Made easy for sTudenTs of allied Medical sciences and Medicine

Authored by osaro erhabor and Teddy charles adias Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine Authored by: Dr Osaro Erhabor (Ph.D, CSci, FIBMS) and Dr Teddy Charles Adias (Ph.D, FIBMS)

Edition 2014

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Meet the authors

Dr Osaro Erhabor is an Immuno-Haematologist, a Chartered Scientist, Registration Portfolio Verifier and a Fellow of the Institute of Biomedical Medical Science, London. He is a seasoned Biomedical Scientist and Lec- turer. He holds a Ph.D in Immuno-Haematology. He has taught best practices in Transfusion Medicine for several years to students in both the United Kingdom and Nige- ria. A recipient of several scientific awards, member of the editorial boards, an article reviewer to several scientific journals, a well published author and speaker in several international scientific conferences. His current research interest includes transfusion safety and alternatives and haema- tology of infectious diseases.

Dr Teddy Charles Adias is currently the Provost of the Bayelsa State College of Health Technology, Og- bia-Town, Nigeria. He holds a Ph.D in Immuno-Haema- tology, as well as a Fellow of the Institute of Biomedical Science (FIBMS), London. He had held several Adjunct academic appointments with various Nigerian univer- sities and had taught at both post graduate and under- graduate levels for over nine years. His current research interest is focus on Transfusion immunology, safety and alternatives, and haematology of infectious diseases. Recent publications have included articles in Journals such as the Journal of Medicine; Transfusion Clinque et Biologique, Pathology and Laboratory Medicine International amongst others.

Contents

Acknowledgements 1

1. History of 2

2. and 4

3. Blood Group Systems and ABO groups 20

4. Anticoagulation and Preservation in Transfusion 49

5. Testing 54

6. Principle and Practice 59

7. Blood Component Preparation 60

8. Challenges of Blood Transfusion in Africa 66

9. Blood Donation and Donor Types 68

10. Advantages of Autologous Blood over Allogeneic Blood 72

11. Transfusion Transmissible Infectious Diseases 79

12. Complications of Blood Transfusion 83

13. Investigation of Blood Transfusion Reactions 90

14. Compatibility Testing 92

15. Red Blood Cells Alloimmunisation 100

16. HDFN and Management of Rh Negative Pregnancies 115

17. Transfusion Alternatives and Exemplary Stewardship in the Management of Blood and 128

18. Blood Components Therapy 133

19. Management of Major Haemorrhage 143

20. Storage Conditions, Shelf Life Indication and Mode of Transfusion 147

22. Fractionated Plasma Products 158 23. Rhesus Blood Group System 162

24. Lewis Blood Group System 177

25. MNS Blood Group System 181

26. Kell Blood Group System 184

27. Duffy Blood Group System 186

28. Kidd Blood Group System 189

29. Bg 190

32. Lutheran Blood Group System 194

33. Minor Blood Group Systems 194

34. Complement 196

35. The Antiglobulin Test 203 Blood transfusion is a field where there has been, and continue to be, significant advances 36. Good Manufacturing Practice (GMP) 217 in science, technology and most particularly governance. The aim of this book is to provide 37. Principle of Good Laboratory Practice (GLP) and Its Application in students of allied medical sciences, medicine and transfusion practitioners with a compre Transfusion 223 hensive overview of both the scientific and managerial aspects of blood transfusion. The book is intended to equip biomedical, clinical and allied medical professionals with practical tools 38. Quality Issues in Transfusion Medicine 230 to allow for an informed practice in the field of blood transfusion management. 247 39. Management Review Meetings in the Transfusion Laboratory Dr Erhabor Osaro 40. Standard Operating Procedure 249

41. Incident Reporting Procedure in Transfusion 255

42. Laboratory Techniques and Transfusion Sample Requirements 260

275 43. Principle of Informed Consent in Transfusion Medicine The authors are indebted to Prof E.K Uko and Prof E.A Usanga both of the Haematology and 44. Stem Cell Transplantation 279 blood transfusion Department of the University of Calabar in Nigeria for taking time out to review this book. We are also grateful to the publishers InTech. Our sincere thanks goes to 45. Alkaline Denaturation Test 289 members of our families and friend for the encouragement while we put this material that will improve the quality of transfusion medicine training and by extension transfusion serv 291 About the authors ice delivery particularly in Africa. We are eternally grateful to God for this opportunity to in our own little way improve the quality of transfusion medicine training offered to students of biomedical, medical and allied medical sciences. To God alone be all the glory.

© 2012 Stopforth et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine

Dr Erhabor Osaro (Ph.D, CSci, FIBMS) Dr Adias Teddy Charles (Ph.D, FIBMS)

Blood Sciences Department Royal Bolton Hospital UK

Preface

Blood transfusion is a field where there has been, and continue to be, significant advances in science, technology and most particularly governance. The aim of this book is to provide students of allied medical sciences, medicine and transfusion practitioners with a compre- hensive overview of both the scientific and managerial aspects of blood transfusion. The book is intended to equip biomedical, clinical and allied medical professionals with practical tools to allow for an informed practice in the field of blood transfusion management.

Dr Erhabor Osaro

Acknowledgements

The authors are indebted to Prof E.K Uko and Prof E.A Usanga both of the Haematology and blood transfusion Department of the University of Calabar in Nigeria for taking time out to review this book. We are also grateful to the publishers InTech. Our sincere thanks goes to members of our families and friend for the encouragement while we put this material that will improve the quality of transfusion medicine training and by extension transfusion serv- ice delivery particularly in Africa. We are eternally grateful to God for this opportunity to in our own little way improve the quality of transfusion medicine training offered to students of biomedical, medical and allied medical sciences. To God alone be all the glory.

© 2012© 2012 Erhabor Stopforth and etAdias, al.; licensee licensee InTech. InTech. This This isis ana paper open distributedaccess chapter under distributed the terms under of the the Creative terms Commons of the AttributionCreative LicenseCommons (http://creativecommons.org/licenses/by/3.0), Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted which use, permits distribution,unrestricted and use, reproduction distribution, in anyand medium,reproduction provided in any the medium, original provided work is properly the original cited. work is properly cited. 2 Dr Osaro Erhabor (Ph.D, CSci, FIBMS) and Dr Teddy Charles Adias (Ph.D, FIBMS) Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine

1. History of blood transfusion at a meeting of the Royal Society, and without any inconvenience to him.” The recipient was Arthur Coga, “the subject of a harmless form of insanity.” Sheep’s blood was used because of spec The first historical attempt at blood transfusion was described by the 17th century chronicler ulation about the value of blood exchange between species; it had been suggested that blood from Stefano Infessura. Infessura relates that, in 1492, as Pope Innocent VIII sank into a coma, the a gentle lamb might quiet the tempestuous spirit of an agitated person and that the shy might be blood of three boys was infused into the dying pontiff (through the mouth, as the concept of made outgoing by blood from more sociable creatures. Lower wanted to treat Coga several times, circulation and methods for intravenous access did not exist at that time) at the suggestion of but his patient refused. No more transfusions were performed. Shortly before, Lower had moved a physician. The boys were ten years old, and had been promised a ducat each. However, not to London, where his growing practice soon led him to abandon research. only did the pope die, but so did the three children. Some authors have discredited Infessura’s account, accusing him of anti-papalism. In 1667 - Jean-Baptiste Denis in France reported successful transfusions from sheep to humans. In 1678 transfusion from animals to humans, having been tried in many different ways, was confirmed Beginning with Harvey’s experiments with circulation of the blood, more sophisticated re- to be unsuccessful, and was subsequently outlawed by the Paris Society of Physicians because of search into blood transfusion began in the 17th century, with successful experiments in transfu- reactions and associated mortality. In 1795 in Philadelphia USA, an American physician Philip Syng sion between animals. However, successive attempts on humans continued to have fatal results. Physick, performed the first known human Blood transfusion, although he did not publish the de The first fully documented human blood transfusion was administered by Dr. Jean-Baptiste De- tails of his findings. In 1818 James Blundell, a British obstetrician, performed the first successful nys, eminent physician to King Louis XIV of France, on June 15, 1667. He transfused the blood of transfusion of human blood to a patient for the treatment of post partum haemorrhage. Using the a sheep into a 15-year-old boy, who survived the transfusion. Denys performed another transfu- patient’s husband as a donor, he extracted a small amount of Blood from the husband’s arm and sion into a labourer, who also survived. Both instances were likely due to the small amount of using a syringe, he successfully transfused the wife. Between 1825 and 1830 he performed ten docu blood that was actually transfused into these people. This allowed them to withstand the allergic mented transfusions, five of which proved beneficial to his patients, and published these results. reaction. Denys’ third patient to undergo a blood transfusion was Swedish Baron Bonde. He re- He also devised various instruments for performing Blood transfusions. 1840 in London England, ceived two transfusions. After the second transfusion Bonde died. In the winter of 1667, Denys Samuel Armstrong Lane, aided by consultant Dr. Blundell, performed the first successful whole performed several transfusions on Antoine Mauroy with calf’s blood, who on the third account Blood transfusion to treat haemophilia. In 1867 English surgeon Joseph Lister utilized antiseptics to died. Much controversy surrounded his death. Mauroy’s wife asserted Denys was responsible control infection during Blood transfusions. In 1901 - Karl Landsteiner, an Austrian physician, and for her husband’s death; she was accused as well. Though it was later determined that Mauroy the most important individual in the field of Blood transfusion, documented the first three human actually died from arsenic poisoning, Denys’ experiments with animal blood provoked a heated Blood groups (A, B and O). A year later in 1902 a fourth main , AB was found by A. De controversy in France. Finally, in 1670 the procedure was banned. In time, the British Parliament castrello and A. Sturli. In 1907 Hektoen suggested that the safety of transfusion might be improved and even the pope followed suit. Blood transfusions fell into obscurity for the next 150 years. by cross-matching blood between donors and patients to exclude incompatible mixtures. Reuben Ottenberg performed the first blood transfusion using blood typing and cross-matching. Ottenberg Richard Lower examined the effects of changes in blood volume on circulatory function and also observed the ‘Mendelian inheritance’ of blood groups and recognized the “universal” utility developed methods for cross-circulatory study in animals, obviating clotting by closed arteriov- of group O donors. In 1908 - French surgeon devised a way to prevent blood from enous connections. His newly devised instruments eventually led to actual transfusion of blood. clotting. His method involved joining an artery in the donor, directly to a vein in the recipient with Towards the end of February 1665 he selected one dog of medium size, opened its jugular surgical sutures. He first used this technique to save the life of the son of a friend, using the father as vein, and drew off blood, until its strength was nearly gone. Then, to make up for the great donor. This procedure, not feasible for Blood transfusion, paved the way for successful organ trans loss of this dog by the blood of a second, I introduced blood from the cervical artery of a fairly plantation, for which Carrel received the Nobel Prize in 1912. In 1908 - Carlo Moreschi documented large mastiff, which had been fastened alongside the first, until this latter animal showed it the antiglobulin reaction. In 1914 long-term anticoagulants, among them sodium citrate, were devel was overfilled by the inflowing blood.” After he “sewed up the jugular veins,” the animal oped, allowing longer preservation of Blood. In 1915 at Mt. Sinai Hospital in New York City, Richard recovered “with no sign of discomfort or of displeasure.” Lewisohn was documented to have used sodium citrate as an anticoagulant which in the future transformed transfusion procedure from one that had to be performed with both the donor and the Lower had performed the first blood transfusion between animals. He was then requested by receiver of the transfusion in the same place at the same time, to basically the Blood banking system the Honorable Robert Boyle to acquaint the Royal Society with the procedure for the whole in use today. Further, in the same time period, R. Weil demonstrated the feasibility of refrigerated experiment,” which he did in December of 1665 in the Society’s Philosophical Transactions. storage of such anticoagulated Blood. In 1916 Francis Rous and J. R. Turner introduced a citrate- On 15 June 1667 Denys, then a professor in Paris carried out the first transfusion between hu- glucose solution that permitted storage of Blood for several days after collection. Also, as in the 1915 mans and claimed credit for the technique, but Lower’s priority cannot be challenged. Lewisohn discovery allowed for Blood to be stored in containers for later transfusion, and aided in Six months later in London, Lower performed the first human transfusion in Britain, where he the transition from the vein-to-vein method to direct transfusion. This discovery also directly led to “superintended the introduction in a patient’s arm at various times of some ounces of sheep’s blood the establishment of the first Blood depot by the British during World War I. Oswald Robertsonwas Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine 3

at a meeting of the Royal Society, and without any inconvenience to him.” The recipient was Arthur Coga, “the subject of a harmless form of insanity.” Sheep’s blood was used because of spec- The first historical attempt at blood transfusion was described by the 17th century chronicler ulation about the value of blood exchange between species; it had been suggested that blood from Stefano Infessura. Infessura relates that, in 1492, as Pope Innocent VIII sank into a coma, the a gentle lamb might quiet the tempestuous spirit of an agitated person and that the shy might be blood of three boys was infused into the dying pontiff (through the mouth, as the concept of made outgoing by blood from more sociable creatures. Lower wanted to treat Coga several times, circulation and methods for intravenous access did not exist at that time) at the suggestion of but his patient refused. No more transfusions were performed. Shortly before, Lower had moved a physician. The boys were ten years old, and had been promised a ducat each. However, not to London, where his growing practice soon led him to abandon research. only did the pope die, but so did the three children. Some authors have discredited Infessura’s account, accusing him of anti-papalism. In 1667 - Jean-Baptiste Denis in France reported successful transfusions from sheep to humans. In 1678 transfusion from animals to humans, having been tried in many different ways, was confirmed Beginning with Harvey’s experiments with circulation of the blood, more sophisticated re to be unsuccessful, and was subsequently outlawed by the Paris Society of Physicians because of search into blood transfusion began in the 17th century, with successful experiments in transfu reactions and associated mortality. In 1795 in Philadelphia USA, an American physician Philip Syng sion between animals. However, successive attempts on humans continued to have fatal results. Physick, performed the first known human Blood transfusion, although he did not publish the de- The first fully documented human blood transfusion was administered by Dr. Jean-Baptiste De tails of his findings. In 1818 James Blundell, a British obstetrician, performed the first successful nys, eminent physician to King Louis XIV of France, on June 15, 1667. He transfused the blood of transfusion of human blood to a patient for the treatment of post partum haemorrhage. Using the a sheep into a 15-year-old boy, who survived the transfusion. Denys performed another transfu patient’s husband as a donor, he extracted a small amount of Blood from the husband’s arm and - sion into a labourer, who also survived. Both instances were likely due to the small amount of using a syringe, he successfully transfused the wife. Between 1825 and 1830 he performed ten docu blood that was actually transfused into these people. This allowed them to withstand the allergic mented transfusions, five of which proved beneficial to his patients, and published these results. reaction. Denys’ third patient to undergo a blood transfusion was Swedish Baron Bonde. He re He also devised various instruments for performing Blood transfusions. 1840 in London England, ceived two transfusions. After the second transfusion Bonde died. In the winter of 1667, Denys Samuel Armstrong Lane, aided by consultant Dr. Blundell, performed the first successful whole performed several transfusions on Antoine Mauroy with calf’s blood, who on the third account Blood transfusion to treat haemophilia. In 1867 English surgeon Joseph Lister utilized antiseptics to died. Much controversy surrounded his death. Mauroy’s wife asserted Denys was responsible control infection during Blood transfusions. In 1901 - Karl Landsteiner, an Austrian physician, and for her husband’s death; she was accused as well. Though it was later determined that Mauroy the most important individual in the field of Blood transfusion, documented the first three human - actually died from arsenic poisoning, Denys’ experiments with animal blood provoked a heated Blood groups (A, B and O). A year later in 1902 a fourth main blood type, AB was found by A. De controversy in France. Finally, in 1670 the procedure was banned. In time, the British Parliament castrello and A. Sturli. In 1907 Hektoen suggested that the safety of transfusion might be improved and even the pope followed suit. Blood transfusions fell into obscurity for the next 150 years. by cross-matching blood between donors and patients to exclude incompatible mixtures. Reuben Ottenberg performed the first blood transfusion using blood typing and cross-matching. Ottenberg Richard Lower examined the effects of changes in blood volume on circulatory function and also observed the ‘Mendelian inheritance’ of blood groups and recognized the “universal” utility developed methods for cross-circulatory study in animals, obviating clotting by closed arteriov of group O donors. In 1908 - French surgeon Alexis Carrel devised a way to prevent blood from enous connections. His newly devised instruments eventually led to actual transfusion of blood. clotting. His method involved joining an artery in the donor, directly to a vein in the recipient with Towards the end of February 1665 he selected one dog of medium size, opened its jugular surgical sutures. He first used this technique to save the life of the son of a friend, using the father as - vein, and drew off blood, until its strength was nearly gone. Then, to make up for the great donor. This procedure, not feasible for Blood transfusion, paved the way for successful organ trans loss of this dog by the blood of a second, I introduced blood from the cervical artery of a fairly plantation, for which Carrel received the Nobel Prize in 1912. In 1908 - Carlo Moreschi documented - large mastiff, which had been fastened alongside the first, until this latter animal showed it the antiglobulin reaction. In 1914 long-term anticoagulants, among them sodium citrate, were devel was overfilled by the inflowing blood.” After he “sewed up the jugular veins,” the animal oped, allowing longer preservation of Blood. In 1915 at Mt. Sinai Hospital in New York City, Richard recovered “with no sign of discomfort or of displeasure.” Lewisohn was documented to have used sodium citrate as an anticoagulant which in the future transformed transfusion procedure from one that had to be performed with both the donor and the Lower had performed the first blood transfusion between animals. He was then requested by receiver of the transfusion in the same place at the same time, to basically the Blood banking system the Honorable Robert Boyle to acquaint the Royal Society with the procedure for the whole in use today. Further, in the same time period, R. Weil demonstrated the feasibility of refrigerated experiment,” which he did in December of 1665 in the Society’s Philosophical Transactions. storage of such anticoagulated Blood. In 1916 Francis Rous and J. R. Turner introduced a citrate- On 15 June 1667 Denys, then a professor in Paris carried out the first transfusion between hu glucose solution that permitted storage of Blood for several days after collection. Also, as in the 1915 mans and claimed credit for the technique, but Lower’s priority cannot be challenged. Lewisohn discovery allowed for Blood to be stored in containers for later transfusion, and aided in Six months later in London, Lower performed the first human transfusion in Britain, where he the transition from the vein-to-vein method to direct transfusion. This discovery also directly led to “superintended the introduction in a patient’s arm at various times of some ounces of sheep’s blood the establishment of the first Blood depot by the British during World War I. Oswald Robertsonwas 4 Dr Osaro Erhabor (Ph.D, CSci, FIBMS) and Dr Teddy Charles Adias (Ph.D, FIBMS) Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine

credited as the creator of the Blood depots. In 1925 - Karl Landsteiner, then working in New Antigen-antibody reaction occurs in 2 stages; sensitization and agglutination. The characteristics of York City, in collaboration with Phillip Levine, discovered three more Blood groups: M, N an antigen and antibody reaction include; the antigen reacts with thegroup specific antibody and and P. View Nobel Biography. In 1926 the British Red Cross instituted the first human Blood the reaction occurs in optimum proportion. Factors affecting antigen –antibody reaction includes: transfusion service in the world. In 1932, the first facility functioning as a was es- tablished in a Leningrad Russia hospital. 1937, Bernard Fantus, director of therapeutics at the Factors affecting antigen-antibody reaction Cook County Hospital in Chicago, Illinois (U. S.), established the first hospital Blood bank in the United States. In creating a hospital laboratory that could preserve and store donor Blood, Specificity (good fit between antigen and antibody) Fantus originated the term ‘Blood bank. In 1939 and 1940 - The was discovered by Karl Landsteiner, Alex Wiener, Philip Levine and R. E. Stetson and was soon recognized as the cause of the then majority of transfusion reactions. Known as the Rhesus Optimum pH of the medium (Rh) system, once this reliable test for this grouping had been established, transfusion reac- tions became rare. Identification of the Rh factor has stood next to ABO as another important Optimum temperatures (IgG = 37˚C, IgM = 4˚C). breakthrough in Blood banking. Number of antigenic determinants (binding sites) 2. Antigen Resolution of discrepancy in ABO An antigen is a substance which in an appropriate biological circumstance can stimulate the production of an antibody. Such substances will react specifically with the antibody in an Factors that play a role in antigen antibody reactions observable manner. Such observable ways includes;agglutination(the clumping of red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles Techniques used in identification. ABO blood group antibodies bind red cells (containing and joins them, creating a large complex) and precipitation (the coalescing of small particles the group specific antigen) suspended in saline. ABO blood group antibodies are IgM anti that are suspended in a solution; these larger masses are then (usually) precipitated. Blood bodies. They are high molecular weight antibodies that can span the distance that red cells group are located within the red cell membrane. Antigens are made up of antigenic keep apart (zeta potential) when suspended in saline whereas Rh antibodies are IgG antibod determinants (antigen binding sites). There are more antigenic determinants on a red cell of ies and will require antihuman globulin (AHG) and or enzyme techniques for its detection. an individual who is homozygote for a particular antigen compared to a heterozygote. For Effect of enzymes. Enzymes like papain (from paw paw) and ficin (from figs) and bromelin example a homozygote (DD) individual has about 25-37,000 Rh (DD) antigenic determinants (pineapple) can either enhance the reactivity of antigen-antibody reaction (Rhesus) or destroy compared to 10,000-15,000 for a heterozygote (Dd). Similarly a homozygote show a stronger (remove) antigen structures of some antigens (Duffy). Characteristics of an antigen includes; reaction with the corresponding group specific antibody compared to a heterozygote. This is foreign (not found in the host) and react specifically with corresponding antibody. the reason why red cells with homozygous antigen expression is preferred as a red cell rea- gent used for antibody detection and identification. Factors determining the effectiveness of an antigen Characteristics of antigens. In order for a substance to be an antigen to you it must be foreign (not found in the host). The more foreign a substance the better it is an antigen. Antigens can either be autologous or homologous. Autologous antigens are your own antigens (not foreign to you). Homologous, or allogeneic, antigens are antigens from someone else (within the same species) that may be foreign to you.

Antigens must be chemically complex. and polysaccharides are antigenic due to their complexity. On the other hand, lipids are antigenic only if coupled to or sugar. Be - sides being chemically complex, antigens must also be large enough to stimulate antibody production. Their molecular weight needs to be at least 10,000. Due to the complexity of these molecules there are specific antigenic determinants (antigen sites) which are those portions of the antigen that reacts specifically with the antibody. Factors determining the effectiveness or whether an antigen will stimulate an antibody response: Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine 5 credited as the creator of the Blood depots. In 1925 - Karl Landsteiner, then working in New Antigen-antibody reaction occurs in 2 stages; sensitization and agglutination. The characteristics of York City, in collaboration with Phillip Levine, discovered three more Blood groups: M, N an antigen and antibody reaction include; the antigen reacts with thegroup specific antibody and and P. View Nobel Biography. In 1926 the British Red Cross instituted the first human Blood the reaction occurs in optimum proportion. Factors affecting antigen –antibody reaction includes: transfusion service in the world. In 1932, the first facility functioning as a Blood bank was es tablished in a Leningrad Russia hospital. 1937, Bernard Fantus, director of therapeutics at the Factors affecting antigen-antibody reaction Cook County Hospital in Chicago, Illinois (U. S.), established the first hospital Blood bank in the United States. In creating a hospital laboratory that could preserve and store donor Blood, Specificity (good fit between antigen and antibody) Fantus originated the term ‘Blood bank. In 1939 and 1940 - The Rh Blood group system was discovered by Karl Landsteiner, Alex Wiener, Philip Levine and R. E. Stetson and was soon recognized as the cause of the then majority of transfusion reactions. Known as the Rhesus Optimum pH of the medium (Rh) system, once this reliable test for this grouping had been established, transfusion reac tions became rare. Identification of the Rh factor has stood next to ABO as another important Optimum temperatures (IgG = 37˚C, IgM = 4˚C). breakthrough in Blood banking. Number of antigenic determinants (binding sites)

Resolution of discrepancy in ABO An antigen is a substance which in an appropriate biological circumstance can stimulate the production of an antibody. Such substances will react specifically with the antibody in an Factors that play a role in antigen antibody reactions observable manner. Such observable ways includes;agglutination(the clumping of red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles Techniques used in identification. ABO blood group antibodies bind red cells (containing and joins them, creating a large complex) and precipitation (the coalescing of small particles the group specific antigen) suspended in saline. ABO blood group antibodies are IgM anti- that are suspended in a solution; these larger masses are then (usually) precipitated. Blood bodies. They are high molecular weight antibodies that can span the distance that red cells group antigens are located within the red cell membrane. Antigens are made up of antigenic keep apart (zeta potential) when suspended in saline whereas Rh antibodies are IgG antibod- determinants (antigen binding sites). There are more antigenic determinants on a red cell of ies and will require antihuman globulin (AHG) and or enzyme techniques for its detection. an individual who is homozygote for a particular antigen compared to a heterozygote. For Effect of enzymes. Enzymes like papain (from paw paw) and ficin (from figs) and bromelin example a homozygote (DD) individual has about 25-37,000 Rh (DD) antigenic determinants (pineapple) can either enhance the reactivity of antigen-antibody reaction (Rhesus) or destroy compared to 10,000-15,000 for a heterozygote (Dd). Similarly a homozygote show a stronger (remove) antigen structures of some antigens (Duffy). Characteristics of an antigen includes; reaction with the corresponding group specific antibody compared to a heterozygote. This is foreign (not found in the host) and react specifically with corresponding antibody. the reason why red cells with homozygous antigen expression is preferred as a red cell rea gent used for antibody detection and identification. Factors determining the effectiveness of an antigen In order for a substance to be an antigen to you it must be foreign (not found in the host). The more foreign a substance the better it is an antigen. Antigens can either be autologous or homologous. Autologous antigens are your own antigens (not foreign Degree of foreignness to you). Homologous, or allogeneic, antigens are antigens from someone else (within the same species) that may be foreign to you. Size and complexity

Antigens must be chemically complex. Proteins and polysaccharides are antigenic due to their complexity. On the other hand, lipids are antigenic only if coupled to protein or sugar. Be Dose and frequency of exposure sides being chemically complex, antigens must also be large enough to stimulate antibody production. Their molecular weight needs to be at least 10,000. Due to the complexity of these Genetic makeup of host molecules there are specific antigenic determinants (antigen sites) which are those portions of the antigen that reacts specifically with the antibody. Factors determining the effectiveness or whether an antigen will stimulate an antibody response: 6 Dr Osaro Erhabor (Ph.D, CSci, FIBMS) and Dr Teddy Charles Adias (Ph.D, FIBMS) Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine

Red cell agglutination. Agglutination is the clumping of particles. The word agglutination tion of the ionic strength reduces the interfering effect of the electrostatic comes from the Latin word agglutinare, meaning to glue. Red cell agglutination occurs when barrier and facilitates better attraction between the antigen and antibody. antigens on the red cell membrane of the red cells are cross-linked with their group spe- Lower ionic strength saline (LISS) (0.003M saline plus glycine) produces an cific antibody to form a three–dimensional lattice structure (clumps). Agglutination occur in isotonic environment due to the reduced Na+ and Cl- ions concentration. 2 phases; primary (antibody sensitization) and the secondary phase (agglutination). Each of LISS facilitate better agglutination and thus shorter incubation times com these phases are affected by certain factors. pared to normal saline. LISS is not a potentiating medium (does not reduce the ionic cloud that exist between red cells suspended in saline and thus Primary phase (Sensitization). Sensitization is a chemical reaction (interaction) between an does not reduce the distance between red cells like Bovine Serum Albu antigen and the group specific antibody. It is the coating of the antigen by the group specific min. It merely facilitates the non-specific interaction between red cells and antibody. It is a reaction in which antigen and antibody associate and dissociate until equilib- antibody. This is why the the ionic strength and the optimum antigen and rium is reached. Sensitization is governed by the law of mass action and it is concentration antibody ratio are most important factors in agglutination reaction. dependent. The higher the concentration of the antigen and antibody the more the AG-AB complexes formed and the stronger the agglutination. These complexes are held together by ionic, hydrogen, hydrophobic bonds as well as covalent van der Waal’s forces. Sensitization is affected by factors such as;

1. Temperature. The type of antigen-antibody bonding determines the opti- mum reactive temperation. Some antigens particularly carbonhydrate an- tigens (A, B, P1 H, Lea, Leb and I) form hydrogen bonds which dissipitate the heat generated during Ag-Ab reaction. These antigens reacts optimally at a cold (exothermic) temperation of 4-20°C. Non exothermic protein anti- gens (Rh, Duffy, Kell, Kidd and Lutheran) non-hydrogen bonding antigens react optimally at a warmer temperature of 37°C. Most IgM antibodies Figure 1: Demonstration of the effect of zeta potential on agglutination reaction (ABO) reacts optimally at cold temperature while IgG antibody (Rh) react optimally at 37°C. 3. . Since the immu noglobulins and the red cell membranes both have an electrical charge, there 2. Ionic strength of the medium. Red cells when suspended in saline becomes is an optimum pH. pH differences cause differences in chemical structures negatively charged and repel each other. Antigens and antibody molecules of antigens/antibodies, affecting the “fit”. are themselves charged molecules. Reduction of the charge (reduced Na+ and Cl - ions per unit volume) of the medium in which the red cells are 4. Shape and structure of antigen and antibody (fit). Specificity between an suspended reduces the electrostatic barrier that exist between red cells sus - tigens and antibodies depends on the spatial and chemical “fit” between pended in saline (Zeta potential) facilitates faster antigen-antibody reac - antigen and antibody. The better the fit between the antigenic determinants tion. The surface of red cells carry a negative charge due to the ionization (antigen site) and the antibody combining sites, the better the agglutination. of the carboxyl group of NeuNac (N-acetyl neuraminic acid), also called NANA or sialic acid. In saline, red cells will attract positively charged Na+, Effect of pH and an ionic cloud will form around each cell. Thus the cells will be re - pelled and stay a certain distance apart. Zeta potential is a measure of this repulsion and is measured in microvolts at the boundary of sheer or slip - ping plane. Zeta potential is measured at the “slipping plane” and results from the difference in electrostatic potential at the surface of the RBCS and the boundary of shear (slipping plane). When zeta potential decreases, the RBCS can come closer together, allowing them to be agglutinated by the small IgG molecule. For IgG molecules to span the distance between red cells in saline, the ZP must be reduced so the cells can come closer. Reduc - Demonstration of the effect of pH of medium of cell suspension on agglutination reaction Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine 7

Agglutination is the clumping of particles. The word agglutination tion of the ionic strength reduces the interfering effect of the electrostatic comes from the Latin word agglutinare, meaning to glue. Red cell agglutination occurs when barrier and facilitates better attraction between the antigen and antibody. antigens on the red cell membrane of the red cells are cross-linked with their group spe Lower ionic strength saline (LISS) (0.003M saline plus glycine) produces an cific antibody to form a three–dimensional lattice structure (clumps). Agglutination occur in isotonic environment due to the reduced Na+ and Cl- ions concentration. 2 phases; primary (antibody sensitization) and the secondary phase (agglutination). Each of LISS facilitate better agglutination and thus shorter incubation times com- these phases are affected by certain factors. pared to normal saline. LISS is not a potentiating medium (does not reduce the ionic cloud that exist between red cells suspended in saline and thus Primary phase (Sensitization). Sensitization is a chemical reaction (interaction) between an does not reduce the distance between red cells like Bovine Serum Albu- antigen and the group specific antibody. It is the coating of the antigen by the group specific min. It merely facilitates the non-specific interaction between red cells and antibody. It is a reaction in which antigen and antibody associate and dissociate until equilib antibody. This is why the the ionic strength and the optimum antigen and rium is reached. Sensitization is governed by the law of mass action and it is concentration antibody ratio are most important factors in agglutination reaction. dependent. The higher the concentration of the antigen and antibody the more the AG-AB complexes formed and the stronger the agglutination. These complexes are held together by + + slipping plane or - + boundary of sheer - + + ionic, hydrogen, hydrophobic bonds as well as covalent van der Waal’s forces. Sensitization - + + + + - + + + + - - + - - - - is affected by factors such as; - + + NeuNac* + - - + + - 1. . The type of antigen-antibody bonding determines the opti RBC - + + + - - + + + - RBC mum reactive temperation. Some antigens particularly carbonhydrate an + - + - - tigens (A, B, P1 H, Lea, Leb and I) form hydrogen bonds which dissipitate + repulsion - + + - -- + + + - the heat generated during Ag-Ab reaction. These antigens reacts optimally + - + + + - - at a cold (exothermic) temperation of 4-20°C. Non exothermic protein anti Na¯ CI¯ ionization of carboxyl + + groups of NeuNac gens (Rh, Duffy, Kell, Kidd and Lutheran) non-hydrogen bonding antigens [COO¯] react optimally at a warmer temperature of 37°C. Most IgM antibodies Figure 1: Demonstration of the effect of zeta potential on agglutination reaction (ABO) reacts optimally at cold temperature while IgG antibody (Rh) react optimally at 37°C. 3. pH of the medium in which the red cells are suspended. Since the immu- noglobulins and the red cell membranes both have an electrical charge, there 2. . Red cells when suspended in saline becomes is an optimum pH. pH differences cause differences in chemical structures negatively charged and repel each other. Antigens and antibody molecules of antigens/antibodies, affecting the “fit”. are themselves charged molecules. Reduction of the charge (reduced Na+ and Cl - ions per unit volume) of the medium in which the red cells are 4. Shape and structure of antigen and antibody (fit). Specificity between an- suspended reduces the electrostatic barrier that exist between red cells sus tigens and antibodies depends on the spatial and chemical “fit” between pended in saline (Zeta potential) facilitates faster antigen-antibody reac antigen and antibody. The better the fit between the antigenic determinants tion. The surface of red cells carry a negative charge due to the ionization (antigen site) and the antibody combining sites, the better the agglutination. of the carboxyl group of NeuNac (N-acetyl neuraminic acid), also called NANA or sialic acid. In saline, red cells will attract positively charged Na+, Effect of pH and an ionic cloud will form around each cell. Thus the cells will be re pelled and stay a certain distance apart. Zeta potential is a measure of this repulsion and is measured in microvolts at the boundary of sheer or slip ping plane. Zeta potential is measured at the “slipping plane” and results from the difference in electrostatic potential at the surface of the RBCS and the boundary of shear (slipping plane). When zeta potential decreases, the pH 7.5 pH 7.0 RBCS can come closer together, allowing them to be agglutinated by the small IgG molecule. For IgG molecules to span the distance between red cells in saline, the ZP must be reduced so the cells can come closer. Reduc Demonstration of the effect of pH of medium of cell suspension on agglutination reaction 8 Dr Osaro Erhabor (Ph.D, CSci, FIBMS) and Dr Teddy Charles Adias (Ph.D, FIBMS) Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine

Antibody combining site tion formed is often super-imposed by the large masses of unagglutinat ed antigens. This can cause a false negative reaction.

The second phase of the agglutination process involves the cell to cell cross linking by anti bodies. The level of agglutination observed is affected by the rate at which red cells sensitized Antigen determinant with antibody collide with each other. Red cell collision (attraction) is dependent on the fol

Good fit Poor fit lowing aggregating forces:

Demonstration of the effect of shape and structure on agglutination 1. . Red cells are attracted together by gravity. This attraction can be facilitated by centrifugation. Centrifugation of the cells attempts to bring 1. The antigen-antibody ratio. The greater the antibody amount for a given the red blood cells closer together, but even then the smaller IgG antibod antigen the more antibodies will be bound to the corresponding antigen ies usually can not reach between two cells. The larger antibodies, IgM, can and the greater the agglutination reaction. The more the antibody bound reach between cells that are further apart and cause agglutination. The to a red cell (sensitization) and more the agglutination. Antigen and an - second phase of agglutination involving an IgG antibody can only be en tibody reaction occur in optimum proportion. If the antibody concentra - hanced either by altering the suspending environment by using an aggre tion is high (excess) and the antigen concentration is low, the antigen sites gating or potentiating mwdium (20% BSA) or by altering the red cell mem (antigenic determinants) becomes saturated with more antibodies com - brane of the red cells using enzyme treatment (papain, ficin or bromelin) peting for the few antigen sites present resulting in few agglutination or by using an additional cross linking reagent (anti- human globulin) to (Prozone effect). The optimum ratio is 80 parts antibody to 1 part antigen. facilitate agglutination. There are specific terms for variations in this ratio. In order to get opti - mum antigen-antiboy concentration in Blood Banking we make washed 2. The concept Zeta potential is important to understand why 3% saline suspension of red cells to mix with our reagents. the cells will maintain a certain distance from each other. Zeta potential re fers to the repulsion between the red blood cells. It is due to an electric charge surrounding cells suspended in saline. It is caused by sialic acid groups on the membrane which gives the cells a negative charge. The positive ions in saline are attracted to the negatively charged red blood cells. The net positive charge surrounding the cells in saline keeps them far apart due to repulsion from electric charges. Smaller antibodies (IgG) cannot cause agglutination when zeta potential exists. To overcome the effect of the zeta potential, there is the need to neutralize these charges. One of the commonest technique is to add a potentiating medium (Bovine Serum Albumin 22%) to the mixture. The hydroxyl group (OH-) neutralizes the net postitive charge and and draw the red cells closer to each other reducing the gap between

Demonstration of the effect of antigen-antibody ratio on agglutination reaction the red cells. This facilitate the ability of low molecular weight IgG antibody to bridge the gap between red cells and cause agglutination. The effect of 2. Prozone effect. Excess antibodies saturates all the antigen sites leaving no these aggregating forces are ofter resisted by the zeta potential (occurs when room for the formation of cross-linkages between sensitized cells. Thus negatively charged red cells suspended in saline repel each other creating an even though there are antibodies in the plasma that are specific against ionic cloud between themselves). The minimum distance between red cells the corresponsing antigens on the red cells suspended in saline a false suspended in saline is > 14nm. Thus the closest the cells can approach each negative reaction with no agglutination observed may be evident. Zone other is the edge of their individual ionic clouds (slipping plane). IgG anti of equivalence: Antibodies and antigens present in optimum proportion bodies are low molecular weight antibodies (150,000) and thus are unable and significant agglutination is formed. Zone of antigen excess: Too many to span the slipping plane that exist beween cells suspended in saline. IgM antigens are present to bind with fewer antibodies. Thus the agglutina- antiboies on the other hand are a high molecular weight (900,000) molecule Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine 9

tion formed is often super-imposed by the large masses of unagglutinat- ed antigens. This can cause a false negative reaction.

Secondary stage of agglutination reaction

The second phase of the agglutination process involves the cell to cell cross linking by anti- bodies. The level of agglutination observed is affected by the rate at which red cells sensitized with antibody collide with each other. Red cell collision (attraction) is dependent on the fol-

Good fit Poor fit lowing aggregating forces:

Demonstration of the effect of shape and structure on agglutination 1. Gravity. Red cells are attracted together by gravity. This attraction can be facilitated by centrifugation. Centrifugation of the cells attempts to bring 1. The greater the antibody amount for a given the red blood cells closer together, but even then the smaller IgG antibod- antigen the more antibodies will be bound to the corresponding antigen ies usually can not reach between two cells. The larger antibodies, IgM, can and the greater the agglutination reaction. The more the antibody bound reach between cells that are further apart and cause agglutination. The to a red cell (sensitization) and more the agglutination. Antigen and an second phase of agglutination involving an IgG antibody can only be en- tibody reaction occur in optimum proportion. If the antibody concentra hanced either by altering the suspending environment by using an aggre- tion is high (excess) and the antigen concentration is low, the antigen sites gating or potentiating mwdium (20% BSA) or by altering the red cell mem- (antigenic determinants) becomes saturated with more antibodies com brane of the red cells using enzyme treatment (papain, ficin or bromelin) peting for the few antigen sites present resulting in few agglutination or by using an additional cross linking reagent (anti- human globulin) to (Prozone effect). The optimum ratio is 80 parts antibody to 1 part antigen. facilitate agglutination. There are specific terms for variations in this ratio. In order to get opti mum antigen-antiboy concentration in Blood Banking we make washed 2. Surface tension. The concept Zeta potential is important to understand why 3% saline suspension of red cells to mix with our reagents. the cells will maintain a certain distance from each other. Zeta potential re - fers to the repulsion between the red blood cells. It is due to an electric charge surrounding cells suspended in saline. It is caused by sialic acid groups on the red blood cell membrane which gives the cells a negative charge. The positive ions in saline are attracted to the negatively charged red blood cells. The net positive charge surrounding the cells in saline keeps them far apart due to repulsion from electric charges. Smaller antibodies (IgG) cannot cause agglutination when zeta potential exists. To overcome the effect of the zeta potential, there is the need to neutralize these charges. One of the commonest technique is to add a potentiating medium (Bovine Serum Albumin 22%) to the mixture. The hydroxyl group (OH-) neutralizes the net postitive charge and and draw the red cells closer to each other reducing the gap between

Demonstration of the effect of antigen-antibody ratio on agglutination reaction the red cells. This facilitate the ability of low molecular weight IgG antibody to bridge the gap between red cells and cause agglutination. The effect of 2. Prozone effect. Excess antibodies saturates all the antigen sites leaving no these aggregating forces are ofter resisted by the zeta potential (occurs when room for the formation of cross-linkages between sensitized cells. Thus negatively charged red cells suspended in saline repel each other creating an even though there are antibodies in the plasma that are specific against ionic cloud between themselves). The minimum distance between red cells the corresponsing antigens on the red cells suspended in saline a false suspended in saline is > 14nm. Thus the closest the cells can approach each negative reaction with no agglutination observed may be evident. Zone other is the edge of their individual ionic clouds (slipping plane). IgG anti - of equivalence: Antibodies and antigens present in optimum proportion bodies are low molecular weight antibodies (150,000) and thus are unable and significant agglutination is formed. Zone of antigen excess: Too many to span the slipping plane that exist beween cells suspended in saline. IgM antigens are present to bind with fewer antibodies. Thus the agglutina antiboies on the other hand are a high molecular weight (900,000) molecule 10 Dr Osaro Erhabor (Ph.D, CSci, FIBMS) and Dr Teddy Charles Adias (Ph.D, FIBMS) Transfusion Medicine Made Easy for Students of Allied Medical Sciences and Medicine 11

that is large enogh to bridge this slipping plane and cause agglutination. IgM portion of the anti-human globulin cross link with the Fc portion of the IgG can agglutinate cells suspended in saline while IgG antibodies cannot. IgG molecule and help overcome the challenge caused by the zeta potential al antibody will however require an alteration to the environment by a poten- lowing the reaction links between the antigens on the red cells and antibod tiating medium to be able to agglutinate cells containing the group specigen ies in the plasma to be visualized in the form of agglutination. Antiglobulin antigens suspended in saline. test is one of the most important serological tests done in a routine blood transfusion laboratory. It utilizes the anti-human globulin (AHG) reagent to 3. Antigen-antibody ratio: Antigen- antibody reaction occurs in optimum bring about agglutination of red cells coated with immunoglobulin or com - proportion. The optimum ratio is 80 parts of antibody to 1 part of an plement component, which do not show any agglutination in saline. R