prevention of hepatitis C prior to the introduction of HCV specific assays but presently the value of these tests remain inconclusive and controversial. In a study reported from India very little co-relation was found between the surrogate markers i.e. (anti-HBc & ALT ) and anti - HCV reactive donors. It is too early to comment whether hepatitis C (HCV) is the only cause of parenteral NANB hepatitis as the current HCV antibodies assay fails to detect 100 % NANB hepatitis.

Community acquired or Sporadic NANB hepatitis.

Sporadic community or acquired NANB hepatitis due to hepatitis C virus (HCV) with no history of parenteral exposure has been identified.

HEPATITIS D (Delta virus)

It is also known as delta hepatitis. It is caused by defective RNA virus that is unable to produce its own protein coat and thus coats itself with HBV surface antigen. The delta virus is dependent on preexisting or concomitant HBV infection for propagation. Co-infection causes acute viral hepatitis which often becomes fulminant and is usually fatal, while superinfection causes acute hepatitis progressing to chronic active hepatitis and cirrhosis in 75 % of carriers.
Delta hepatitis has parenteral mode of transmission and usually follows HBV infection.
Although tests for delta antigen and antibody exist but should not be used in blood donor screening as all infections with delta virus are positive for HBsAg. So routine testing for HBsAg will eliminate the risk of its transmission.

HEPATITIS G VIRUS (HGV)

Hepatitis G virus is an enveloped RNA virus and belongs to the flavivridae family. The virus was discovered in 1995. The virus genome is similar to that of HCV with which it shares a 25 % homology at the nucleotide level. The HGV appears less variable although variants have been described. HGV replicates in peripheral blood cells but its replication in the liver is still questionable. The mode of spread of HGV is almost same as HCV. The HGV can be detected in the serum by nucleic acid technology (PCR). Recently an immuno-assay has been developed to detect antibody to HGV.

The prevalence of HGV in blood donor population by doing antibody test in the developed countries ranges between 1 to 5% but the detection of antibody generally indicates recovery from an infection and probably immunity to the HGV agent.

CYTOMEGALOUS VIRUS (CMV) INFECTION

CMV is known to be carried in leukocytes, therefore blood components containing white blood cells are more likely to transmit CMV infection. Plasma-derived components and derivatives are not likely infectious for CMV. Transfusion-transmitted CMV can cause severe disease in seronegative premature baby of low birth weight and seronegative immunosuppressed patients e.g. bone marrow transplant recipients or patients with HIV infction.

Since more than 50-80% of adults in developed countries and more than 90% in underdeveloped countries have antibodies to CMV, so it is not practical or advisable to screen donors blood for CMV infection. However, blood transfusion services should have panel of CMV negative donors. In high risk group seronegative patients it is advisable to use CMV negative blood or use leukocytes depleted blood.

Screening tests for CMV

             Latex agglutination

             Enzyme immunoassay

             Complement fixation

The latex agglutination test is mostly used in blood transfusion centers because it is simple and rapid.

Human T- cell lymphotropic virus type I & II

HTLV I & II are retroviuses. Both are cell associated and are not present in plasma. HTLV-1 has been shown to be associated with adult T-cell leukemia-lymphoma and various neurological conditions originally called tropical spastic paraparesis but now often called HTLV-associated myelopathy (HAM). Incubation period for HTLV-1 infection is very long and 2-4% of sero-positive persons develop the disease.

The importance of HTLV-11 is not clear. It is usually associated with intravenous drug abuse and has been found in few cases of Hairy cell Leukemia.

HTLV-1 infection can be transmitted by blood, which has caused concern in those parts of the world where the virus is prevalent, notably in some countries in the Western Pacific and the Caribbean basin. Surveys conducted in Europe and North America indicate that HTLA-1 infection is very rare.

Transmission

             It is transmitted with transfusion of cellular components of blood.  After refrigerated storage for 10 days or more, red cells from an infected donor are far less likely to cause sero-conversion

             Transmission by sexual contact (predominantly male to female)

             Mother-to-child transmission through breast milk

Donor screening should be considered in those countries where HTLV-1 is endemic. Preliminary epidemiological studies are therefore necessary ( WHO, 1992, Guidelines for the organization of a Blood Transfusion Service).

ELISA techniques are used for screening HTLV-1 antibodies, and Western blot, immunofluorescent, or radioimmune precipitation techniques are used for confirmatory testing. It is difficult to differentiate between HTLV-1 and HTLV-11 unless advanced techniques such as polymerase chain reaction are available.

BACTERIA

Bacteria consist of distinct cells which possess cell walls, and have a very simple structure and lack a true nucleus. Bacteria may have capsules which are often important in the immune response.

Example of common bacteria which are known to be transmitted through blood is Treponema pallidum.

Syphilis

Etiological agent for syphilis is Treponema pallidum. Blood and its components may transmit syphilis. The incubation period for transfusion transmitted syphilis is 1 to 4 months, the blood recipient exhibiting the signs and symptoms of secondary syphilis.  Transfusion-transmitted syphilis is not a major hazard of modern transfusion therapy, its chief reasons are:

 

             The spirochetes do not survive in citrated blood stored at 4°C for 72 hours.

             Now the blood is mostly collected from the voluntary donors and sexually promiscuous persons are excluded from blood donation.

             Most patients who need transfusion of blood or its components receive antibiotics therapy because of their clinical condition.

             Widespread use of penicillin and other antibiotics to treat syphilis and its carrier might account for the rarity of infected cases.

Role for syphilis in prevention of posttransfusion syphilis:

Spirochetemia usually is common in the early stages during the invasion of lymphnodes. The screening tests used for blood donors often are negative in early syphilis when spirochetes could be transmitted by blood transfusion. Only 25% of patients with primary syphilis have a reactive serological test for syphilis (STS). and the rest do not become positive until the 4"' week after theonset of primary syphilis. By the time the person develops a positive STS, the spirochetemia has typically cleared.

In addition, a high proportion of healthy people have biological false positive reactions even they do not have circulating spirochetes. It may be due to viral infections, immunization, lupus erythematosus and dysproteinemias.

The prevention of spirochete transmission is not well accomplished by STS test. Anyhow it is considered safe to test blood donation for syphilis due to the following reasons:

             There is possibility of transmitting syphilis.

             Demand of fresher blood for exchange transfusion, and platelets.

             Screening for spirochetes helps to exclude donors who are in high risk group for HIV and HBV infections.

             Cost for STS is low.

Tests

The serological tests are of two types:

            Non-specific tests

            Specific test

Non-specific test:

The most commonly used test in blood donor screening are non-specific tests because they are simple, rapid and economical, They are:

            Venereal Disease Research Laboratory (VDRL) test

            Rapid plasma reagin (RPR) test

Specific tests:

These tests are mainly confirmatory tests and have lengthy procedures. They are not suitable for routine screening of donors blood. Tests are:

            Treponema pallidum hemagglutination test (TPHA)

            Fluorescent treponemal antibody absorption test (FTA-ABS)

            Treponema pallidum immobilization test (TPI)

            Enzyme Immuno Assay

PROTOZOA

Protozoa are unicellular organisms. They have a well-defined cellular structure with a clear nucleus and other organelles. A typical cell is enclosed by a cytoplasmic membrane. This may be covered in an outer cytoplasmic layer. Proteins present in the membranes are important in immune response.

Examples of common protozoal infections, which can be transmitted due to blood transfusion are Plasmodium species.

Malaria

Malaria is caused by intra-erythrocytic protozoan parasites. Plasmodium vivax, P. falciparum, P. ovale or P.malariac. The usual mode of transmission is via the bite of anopheles mosquito.


Blood infected with malarial parasites can also transmit the infection. Transfusion-transmitted malaria may become evident a week to several months following the transfusion of infected blood. The incubation period of plasmodium vivax and P. falciparum is shortest e.g. one week to one month, while that of P. malariae is the longest e.g. many months.


Malarial parasites survives for at least one week at 4°C in whole blood and red cells concentrates, and at least for one week at room temperature in platelets

Screening tests for the malaria

(1)     Microscopic examination of thick and thin blood smear.

It is difficult to find parasites in blood film in short time especially if the density of parasites is less than 100 per microlitre of blood. However it is best practical method for testing malarial parasites

 
(2)     Tests for antibody to malarial parasites are:

             Indirect fluorescent antibody test (IFA)

             ELISA

 
3)       Immuno-diagnostic testing, including EIA methodology

(4)       Nucleic acid probe methodology, including polymerase chain reaction (PCR).

None of the above methods except microscopic examination of blood smear is practical and possible.

Since there is no appropriate method for screening malarial parasites in blood donation, it has been suggested that chemoprophylaxis therapy for malaria should be given to all recipients of blood in highly endimic areas.

VIRAL INACTIVATION

With the emergence of HIV/AIDS there is great demand for guaranteed safe blood and blood products. Today there is rigorous screening of blood to prevent transmission of blood borne infection, the transfusion of blood products have already achieved high level of safety. The "window period" viraemia can be further reduced by screening the donated blood for nucleic acid testing methods. Thus further reducing the risk and increasing the already high cost of testing.

METHODS OF VIRAL CLEARANCE

Several methods of removing or inactivating viruses that may be present in plasma pools from the source or recovered plasma donations are being tried. These steps could be divided into two broad categories: removal, or partitioning, is the physical separation of the viruses or viral particles from the blood and blood products; and inactivation, which destroys the virus so that the remaining viral fragments lack the structure and components needed to infect an individual receiving the blood or blood product.

Removal processes include filtration, affinity chromatography, ion-exchange chromatography, and polyethylene glycol fractionation. Inactivation of viruses has been attempted by heating.

Further, some processes, such as ethanol fractionation results in both removal and inactivation of viruses. Finally, a number of new methods are under development, such as irradiation, photoinactivation and treatment with a variety of chemicals. Several of these novel techniques  have been incorporated into the production of investigational products. In order to be effective, viral inactivation techniques must destroy at least one viral element essential to replication. Photosensitizing techniques use light - activated dyes that are irradiated, causing the dyes to convert to molecules that can alter DNA or membrane lipoproteins. Heat treatment denatures viral proteins and nucleic acids, rendering viruses incapable of replication. Irradiation processes may destroy viral nucleic acids by inducing breaks and linkages. Solvent detergent techniques destroy the viral envelop on lipid - enveloped viruses.

Ultra Violet(UV) inactivation was one of the method thought to be useful for this purpose, as most viruses are quite sensitive to UV, however, current expert opinion is that viral inactivation sufficient for the purpose is not feasible without intolerable level of damage to the different components of the blood.

Photochemical inactivation of viruses in platelet concentrates by use of novel Psoralen and long - wave length U V light, significantly reduces the viral load but cannot be relied on for total removal. This process is based on photochemical reaction of novel Psoralen ( S - 59 ) with nucleic acid, upon illumination with long wave length UV light (UVA, 320 - 400 nm).

Recently (Nov. 2000) Gambro BCT announced inactivation of pathogens in blood products  like packed Red Blood Cells by Riboflavin and UV light. Riboflavin can inactivate pathogens in all the three major blood components that is Red Blood Cells, platelets and fresh frozen plasma.

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