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CHAPTER ONE Definition Blood transfusion Science is a Science that deals with the study of the various Blood Group antigens, their Medico- legal significance and reactions. The Science stems from the importance of Blood as a tissue fluid. History of Blood Transfusion The transfer of blood from a healthy individual to a sick one is an ancient idea. Drinking of animal blood was practiced during medieval times, but this did not produce the desired results. In 1916 William Harvey advanced the story of blood circulation. Within that same period Christopher Wren attempted the first transfusion. Using an intravenous needle he transfused blood from one dog to another. In 1667 , a French Physician had performed a transfusion of Lamb’s blood to a human patient , but after the death of the second patient no further transfusion were attempted. A scientist by name James Blundell showed that blood from one specie could not successfully be transfused to another. This Scientist was the person to perform a successful blood transfusion between members of the same species. His exercise also resulted into some problems such a clotting of Blood after transfusion. This problem got a break through in 1900 when Karl Land Steiner demonstrated the ABO Blood grouping system. Land Steiner and Levine went ahead to discover another blood group system in 1927 known as the MN and P system. They also in 1939 discovered the Rhesus System. Since then a lot of different Blood Group Systems have been discovered e.g the Lutheran , Kell , Lewis , 1 Duffy and Kidd Systems. This scientific advancement continues today. BLOOD CENTRES The Blood transfusion Science has explored the Biochemistry nature of Blood groups antigens. The Science has greatly increased our knowledge about Blood group systems. A Blood transfusion Laboratory is thus a Laboratory mainly concerned with the Blood Grouping of patients, Provision of suitable Blood for Transfusion and detection of a typical antibodies which may complicate blood transfusion and for Pregnancy. Blood transfusion centers are concerned with the bleeding and testing of blood donors and provisions of suitable blood , blood products and reagents to Hospital Laboratories. These Centres may also act as reference and confirmatory centres for Hospital Laboratory findings and to perform test and solve problems beyong the scope of a Hospital Laboratory. 2 CHAPTER TWO Blood Group Immunoglobulins Man lives in an environment in which he is surrounded by potentially dangerous bacteria and Viruses. It is necessary that , the body has suitable defense in order to remove foreign organism, which enter it. This is carried out in several ways . One of which is the production of an antibody ( Immunogloblins) which reacts with the foreign organism/s or antigens. Blood group Immunoglobulins are high molecular weight proteins produced as a defense to remove foreign substances in circulation. Their Production is govern by the presences of an antigen/s. These Immunoglobulins are referred to as Antibodies. Definition of Antigen: An Antigen is a Substance which stimulates the production of antibodies , and when missed with the antibody reacts in some observable way. Definition of Antibodies : An Antibody is a protein formed in the spleen or lymph nodes in response to the Presences of an antigen, and reacts specifically with that particular antigen in some observable way. Basic Structure of Immunoglobulins All type of Immunoglobulins have the same basic structure . This consist of four polypeptide chains , two long heavy chains and two short chains which exist in two forms ( Kappa or lambda). The heavy and light chains are joined to each other by disulphide bonds. The disulphide bonds confer the molecule strength but with flexibility. 3 The variable region occurs at the end of both light and heavy chains, made of a sequence of amino acids that gives the antibody , the antigen specificity. The more the amino acids the more the antibody specificity. Classes of Immunoglobulins Immunoglobulins are classified base on the Structure of the heavy Chain. The Classes include : IgG, IgA, IgM, IgE, IgD. Blood group antibodies exclusively are made of IgG , IgM and IgA. Structure of Immunoglobulin Molecule 4 Basic Differences Between the Immunoglobuins Designation Heavy Chain Light Chain Molecular Wt Sedimentation Coef Content in Normal Serum per 100ml Percent of Normal Igm Placental Crossing IgG y K-λ 150.000 7s IgA α K-λ 160.000 7s -gs IgM µ K-λ 900.000 19s 8001600mg 140400mg 50 -200mg 0 – 40mg 10 -70mg 13 6 1 0.002 None None 80 Free None None IgD ∂ K-λ 180.000 7s IgE € K-λ 200.000 8s 5 CHAPTER THREE The Immune Response When an antigen is introduced into an individual, antibodies are produced in response to the stimulation. The production and increase in antibody is known as the Immune response. The Peak of this reaction usually occurs at an interval of 10- 20 days after the initial stimulation . The response to the antigen depends on whether the individual has previously been exposed to that particular antigen. The response after the first dose known as the Primary or sensitizing dose , is usually slow and weak , but subsequent exposure to the same antigen produces a strong response with large amounts of antibody being produced quickly. These may remain in the Circulation for many years. The antibodies initiated by the Primary response are Predominantly IgM. While subsequent exposure to the antigen results in the production of IgG antibodies. The Immune Response Curve. 6 CHAPTER FOUR ANTIGEN- ANTIBODY REACTIONS The Forces holding an antigen- antibody complex together are : Ionic bonds Hydrogen bonds Vander waal forces Hydrophobic bonds In Blood grouping the two most commonly observed results of antigen- antibody reactions are : 1) Agglutination or Clumping of Red Cells caused by cross linking of a multivalent antibody. 2) Haemolysis , where antigen antibody reaction results in the breakdown of break down of cells. Red cells when suspended in Saline, although appearing to touch each other when viewed under the microscope , in fact do not as they are surrounded by what is referred to as an ionic cloud. Red Cells have a net negative electrostatic charge on their surface due to the ionization of the carboxyl group of sialic acid present at the cell membrane which attracts positive ions from the surrounding medium. This positive layer subsequently attracts negatively charged ions to its surface . This process continues in layers until there is insufficient force to attract more ions and the outer edge is called the surface of shear. These electrostatic charge is referred to as the Zeta Potentials. The relative size of the Immunoglobulin and the techniques for demonstrating specific red cell antigens and antibodies takes into 7 account the surrounding ionic cloud. The IgG antibodies has two binding sites which are not enough to bind with the corresponding antigen in saline. While the IgM antibodies has five binding sites which make it easily reactive to its corresponding Antigen. Fig : Illustrated Diagram Application of Zeta Potentials The relative size of Immunoglobulins and the techniques for demonstrating Specific Red Cell Antigens and antibodies takes into account the surrounding Ionic Cloud. The IgM or Complete antibodies are large enough to bridge the ionic cloud and therefore agglutination can occur in a saline medium. The IgG or incomplete antibodies are much smaller and because of this , the majority are unable to straddle the ionic cloud , producing blocking of the receptor site , but no agglutination in saline. These Red Cells can be said to be Sensitized . Each molecule of IgG and IgA has two antigen binding sites, whereas IgM has ten . This may facilitate agglutination in saline by IgM antibodies. In order for an IgG antibody to agglutinate two red cells , it would have to bind each each of its two binding sites onto separate Red cells. An IgM antibody having many more antigen binding sites , would be able to bind more than one antigen binding site to each red cell. 8 Fig : Ilustration. CHAPTER FIVE THE ABO BLOOD GROUP SYSTEM Land Steiner observed that the Red Cells of some individuals were agglutinated by the serum of other individuals. He demonstrated that these people could be classified into four groups according to which of the two antigens were detectable A , B, AB and O. He also showed that an individual possesses antibodies against the antigen or antigens that he lacks on his Red cells. For Example : Antigen on Red Cell Antibody in Serum A Anti- B B Anti-A AB None O Anti- A +B 9 Because of the presence of these antibodies , the ABO system is of major importance when transfusing blood in an individual. And whenever possible blood should not be transfused d if it carries an ABO antigen which the recipient lacks. The presences of these anti A and anti B agglutins means that ABO grouping can be performed on both cells and serum. This acts as a double check to ensure that the Correct ABO group has been determined. ABO Antigens The A and B antigens can be detected at an early stage in the foetus, but are still not fully developed at birth. There is no problem grouping cord blood cells with potent anti-sera , but weaker sub groups may be difficult to detect. There are many antigens on a single red cell which express the presence or absence of the various blood group. For example Antigen A on Red cell has approximately 1.000.000 antigen sites Antigen B on Red Cell has approximately 700.000 antigen sites Antigen AB on Red cell has approximately 500.000 antigen sites. These ABO antigens are also present in White Blood cells , Platelets and Tissue cells. GROUP SPECIFIC SUBSTANCES Blood group Substances are carbohydrate substances called hapten .They are non antigenic and are found in tissue cells and body fluids. These substances are present in the saliva of certain individuals. Individuals whose saliva contains appropriate ABO substances are called Secretors. Secretion of these Substances are 10 determined by a pair of allelomorphic genes, Se and se giving rise to three genotypes. Se Se Se se 80% se se ----20% These blood groups specifc Substances appear in two forms : 1) Water Soluble form present in body fluids and tissues 2) Alcohol –Soluble forms present in Red Cells , but absent in body fluids. ABO SUB- GROUPS In 1911 , Van Dungern and Hirszfeld described two different types of A antigens known as A1 and A2. Nearly all anti-A produced by group B individuals contain two anti A antibodies i.e Anti A and Anti A 1. Anti A agglutinates red cells of the group A1 , A2 , A1B and A2B1. But anti-A1 agglutinates only the red cells of group A and A1B. The A2 antigens react more weakly than the A1 antigen when mixed with anti A. This is because there are fewer antigen sites of which the anti A can become attached. Another Subgroup of A cells include A3, Ax , Aint , Am and Aend. INCIDENCE OF THE ABO GROUPS The incidence of the ABO groups varies strikingly in different parts of the world and certain races have a predominance of different groups to others. For example Negroes have a higher Percentage of Group B within their ethnic groups. 11 ABO ANTIBODIES They are also referred to as naturally occurring antibodies or allo -antibodies. They are said to be formed naturally circulation following stimulation from ABO antigens. The production of ABO antibodies in infants does not begins until about 4 months of age. Which means that it may only be possible to determine an infants ABO type from a red cell group. However antibodies may be detected in the cord blood which has been transferred via the placenta from the mother. Characteristics of Natural Antibodies They have the following properties. React maximally at 4.0C , but the thermal range of activity includes 37.0c Agglutinate cells suspended in Saline Are Absorbable The agglutinated Cells adhere very strongly and agglutinates are difficult to break. Anti- A and Anti- B levels are highest between the ages of 5 – 10 , after which the decrease and are sometimes difficult to detect in elderly patients. ABO antibodies like other antibodies are found in the globulin fraction of plasma. They can be demonstrated in other body fluids which contain plasma globulins such as lymph , exudates and milk. 12 ABO GENES The presences of antigens on the RBC is determined by genes. The genes are carried on Chromosomes which are present in the nucleus of all Red Cells of the body. There are 46 Chromosomes arranged in 23 pairs in each nucleus , with exception of the sex chromosomes which has only 23 chromosomes. So that only one chromosome from each pair is presence in the ovum and spleen. The fusion of the ovum and sperm brings the total number back again to 23 pairs. The two genes ( one from each Parent ) which control the ABO Group can be the same or different . if the genes are the same , the person is called Homozygous that character and if different is called heterozygous . The gene which can occupy the same site or locus on the chromosomes are called Allemorphy or alleles. Ignoring the question of subgroups , the inheritance of the ABO Group depend upon their genes A, B and O. The O gene is codominant while the AB are dominant. The O gene is an amorph which is a recessive gene , showing no observable change when in the homozygous form. An individual inherits the A,B and O genes from one parent and the A, B or O gene from the other thereby making a pair of gene called the genotype. Therefore six genotype can occur Viz : AA , AB, BB, AO, BO and OO. It is not possible to differentiate Red cells of genotype AA or AO, BB or BO and so the term Phenotype is used to describe the observed reaction . AB Red cells and OO red cells are both a phenotype and the genotype. 13 Genotype of Parents Permutation A O B AB BO O AO OO Possible genotype AB , AO BO and OO Possible Phenotype AB , A O and B 14 CHAPTER SIX ABO GROUPING SERUM These serum are usually obtained from selected donors whose antibody levels are suitable for us as Laboratory Reagent. Standard Anti A serum should have a titre of 1 in 512 and anti B a titre of I in 256, when titrated against A and B cells. Several dilution of the serum are made in Saline and Red cells of the appropriate group added. The titre is the reciprocal of the highest dilution at which agglutination occurs . Although some sera may confirm to these requirements , the avidity of the antibody may not be suitable. Avidity is the power of the antibody to agglutinate quickly and strongly. LECTINS These are plant extracts that can react with red cells and antigens to form agglutination .Their use can be extended to the identification of Blood Types . For Examples : 1. Dolichos biflorus : This can react specifically with A1 antigen. It can be used to differentiate A1 and A1B red cells from A2 and AB Red cells. 2. Ulex Europaeus : This has anti A Specificity and agglutination of A2 , A2B cells are more strongly than A1B1 or B red cells. General Characteristics of Antibody and Antigen Reactions Potency of antibody 15 Titre ( Level of Antigen and Antibody Binding ) Specificity ( Exact Antigen binding Sites ) Detection of Subgroups No Rouleaux Formation CHAPTER SEVEN LAW OF HEREDITY Two Laws of inheritance have been proved in accordance with Bernstein theory. 1. The Off- Spring cannot possess the antigen A or B alone or in Combination except that it has been inherited from one or both parents. 2. The Parents of group AB cannot produce an offspring of group O , nor can parents of group O give rise to a child of group AB. This is because the group AB is heterozygous , so that the A gene must come from one parent and the B gene from the other. Phenotypes of Parents Possible Phenotype of Offspring O X O --------------------------------------------------------------------O O X A ------------------------------------------------------------------O or A O X B------------------------------------------------------------------O or B O X AB ----------------------------------------------------------------A or B 16 CHAPTER EIGHT THE RHESUS SYSTEM In 1940 Landsterier and Wiener injected the red cells of the Rhesus Monkey into rabbits thereby producing an antibody which not only agglutinates Rhesus Monkey red cells but also the red cells of some human beings. As these people apparently possessed an antigen similar to the Rhesus Monkey , these individuals were called Rhesus Positive or Rhesus Negative. The antigen was called D and the antibody anti- D The Rhesus system is very complex and contain more than 40antibodies. At the level of general use, six common Rhesus genes exist C, D and E with their allelomorphs c d and e. THE Du ANTIGENS It has been postulated that the D antigen is in fact made up of a mosaic of 4 parts, Rh A, Rh B, Rh C and Rh D. An individual who is Rh D positive possesses all 4 parts of the mosaic whereas none of these parts is present in Rh D negative individuals, The D u antigen is the weaker form of the D antigen. The D u antigen does not usually react with complete anti D but will react with varying amount of different incomplete anti D sera depending on whether it is high grade or low grade D u antigen. When the D u antigen is present , any anti D serum which does not agglutinates the red cells will have sensitized the cells. That is to say the red cells will have been coated with the antibody and this may be demonstrated using the anti human globulin test. 17 Diagram of D Antigen A Du person if given RhD positive blood may although rarely , form an anti-D and similarly Du Blood given to a Rhesus Negative person may well stimulate the formation of Anti-D. It is therefore accepted that a Du individual is considered as Rhesus Positive as a donor and Rhesus negative as a recipient . HAEMOLYTIC DISEASE OF THE NEW BORN ( HND) In 1939 , it was demonstrated by Levine and Stetson that maternal antibodies crossing the placenta could damage foetal red cells possessing the antigen specific for the maternal antibody. Since only IgG antibodies are able to cross the placenta . IgM antibodies being too large . It is these IgG antibodies when active at 37.0c can cause HND. In the majority of cases the causative antibody is anti-D. The mother being RhD negative and the foetus having inherited the D antigen from the father. The first child is seldom affected by HND , since the stimulation of the antibody is frequently due to as transplacental hemorrhage from the foetus to the mothers during delivery. If a Pregnancy with a second Rh Positive foetus occurs , then small bleeds from foetus to mother may further stimulate antibody production. The antibody produced as a result of the 18 primary dose of the antigen will be mainly IgM ,but on subsequent exposure to the antigen, IgG Production replaces IgM. It is the IgG antibodies which are able to cross the placenta, enter the foetal circulation and destroy red cells. The affected infant usually presents with anaemia and jaundice. The Anaemia usually have an increase in reticulocytes and a high nucleated red cell count. DANGERS OF HND Massive infant anaemia Massive Jaundice that can lead to irreversible brain damage. Still birth in High sensitization SOLUTIONS Treat for Anaemia Removal of sensitized Red Cells in Circulation Infusion of Compatible ABO Rh – ve Blood Antenatal Screening of antibody levels Exchange transfusion Injection of anti- D Immunoglobulins to Rh negative woman bearing a Rh D positive Children. OTHER BLOOD GROUP SYSTEMS Over 100 blood group antigens maybe demonstrated using the specific antisera and these have been classified into blood group systems. Many of these antigens fortunately have no clinical significance. But as the blood group systems are inherited quite independently from each other, they are of immene value as genetic 19 markers. Some of the other blood group systems apart from ABO are : MNs, P , Kell , Lewis , Lutheran , Duffy , Kid and I. MEDICO-LEGAL ASPECT OF BLOOD GROUP In paternity dispute , the blood grouping of all parties concerned can do no more than exclude one of the parents. Usually it is the father who is a dispute and he is excluded if antigen which he genetically must pass are not present in the child. Also if the child possesses an antigen which both he and the mother lack , the disputed father must be excluded. This type of work is not usually carried out in the Hospital Laboratory because of the legal implication. FORENSIC ASPECTS The determination of the Blood group antigen of an individual are nearly as exclusive as the finger print. This fact is used frequently by Police Departments to investigate criminal cases. Blood groups antigens can be detected from dried stain saliva and other body fluids inclusive. 20 CHAPTER NINE COLLECTION AND STORAGE OF BLOOD. BLOOD DONATION CRITERIA Donor must be within the ages of 18 and 65 of both sexes Females Donors must have HB values greater than 12.5 g/dl Male Donors must have HB Values greater than 13.5 g/dl Donors should be absolutely well devoid of malaria, Hepatitis B, Hepatitis C , HIV , Syphilis and other diseases. Donors should not have any history of jaundice or allergy BLOOD COLLECTION Blood is collected in plastic bags or Medical Research Council (MRC) Bottles. The said container contain the relevant anticoagulant. The anticoagulant commonly used are : Acid Citrate Dextrose ( ACD) Citrate Phosphate Dextrose Adenine ( CPDA) The CPD Adenine anticoagulant has a better preservation of red cells enzymes, Oxygen carrying capacity than ACD. Blood stored at 4.0c is well preserved in CPD- adenine and maybe used for up to 35 days after the date of collection. FUNCTION OF THE COMPONENTS OF THE ANTICOAGULANT Citrate - Prevents the Blood Clotting by acting on Prothrombin preventing its conversion to thrombin. Phosphate – Act a buffer , thus buffering the Blood Electrolytes Dextrose – Provides the energy needed by the Blood Cells Adenine - Amino acids helping the maintaining the internal structure of the Hb 21 BLOOD BANK Since Blood deteriorates rapidly if not kept under ideal condition, it is very necessary to store blood in a specially constructed refrigerator which have a high insulator. Most maintain a temperature of 4.0c with a Maximum range of 2 – 6.0C. It is essential that the temperature does not exceed 6.0c or fall below 2.0c. If this occurs damage to the red cell may occur. To prevent this, the Blood bank must have a temperature recorder so that it is possible to tell the temperature at a glance and also to be able to keep a record of the stability of temperature. The Blood Bank must also be connected to an alarm system. Preferably a laud bell or buzzer which will give an audible signal, If the temperature rises or falls outside the prescribed units. The alarm must also sound in a place where the staff are always on study thereby ensuring that a responsible person will take the predetermined action even when the Laboratory is closed. The alarm should also sound if the electricity supply fails. It therefore follows that the alarm system must be battery operated. On no account must blood be stored in a domestic refrigerator because of its temperature fluctuation. If there is any doubt regarding the storage of blood, once it has left the Laboratory blood blank, it must be discarded and not returned to the stock to be re-cross matched for another patient. TRANSPORTATION OF BLOOD Blood should be transported in an insulated box which keeps at a temperature of 4-6.0c for at least 6 hours. STORAGE OF BLOOD 22 Apart from a 21 -35 days storage duration at 4.0c. Blood can also be used in frozen form. For example Red Cells can be frozen for 2 years at - 80.0c in the present of glycerol. Blood can also be stored in the presences of liquid nitrogen at - 196.0c, for 10 years. The Blood cells can be recovered by thawing and washing of the cells. BLOOD PRODUCTS AND SUBSTITUTES With the advent of more intensive and sophisticated methods of Clinical management, the demand for blood have greatly increased. In order to keep up with these demands , it has been necessary to split blood into various components such as red cells , Platelets , white cells etc. In this way the same amount of blood may be used to treat many more patients. 1. Fresh Frozen Plasma This is plasma that has been separated from red cells on collection and stored at – 20. 0C. The plasma contain clotting factors and hence can be used for the treatment of multiple clotting factor deficiencies. 2.Plasma Protein Fraction This is a solution of the protein of human plasma and is a clear amber fluids. It is prepared from pooled plasma which has been precipitated with suitable organic solvents and re-dissolved in water. This may be used to replace depleted plasma volume and may be used in place of blood in an emergency while awaiting issues of blood. It is also called Human Albumin solution 4.5%. 3. Dried Human Plasma This is plasma that has been freezed dried and can be reconstituted with sterile isotonic saline for usage. 4. Albumin Solution 23 These are used to give colloid. They are pasteurized at 60.0C for one hour and have no risk of transmitting Hepatitis B or HIV. 4% and 2% solution are the most common. 5. Cryoprecipitate This is prepared from fresh Plasma which is frozen solid in and a mixture of solid CO2 ( DRY ICE) and ethanol is then thawed slowly at 4.0c for 24 hours. This contains clotting factors including factor V111. It is indicted in coagulation failure. Treatment of haemophilia and other bleeding disorders. BLOOD SUBSTITUTES These are solutions which are used in the absence of blood to replace blood volumes. 1. Dextran 6 % 2. Gelatin 3 -5 % 3. 4% Glucose Saline 4. Hartmann’s Lactate Solution and Isotonic Saline. All these solutions are predominantly used to maintain the blood volume and prevent dehydration after surgery. The development of synthetic compounds called perfluorocarbons are being developed . These are somehow capable of carrying Oxygen to the tissues. CHANGES TO BLOOD DUE STORAGE 1. Oxygen carrying capacity is reduced due to deterioration of red cell function, during storage. Fresh blood has the best Oxygen carrying capacity. 2. Platelets function declines rapidly so that after 48 - 72 hours of storage the platelets will not assist in Coagulation. 3. HyperKalaemia 24 During Storage potassium is released from damaged red cells. Red cells membrane become less efficient with increasing length of storage. 5. Deterioration of Coagulation factors 6. Acidosis from stored blood 7. Citrate toxicity above 35 days storage. 8. Hypothermia : Since blood is stored at 4.0C , large transfusion may produce hypothermia. Blood can be warmed by placing a sealed bag or bottle in water at 35 40.0c for 7 minutes. If temperature exceeds 45.0C heamolysis will occur. 25 CHAPTER TEN FACTORS AFFECTING ANTIGEN/ANTIBODY REACTIONS The speed at which Antigen -Antibody reaction takes lace and the strength of the reaction are affected by many factors. 1) Concentration : The sensitivity of blood grouping tests is dependent on the use of Optimum Antigen-Antibody concentrations. An increase in the number of antibodies that are bound to each red cells results in an increase in the strength of the reaction. 2) PH : Most blood group antibodies show optimum activity between PH 6.5 and 7.5. 3) Temperature : The Maximum rate of formation of AntigenAntibody complex is at 37.0c, with a reduction in this rate at lower temperatures. 4) Ionic Strength : An increase in the rate of association between Antigen-Antibody can be observed if the ionic strength of the medium is decreased. This results in an increase in Zeta potential, causing an increased attraction between the negatively charged cells and Antibody molecules, most of which are positively charged. Since the titre of most antibodies can be increased by diluting the serum in low ionic strength saline, and because of the increased rate of Antigen –Antibody association, it is possible to reduce incubation time of blood grouping and cross matching tests without any loss of sensitivity. This fact has led to the widespread routine use of low ionic strength saline in Hospital Laboratories. A marked reduction in 26 ionic strength has been associated with non specific antibody uptake onto red cells. A low ionic strength of saline with glycine with a molarity of 0.03 has been found to combine maximum with increase sensitivity without large false positive results. Physiological Saline has a molarity of 0.17. 27 CHAPTER ELEVEN COMPLEMENT Complement is a complex group of serum globulins which are present in fresh normal serum and are able to lyse red cells and destroy certain bacteria. Eleven components of complements are recognized C1-C9, C1 having three subunits C1q , C1r and C1s. Complement activation can occur in at least two ways. a) The Classical Pathway : In this pathway antibody binding leads to haemolysis of the Cell by the later stages of the sequence. b) The Alternate Pathway : In this pathway Antibodies are not essential for activation to take place. The activation of the Classical Complement pathway can be divided into three main steps. 1) The Recognition Stage where the first component of the Complement C1 is activated by the exposed complement binding site on the FC portion of the Immunoglobulin molecule. 2) The activation stage which leads to the formation of a C2C4 complex which acts on C3. Parts of C3 and its activating enzyme forms another enzyme which , in turn bring about formation of C5. 3) The final stage or membrane attack sequence involves component C6-9 which interact to produce cell lysis by means of circular holes in the red cell membrane. It is possible to detect the complement bound onto the red cell by using anti-complement antibodies. 28 When Antigen-Antibody reactions occur on the cell surface, some are capable of binding complement to the red cell. During binding the Antibody is thought to undergo configurational change , exposing a complement activation site on the Antibody molecule. The complement activation site is located on the FC position of an Immunoglobulin molecule. IgM antibodies are therefore better activating complement because of their many bind sites than IgG molecules which have only two binding sites. CHAPTER TWELVE TECHNIQUES USED IN BLOOD TRANSFUSION Although IgG Antibodies are mostly unable to cause agglutination of red cells in a saline medium they are of significance , when grouping and cross matching. Several techniques may be used to demonstrate these antibodies. 1) Use of Bovine Albumin The use of a medium which is able to dissipate electric charge( and therefore disperse the ionic cloud surrounding the Red Cell) allows the cell to become more closely associated . Agglutination of the cells by IgG or incomplete Antibodies is then Possible. 2) Use of Proteolytic Enzymes The net negative charge which the red cell carries is due to the ionization of carboxyl group of Sialic acid present at the cell surface. As a result of Proteolytic action, some enzymes are able to librate Sialic acid residues from the cell membrane. This has the effect of decreasing the negative charge at the red cell surface, thus 29 allowing cells to approach one another more closely. IgG antibodies are then able to bring about agglutination of the cells. Type of Enzymes Papain extracted from paw-paw fruits Bromelin extracted from Pineapples Ficin extracted from Figs Trypsin extracted from Pancreas. TWO TYPES OF ENZYME TECHNIQUES a) Two Stage Technique In this technique the cells are pre-treated with enzymes before setting up the test. This method is more sensitive and very ideal tests where the cells are tested against a number of sera ( Antibody Screening and Antibody Identification ). b) One Stage Technique In this technique the serum , enzymes and cells are layered in the tube and the cells become enzyme treated as they fall through the enzyme layer into the serum. This stage is frequently used because of less consuming time. NB. Using enzyme techniques , by Proteolytic action on the Sialic acid ,more of the Red Cell antigen can be destroyed e.g M , N, and Fya antigen. COMPATIBILITY TESTING - CROSS MATCH In most blood transfusion cases even though the blood donor and the recipient ABO and Rhesus Groups are the same . It is essential that Cross match techniques be performed. This is to ensured that the donor blood will not give rise to any reaction from antibodies the recipient may have formed to any other blood group systems. 30 As it is true to say that no blood given will exactly match the antigenic structure of the recipients red cells. It is vital that every precaution is taken to prevent harm to the patient. The Cross matching is performed by testing the donor red cells against the recipients serum using several different techniques. SOME TECHNIQUES INCLUDE : 1. Saline technique at room temperature for detection of cold antibodies. 2. Saline at 30. 0C or 37.0c detect antibodies with a wide thermal ranges. 3. Albumin addition Technique 4. Enzyme technique to detect incomplete warm antibodies that react at high temperature above 6 - 37. 0C 5. Indirect antihuman globulin Technique IMPORTANT NOTE 1. Blood should ideally never be given to a patient without cross matching 2. If Blood is required in an Emergency and it is necessary to issue uncross-matched blood , the requesting clinician then assumes responsibility. 3. An emergency Cross match can be performed using reduced incubation time , the majority of Clinically significant antibodies will be detected by thes methods. 4. Group O Rhesus negative blood should be given in cases of emergency. This flying squad blood can be issued in cases where there is insufficient time to perform a rapid group on the patient. 31 ASSESSMENT 1: ON BLOOD BORNE PATHOGENS All Questions are to be answered: Answer All Questions Placing an “X” against the correct answers. Q.1 Bloodborne Pathogens organisms carried in what way? Q.2 a) By mosquitoes b) By contaminated water c) By blood to blood contact d) All of the above Which of the following are not types of bloodborne pathogens? Q.3 a) Malaria b) Hepatitus B c) HIV Is the following statement true or false? “Hepatitus B is transmitted primarily through blood to blood contact” True ………….. False …………….. Q.4 Is there a cure for Hepatitus B? Yes ………… No ………………… Q.5 Personnel at risk of exposure can be vaccinated against this disease. True or False? True ………….. False …………….. Q.6 Q.7 Is it true that HIV may take several years to develop into AIDS? Yes ………… No …………. AIDS is a fatal disease but it also weakens the body such that it cannot resist other dangerous diseases. Is this statement true or false? True ……….. False ………….. Q.8 Is there a cure for HIV / AIDS? Q.9 Yes …………. No …………… Can the HIV virus last for long outside the body? 32 Yes ……….. No …………. Q.10 “The HIV virus can easily be transmitted through sexual contact”. True or false? True …………. False …………. Q.11 HIV infected blood can enter the body through which route? a) b) c) d) Cuts Abrasions Open sores All of the above Q.12 Bloodborne pathogens may be transmitted through the mucous membranes of : a) b) c) d) Mouth Nose Eyes All of the above Q.13 Which of the signs shown below must be displayed on containers of blood contaminated waste (regulated waste)? Please put an X against the correct picture. Danger Hazardou s Substanc e DANGER BLOOD Q.14 Is it true or false that when dealing with a casualty who is bleeding you should wear PPE to minimize exposure to bloodborne pathogens? Yes ………….. No ……………. Q.15 If your skin is exposed to another persons blood you should wash your hands with soap and water? Yes …………. No …………. 33 Q.16 If your eyes should get splashed with another persons blood you should: a) b) c) See a doctor as soon as it is convenient. Do nothing Flush your eyes out with water for fifteen minutes. Q.17 If blood is present following an accident you should: a) b) Treat the blood as potentially infectious. Assume that there is no risk if you know the injured person. Q.18 If decontaminating equipment contaminated with blood, approximately how much household bleach should you mix with a gallon of water? a) b) c) One cupful Four cupful One quarter cupful Q.19 Is it ok to pick up blood contaminated glass with your hands if you are careful? Yes ……….. No ………. Q.20 Is it true that needles and broken glass which may be contaminated with blood must be disposed of in a specially marked “sharps” container? Yes ……….. No ………... 34