Free download transfusion medicine technical manual. Handbook of Blood Banking and Transfusion Medicine

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Free download transfusion medicine technical manual

The challenge is ever present. It should be done carefully. The donor should be in good health in order to avoid any untoward effect to the donor or the recipient. Male homosexual or bisexual 2.

Promiscuous men or women 3. Male or female sex entertainers 4. Drugs abusers 5. Individuals who have had sexual intercourse with any one in any of these groups. Unexpected weight loss 2. Night sweats 3. Swollen lymph nodes for more than 1 month 5.

Persistent diarrhea 6. Persistent cough with shortness of breath 7. The donor is accepted by a suitably qualified person trained to follow prescribed guidelines for the selection of blood donors.

This person works 8 Donors Selection and Blood Collection under the instructions of a physician of the blood bank. A donor with any abnormal condition is referred to the physician of blood bank, who takes the final decision on whether blood should be collected from such a donor or not.

In doubtful cases the donor should be deferred. When did you east last? Are you taking any medicine? Have you been vaccinated or immunized recently? Have you ever suffered an epileptic fits, convulsions or mental disorder? Have you ever had jaundice or hepatitis? Have you been in contact with a person suffering from jaundice hepatitis during the past 6 months?

Do you know about AIDS? Have you if male had sex with another man? Have you had unsafe sex with an individual at increased risk for AIDS? Have you lost significant weight in last 6 months? Have you ever been positive for HIV? Occupational hazards: Air Crews, drivers of long-distance heavy-duty vehicles and construction workers on high buildings are advised not to give blood within 12 hours of going on duty.

Permanently defer Infectious Diseases Donors should be free from infectious diseases known to be transmissible by blood, so far as can be determined by usual examination and history. Immigrants, refugees, or citizens coming from a country endemic for malaria may be accepted as blood donors three years after departure from endemic area, if they have been asymptomatic in the interim.

In a region endemic for malaria, it is not practicable to reject donors who have history of malaria infection or who have taken antimalarial drug s. Patients may be given anti-malarial drugs. Two-weeks deferral from time of vaccination. Smallpox two weeks after scab falls off Polio oral sabin vaccine, oral Measles rubeola Mumps Yellow fever 3.

Four-weeks deferral from time of vaccination Anti-tetanus serum Anti-venom serum Anti-diphtheria serum Anti-gas gangrene serum Rubella German measles 4. Proscar used to treat benign prostate hyperplasia Oral anti-diabetic drugs with no vascular complication. Tegison to treat psoriasis. It is teratogenic. Persons with thalassemia trait may be accepted as donors, provided they have normal Hb concentration and normal reticulocyte count.

Polycythemia Vera Blood from people suffering from the polycythemia vera is not used for transfusion as it is a precancerous condition and its recipient sometimes develops leukaemia or other malignant cells, though it is rare. GlucosePhosphate Dehydrogenase GPD deficiency The use of donor blood with this abnormality imposes a small but definite risk to the recipient.

Although the survival of GPD deficient red cells is almost normal for about days. On storage, the viability of red cells from GPD deficient donor is less than that of normal red cells.

Those weighing 60 kg or more may give ml blood as well as pilot samples. Blood Pressure: The systolic blood pressure should be between and mm of Hg and the diastolic pressure between 50 to mm of Hg. Pulse: The pulse should be between 80 to beats per minute and regular. Temperature: Oral temperature should not exceed Donation Interval The interval between the donation of a unit of blood should be at least 12 weeks except in unusal circumstances.

Whole blood donation must be deferred for atleast 72 hours after plasma- or platelet-apheresis. Systemic Examination: Clinically heart, lungs and abdomen should be normal.

Liver and spleen should not be palpable. The Hb should not be less than See chapter "Preparation of Solutions and Methods" b Sahlis method c Cyanmethaemoglobin method using spectrophotometer or photoelectric colorimeter.

See chapter "Preparation of Solutions and Methods" d Hemo-Cue method See chapter "Preparation of Solutions and Methods" The copper sulphate specific gravity test is generally used as a Hb screening procedure. It is simple, quick and inexpensive test.

Out-door blood collection Out-door blood collection sites can present special problems. An individual trained in safety principles should make an advance visit to the collection site to ensure that hazards are minimized. All personnel in out-door blood collection camps should be trained to recognize unsafe conditions and understand infection control policies and procedures. The responsibility for site safety should be assigned to a senior-level employee. Hand washing access is essential at all collection sites.

Carpeted or difficult-to-clean surfaces can be protected with a clean suitable overlay. Portable screens are helpful to protect and maintain safe work areas. Refreshment service areas should be separated from areas of blood collection and storage.

Blood-contaminated waste must be packaged and returned to a central location for disposal using thermal autoclave, incinerator or chemical disinfectant in accordance with local regulations for medical wastes. A physician should be present on the premises when blood is being collected. Equipment and Materials a Blood Containers Blood containers are polyvinyl chloride PVC plastic bags which are closed system of single, double or triple bags for collection of ml or ml blood.

Currently CPDA-1 anticoagulant solution is mostly used. It should be sterile, pyrogen free. Identification Blood bag and pilot tube s are identified by a donation number at the time of collection of blood, so that it can be traced back to the donor. The dates of collection and expiry, and ABO, Rh D group are written on the label of bag for blood collection.

If less blood is to be collected. If less amount of blood is to be drawn two factors must be calculated: 16 Donors Selection and Blood Collection 1 How much blood can be safely drawn from the donor. Inspect the bag for leakage or any other defect. The anticoagulant solution must be clear. Check the donor name, donation number on the form and the bag.

Donation number on the bag and form should be same. Place the bag on a balance and ensure that it is below the level of the arm. Choose the site of venipuncture in the anticubital area of the arm. Asking the donor to close the fist usually helps in bringing the vein into prominence.

Allow it to dry. Inflate blood pressure cuff to maintain pressure mm of Hg. Ask the donor to close the fist. Uncover the sterile needle and perform venipuncture immediately, using aseptic procedure. Ask the donor to open and close hand or to squeeze a rubber ball. The donor should be under constant observation throughout the phlebotomy and should never be left unattended.

Mix the blood and anticoagulant gently and periodically during collection of blood. The flow of blood should be uninterrupted and constant, otherwise, it will not be suitable for preparation of platelet concentrate, fresh frozen plasma or cryoprecipitate.

One ml of blood weighs 1. Thus, ml of blood weighs g. The initial weight of the bag and the anticoagulant solution should be taken into account while measuring the total weight. As soon as the required amount of blood is collected, clamp the tubing of the bag with plastic clip. Deflate the cuff or release the tourniquet. Place the sterile swab at the venipuncture site, apply light pressure and withdraw the needle.

Remove blood pressure cuff or tourniquet. Ask the donor to put the fingers of the other hand on the swab at the venipuncture site and to raise the arm. Take the bag to the processing table. Seal the tube with di-electric tube sealer and separate the needle.

Strip blood bag tubing, starting at seal, pushing blood into bag. Do it quickly, to avoid allowing the blood to clot in the tubing. Invert bag several times to mix blood thoroughly; then allow tubing to refill with anticoagulated blood from the bag. Repeat the process a second time. Seal the tubing attached to the bag into segments with di-electrictube sealer. Platelets should be separated within hours after the collection of the whole blood. The donor should remain on the bleeding couch for minutes under the observation of the staff.

Then the donor is allowed to sit up and go for refreshment. Check the arm and apply band-aid after bleeding stops. The donor should be given donation card and thanked for the contribution and encouraged to donate again. Personnel in the phlebotomy area must be trained and prepared to respond quickly to those reactions. Put the sterile swab at the venipumcture site and apply pressure with thumb. Donor adverse reactions are: Syncope fainting or vasovagal syndrome : The symptoms may include sweating, weakness, dizziness, pallor, loss of consciousness, convulsions, and involuntary passage of urine and feces.

The skin is usually cold, blood pressure falls and the pulse becomes thready. Tetany Twitching or Muscular spasm Anxiety and deep breathing may cause the excited donor to loose excess of carbon dioxide, which may cause tetany characterized by twitching or muscular spasm due to hyperventilation. Management The donor is asked to breath into a paper bag which brings prompt relief.

Nausea and vomiting. Management: a Make the donor as comfortable as possible. If donor vomits, provide suitable receptacle and towel or cleansing tissues. Hematoma Management a Deflate the blood pressure cuff. Ask the donor to open the fist and withdraw the needle from the vein. Cardiac problems Serious cardiac problems are extremely rare in the blood donor. If the donor is in cardiac arrest, begin cardiopulmonary resuscitation and continue until medical aid arrives.

Instructions to Donors after donation 1. Drink more fluids than usual in the next 4 hours. Do not remain hungry. Do not smoke for half an hour. Do not take alcoholic drinks for at least 6 hours. If there is bleeding from phlebotomy site, raise the arm and apply pressure. If there is feeling of faintness or dizziness, either lie down or sit with head between knees. If symptoms persist, ask for help, return to the blood bank or consult a doctor.

Number of donation on the blood bag. Test with anti-AB serum is optional if monoclonal anti-A and anti-B are used. Blood must not be released for use until the discrepancies, if any, arc not resolved. Rh group must be tested for Rh D only with anti-D serum. Rh D negative units must be tested for weak Rh D i. Du by anti-human globulin AHG serum. Routine testing for other antigens of Rh system is not recommended.

Tests using monoclonal antibodies for detection of malarial antigens are available but they are costly. As there is no appropriate test which can be done easily in screening blood donor for malaria, it has been suggested that anti- malarial drugs may be given to the recipients of blood in highly endemic areas.

It consisted of a citrate-glucose solution in which blood from rabbits was stored for two weeks, which prevented anemia when transfused in other rabbit who had suffered from blood loss. The next important development occurred in during the Second World War when acidified citrate dextrose ACD solution was introduced for clinical use by Loutit and Mollison.

In citrate-phosphate-dextrose with adenine CPDA-1 preservative was developed. Giycosis results in the production of lactate, with subsequent decrease in pH. Whole blood collected in CPD has a pH 7. Preservative solutions provide buffering capability to minimize pH changes and optimize the storage period. Loss of ATP causes increase in cellular rigidity and decrease in red cell membrane integrity and deformability. Decline of 2, 3-Dipnosphoglycerate 2,3-DPG A fall in pH in the stored blood results in a decrease in red cell 2,3-DPG level, which results in increase in hemoglobin-oxygen affinity.

DPG-depleted red cells have impaired capacity to deliver oxygen to the tissues. The degree of reduction of 2,3-DPG levels depends upon the preservative solution used. The pathological effects of the transfusion of red cells with low 2,3-DPG levels and increased affinity with oxygen include increase in cardiac output and a decrease in mixed venous PO2 tension.

Myocardial functions improve following transfusion of blood with high 2,3-DPG levels during cardiovascular surgery. In patients with shock the transfusion of DPG-depleted red cells makes a significant difference in recovery. After transfusion, the red cells continue to synthesize 2,3-DPG and levels return to expected normal values within 24 hours. The acid-base status of the recipient, phosphorous metabolism and degree of metabolism influence the rate of restoration of 2,3-DPG.

Adenine Simon showed that in CPD solution supplemented with 17 mg 0. Adenine synthesizes ATP and its level is This amount is present in 30 units of fresh CPD adenine 0. Temperature The lower temperature keeps the rate of glycolysis at lower limit and minimizes the proliferation of bacteria that might have entered the blood unit during venipuncture or from atmosphere.

They must be considered in the differential diagnosis of hemolytic transfusion reactions. Transfusion reactions due to bacterial contamination are commonly caused by endotoxins produced by bacteria capable of growing in cold temperature such as Pseudomonos species. Escherichia coli and Y. They are commonly attributed to platelet transfusion because platelets are stored at 20 - C.

Hyperkalemia in massive transfusion After massive transfusion, a life-threatening load of potassium may be released into the plasma and causes signs of hyperkalemia particularly arryhthmias.

Signs and symptoms of contaminated products or hemolysed units : They appear rapidly during transfusion or with in 30 minutes after transfusion. In case of bacterial contamination there may be change in the color of blood in the bag and the color of blood in the tubing of the bag is not changed and there may be clots in the blood. Using a new infusion set, keep the intravenous IV line open with a drip of normal saline. Check the label on the blood bag, cross-matching report and identify the patient to confirm that the patient received the correct unit of blood or component..

Notify the blood bank and describe the signs and symptoms. A post-transfusion fresh blood sample 10 ml of blood in plain test tube and 2 ml in EDTA , taken from another vein, should be sent to the blood bank along with blood bag and transfusion set and transfusion reactions report. First voided urine should be sent for to the laboratory for the analysis of free hemoglobin Laboratory Investigations 1.

Check identity of patient, donor blood and all relevant papers to ensure that there was no clerical error. Check the color of bag i purple color and clots in blood bag no change in color in the segments of tubing of the bag may be due to bacterial contamination ii Change in color both in the bag and the tubing of the bag may due to hemolysed blood. Interpretation of Laboratory findings 1. If nothing abnormal is found in the above findings, it indicates that there has not been an acute hemolytic reaction.

Optional Tests for Clinical Evaluation of Patients: 1. Serum non-Conjugated bilirubin is tested in pre and post transfusion blood samples of the patients. Peak levels of bilirubin occur 5 to 7 hours after transfusion and disappear with in 24 hours if kidney function is normal. Measure Haptoglobin in pre and post transfusion blood samples of the recipient.

If recipients blood sample is taken after 1 -2 days, its value is diminished in post transfusion blood sample. Visible hemoglobinemia results only when haptoglobin reserves are depleted, so there is no point in measuring haptoglobin level.

Haptoglobin can rapidly regenerate when depleted, so if measurements are made several days after hemolytic reaction, normal levels may already been restored. Estimation of free hemoglobin is done in post-reaction blood sample. If sample is taken after some time the plasma may be clear of hemoglobin and it may not be visible. Therapy for hypotension 0. Such antibody-antigen reaction can activate complement and stimulate cytokines production, which results in the release of endogenous pyrogens.

In some cases, cytokines accumulating in stored blood may directly activate endogenous pyrogens. The antibody antigen reaction initiate histamine release which causes hives, itching and rearly laryngeal edema. Signs and symptoms Mostly allergic reactions are mild and not life threatening. If the only symptoms is skin rash or hives, and if the symptoms resolve with 30 minutes of the treatment the transfusion may be restarted.

Anaphylactic Reactions Anaphylactic and anaphylactoid reactions are due to immediate hypersensitivity of immune system. They may begin after infusion of only a few ml of plasma or plasma-containing blood products. They are usually mild at first but can progress to loss of conciousness, shock and in rare cases death. Good transfusion practice calls for close observation during the first quarter hour of infusion and less intensive afterwards but nonetheless continuing surveillance is kept throughtout and after the transfusion.

Causes Anaphylactic and anaphylactoid reactions are generally caused in patients who are congenitally IgA deficient and have developed anti-IgA antibodies by the sensitization from transfusion or pregnancy. Anaphylatic transfusion reactions, however, are quite rare. Endotracheal intubation may be necessary.

Transfusion-related Acute Lung Injury: TRALI Also known as non-cardiogenic pulmonary edema, this unusual, life threatening complication is associated with altered permeability of the pulmonary capillary bed from activation of complement, histamine-mediated events, or prostaglandins, which leads to fliuds accumulation, inadequate oxygenation, and reduced cardiac return. The reaction is also attributed to anti-leukocyte antibodies in donor or patient plasma that react with leukocyte in the pulmonary microvascular system, resulting in leucocyte emboli aggregating in the lungs capllaries.

Other suggested cause includes cytokines in the donor units. Manifestation Transfusion-related acute lung injury TRALI is manifested by an acute onset of respiratory distress, dyspnea, cyanosis, fever, and chill. An X-ray of chest will show bilateral pulmonary infilterates, but no other signs of left heart failure are seen. Potentially fatal hypoxia may occur and persist for hours. Oxygen therapy and sometimes ventilatory assitance i. Usually pulmonary sufficiency returnes within 10 - 12 hours.

Donor who have been implicated in a case of TRALI and who possesses potent leuko-agglutinins can trigger reactions in other patients and should be deferred from donating blood. Circulatory overload Patients who are very young or very old, and have underlying congestive heart failure or have chronic normovolumic anaemia and an expanded blood volume are at greatest risk from circulatory overload.

Primary Alloimmunization This reaction occurs several weeks after transfusion and is mostly mild. It is the result of the primary allo-immunization due to the incompatibility of Rh, Kell, Duffy, Kidd, and other systems. It may be due to blood transfusion, tissue transplantation or pregnancy.

It is also due to the incompatibilty of Rh, Kell, Duffy, Kidd and other blood group systems. Few days after transfusion, the antibody ies concentration increases and they destroy red cells causing delayed hemolytic reactions.

There is no reaction at the time of transfusion. Afterwards there may be. Posttransfusion purpura occurs in cases who are already immunised by earlier transfusions of platelets. Post-transfusion thrombocytopenic purpura is a rare event occuring usually in multiparous females. Some patients develop a platelet specific allo-antibody anti-PlAl.

The antibody destroys not only the transfused P1A1 positive platelets but also causes non-specific destruction of the patients P1A1 negative platelets Antibodies to platelet in multi-transfused patients may cause reaction such as: chills, fever and dyspnea etc.

Management The thrombocytepenia is usually self-limiting and platelet transfusion is usually not beneficial. GVH disease occurs if donor-functional lymphocytes engraft and multiply in a severly immunodeficient recipient. These engrafted donor cells react against the foreign tissue of the host recipient. Death is usually caused by infection or hemmorhage secondary to bone marrow aplasia. The functions of red cells, granulocytes and platelets are not affected by such irradiation.

Transfusion is thuoght to have other, less benificial effects in other clinical settings, including increase rates of solid tumor recurrence and increased rates of postoperative bacterial infections.

These effects are controversial but suggest that the relationship between transfusion and the immune system is more complex than previously considered. No specific mechanism has been definitly proved for post transfusion immunodulation immunosuppression No specific therapy regimen is available.

Transfusion of appropriate blood component s is advised to have beneficial effects. Chronically transfused patients specially those with hemoglobinopathies like beta-thalassemia major, congenital hemolytic anemias, or aplastic anemia have progressive accumulation of iron and no physiological means of excreting it. Storage occurs initially in reticulo endothelial sites, but when these are saturated there is deposition in parenchymal cells. Iron deposition interferes with the functions of the heart, liver and endocrine glands.

Metered subcutaneous infusion of desferrioxamine, an iron chelating agent is valuable for reducing body iron stores in these patients. Transfusions of neocytes young red cells have been effective in longer survival of neocytes, causing longer transfusion interval, and lower amount of iron accumulation.

Malaria Incubation period for transmission of malaria depends on the number and strains of plasmodia transfused. 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.

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. 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. 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.

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.

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. 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. Strategies to reduce the risk of transfusion- transmitted infections. 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.

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.

This syndrome was recognized in , well before the discovery of the causative virus. The virus was subsequently identified as the causative agent of AIDS. It is morphologically similar to HIV 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. 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. There is no DNA present. This is Blood Transfussion Transmitted Diseases more commonly known as reverse transcriptase. 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. These are abbreviated to p7 and p9 respectively. The reverse transcriptase enzyme is a 66kDa protein, which is abbreviated to p The core is totally enclosed in a cone-shaped shell of p24 protein.

The whole unit is called the viral capsid. Fig 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 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 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.

Gene products marked with asterisk are not usuallay visible on a Western Blot. 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.

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. 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. These cancers are usually aggressive and do not respond very well to standard chemotherapy.

The presence of opportunistic infections or secondary cancers is only determined following clinical and laboratory investigation. Within few weeks antibodies to both envelope gp41 and core p24 proteins appear in almost all infected individuals. 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 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 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. It has also been found to be the best means of monitoring the progress of infection. 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 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. Soon after antigen has appeared, antibody emerges corresponding with a decrease in free antigen. The levels of antibodies to p and gp 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.

Whilst virus can be isolated from many body secretions, infection is transmitted in only a limited number of ways. Unprotected penetrative sexual contact with an infected person, either between men or between man and woman.

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. 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.

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. 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.

Manufacturers often use the terms first generation, second generation and third generation assay. This method needs specialized equipment. See figure Positive and negative controls are added to a number of wells on each plate run. 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.

Conjugate solution contains antihuman 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 antiHIV sera bound to antigen. Conjugate Enzyme labeled human globulin antibody Fig 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. The acid inactivates the enzyme and fixes the color. The color change can be read visually.

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

The wells are incubated for the defined period and at correct temperature. During this Blood Transfussion Transmitted Diseases 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 antiglobulintype assay. Substrate solution is immediately added to all wells and incubated in dark for the defined period and at correct temperature. At the end of the incubation period, dilute acid 1 N H2SO4 is added to all wells to stop the reaction.

An intense color signifies a nonreactive sample, while lack of color signifies a reactive specimen. 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. Sera or diluted sera are added to antigen bound wells. 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. 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.

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

At the end of incubation period, diluted acid 1 N H2SO4 is added to all wells to stop the reaction. Color develops in the wells having sera containing anti-HIV and no color will develops in the wells having sera with no anti-HIV. The optical densities OD values of the solutions are read by the ELISA reader after calculating cut-off value, and the results are recorded.

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. Test samples and controls are diluted in microwells with test diluent which is provided with assays. The HIV coated particles are added to the diluted samples and controls. During incubation, the particles are agglutinated by anti-HIV present in the serum. 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. 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.

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. It is incubated for 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. The membrane is rinsed with the buffer solution to remove unbound antibodies. Then conjugate is added. The composition of the conjugate varies between assays. 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. The final result is read visually and compared with the expected results described by the manufacturer. Although control is not required, it is good practice to set up a negative control and a weak positive control. 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 The non-reactive and weakly reactive controls are included with each run, regardless of the number of test specimens. Figure The binding of this tracer antibody to the Blood Transfussion Transmitted Diseases 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. Location or position at which antibodies in specimen attach to viral antigen on strips indicate whether antibody is specific for viral antigen or not. Figure , indicates the bands on the strip used with the strongly positive control. 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 , and gp 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.

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.

Methods for HIV antigen testing. The difference between HIV-antigen and anti-HIV antibody screening is that HIV-antigen test uses a sandwich of antibody-antigenantibody, unlike HIV antibody screening which comprises a sandwich of antigenantibody-antigen. At the end of incubation period, the excess serum is washed off. The conjugate is an enzyme-labeled specific antibody usually monoclonal. A sandwich is formed of antibody-antigen-antibody. 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. 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.

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. Saran RK. Transfusion medicine technical manual. India: Ministry of Health and Family Welfare; Background and objective: Platelet rich plasma-platelet concentrate PRP-PC , were prepared and their quality parameters were assessed.

Materials and methods: This was a prospective cross-sectional study carried out from February and May to study various quality determinants of platelets prepared at the Kenyatta National Hospital. The objectives were to assess their quality using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH.

Results: A total of 78 platelet concentrates were analyzed. The majority 54,