A comparative study between antiglobulin crossmatch and type and screen procedures for compatibility testing

by

Vanessa Zammit

Supervisor: Dr. M. Caruana M.D.

A project submitted in partial fulfillment of requirements for the B.Sc. (Hons.) in Health Science (M.L.S.)

May 2004

Abstract

Background and purpose  Blood transfusion is the cornerstone of therapy for many serious and common diseases. Indeed, without blood products it would be impossible to implement many of the modern regimen used for the treatment of malignant diseases and to perform the complex surgery now regarded as routine. Every medical procedure bears potential benefits to the patient as well as potential risks. These must be evaluated whenever transfusion of blood or blood components is considered. The main objective of this dissertation is to demonstrate whether the type and screen procedure is a safe method of pretransfusion testing, when compared to the antiglobulin crossmatch currently in use.

Method  For this test analysis a batch of 511 randomly selected samples was considered. For each sample, a blood group determination, consisting of a forward and reverse grouping, including testing for the rhesus D antigen, was performed. An antibody screen was also performed on all samples. Necessary controls were included during the analysis. The actual crossmatch was carried out routinely and the screening was carried out blindly, without knowing the result of the compatibility test. The two were later compared.

Results  Taking the number of incompatible crossmatches as 100%, it is possible to calculate the percentage of the number of incompatible crossmatches in which an antibody was detected. This calculates the percentage safety of the type and screen as compared to AHG compatibility testing. In this study, one result in particular gave a negative screen with an incompatible crossmatch.

Conclusion  This study did not show the type and screen to reach the expected safety level of 99%, taking that of the full antiglobulin crossmatch to be 100%. A more extensive study should be considered.

Chapter 1

Introduction

1.1 Transfusion History

It was recorded in the Bible that the "life of the flesh was in the blood" (Greenwalt 1997). So from ancient time blood has been intimately associated with life. The most obvious proof of this to the ancients was acute blood loss due to injury or weapons.

The major ancient civilizations of Greece and Rome deduced from their many wars that acute blood loss was one of the most rapidly lethal consequences of any wound. As a consequence one of the main goals was to stop blood loss. The ancient Greeks were the first recorded to attempt this by the process of using a tourniquet. This involved placing a tourniquet, on a limb, above the wound. The aim was to allow the body to form a stable clot and thereby stop bleeding. The major problem was, that if left on too long, the tourniquet would deprive the tissue below it of oxygen and gangrene would set in.

It was left to the Romans to refine medical treatments of acute blood loss to include not only tourniquets but ligature (the tying off of a blood vessel). The Romans also invented the first surgical clamp that was able to tie off a severed artery while still allowing blood to flow to the limb thereby reducing the risk of fatal gangrene (Gabriel and Metz 1991).

It is interesting to note that after the fall of Rome these same skills were not rediscovered till the seventeenth century.

Ancients only devised ways of stopping acute blood loss. The first suggested case of transfusion was rumoured to have been given to Pope Innocent VIII in July 1492. It was widely believed in the middle age that the drinking of human blood was a method where a person's health could be restored (Gottlieb 1991).

This should be viewed in the context that medieval "medicine" also considered swallowing paper with the Lords prayer or ground bones of saints and bleeding various parts of the body according to the phases of the moon valid and beneficial therapies. In the case of Pope Innocent VIII a Jewish physician Abraham Myere of Balmes suggested sucking the blood of a youth for a restorative tonic for the Pope. As a result three

"volunteer" youths were said to have donated a restorative tonic for the Pope and were paid one ducat each. Whether the Pope drank it or it was transfused (unlikely as the circulatory system was not discovered until 1628 by William Harvey) is dubious, as it was not reported by any court ambassadors of the time. As for the three young donors, they died of blood loss but had the consolation of being considered martyrs (Gottlieb 1991).

The first truly verified accounts of transfusion occurred in the mid seventeenth century. The first transfusions were animal to animal. In 1665 John Wilkins was the first person, via a syringe, to intravenously transfuse two ounces of blood from one dog to another with no harmful effect. These experiments were extended by Richard Lower who in 1666 continued transfusion experimentation in dogs with vein to vein and artery to vein transfusions. He discovered that artery to vein transfusions worked due to the higher arterial blood pressure (Myhre 1990).

A Frenchman Jean Denys performed the first human involvement as a transfusion recipient on the 25 of June 1667. The "scientific" principle behind the use of animals blood was that animals possessed purer blood than humans as they were not prone to the vices of drinking and eating that humans were (Myhre 1990).

To this end a transfusion was used to treat Antoine Mauroy, a newly wed, who was prone to run away to Paris for extended bouts of debauchery and vice. To cure this Jean Denys selected calf’s blood due to the animal’s gentle nature. To consolidate the treatment a second transfusion was performed a week later. One small problem is the immune system is very efficient, and antibodies to the calf cells would have formed. This became immediately evident when Mauroy complained of kidney pain and a heavy sensation in the chest and passing of dark urine. This was undoubtedly the first recorded case of an acute haemolytic transfusion reaction. Later attempts to repeat the treatment were refused by the patient (Greenwalt 1997).

The practice of transfusion remained at this level until the appearance of James Blundell in the early 19th Century. James Blundell was motivated by the consequences of post partum haemorrhages who often went into shock and died from acute blood loss. Unlike earlier attempts he refused to use animal blood based on work by Dr John Leacock whose experiments showed the blood of one species may be harmful to another (Greenwalt 1997).

In 1818 Blundell transfused 12 - 14 ounces of blood via a brass syringe into a patient who temporarily improved but died of their initial disease. However the practice of transfusions from animals did not disappear with papers as late as 1874 Franz Gesellius and Oscar Hasse advocated the use of lamb’s blood. (Greenwalt 1997).

The major problem before the discovery of anticoagulants was that blood collected for transfusion clotted. To get around these two methods were devised. The first was by Jean Prevost and Jean Baptiste Dumas who found that stirring collected blood caused the creation of fibrin and preventing the remaining blood from clotting. They also found that defibrinated blood was just as good as untreated blood for resuscitation (Greenwalt 1997).

The second method invented by Gesellius involved capillary transfusions via a device, which simultaneously punctured the skin in many places. The blood was then sucked into a bowl and transfused. The end of animals being used as donors was accomplished in May 1874 by Ponfick and Landois who studied the death of a 34 year old woman transfused by a sheep and noticed lysed red cells in her serum and haemoglobinuria. This problem of red cell lysis was noted when blood was transfused between species (Greenwalt 1997).

Once the use of animals ceased there was the major problem of deaths resulting even when human blood was used. This was due to the major barrier posed by the ABO blood group system. By pure random chance based upon caucasian frequencies of the ABO blood groups the chance of a compatible transfusion is 64.4 % (Greenwalt 1997). The evolution of safer transfusions that we know today was heralded by the the discovery of ABO blood groups by Karl Landsteiner in 1901

1.1.1 Karl Landsteiner

Karl Landsteiner who was born in Vienna on the 14 / 06 / 1868 discovered the ABO blood group system. He studied medicine at the University of Vienna, graduating in 1891. In 1896 he became an assistant to Max von Gruber in the Hygiene Institute of Vienna and became interested in the mechanisms of immunity and the nature of antibodies. In 1898 he became an assistant in the university Department of Pathological Anatomy in Vienna (http://www.nobel.ki.se/laureates/medicine-1930-1-bio.html ).

Figure 1.1: Karl Landsteiner (http://www.nobel.se/medicine/laureates/1930/l andsteiner-bio.html)

It was during this time he started to investigate whether differences existed between different peoples red cells. This was inspired by work done by Landois and Ponfick who in 1874 discovered that transfused red cells from one species to another and some humans to other humans lysed in the circulation. Death was attributed to the organ damage and hyperkalemia from the rapid release of potassium from the lysed red cells (Greenwalt 1997).

To investigate for any potential differences between human red blood cells Landsteiner in 1901 chose a simple experiment. He mixed the serum and red cells from different people and observed the reaction. As a result of observing the agglutination patterns he described three distinct groups whose serum possessed naturally occurring antibodies, which could react with some other peoples red cells. As a result Landsteiner divided these individuals into three groups called groups A, group B and group C. The group C was later changed to group O (Greenwalt 1997; Uhlenbruck and Prokop 1969).

In 1902 Decastello and Sturli who identified people whose serum did not naturally produce antibodies that agglutinated other human red cells defined the fourth group of the ABO system. This group was called AB (Greenwalt 1997; Uhlenbruck and Prokop 1969).

In 1940, Landsteiner also discovered the rhesus factor in blood, labeling it rhesus positive if the antigen was present in the red blood cells and rhesus negative if not. Today, blood typing can also include testing for different types of enzymes and proteins that perform specific activities in the body, allowing one to differentiate between blood from separate individuals. This is mainly a forensic use (http://www.crimelibrary.com/criminal_mind/forensics/serology/3.html?sect=21).

Landsteiner made numerous contributions to both pathological anatomy, histology and immunology, all of which showed, not only his meticulous care in observation and description, but also his biological understanding. But his name will no doubt always be honoured for his discovery in 1901 of, and outstanding work on, the blood groups, for which he was given the Nobel Prize for Physiology or Medicine in 1930 (http://www.nobel.se/medicine/laureates/1930/landsteiner-bio.html).

Besides making blood transfusion a safe procedure, Landsteiner's work added an important chapter to the development of legal medicine, providing admissible evidence in paternity suits and murder trials. Proof that blood types are inherited through specific genes has provided an effective tool for the study of human genetics and anthropology.

To the end of his life, Landsteiner continued to investigate blood groups and the chemistry of antigens, antibodies and other immunological factors that occur in the blood. It was one of his great merits that he introduced chemistry into the service of serology. Rigorously exacting in the demands he made upon himself, Landsteiner possessed untiring energy. Throughout his life he was always making observations in many fields other than those in which his main work was done (he was, for instance, responsible for having introduced dark-field illumination in the study of spirochaetes). (http://www.nobel.se/medicine/laureates/1930/landsteiner-bio.html)

1.2 Chemistry of the ABO antigens

Karl Landsteiner of the Rockefeller Institute first described the ABO blood-group system in 1900, but it was not until 1953 that Walter Morgan and Winifred Watkins of the Lister Institute demonstrated that the specificity of the major blood types is determined by sugars. For example, the difference between the blood types A and B lies in a single sugar unit that sticks out from the end of a carbohydrate chain of a glycoprotein or glycolipid on the surface of the red blood cell. In blood type A the determinant is acetylgalactosamine, in blood type B it is galactose. The two monosaccharides differ by only a small group of atoms, but that little difference is sometimes a matter of life and death, since using the wrong type of blood in a transfusion can have fatal results. Some researchers as merely ‘icing on the cake’ of glycoprotein structures have dismissed the blood group antigens. The fact that there are no lethal mutations and individuals have been described lacking ABO, H and Lewis antigens seems to lend weight to the argument. Research suggests that these antigens do indeed have function and argues that blood group antigens play important roles in modulation of protein activity, infection and cancer (http://www.dadamo.com/lect9.htm).

Figure 1.2: Synthesis of A and B antigens from the H antigen (http://www.mds.qmw.ac.uk/biomed/kb/cardioresp/blood/blood8.htm)

1.3 Antigens and Antibodies

An antigen is a substance that can, in certain circumstances, excite the production of a corresponding antibody. An antibody is a substance capable of reacting specifically with particular antigens. Blood group antigens are carried on the surface of the red cells (Britannica Encyclopedia 1997).

Humans automatically make antibodies, even without ever having a transfusion, to the blood group antigens that they do not have on their own red blood cells. For instance, people who are group A make anti-B. People who are group O make anti-A and anti-B, since they do not have either antigen on their red cells. For transfusion, it is necessary to transfuse blood that will not interact with these antibodies. Otherwise, the antibody will attack the transfused red cells and cause a serious transfusion reaction. For example, a patient who is group A has anti-B, so transfused blood must not be group B or group AB since these red cells carry the B antigen.

1.3.1 Blood group antigens

An antigen (immunogen) is any substance that can induce a specific immunological response, i.e., cause the production of antibody. Blood group antigens are located in the red cell membrane. Some antigens (such as ABH) protrude from the membrane, and some (such as Rh) are embedded in the membrane. Each red cell has > 1 million antigens. Some antigens are evenly distributed over the red cell surface, and some are distributed in clusters (http://129.128.91.75/de/immunology/70imm-ag.html).

Figure 1.3: Red cell antigens in the red cell membrane (http://129.128.91.75/de/immunology/70imm-figure2-1.html)

In general, antigens are composed of carbohydrates, lipids, and proteins, usually a combination of any two. For example, ABH antigens are glycosphingolipids (sugars attached to a lipid backbone) and glycoproteins (sugars attached to a protein backbone). Rh antigens are lipoproteins (http://129.128.91.75/de/immunology/70imm-ag.html).

The chemistry of many antigens is unknown. The biological role of blood group antigens is unknown, but one is presumed to exist. In a general sense, antigens obviously provide a way to distinguish between self and non-self, and thus play a role in immunity. But this does not explain why so many blood group antigens exist. Some antigens are associated with susceptibility or protection against diseases, but these associations are statistical and the causes are not well understood. For example, group A people have a higher incidence of cancer of the stomach, and group O people have a higher incidence of gastric/duodenal ulcers. On a trivial level, studies have shown that mosquitoes prefer group O blood, and group O people have a higher IQ than group A people, at least in the north of England. One association of importance is that the Duffy phenotype Fy(a-b-), prevalent among West Africans and American blacks (68%), acts to protect against infection by certain malarial parasites (http://129.128.91.75/de/immunology/70imm-ag.html)

The antigens, which determine blood types, belong to glycoproteins and glycolipids. There are three types of blood-group antigens: O, A, and B. They differ only slightly in the composition of carbohydrates.

Figure1.4: Blood group antigens. Structure of terminal sugars, which constitute the distinguishing epitopes, in the A, B, and O blood antigens (http://ntri.tamuk.edu/immunolo gy/blood.html)

All humans contain enzymes, which catalyze the synthesis of the O antigen. Humans with A-type blood also contain an additional enzyme (called A-type enzyme here), which adds N-Acetylgalactosamine to the O antigen. Humans with B-type blood contain another enzyme (called B-type enzyme here), which adds Galactose to the O antigen. Humans with AB-type blood contain both A-type and B-type enzymes while humans with O-type blood lack both types of enzymes.

ABO Blood Antigen A Antigen B Antibody Antibody Type anti-A anti-B A Yes No No Yes B No Yes Yes No O No No Yes Yes AB Yes Yes No No Table 1.1: Possible permutations of antigens and antibodies with the corresponding ABO types (http://anthro.palomar.edu/blood/ABO_system.htm)

1.3.2 Blood group antibodies

The ABO antigens are developed well before birth and remain throughout life. ABO antibodies are acquired passively from the mother before birth, but by three months the infant is making his own--it is believed the stimulus for such antibody formation is from contact with ABO-like antigenic substances in nature (Britannica Encyclopedia 1997) Antibodies are proteins synthesized by the cells of the immune system in response to a foreign (non-self) antigen. A family of different antibody classes are produced by the immune system, depending upon the site of synthesis - IgG, IgM, IgA, IgD and IgE. The archetypal antibody class is the IgG molecule.

The common structure of all antibodies, or immunoglobulins, is a tetramer of two heavy chains (molecular weight 44,000 or more) and two light chains of molecular weight about 22,000. The molecule is symmetrical with one light chain and one heavy chain in each half. The four subunits are held together by noncovalent bonds and by cystine residues (disulfide bridges).

Figure 1.5: IgG Antibody

(http://www.mds.qmw.ac.uk/biomed/kb/cardioresp/blood/blood8.htm)

An immunoglobulin can be split into three parts by partial digestion with papain. This cuts each heavy chain, leaving one fragment, Fc, containing the carboxy terminal parts of both heavy chains and two identical fragments, Fab, each containing one light chain and the amino terminal part of one heavy chain.

The IgG molecule has 2 antigen-combining sites. Antibody-antigen reactions, particularly when the antigen is on the surface of a cell membrane (e.g. human blood groups), can be either complete or incomplete (sensitization). Antibodies are either warm (read at 37°C) or cold (react only at low temperatures). In blood grouping, when an appropriate antibody recognizes its antigen on the surface of red cells, and causes the cells to clump together, the process is called agglutination.

The Fab fragment contains the antigen-binding site. The site is made from the variable domains of both the heavy and light chains and is at the very tip of the molecule. Within this region there are several specific hydrogen bonds and a Gln residue of lysozyme is buried deep inside the antibody-binding site. This structure was determined by X-ray crystal diffraction.

The Fc region, which is very similar for all immunoglobulins, is responsible for the stages of the immune response that follow antibody binding. The foreign body is attacked by one or more of three systems: the complement system; phagocytosis; and antibody– dependent cell–mediated killing.

1.4 Blood group inheritance and Genetics

The fact that the ABO blood group system was inherited was suggested in 1910 by Epstein and Ottenberg (Greenwalt 1997). The confirmation of the ABO system being genetically inherited was by von Dungern and Hirszfeld. Who studied 72 families with 102 children. They found that the inheritance of the A and B agglutinogens obeyed Mendels laws (Greenwalt 1997).

It was discovered that the ABO gene was autosomal (the gene was not on either sex chromosome). Therefore each person has two copies of genes coding for their ABO blood group (one maternal and one paternal in origin). It was observed that the A and B blood groups were dominant over the O blood group. It was also found that the A and B group genes were co-dominant. This meant that if a person inherited one A group gene and one B group gene their red cells would possess both the A and B blood group antigens. These alleles were termed A (which produced the A antigen), B (with produced the B antigen) and O (which was "non functional" and produced no A or B antigen) (Greenwalt 1997; Uhlenbruck and Prokop 1969).

1.4.1 Gregor Mendel and Mendelian Genetics

Gregor Johann Mendel was born on July 22, 1822 to peasant parents in a small agrarian town in Czechoslovakia. During his childhood he worked as a gardener, and as a young man attended the Olmutz Philosophical Institute. In 1843 he entered aAugustinian monastery in Brunn, Czechoslovakia. He was later sent to the University of Vienna to study. By both his professors at University and his colleagues at the monastery, Mendel was inspired to study variance in plants. He commenced his study in his monastery's experimental garden. Between 1856 and 1863 Mendel cultivated and tested some 28,000 pea plants. His experiments brought forth two generalizations which later became known as Mendel's Laws of Heredity. Ironically, when Mendel's paper was published on 1866, it had little impact. It wasn't until the early 20th century that the enormity of his ideas was realized (http://www.biopoint.com/engaging/MENDEL/MENDEL.HTM).

1.4.1.1 Mendel's First Law is the law of "Segregation of Characteristics”

Mendel's hypothesis essentially has four parts. The first states that "Alternative versions of genes account for variations in inherited characters." In a nutshell, this is the concept of alleles. Alleles are different versions of genes that impart the same characteristic. Each human has a gene that controls height, but there are variations among these genes in accordance with the specific height the gene "codes" for. The second, "For each character, an organism inherits two genes, one from each parent", alludes to the fact that when somatic cells are produced from two gametes, one allele comes from the mother, one from the father. These alleles may be the same (true- breeding organisms), or different (hybrids). The third law, in relation to the second, declares that, "If the two alleles differ, then one, the dominant allele, is fully expressed in the organism's appearance; the other, the recessive allele, has no noticeable effect on the organism's appearance." "The two genes for each character segregate during gamete production," is the last part of Mendel's generalization. This references meiosis when the chromosome count is changed from the diploid number to the haploid number. (http://www.biopoint.com/engaging/MENDEL/MENDEL.HTM).

1.4.1.2 Mendel's Law of Independent Assortment

The most important principle of Mendel's Law of Independent Assortment is that the emergence of one trait will not effect the emergence of another. While his experiments mixing one trait always resulted in a 3:1 ratio between dominant and recessive phenotypes, his experiments with two traits showed 9:3:3:1 ratios. Mendel's findings allowed other scientists to simplify the emergence of traits to mathematical probability. A large portion of Mendel's spectacular findings can be traced to his proper usage of the scientific method. His choice of peas as a subject for his experiments is extraordinarily lucky. Peas have a relatively simple genetic structure. Also, Mendel could always be in control of the plants' breeding. When Mendel wanted to cross-pollinate a pea plant he needed only to remove the immature stamen of the plant. In this way he was always exactly sure of his plants' parents. Mendel made certain to start his experiments only with true breeding plants. He also only measured absolute characteristics such as color, shape, and position of the offspring. His data was expressed numerically and subjected to statistical analysis. This method of data reporting and the large sampling size he used gave credibility to his data. He also had

the foresight to look through several successive generations of his pea plants and record their variations. Without his careful attention to procedure and detail, Mendel's work could not have had the impact it made on the world of genetics (http://www.biopoint.com/engaging/MENDEL/MENDEL.HTM)

1.4.2 Blood Group inheritance

Human blood type is determined by co-dominant alleles. An allele is one of several different forms of genetic information that is present in our DNA at a specific location on a specific chromosome.

Each of us has two ABO blood type alleles, because we each inherit one blood type allele from our biological mother and one from our biological father. A description of the pair of alleles in our DNA is called the genotype. Since there are three different alleles, there are a total of six different genotypes at the human ABO genetic locus.

Parent Alleles A B O Table 1.2: The possible ABO alleles for one parent are in the top row and the alleles of A AA AB AO theother are in the left column. (A) (AB) (A) Offspring genotypes and B AB BB BO phenotypes are shown in black. (AB) (B) (B) Phenotypes are enclosed in red parentheses. O AO BO OO (http://anthro.palomar.edu/blood (A) (B) (O) /ABO_system.htm)

Both A and B alleles are dominant over O. As a result, individuals who have an AO genotype will have an A phenotype. People who are type O have OO genotypes. In other words, they inherited a recessive O allele from both parents. The A and B alleles are co dominant. Therefore, if an A is inherited from one parent and a B from the other, the phenotype will be AB. Agglutination tests will show that these individuals have the characteristics of both type A and type B blood. There are however, some exceptions to these straight forward rules of inheritance (http://anthro.palomar.edu/blood/ABO_system.htm).

1.4.3 Blood Group Genetics

Figure 1.6 illustrates the biochemical pathway, which produces the "H" antigen (or the antigen present in O people) and modifies the "H" antigen into either the "A" antigen (present in "A" people) or the "B" antigen (present in "B" people). The "A" enzyme is coded for by the A allele of the ABO gene and the "B" enzyme is coded for by the B allele of the ABO gene. The O allele does not code for an enzyme so a person who is homozygous ii cannot modify the "H" antigen into either "A" or "B" and so is of blood type "O" or zero (none of the above). Individuals who are heterozygous for the A and B alleles (AB) code for both the "A" enzyme and the "B" enzyme and make both the "A" and "B" antigens. The ABO gene is located on chromosome 9 (9q34.1). Other traits associated with genes on chromosome 9 are galactosemia, nail-patella syndrome, and xeroderma pigmentosa (http://www.people.virginia.edu/~rjh9u/abo.html).

Figure1.6: Biochemical pathway (http://www.people.virginia.edu/~rjh9u/abo.html)

ABO genes consist of at least 7 exons, and the coding sequence in the seven coding exons spans over 18kb of the genomic DNA. The single nucleotide deletion found in most (but not all) of the O alleles and responsible for the loss of the activity of the enzyme is located in exon 6. The first of the seven nucleotide substitutions that distinguish the A and B transfersases resides in coding exon 6. Exon 7, the largest of all, contains the other six nucleotide substitutions which result in four amino acid substitutions that differentiate the A and B transferases. Among those, substitutions responsible for alterations at two sites (residues 266 and 268) determine the A or B specificity of the enzyme (Yamamoto and Hakamori 1990).

1.5 Antigen Antibody reaction

When foreign bodies (molecules, viruses, bacteria) enter the bloodstream, they stimulate the production of antibodies that trigger cellular defense mechanisms that attack the foreign bodies. Antibodies bind specifically to their cognate antigen.

Binding of an antigen by an antibody requires each to have a shape complementary to the other. The forces involved include hydrogen bonding, van der Waal's (hydrophobic), and ionic.

Antigen-antibody reactions are the binding processes of an antigen molecule with its specific antibody on the basis of some principles. This reaction is a 2-staged, reversible and chemical reaction and shows a high level of specificity.

There are 4 main forces that play role in these reactions. These are 1. hydrogen bonds 2. electrostatic forces 3. Van der Waals bonds 4. hydrophobic bonds.

The strength of the antigen-antibody binding depends on the valency of antibodies, which are termed as avidity and affinity.

The avidity of an antibody is also related with the maturation of this antibody molecule and has an important role for the diagnosis of primary or secondary immunity. The specific reaction indicates a binding between antigen and antibody with overall configurations and the cross-reaction indicates a binding of an antigen, which shared some epitopes with the original one to the previously formed antibody.

1.5.1 Nature of antigen antibody reaction

1.5.1.1 Lock and Key Concept

The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. X-Ray crystallography studies of antigens and antibodies interacting shows that the antigenic determinant nestles in a cleft formed by the combining site of the antibody as illustrated in Figure 1. Thus, our concept of Ag-Ab reactions is one of a key (i.e. the Ag), which fits into a lock (i.e. the Ab).

1.5.2 Non-covalent Bonds

The bonds that hold the Ag in the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. Multiple bonding between the Ag and the Ab ensures that the Ag will be bound tightly to the Ab.

1.5.3 Reversible

Since Ag-Ab reactions occur via non-covalent bonds, they are by their nature reversible.

Figure 1.7: Stages of the Antigen-Antibody Reaction (http://ntri.tamuk.edu/monoclonal/fda-fig10-2.html)

1.6 Pretransfusion Testing

The objective of pretransfusion testing is to ensure that enough red blood cells (RBCs) carrying selected red cell components will survive when transfused. Yet, while no one questions the need for subjecting donor and patient blood to pretransfusion testing, some do question how to best and most economically do it. In most blood banks, pretransfusion testing involves determining the ABO and Rh types of both patient and donor blood, screening patient and donor sera for RBC alloantibodies, and performing a major crossmatch (testing the patient's serum against the donor's RBCs).

Pretransfusion testing can assure ABO compatibility between donor and patient blood as well as detect most clinically significant RBC alloantibodies that react with antigens on donor RBCs. Unfortunately, it can not always guarantee the normal survival of transfused cells since a minute number of deleterious reactions due to serological incompatibility can still occur.

Over the past 30 years, pretransfusion tests have undergone considerable modification (Masouredis 1982; Oberman 1981).

In the early 1960s, many blood banks carried out minor crossmatching (testing donor's serum against patient's RBCs) in addition to major crossmatching. It was only in the mid-1970s that the minor crossmatch was finally abandoned as antibody screening of donor blood became routine (American Association of Blood Banks 1976).

The antiglobulin test was first introduced into clinical medicine in 1945 by R.R. Coombs who showed that it could be used to detect non-agglutinating red cell antibodies (indirect antiglobulin test, IAT) or sensitized red cells (direct antiglobulin test, DAT). Most non-agglutinating (incomplete) antibodies are IgG, although some antibodies are IgM. These antibodies do not spontaneously cause agglutination due to a strong electronegative charge on the red cell surface that prevents the cells from coming into close proximity. The antiglobulin reagent is able to bridge these negative forces. Current antiglobulin reagent (Coombs reagent) preparations contain a "cocktail" of monoclonal antibodies directed against human IgG and C3. The latter is more effective than an anti-IgM reagent for detecting IgM antibodies because a single IgM molecule will

bind numerous complement molecules to the red cell surface and IgM antibodies tend to spontaneously dissociate from the red cell membrane (Darrell and Triulzi 1994) Most of the early methods for antibody screening and crossmatching involved testing at room temperature. In 1978, the American Association of Blood Banks (AABB) deleted the room-temperature requirement from its Standards (American Association of Blood Banks 1978).

In the past, the prevailing tendency in blood banking was to use to the utmost, and no matter the cost, all available highly sensitive techniques to detect any serological incompatibilities. Yet, as blood-banking and other health care institutions have become more aware of the economy of health care delivery, there has been an increasing trend toward abbreviating the crossmatching procedure in pretransfusion testing ((Masouredis 1982).

Obviously, the main reason for abbreviating crossmatching is to save the cost of reagents and labor. The estimated savings from eliminating the anti-human globulin (AHG) phase of a crossmatch is approximately $1.00 in cost and 30% in technologist's time. With approximately 12,000,000 units of blood transfused annually in the United States, the cost and time savings would be substantial (Masouredis 1982; Oberman 1981).

Pretransfusion testing is intended to guarantee the normal survival of transfused RBCs at minimum cost, yet how best to do this remains a matter of controversy. Because of the increasing awareness of economy and efficiency in health care delivery, the blood-banking community is now reexamining its rationale for performing some steps in pretransfusion testing.

While no one questions the importance of careful clerical checks, ABO and Rh typing, and antibody screening, some do question the extent of crossmatching procedures. In fact, there has been an increasing trend toward abbreviating the crossmatching procedure or even eliminating it altogether.

1.6.1 Pretransfusion testing in Malta

Dr. Schembri Wismayer (2003) stated that: [ In the late 1980’s, the type and screen technique was being introduced, with the aim of including such a procedure as part of the compatibility tests at the National Blood Transfusion Centre (NBTC) – Malta.

However, various problems arose. One being the increase in workload for the staff. Another being the availability of the reagents, since these are composed of red cells, the shelf life is less than one month. The supply of these reagents were not regular, resulting in outdated reagents being used at the end of the month. It was therefore, eventually decided to perform an antihuman globulin crossmatch on all routine cases, leaving the antibody screen for problematic cases only.]

Beginning a type and screen procedure instead of an antiglobulin crossmacth will produce an increase in the cost of pretransfuion testing. However, it is postulated that this may allow us to work with a smaller reserve blood supply since units will not be reserved for a particular patient.

1.6.2 Blood Sampling and Clerical Checking

In pretransfusion testing the importance of careful clerical checking cannot be overemphasized. From the out set collecting and properly labeling blood samples from the correct patient is critical to accurate serological testing and safe blood transfusion. When a sample is received in the transfusion service laboratory a medical technologist must confirm that the information on the label and on the transfusion request form are identical. The patient's serological and transfusion history must also be checked and the results of current testing compared with those of previous tests. In short any discrepancies must be resolved before blood can be released for transfusion.

1.6.3 Blood Grouping

The ABO group and Rh type must be determined for the blood of both the donor and the intended recipient. ABO group is determined by testing the RBCs with anti-A and anti-B reagents and by testing the serum with A1 and B RBCs. Rh type is determined with anti-Rh (anti-D): if the initial test of the donor blood with anti-D is negative, then that blood is tested by a method designed to detect weak D (Du). When either Rh test is positive, the blood sample label reads "Rh positive." Conversely, when the tests for both D and Du are negative, the label reads "Rh negative." It is not necessary to determine whether a patient's RBCs are of the Du type because no harm results from transfusing Rh-negative blood.

1.6.4 Antibody Detection Tests

Besides clerical checking grouping and typing of donor and patient blood the serum or plasma of the patient must be tested against a single donor suspension of unpooled group O reagent RBCs. This meets AABB Standards (Standards for Blood Banks and Transfusion Services 1993).

Current antibody screening tests cannot detect all clinically significant antibodies. For instance, antibodies against low-incidence antigens are likely to be missed. Other antibodies manifest a dosage effect dependent on the homozygosity of genes controlling expression of antigens on the reagent screening cells. Examples of such dosage-effect antibodies are anti-Jka, anti-Jkb, anti-Fya, anti-Fyb, anti-C, anti-E, and anti-c.

Using three types of screening cells improves the chances of having all major RBC antigens present in their homozygous forms during testing. In cases of positive antibody screening, further serological testing with an expanded panel of reagent RBCs for the identification of clinically significant antibodies is required. Then, once the specificity of the antibody is known, donor units must be screened for the corresponding antigen to select those units that lack the antigen.

1.6.5 Competibility testing

The compatibility between donor and recipient must be assured in transfusions of components containing amounts of red cells visible to the naked eye. Ideally compatibility testing should be carried out on repeat samples other than the one used in initial blood typing but should, in any case, be carried out on a sample taken no more than 4 days before the proposed transfusion.

The basis for compatibility is a correctly determined ABO and Rh (D) blood type in donor and recipient. When irregular erythrocyte antibodies are present in the patient's circulation, only red cells, which lack the corresponding antigens, should be selected for transfusion.

Compatibility testing between donor red cells and recipient's serum shall be done in all cases with irregular erythrocyte antibodies. It is recommended as a routine procedure even when no antibodies have been found but may be omitted if other measures (e.g. type and screen, see below) are taken to guarantee safety. The compatibility testing shall include a sufficiently reliable and validated technique to guarantee detection of irregular erythrocyte antibodies, such as the indirect antiglobulin technique.

Figure 1.8: Crossmatch testing (http://www.med.umich.edu/trans/ public/ttbrochure/image4.gif_)

A sample of the serum used for crossmatching or antibody screening should be retained in a frozen state for a period of time determined by national regulations.

1.6.5.1 Types of crossmatches

The crossmatch procedure determines whether donor blood is compatible (or incompatible) with recipient blood. A crossmatch is performed prior to administration of blood or blood products (e.g. packed red blood cells). The purpose of the crossmatch is to detect the presence of antibodies in the recipient against the red blood cells of the donor. These antibodies attach to the red blood cells of the donor after transfusion. An incompatible transfusion can result in a severe hemolytic anemia and even death. There are two types of crossmatches.

The crossmatch (CM) represents a special form of the IAT in which the red cells used for testing are from the unit intended for transfusion. The purpose is to establish in vitro compatibility in the expectation that the transfused cells will exhibit normal in-vivo survival. Historically, a major CM involving patient serum and donor red cells was performed on every unit intended for transfusion.

More recently it has been recognized that patients with a negative antibody screen and no history of red cell antibodies do not require a complete 20-30 minute crossmatch. The chances of a clinically significant red cell antibody being missed in a patient with a negative antibody screen (false negative) are 1-4/10,000. Approximately 95% of transfusions occur in patients with a negative antibody screen. Such patients can undergo abbreviated CM testing in which only ABO compatibility of the unit need be established. There are two methods for abbreviated CM testing.

1.6.5.1.1 Major crossmatch

This is the most important crossmatch, comparing donor erythrocytes to recipient serum (i.e. you are checking for preformed (acquired or naturally occurring) antibodies in recipient serum against donor erythrocytes. For the major crossmatch, you need red blood cells from the donor (this can be whole blood from a donor animal or packed red blood cells) in EDTA or citrate and serum from the recipient (non-anticoagulant tube).

The major crossmatch takes 20-30 minutes and is now essential only for patients with clinically significant red cell antibodies.

The “immediate spin” crossmatch (IS-XM) requires only 5 minutes incubation at room temperature with patient serum and donor red cells.

1.6.5.1.2 Minor crossmatch

This compares donor serum to recipient erythrocytes and checks for preformed antibodies in donor serum that could hemolyse recipient red cells. This crossmatch is less important as usually the donor serum is markedly diluted after transfusion and is unlikely to produce a significant transfusion reaction. This type of crossmatch could be important if transfusing small patients, in which hemodilution is less likely to occur.

The minor CM involving patient red cells and donor plasma has not been performed for more than 20 years because packed red cells have <70 ml of plasma.

1.6.5.1.3 Computerized Crossmatching

In recognition of the improved capabilities of computer systems, the AABB has stipulated that blood banks may use computerized crossmatching, instead of serological crossmatching, to detect ABO incompatibility prior to transfusion and so prevent the release of ABO incompatible blood components for transfusion (Standards for Blood Banks and Transfusion Services 1993).

However, the AABB also stresses that such computerized crossmatching only be done if the following conditions have been met: ∼ the patient's ABO group has been determined twice (once on a current sample; a second time on the same sample, on a second current sample, or by comparison with previous records); ∼ the computer system's database contains the donor unit number, the component name, the ABO group and Rh type of the component, blood group confirmatory test interpretation and identification, and the ABO group and Rh type of the patient; ∼ a method is in place to ensure correct entry of data; and ∼ the system can alert the user to discrepancies between donor unit labeling and blood group confirmatory test interpretation and to ABO incompatibilities between the patient and donor blood

Advantages of the computer crossmatch includes: faster turn-around, computer prevents release of ABO incompatible units, lower reagent costs, and improved quality control.

1.6.5.2 Crossmatching Tests

The issue of whether to omit anti-human globulin crossmatch (AHG-XM) for patients screened as negative for RBC alloantibodies remains controversial (Masouredis 1982).

According to a survey conducted by the in 1987, only 11% of hospitals at the time routinely used immediate spin crossmatch (IS-XM) without AHG-XM for patients with

negative screening for RBC alloantibodies; all others surveyed used AHG-XM (College of American Pathologists 1987).

Any policy decision by a hospital to omit AHG-XM from crossmatching procedures must be made by a transfusion service's medical director and only after several factors have been considered: ∼ the possibility of incompatible crossmatches or hemolytic transfusion reactions due to RBC alloantibodies not detected by antibody screening, ∼ the potential cost- and labor-saving benefits of omitting AHG-XM, and ∼ the sensitivity of the antibody detection test used in the laboratory.

In 1985, at M. D. Anderson Cancer Center began to use IS-XM testing without the 37 degrees Celsius-AHG phase for patients screened as negative for RBC alloantibodies. A recent retrospective study of our experience over the ensuing 4 yr period (September 1985-August 1989) verified the safety of using IS-XM alone in screening incompatible blood at this institution (Havemann and Lichtiger 1992).

The study showed that among 92,759 units of packed RBCs transfused, there were only two immediate hemolytic transfusion reactions and eight cases of RBC alloimmunization. The two immediate hemolytic transfusion reactions were caused by transfusion of ABO-incompatible blood resulting from clerical error and would not have been prevented by AHG-XM. All eight cases of RBC alloimmunization were discovered during subsequent routine serological testing prior to additional transfusions to patients. The specificity of the antibodies was anti-Jka in three cases; anti-Fya in two cases; and anti-Jkb, anti-E, and anti-Fyb in one case each. No significant untoward effects of the tranfusions were documented in the records of these eight patients. In effect, the results of our retrospective institutional study agreed with those of previous studies in suggesting a very low risk of transfusing incompatible blood when the 37 degrees Celsius-AHG phase was eliminated from crossmatching procedures as seen in the table (Oberman et al. 1978; Mintz et al.1980; Taswell et al. 1981).

1.7 Difference between type and screen and crossmatch

A type and screen (T&S) consists of a group of tests performed on a patient's blood specimen as part of pretransfusion testing. It includes typing the patient's red cells for ABO and Rh blood group. The ABO group is determined by typing the patient's red cells using anti-A and anti-B reagents and by reverse-typing the patient's serum against A1 and B reagent cells. The patient's serum is screened for the presence of unexpected antibodies by incubating it with selected reagent red cells (screen cells) using an antihuman globulin (AHG) technique (indirect antiglobulin or Coombs test). These screen cells, usually two to four, are selected in such a way that all common red cell antigens capable of inducing clinically significant red cell antibody reactions are represented on at least one cell. This group of tests takes approximately 45 minutes.

If the antibody screen is negative and the patient has no past history of unexpected antibodies, it can be predicted that more than 99.99 percent of ABO- compatible red blood cell units would be compatible in an AHG crossmatch. ABO- and Rh-compatible blood can be selected from the stock and issued within five to 10 minutes. If the antibody screen is positive (approximately 1 percent of patients), the unexpected antibody or antibodies must be identified before antigen negative-compatible Red Blood Cells can be found. This usually takes several hours.

A type and screen procedure, where used as a replacement for crossmatch testing, must include: ∼ a reliable and validated, preferably by computer, checking procedure when the blood units are delivered; ∼ test cells which cover all antigens, preferably homozygous, corresponding to the vast majority of clinically important antibodies; ∼ sufficiently sensitive techniques for the detection of erythrocyte antibodies; ∼ laboratory records of tests performed and of the disposition of all units handled (including patient identification

In a crossmatch, the patient's serum is incubated in a test tube with an aliquot of red cells from a specific donor unit to verify in vitro compatibility. A crossmatch is performed either as a short (immediate spin) incubation intended solely to verify ABO compatibility or as a long incubation with an AHG technique intended to verify

compatibility for all other red cell antigens. The immediate spin crossmatch takes five to 10 minutes, while the antiglobulin crossmatch takes at least 45 minutes. A crossmatch is only performed if a valid (less than 72 hours old) T&S result is available and should be ordered only if the likelihood of transfusing red cells to the patient is high. In many institutions, the immediate spin crossmatch is used for patients without unexpected antibodies. An AHG crossmatch is only required for patients with a current or past history of clinically significant unexpected antibodies in their serum.

1.7.1 Type and Crossmatch (T&C)

Type and Crossmatch involves the same testing as type and screen with the addition of crossmatching. Crossmatching involves testing the recipient’s serum for antibodies against red cell antigens present on the donor unit. Once completed the number of units requested will be set aside. The patient will be charged a fee for each unit crossmatched, even if the unit is not transfused.

1.7.2 Type and Screen VS Type and Crossmatch

When faced with ordering T&S or T&C, the likelihood of the patient being transfused should be considered. If the patient has a very high chance of needing a transfusion Type and Crossmatch is appropriate, If the patient may need to be transfused, order a Type and Screen. If the antibody screen is negative and the patients status changes products can be re-leased from the blood bank in about 20 minutes (please note this does not include transporter or tube travel time.)

1.8 The Type and screen procedure

When Type and Screen is ordered the patient’s ABO and Rh “Type” is determined as well as their serum tested for unexpected antibodies (screening.) If the patient’s antibody screen is negative, blood products can be released immediately if needed. If antibodies are detected against red cell antigens during the initial Type and Screen, the blood bank will automatically identify the antibody and find two crossmatched compatible units.

Patients may form antibodies to blood group antigens they do not possess on their red blood cells as a result of exposure to the antigens by prior transfusion and/or pregnancy. Screening for antibodies in patient or donor serum is an established blood bank procedure. The Advantages of systematic screening to detect unexpected antibodies includes: 1. facilitates the selection of “safe” donor blood for transfusion 2. aids in the prognosis and treatment of hemolytic disease of the newborn 3. enables donor blood containing antibodies of potential clinical significance to be recognized.

Before this procedure became popular many units of donor blood were crossmatched and held in reserve for patients who would probably not need it. At times this would cause shortages of the blood supply and unnecessary outdating of donor units. These factors, along with the added expense of crossmatching blood, caused the Type and Screen (T&S) procedure to gain popularity.

This procedure is most frequently used to screen pre-operative or obstetrical patients whose risk of excessive blood loss is minimal. In case of an emergency, where blood is needed for these patients, uncrossmatched ABO and D compatible blood can be released with 99.9% assurance of safety, as long as the patient has no unexpected antibodies. If the antibody screen is positive the patient is not a T&S candidate, the antibody present in the serum must be identified and antigen negative donor units must be crossmatched. This procedure consists of two stages: 1. Performing a complete ABO and D typing on the patient's blood sample. 2. Screening the patient's serum for atypical antibodies.

If an antibody is detected and identified during a routine work-up, the identification will be reviewed. When antibodies against red cell antigens are found, multiple donor units must be tested in order to find units that lack the corresponding antigens. This process can be very time consuming. For example if Anti-e is identified (the e antigen is present on 98% of red cells and only 2% lack the antigen) the blood bank would have to test one hundred units to find two that lack the e-antigen and are compatible.

1.8.1 ABO and D Typing

As has been briefly examined, transfusion success was a very hit and miss affair even when humans were used as blood donors. This was directly due to the major barrier posed by the ABO blood group system. The consequences of an ABO incompatible blood donation are acute and potentially lethal intravascular destruction of the transfused red cells.

ABO typing is accomplished by testing the patient’s red cells with anti-A and anti- B antisera and testing the patient’s serum for Anti-A and anti-B. The ABO system is unique in that a subject’s serum has naturally occurring antibodies, to the ABO red cell antigens that are absent from self red cells. These antibodies are the basis for ABO compatibility criteria when selecting red cells and plasma for transfusion.

If you are: You can donate to: You can receive from: O A, B, AB, O O Group A A, AB O, A B B, AB O, B AB AB A, B, O, AB Rh + Rh + Rh +, Rh - Rh status Rh - Rh +, Rh - Rh - Table1.3: Compatibility chart (http://www.mckinley.uiuc.edu/health-info/hlthpro/bloodtyp.html)

The presence or absence of the D antigen in the Rh blood group system defines whether a person is Rh-positive or Rh-negative. In contrast to the ABO system, patients with D-negative red cells will not make anti-D unless they have been immunized previously by exposure to D-positive red cells via fetomaternal transfer during pregnancy or prior transfusion. Rh-positive recipients can receive Rh-positive or Rh-negative RBC, but Rh-negative recipients should only receive Rh-negative blood.

1.8.1.1 Forward blood grouping

ABO forward typing determines which ABO antigens are present on the recipient’s red cells, whereas reverse typing looks for the presence of corresponding antibodies in the recipient’s serum.

1.8.1.2 Reverse blood grouping

The reverse (or back) type, on the other hand, tests the recipient’s serum with reagent RBCs to determine what antibodies are present in the patient’s serum. This, then, is used to con-firm (or more correctly act as a control for) the forward type. An individual who is group A should have anti-B but not anti-A in his serum, whereas a B patient should have anti-A but not anti-B. An O patient should have both anti-A and anti- B and an AB individual should demonstrate the presence of neither anti-A nor anti-B.

1.8.1.3 Forward and Reverse Typing Discrepancies

Whenever there is a discrepancy between the forward and re-verse type, it is important to determine the cause of the discrepancy before transfusing the patient with type-specific RBCs. In an emergency, if the discrepancy cannot readily be re-solved, group O Rh-compatible RBCs should be issued until the recipient’s ABO group can be reliably ascertained. The most common cause of ABO forward-reverse discrepancies is a patient who belongs to a subgroup of A and who has formed anti-A1. Approximately 80% of A patients are subgroup A1 (and 80% of AB patients are A1 B); the vast majority of the remainder are A2 (or A2 B).

1.8.2 Antibody Screening

The antibody screen, involves testing the patient’s serum against reagent screen cells selected to possess relevant blood group antigens for detection of clinically significant, unexpected antibodies.

Immunoglobulins are the antibodies formed as a result of immune stimulus (exposure to foreign antigen). Most attention is given those antibodies that are known to cause transfusion reactions and hemolytic disease of the newborn. Except for anti-A, anti-B, and anti-A,B these are usually IgG immunoglobulins. An old term that referred to IgG antibodies that can not cause agglutination of red cell antigens in saline is ncomplete antibodies.

The A and B antibodies are naturally occurring since they are formed without previous exposure to foreign blood cells. These antibodies are expected and can be used to confirmed the antigen typing for ABO grouping.

The antibody screening cells are used to detect unexpected antibodies. In most cases these are alloantibodies, which formed to foreign antigens on cells from other individuals within the same species. Therefore for an individual to make an alloantibody they would lack the antigen for which they made a specific antibody due to its foreignness to the individual.

Autoantibodies can also be detected with the antibody screening procedure. These antibodies are ones a person makes toward their own antigens. These are not a normal occurrence and may indicate autoimmune hemolytic anemia. The autocontrol tube within the antibody screening will detect this type of antibody and other causes of a positive direct antiglobulin test.

Clinically significant antibodies are those antibodies known to cause transfusion reactions and hemolytic disease of the newborn. Other the AB antibodies, which are saline agglutinins and IgM, the rest of clinically significant antibodies are IgG antibodies that are warm-acting and may only be demonstrated at the antiglobulin stage of testing.

In vitro hemolysis and/or agglutination of the screen cells by the patient serum at any of the various stages of the test constitutes a positive test result and indicates the presence of unexpected antibodies in the patient serum which could possibly cause in- vivo hemolysis or decreased cell survival of transfused donor cells.

Antibodies that show up at the immediate spin phase are most likely nuisance antibodies that won't cause transfusion reactions. These would also be referred to as saline agglutinins since it is an antibody capable of causing direct agglutination of

antigens suspended in a saline medium without requiring any enhancement techniques. Most clinically significant antibodies are warm antibodies whose optimal temperature of reactivity is greater than 35oC.

Antibodies that are reactive at 37°C and/or in the antiglobulin test are more likely to be significant than those reactive at room temperature or below. If a positive result is obtained, further testing is required to determine the specificity of the antibody so that appropriate antigen negative donor units can be selected.

The absence of hemolysis and/or agglutination constitutes a negative test indicating the serum being tested does not contain detectable antibodies directed to any of the wide variety of antigens present on the reagent screen cells.

1.8.2.1 Uses of Antibody screening test

Donor plasma is tested to make sure no unexpected antibodies will be transfused. Since an unit of blood is usually divided into various components (red cells, fresh frozen plasma, and platelets), blood centers need to make sure none of these unexpected antibodies are present in the fresh frozen plasma. If these antibodies are present, they could attach to the recipient's cells cause a positive direct antiglobulin test and potentially a transfusion reaction.

Patient serum is screened before transfusion to make sure patient has no unexpected antibodies to react with donor cells. In this case we do not want the recipient's antibodies to attach to the donor cells causing a transfusion reaction.

Maternal serum is screened to make sure pregnant mother has no antibodies to react with fetal cells. Hemolytic disease of the newborn is caused by the mother's IgG antibodies crossing the placenta and attaching to the baby's red blood cells. The physician wants to know as early as possible in the pregnancy whether HDN can be a possibility. This is also necessary if the mother is Rh negative and would be a candidate for RhoImmune Globulin. Since Rho Immune Globulin is anti-D given to the Rh negative mother who do not already have anti-D so we need to rule out the presence of anti-D already.

1.8.3 Antibody Identification

The presence of irregular antibodies is detected by a direct agglutination test at 37°C and an indirect antiglobulin test. If the antibody screen gives negative results, no blood will be crossmatched for reserve. Should transfusion become necessary, an abbreviated cross-match is done by using the immediate-spin method to demonstrate ABO compatibility. If, however, the re-cipient has blood-group alloantibodies, an FXM test using antigen-negative donor blood is performed before the unit of blood is issued for transfusion. In the conventional FXM procedure, the recipient’s serum is incubated with erythrocytes from the donor at 37°C; a direct agglutination test and an indirect antiglobulin test are performed after ABO and Rh D blood-group typing. In contrast to the TS test practice,units of compatible blood are reserved for the specificpatient, as requested by the clinician.

Determining the specificity of an unexpected alloantibody is important in pre- transfusion and prenatal testing. If the antibody specificity is known, it is possible to test donor blood for the absence of the corresponding antigen. Antibodies should also be identified in donor blood so that this blood is not transfused to antigen-positive recipients. In prenatal testing, knowledge of the specificity of the antibody helps predict the likelihood of hemolytic disease of the newborn.

Once an unexpected antibody is detected, its specificity should be determined and its clinical significance assessed. A clinical significant antibody is one that shortens the anticipated survival of transfused RBCs or has been associated with haemolytic disease of the new born.

The serum under investigation should be tested by the desired techniques with a panel of eight or more group O reagent RBC samples of known blood group phenotype. To be functional, a reagent RBC panel must make it possible to identify with confidence those clinically significant alloantibodies that are most frequently encountered such as anti-D, -E, -K and –Fya. When a serum contains only one of these antibodies, the reagent RBC phenotypes should be such that the presence of most other common alloantibodies can be at least tentatively excluded.

It is important to know how the serum investigation reacts with the autologous RBCs. This helps determine whether alloantibody, autoantibody or both are present.

1.8.3.1 Antibody identification systems

Determining the specificity of an unexpected alloantibody is important in pre- transfusion and prenatal testing. If the antibody specificity is known, it is possible to test donor blood for the absence of the corresponding antigen. Antibodies should also be identified in donor blood so that this blood is not transfused to antigen-positive recipients.

Historically the naming of Blood grouping systems has been disorganized. The common conventions stipulating that dominant traits be given capital letters and recessive traits be designated with lower case letters have not been followed. Also by tradition, red cell antigens were given alphabetical designations or were named after the family of the antibody producer. The International Society of Blood Transfusion (ISBT) has instituted a numerical system of nomenclature to help standardize red cell Blood group terminology. This convention mandates that each system and collection has been given a number and letter designation, and each antigen within the system is numbered sequentially in order of discovery. As of this writing, over 20 Blood group systems and seven antigen collections have been defined. High-prevalence or "public" antigens and low-prevalence or "private" antigens that are not associated with known systems or collections also are delineated in numbered series (http://www.bloodbook.com/type- sys.html).

Some systems (i.e. H, Ii, Lewis) delineate naturally occurring antibodies, but most of the other systems give rise to isoantibodies, which result from incompatible transfusions and pregnancy.

Blood Group Systems Conventional Name ISBT Symbol ISBT Number Antigens ABO ABO 001 4 MNSs MNS 002 37 P P1 003 1 Rh RH 004 47 Lutheran LU 005 18 Kell KEL 006 21 Lewis LE 007 3 Duffy FY 008 6 Kidd JK 009 3 Diego DI 010 2 Cartwright YT 011 2 Xg XG 012 1 Scianna SC 013 3 Dombrock DO 014 5 Colton CO 015 3 Landsteiner-Wiener LW 016 3 Chido/Rogers CH/RG 017 9 Hh H 018 1 Kx XK 019 1 Gerbich GE 020 7 Cromer CROMER 021 10 Knops KN 022 5 Indian IN 023 2 Ok OK 024 - - Raph RAPH 025 - - JMH JMH 026 - - Antigen Collection Cost COST 205 2 Ii I 207 2 Er ER 208 2 P, P1, LKE GLOBO 209 3 Lewis-like: Le-c, Le-d - - 210 2 Wright WR 211 2 Low Prevalence Low Prevalence - - 700 36 High Prevalence High Prevalence - - 700 36 Table1.4: ISBT Type/Class Chart (http://www.bloodbook.com/type-sys.html)

1.8.4 The efficacy of type and screen to reduce unnecessary cross matches

One of the best means of evaluating transfusion practices is to determine the ratio of units cross matched to units transfused (cross match/transfusion or C/T ratio); a C/T ratio of 2.5/1 is considered optimal for the elective surgical procedures (Henry et al 1978).

The type and screen procedure has proved effective in reducing unnecessary cross matches for effective surgery procedures. By examining transfusion practices, a list of surgical procedures with high C/T ratios can be determined where blood transfusion is possible but unlikely.

Boral and Henry have shown that less than one patient in 10,000 will have a positive cross match if the antibody screen is negative

1.8.4.1 Advantages of type and screen

The type and screen offers several advantages over the older practice of crossmatching and reserving specific donor units for patients: ∼ Better use of donor blood, as it is not tied up by being crossmatched and held for patients who probably will not need it. ∼ More efficient service for patients, as blood bank personnel are not tied up crossmatching needlessly or removing tags from unused products but rather are available for more useful purposes. ∼ Potential for a more economic transfusion service due to decreased blood inventory requirements, decreased reagents, and more efficient use of technologist time.

1.8.4.2 Limitation of type and screen

The type and screen, in which specific donors are not crossmatched for patients, can be used in only particular circumstances: ∼ It is indicated only for patients undergoing surgery in which blood transfusion is unlikely to be required. ∼ The antibody screen must be sensitive and reliable or the patient's well-being can be put in jeopardy, e.g., if clinically significant antibodies go undetected in the type and screen, they will not be detected by an IS or electronic crossmatch. ∼ If an unexpected antibody is detected during the type and screen, it is identified and donor blood must be antigen typed and crossmatched (by tube IAT) so that it will be available if required during surgery.

1.9 Type and Screen test policy

1.9.1 The effect of the type and screen test policy on the clinicians

When the traditional FXM test was practised, blood units were reserved for a designated patient for 2 days. If the reserved units had been depleted or had exceeded the reservation date, repeat blood sampling and crossmatching would have been required if additional units were needed. This arrangement led to additional blood-taking by the front-line clinicians. In addition, as a repeat crossmatch required at least another 1 to 2 hours, depending on the FXM method-ology used, there was a tendency to overestimate the number of units that would be required. Hence, not only was the workload of the blood bank staff increased, but the blood stock needed for emergency use was also jeopardised.

Under the TS test policy, blood units are no longer reserved for a patient if the results from the antibody screen are negative. Instead, a validity period is given to an individual for their negative antibody screen status, so that within such a time period, as many units as possible can be issued after performing an abbreviated crossmatch, depending on the amount of serum available. For patients who have received a transfusion or who have been pregnant within the preceding 3 months of the transfusion, or whose history is unknown, the validity period given is 3 days, because antibodies may develop within that time (Walker, 1993).

The TS test policy also avoids the need for repeated crossmatching of neonatal blood when blood trans-fusion is required to replace blood drawn from the newborn for laboratory studies, including cross-matching. Because the immune system of the newborn is immature and relatively unresponsive to antigenic stimulation during the first 4 months of life, AABB standards permit a reduction in the stringency of pretransfusion compatibility for neonates. If the antibody screen and direct antiglobulin tests are both negative, compatibility testing may be omitted during any one hospitalisation, provided that the red blood cells transfused are group O- or ABO-identical, or compatible with both the mother and child (Klein, 1994).

This procedure reduces unnecessary blood taking for crossmatching and allows the freshest units to be selected when transfusion is required.

1.9.2 The effect of the type and screen test policy on patients

The implementation of the TS test policy provides many benefits to patients. The blood issued is safe and compatible. According to the conventional FXM policy, blood units are randomly crossmatched without information about the patient’s antibody status (Cheng et al 1995).

The presence of weak antibodies may be missed if donor blood is heterozygous for an antigen. This deficiency is corrected by the TS test policy, which stipulates that antibodies must be systematically screened using selected group-O reagent erythrocytes that harbor representative antigens. Patients who require a massive transfusion will benefit most from the TS test, because as many additional compatible blood units as required can be issued quickly without the need for taking a new blood sample for repeating the crossmatch. The use of unmatched group-O or group-specific blood is no longer practiced in this group of patients.

Neonates who are younger than 4 months are another group of patients who can benefit significantly from the TS test policy. For neonates with negative antibody screens, blood units can be issued without further crossmatching during the entire hospital stay up to 4 months of age. The problem of requiring maternal blood samples for repeated crossmatching now no longer exists.

For patients with positive antibody screens, anti-body identification will be performed. When specific antibodies are identified, antigen-negative units will be selected for the FXM. These antigen negative crossmatch compatible units can usually be reserved for up to 7 days and will not compromise the daily blood stock that has been allocated for emergency use. Previously, when the traditional FXM test was practiced, blood stock control was inflexible; thus, a blood unit could be reserved for a patient for only 3 to 4 days, depending on individual hospital policy.

For patients with multiple alloantibodies or autoimmune hemolytic anemia, the TS test policy offers the opportunity to promptly detect the existence of alloantibodies or

autoantibodies. Alloantibodies can be correctly identified or excluded and phenotype- specific units can be transfused. In contrast, according to the conventional FXM test, antibodies may not be suspected until blood units are repeatedly incompatible, a situation that would have resulted in at least a 3- to 4-hour delay. Finding compatible units thus remains difficult and if the implicated antibodies are not identified, patient care becomes compromised.

In a 2 year study, a total of 468 positive antibody screens were detected among 25471 crossmatch requests, giving a positive antibody rate of 1.83%. This rate includes false-positive antibody screens as well as antibodies that are rarely clinically significant, a b such as anti-Le , anti-Le , anti-P1, and other ‘cold’ antibodies. As for the clinically significant antibodies encountered, the most common is anti-M, followed by anti-E. These findings agree with those reported among Taiwanese (Broadberry and Lin, 1994) and Thai patients (Chandanayingyong and Bejrachandra, 1975).

In fact, the number of clinically significant antibodies that are detected among the Chinese population is less than 1% (Chan et al. 1996) whereas for Caucasians the rate of obtaining positive antibody screen is between 3% and 5%, with anti-K, anti-E, and anti-D being the most commonly detected antibodies (Heddle et al. 1992; Cordle et al. 1990; Hoeltge et al. 1995)

This finding illustrates the fact that the TS procedure is even more cost-effective among the Chinese population due to their unique antigen frequencies.

1.9.3 The effect of the type and screen test policy on the blood bank

There may be also a general fear among the technical staff towards the use of the abbreviated crossmacth test, since the test requires them to be quick (within 10 minutes) (within 10 minutes) and accurate. This apprehension was the major obstacle of initiating the use of the TS test.

From the management’s point of view, the ability of the blood bank to issue blood quickly is essential, so as to gain clinicians’ confidence in the TS method. Thus, as well as giving blood bank staff educational seminars and training about the antibody screen

and abbreviated crossmatch techniques, much emphasis should be put on the development of skills needed to perform the abbreviated crossmatch test.

By implementing the TS test policy, the number of unnecessary crossmatch tests is reduced, as is the number of blood returns and extensions. Human resources can be redirected to providing ward consultation and better transfusion services, especially to patients who have special needs (example major transfusion or treatment for trauma). For some hospitals, the use of the TS test has necessitated extra financial and human resources to investigate serological problems derived from positive antibody screens. Both the American and British standards stipulate that when an irregular antibody is detected, its specificity should be determined and its clinical significance assessed (Walker 1993).

Furthermore, for transfusion purposes, antigen negative blood should be selected for crossmatching if the recipient has a clinically significant antibody (Walker 1993).

Based on these standards, it is clear that any hospital providing the TS test should have in place means for investigating serological problems.

1.9.4 The effect of the type and screen test policy on the hospital

The aim of the TS test policy is to raise efficiency without compromising patient safety. The latter has been validated in a number of studies (Boral and Henry 1977; Oberman et al, 1978; Friedman 1979).

Boral and Henry (Boral and Henry 1977) examined 12848 blood specimens using the TS and FXM tests; 283 types of antibodies were detected in 247 patients. The screening cells used were able to detect 96.11% of the antibodies.

If the antigen frequencies corresponding to the antibodies that were not detected by the screening cells were also taken into consideration, the TS test was calculated to be 99.99% effective in preventing the transfusion of serologically incompatible blood (Boral and Henry 1977).

Reports of data concerning the safety of abbreviated compatibility testing have recently suggested that the FXM could be omitted from the pretransfusion testing without putting patients at risk (Heddle et al. 1992; Oberman et al. 1982; Shulman et al. 1985).

The efficiency of the TS test may be measured by calculating the crossmatch to transfusion (C/T) ratio. The more accurately that clinicians predict a patient’s blood needs, the closer the C/T ratio will approach 1:1. Thus a low C/T ratio signifies efficient hospital transfusion policy and practice, and vice versa.

1.9.5 The effect of the type and screen test policy on the future

Based on the foundation of the TS test policy, the 15th edition of the AABB Standards for Blood Banks and Transfusion (Widman 1993) described radical changes in the requirements for serological confirmation of ABO compatibility. Specific guidelines were outlined such that a computer can be used to determine which units of red blood cells can be given to a patient without having to perform an abbreviated crossmatch test. This procedure is commonly referred to as a ‘computer crossmatch’ or an ‘electronic crossmatch’ (EXM).

The EXM may be economically advantageous pro-vided that the computer system has been fully validated to prevent the issue of ABO-incompatible blood units. Significant time savings can be accrued by replacing the immediate-spin crossmatch with a computer crossmatch, as less time will be needed to prepare donor and recipient cells for testing (Widman 1993).

This is especially advantageous to patients who require a large amount of blood in a short time - for example, liver transplant recipients - as unlimited number of blood units can be issued efficiently without the need for a new sample to be taken. The EXM is currently being practiced in several local hospitals. The concept of the EXM can be adapted to a centralised transfusion program that uses the electronic allocation of blood at a site remote from the blood bank. This can be achieved through a networked electronic blood release system (Cox et al. 1997) or through a computer-generated list of crossmatch compatible blood units for a patient (Cheng et al. 1996).

With the electronic blood release system, an out-of-hours blood bank service can be made available to small hospitals without the need for staff. In addition, blood availability can be improved, and the C/T ratio and laboratory workload reduced (Cheng et al. 1996).

The principles of the TS method, other than EXM, can be extended to allow preadmission pretransfusion work-up, especially for patients undergoing elective surgery, who can be admitted on the morning of the operation and still have blood available when required. The same-day admission policy is commonly used for patients undergoing minor surgical operations that do not require blood crossmatching. As for other operations that may necessitate a blood transfusion, patients are commonly admitted on the preceding evening for pretransfusion work-up. According to the TS test policy, the need to obtain a blood sample within 3 days of the intended transfusion date can be avoided if the patient has not been transfused or become pregnant within the preceding 3 months, because the antibody status is expected to remain unchanged in the absence of alloimmunisation (Marosszeky et al. 1997)

A modification of TS test policy is to give patients their pretransfusion test while they attend the preadmission clinic, which can be 2 to 4 weeks before the planned date of operation.

If the antibody screen is negative, the patient can be admitted to hospital on the morning of the operation and the antibody validity period extended such that blood units can be released immediately if required. On the other hand, if the antibody screen is found to be positive during the preadmission work-up, ample time will be available for resolving the antibody.

Antibody-positive patients may also be admitted on the day of surgery with antigen-negative units readily available for a crossmatch test. The incorporation of the TS protocol into the preadmission work-up will enable a larger group of patients with elective operation to enjoy the benefit of same day admission. Furthermore, allowing more same-day admissions will no doubt be economically advantageous.

The TS test policy has proven to be safe, efficient, and beneficial to the transfusion practice. Hospitals that are currently experiencing a high C/T ratio and blood

expiry rate or that have a large workload of elective surgeries should consider adopting such a policy to allow better transfusion management.

Chapter 2

Materials and Methods

2.1 The gel card technology

The gel test, as developed by Lapierre, is an innovative approach to blood group serology. This technology addresses the issue of standardization and incorporates sensitivity, specificity and efficiency. The gel test (ID-MTS; Ortho-Clinical Diagnostics, Raritan, NJ) was released in Europe in 1988 and was available in the United States in 1995 (Lapierre et al. 1990).

By employing gels premixed with reagents, specific volumes and a no-wash antiglobulin test that eliminates resuspension of red cell buttons, the gel test reduces the variation inherent in conversional techniques.

The DiaMed-ID Micro Typing System utilises a sephadex gel to capture agglutinates in a semi-solid medium. This enhances visualisation of agglutination as compared to the traditional tube techniques. In the latter, the agglutinate, particularly in weak reactions, mixes with the free cells at the bottom of the tube, making visualisation difficult (http://www.sanbs.org.za/Congress/abstract75.html).

The capacity of the gel test to separate RBCs from their surrounding fluid permits an antiglobulin test to be performed without washing. At the beginning of centrifugation, the RBCs are pulled away from the suspension medium (unbound serum globulin) and enter the gel first. The surrounding medium remains above the gel and the characteristics of the gel prevent the medium from interfering with the antiglobulin reaction. Sensitized RBCs agglutinate as they come in contact with the antiglobulin reagent in the gel and are trapped. Unsensitized RBCs are not agglutinated and pass through the gel to pellet at the bottom of the microtube.

The gels may also contain other elements: preservatives such as sodium azide, sedimenting agents such as bovine serum albumin, and, in some cases, specific reagents such as anti-IgG or other RBC-specific antisera (ABO and D). When gels are to contain specific reagents, the manufacturer adds the reagents to the gel during preparation, before the microtube is filled. Thus, the reagent is dispersed throughout the length of the gel column. The gel column is about 75 percent packed gel and 25 percent liquid. Six of these microtubes are embedded in a plastic card to allow ease of handling, testing, reading, and disposal (Malyska and Weiland 1994).

2.1.1 Principle

The basic principle of the gel test is that, instead of a test tube, the serum and cell reaction takes place in a microtube consisting of a reaction chamber that narrows to become a column about 15 mm long and 4 mm wide.

Six microtubes are held together in a plastic card about the same size as a credit card, thus minimizing labeling and handling. Each microtube within a card may contain the same gel or a variety of different gels, depending on the application. The cards are filled with the respective gels at the point of manufacture and then sealed.

The gel test uses the principle of controlled centrifugation of red blood cells through a dextran-acrylamide gel and appropriate reagents pre-dispensed in a specifically designed microtube. Measured volumes of serum or plasma and/or red blood cells are dispensed into the reaction chamber of the microtube. The "card" is incubated and then centrifuged. If agglutination is present, the red cells are trapped in the gel and cannot travel through the gel during centrifugation. Agglutinated red cells that are present remain fixed or suspended in the gel. Unagglutinated red cells travel unimpeded through the length of the microtube, forming a pellet at the bottom. Unlike agglutination observed with traditional test tube hemagglutination methods, the gel test reactions are stable, allowing observation or review over an extended period of time (http://www.bloodlink.bc.ca/newsletter/bm-oct99.htm).

2.1.2 Advantages of the gel test

Advantages of the gel test include small sample size, decreased variation in volume delivery, greater uniformity between repeat tests, no cell washing step and decreased technique dependence.

Applicable to a broad range of tests routinely performed in the blood bank, such as antibody screening, antigen typing, and crossmatching, gel test procedures are easy to perform and yield clear-cut end points that are stable; besides, they can be reviewed at a later time.

The gel test is also easy to learn, since there are relatively few steps in the procedure and it provides a clear, easy-to-read, stable endpoint. When the microtubes are covered and refrigerated, the gel cards can be read with accuracy for at least 24 hours after testing.

Sensitivity and specificity of gel testing have been found to be comparable to the tube LISS-IAT. However, there are a few disadvantages. For reference laboratories, batch testing is less of an option because of urgency, timing and variation in samples. Therefore, the efficiency made possible by batch testing is often lost.

Complexity of antibody identification usually requires multiple runs of selected cells. Although a 0.8 percent panel of RBCs is commercially available, any other test cell that needs to be tested must be prepared as a 0.8 percent concentration in the appropriate diluent prior to use. This is more cumbersome than adding the cells right from the vial into a test tube.

The ABO and Rh typing may also have a disadvantage, depending on the laboratory’s needs. The RBCs must be incubated in Diluent 1 for 10 minutes prior to centrifugation in the appropriate card. Therefore, it takes a minimum of 20 minutes (10 minutes incubation and 10 minutes centrifugation) to perform an ABO and Rh test. The test-tube method still wins on this one with ease and speed. Although direct antiglobulin tests (DATs) can be done on the gel system, only a DAT using anti-IgG is available.

Finally, rouleaux and incompletely clotted samples may cause patterns that resemble positive reactions.

2.1.3 Interpretation of results

After centrifugation, positive reactions are indicated by RBC agglutinates trapped anywhere in the column of the gel. Positive reactions can be graded from 0 to 4+. 1. A 4+ reaction is indicated by a solid band of RBCs on top of the gel. 2. A 3+ reaction displays agglutinated RBCs in the upper half of the gel column. 3. A 2+ reaction is characterized by RBC agglutinates dispersed throughout the length of the column. 4. A 1+ reaction is indicated by RBC agglutinates mainly in the lower half of the gel column with some unagglutinated RBCs pelleted at the bottom. 5. Negative reactions display a pellet of RBCs at the bottom of the microtube and no agglutinates within the matrix of the gel column.

Mixed-field reactions can also be observed in the gel test. These reactions are more commonly encountered during RBC typing procedures, rather than during serum or plasma testing methods, but in either case they are easy to recognize. Antigenpositive RBCs, in this case, are completely agglutinated by the specific antisera present and they lie at the top of the gel, whereas the remainder of the RBCs that are antigen negative do not agglutinate and pellet at the bottom.

Figure 2.1: Agglutination strengths (Serology SOP NBTC Labs – Malta)

2.2 Materials

2.2.1 Apparatus

The basic pieces of equipment and materials required for the gel test are the dedicated incubator, centrifuge and RBC diluents. Accessories are also available to make the testing run smoothly. These include specially designed workstations, automatic pipettes with disposable tips and an automated reader.

2.2.1.1 Micro Typing System working station

Figure 2.2: Micro Typing System card rack (www.ld.ru/catalog/firms/diamed/images/id-working-table.jpg)

2.2.1.2 ID-dispensers and automatic pipettes

Figure 2.3: ID-Dispensers (http://cgi.diamed.de/geraete_pix/aplatz_gesamt_r1_c3.gif)

Figure 2.4: ID-Automatic pipettor (http://diamed.com/product_detail.asp?id=610)

2.2.1.3 The ID-Incubator

Figure 2.5: ID-Incubator (http://cgi.diamed.de/geraete_pix/aplatz_gesamt_r1_c2.gif)

2.2.1.4 The ID-Centrifuge

The only special equipment that is required for the gel test is the centrifuge.

Figure 2.6: ID-Centrifuge (http://cgi.diamed.de/geraete_pix/aplatz_gesamt_r1_c1.gif)

A specific centrifuge has been developed to ensure that the correct centrifugation parameters are reliably achieved. Pipetting and dispensing devices capable of accurately measuring the designated volumes are needed. In addition, a 37°C incubator is required for certain applications. Appropriate gel test cards, diluents, reagent red cells and test samples are also necessary to perform the assays. Each laboratory's specific requirements for equipment depends on the workflow, existing equipment and applications selected.

Centrifugation plays a key role in the gel test and the centrifugation criteria are extremely important.

The axis of the microtube during centrifugation must be held strictly in line with the direction of the centrifugal force. In addition, the speed and time of centrifugation are critical. If the microtubes are centrifuged too long or too fast, the weakly agglutinated RBCs may be driven through the gel, thereby causing false-negative results.

Alternatively, if centrifugation is too slow or too short, all of the unagglutinated RBCs may not reach the bottom of the microtube, potentially leading to a false-positive result.

The optimal centrifugation parameters are a relative centrifugal force of 80 applied over 10 minutes.

2.2.1.5 ID-Reader M

Figure 2.7: ID-Reader M (http://cgi.diamed.de/geraete_pix/readerM.jpg)

The Reader M instrument utilizes advanced image analysis to digitally read gel cards within seconds. Results can be archived or printed on customized reports

2.2.2 Reagents

2.2.2.1 Test cells for reverse grouping (ID-DiaCell ABO) and for antibody screening (ID-DiaCell I-II-III)

Test cell reagents are routinely used in blood group serology to detect the presence of anti-A and anti-B isoagglutinins (in reverse grouping) and for the detection of irregular antibodies (in antibody screening and identification purposes).

For reverse grouping, test cell reagents of group A1 and B/ A1, A2 and B/or A1, A2, B and O are used, following various requirements and guidelines.

For the detection of irregular antibodies, the requirements for antigen configuration are stringent. Since some of the antibodies show a dosage effect, screening cells should posses antigens in presumed double dose form within the Rh, Duffy, kidd and MNS blood group systems. Certain, guidelines require that the Lewis antigen be present, as should the rare antigen Kpa Kel3.

Considering these requirements, DiaMed provides a complete line of test cell reagents for reverse grouping, for antibody screening and identification by the indirect antihuman globulin test (IAT), by the NaCl test procedure and by the two-stage enzyme technique which can enhance the reaction of certain antibodies, notably in the Rh, Kell and Kidd system.

The test cell reagents are specially designed for the ID-Micro Typing System, ready-to-use in a 0.8% suspension.

2.2.2.1.1 Reagent content

All cells are of human origin, in buffered suspension medium at 0.8% (±0.1%). Preservatives: the antibiotics trimethoprim and sulfamethoxazole.

1. For reverse grouping ID-DiaCell ABO: A1 B

2. For antibody screening (single donors, blood group O) w 2 2 ID-DiaCell I-II-III: R1 R1+R R +rr for IAT and NaCl test ID-DiaCell I-II-III P: Papainized, for enzyme technique

Store at 2-8°C and for stability, see expiratory date label.

2.2.2.1.2 Sample material

Draw blood samples using acceptable phlebotomy techniques. Preferably, blood samples should be drawn into Citrate, EDTA, Heparin or CPD-A. For reliable results, use of freshly collected blood is indicated. When the use of serum instead of plasma is required, the serum must be well cleared, by centrifugation at 1500g for 10 minutes, before use to avoid fibrin residues, which may interfere with the reaction pattern.

2.2.2.1.3 Controls

Controls should be included in accordance with the relevant guidelines of quality assurance.

2.2.2.1.4 Use of the ID-Test cell reagents

• All test cell reagents are for use with the ID-Cards of the DiaMed-ID Micro Typing System only • Strictly follow the test procedures as described in the specific package inserts of the ID-Cards to be used. • Always gently resuspend the red cells, by inverting the vial several times before use and also before placing the vials into a pipetting automate. • Make suree that the test cells are at room temperature (18-25°C) when in use. • During the working procedures, check that the test cell reagents remain in suspension. If there is settling of the cells, resuspend them again. • For the ID-System, precise pipetting is of importance. Use the ID-Pipetors for serial pipetting. Except for an emergency single test, avoid using vial dropper which may not provide reproducible and exact dispensing volumes. • Avoid contamination of the test cell reagents. • When recording the reactions, ensure that the lot number of the antigram corresponds with the lot number of the reagent vials. • After use, close the vials and replace them in the refrigerator.

2.2.2.1.5 Limitations

• Bacterial or other contamination of materials used can cause false positive or false negative results. • Strict adherence to the procedures and recommended equipment is essential. The equipment should be checked regularly according to GLP procedures.

2.2.2. ID-DiaPanel test cells

The reliability of antibody detection is largely dependent on the availability of test cells with appropiate antigens and on the sensitivity of the test method used.

The requirements for antigen configuration are stringent: it must allow the safe detection of all clinically significant antibodies. For the Rh system, MNS, Duffy and Kidd, the antigens must be in homozygous form. The Lewis antigens most be present, as should the rare antigen Kpa.

It is generally considered most effective to perform a screening test by both anti- human globulin (AHG) and enzyme test procedures. Due to the high sensitivity of the indirect anti-human globulin test (IAT) with new procedures such as the DiaMed Micro typing Syetm, some scientists in various countries have formed the opinion that the enzyme test has become somewhat less important.

However, enzyme techniques are useful when increased sensitivity in antibody screening is desired or where more than one antibody may be present. They enhance the reactions of certain antibodies, notably in the Rh, Kell and Kidd system, whereas antibodies to the enzyme-sensitive antigens may not be detected, notably the Duffy and MNS system.

The red cells are specifically designed for the ID-Micro Typing System.

2.2.2.2.1 Reagent content

All cells are of human origin, in buffered suspension medium at 0.8% (±0.1%), with preservatives the antibiotics trimethoprim and sulfamethoxazole.

1. For antibody identification, single donors, blood group O ID-DiaPanel: 11 test cells for IAT and NaCl test ID-DiaPanel P: 11 test cells papainized, for enzyme technique.

Store at 2-8°C and for stability, see expiratory date label.

2.2.2.2.2 Sample material

For optimal results, the determination should be performed using a freshly drawn sample, or in accordance with the local laboratory procedures for sample acceptance criteria. Preferibily, blood samples should be drawn into citrate, EDTA or CPD-A anticoagulants. Samples drawn into plain tubes (no anticoagulant) may also be used.

2.2.2.2.3 Controls

Controls should be included in accordance with the relevant guidelines of quality assurance.

2.2.2.2.4 Use of the ID-test cell reagents

• All test cell reagents are for use with the ID-Cards of the DiaMed-ID Micro Typing System only • Strictly follow the test procedures as described in the specific package inserts of the ID-Cards to be used. • Always gently resuspend the red cells, by inverting the vial several times before use and also before placing the vials into a pipetting automate. • Make sure that the test cells are at room temperature (18-25°C) when in use. • During the working procedures, check that the test cell reagents remain in suspension. If there is settling of the cells, resuspend them again. • For the ID-System, precise pipetting is of importance. Use the ID-Pipetors for serial pipetting. Except for an emergency single test, avoid using vial dropper which may not provide reproducible and exact dispensing volumes. • Avoid contamination of the test cell reagents. • When recording the reactions, ensure that the lot number of the antigram corresponds with the lot number of the reagent vials. • After use, close the vials and replace them in the refrigerator.

2.2.2.2.5 Limitations

• Bacterial or other contamination of materials used can cause false positive or false negative results. • Strict adherence to the procedures and recommended equipment is essential. The equipment should be checked regularly according to GLP procedures

2.2.2.3 ID-Diluent 1

Proteolytic enzyme modifyed red blood cell antigens and thus enhances the reactivity of some antigen/antibody systems and suppress others such as M, N, S, Fya and Fyb.

The most commonly used enzymes in blood group serology are papain and bromelin.

Papain is generally used for enzyme treatment of red blood cells prior to their use, whereas bromelin is often used as an additive reagent.

Enzyme reagents produced within an immunohaematology laboratory are difficult to standerdise with regard to activity and concentration of the enzyme. Standerdisation is important for consistent reliable results.

“ID-Diluent 1” is a modified bromelin solution in which the enzyme activity is standerdised and stabilised for a long period, specially prepared for the DiaMed-ID Micro typing System. “ID-Diluent 1” is used for preparing suspensions of red cells for blood grouping and as an additive for enzyme tests with untreated red cells for antibody detection and crossmatching.

2.2.2.3.1 Reagent content

1. ID-Diluent 1 Modified bromelin solution for red cell suspension, in 100 and 500ml vials. Preservative: the antibiotics trimetoprim and sulfamethoxazole.

Store at 2-8°C and for stability, see expiratory date label.

2.2.2.3.2 Sample material

Draw blood samples using acceptable phlebotomy techniques. Preferably, blood samples should be drawn into Citrate, EDTA, Heparin or CPD-A. For reliable results, use of freshly collected blood is indicated.

2.2.2.3.3 Limitations

• Bacterial or other contamination of materials used can cause false positive or false negative results. • Strict adherence to the procedures and recommended equipment is essential. The equipment should be checked regularly according to GLP procedures

2.2.3 Cards

2.2.3.1 DiaClon ABO/D + Reverse Grouping Card

ABO Blood typing, using anti-A and anti-B test sera, is known as direct or forward grouping. Serum (reverse) grouping, using A and B red cells to detect the presence or absence of anti-A/or anti-B. (From kit)

Determination of blood groups must be based on both forward and reverse grouping.

The ID-Card “DiaClon ABO/Rh+ + Reverse Grouping” allows combined testing of forward and reverse grouping as well as Rh D determination.

2.2.3.1.1 Card reagent content

ID-Card “DiaClon ABO/D + Reverse Grouping” contains monoclonal anti-A and anti-B and anti-D, within the gel matrix. The microtube “ctl” is the negative control. Two microtubes with “neutral” gel for reverse grouping A1 and B cells

Store at room temperature (18-25°C) and do not store near any heat, air conditioning sources or ventilation outlets. For stability, see expiratory date.

2.2.3.1.2 Sample material

1. For blood grouping Draw blood samples using acceptable phlebotomy techniques. Preferably, blood samples should be drawn into Citrate, EDTA, Heparin or CPD-A. For reliable results, use of freshly collected blood is indicated.

2. For reverse grouping When the use of serum instead of plasma is required, the serum must be well cleared, by centrifugation at 1500g for 10 minutes, before use to avoid fibrin residues, which may interfere with the reaction pattern.

Where samples are not for immediate testing they should be stored at 2-8°C after separation for a maximuim of 48hours, thereafter at –20°C.

2.2.3.1.3 Controls

Known positive and negative samples should be included for validation of the results obtained.

2.2.3.1.4 Remarks

Do not use ID-Cards which show signs of drying, have bubbles or damaged seals. Allow the test cell reagents and samples to reach room temperature before use and resuspend gently.

2.2.3.1.5 Observation on reaction

The negative control (ctl) must always show a neagtive reaction. If the negative ctl is positive proced as follows: • Wash the red cells first with NaCl 0.9% (or ID-Diluent 2) before preparing the 5% cell suspension • Then proceed as under according to the normal procedure.

Where a discrepancy occurs between the results of ABO typing and reverse grouping, consult the “DiaMed ABO discrepancy chart” for appropiate information.

Full forward and reverse grouping requires the use of anti-A, -B, -AB, resp. A1, A2, B and O cells. The “DiaClon ABO/D + Reverse Grouping” does not contain anti-AB and allows the use of A1 and B cells only. It should be used only for confirmation previously fully tested samples with established blood group status.

2.2.3.1.6 Limitations

• Bacterial or other contamination of materials used can cause false positive or false negative results. • Fibrin residues in the serum or red cell aggregates in the red cell suspension may trap non-agglutinated cells presenting a fine pink line on top of the gel while most of the cells are on the bottom of the microtube after centrifugation. • Strict adherence to the procedures and recommended equipment is essential. The equipment should be checked regularly according to GLP procedures. • Use of suspension solutions other than ID-Diluent1 may modify the reactions. • Too heavy or too light red cell suspension can cause aberrant results.

2.2.3.2 ID-Card “Reverse Grouping with Antibody Screening” card

ABO Blood group determination requires that reverse grouping using plasma or serum should be carried out to confirmthe accuracy of ABO red cell typing.

Reverse grouping uses red cell reagents of known ABO antigen specificity to indicate the presence or absence of anti-A and anti-B isoagglutinine, the results of which determine the reverse grouping. Discrepancies between forward and reverse grouping required further investigation.

Good laboratory practice indicates that ABO grouping is incomplete without a screening procedure to detect the presence of irregular antibodies.

The ID-Card “Reverse Grouping with Antibody Screening” is used with the test cell reagents “ID-DiaCell ABO/I-II” or “ID-DiaCell ABO/I-II-III”, allowing reverse grouping and antibody screening (indirect antiglobulin test at room temperature) in an easy single test procedure.

2.2.3.2.1 Card reagent contents

ID-Card “Reverse Grouping with Antibody Screening” card consisting of 3 microtubes containing neutral gel and 3 microtubes containing anti-human globulin serum (rabbit anti-IgG and monoclonal anti C3d, within the gel matrix. Preservative:

<01%NaN3.

Store at room temperature (18-25°C) and do not store near any heat, air conditioning sources or ventilation outlets. For stability, see expiratory date.

2.2.3.2.2 Sample material

Draw blood samples using acceptable phlebotomy techniques. Preferably, blood samples should be drawn into Citrate, EDTA, Heparin or CPD-A. For reliable results, use of freshly collected blood is indicated.

When the use of serum instead of plasma is required, the serum must be well cleared, by centrifugation at 1500g for 10 minutes, before use to avoid fibrin residues, which may interfere with the reaction pattern.

Where samples are not for immediate testing they should be stored at 2-8°C after separation for a maximum of 48hours, thereafter at –20°C.

2.2.3.2.3 Controls

Known positive and negative samples should be included for validation of the results obtained.

2.2.3.2.4 Remarks

Do not use ID-Cards which show signs of drying, have bubbles or damaged seals. Allow the test cell reagents and samples to reach room temperature before use and resuspend gently.

2.2.3.2.5 Observation on reactions

• Negative reactions with all antibody screening cells indicate the absence of detectable antibodies. • Positive reactions with one or more antibody screening cells suggest the presence of irregular antibodies, and further investigation is required to determine the antibody specificity. • When there is a positive reaction with each antibody screening cell, it is advised to perform an autocontrol and to proceed to further tests (complete antibody screening at 37°C).

2.2.3.2.6 Limitations

• The optimal reaction conditions may vary considerably from one type of antibody to another. Consequently, one simple test method may be insufficient to detect all antibodies and combined test procedures should be applied. • Certain drugs are known to cause a positive reaction in anti-himan globulin procedures. • Some pathological conditions are also reported as causing positive reactions in anti-human globulin procedures. • Bacterial or other contamination of materials used can cause false positive or false negative results. • Fibrin residues in the serum or red cell aggregates in the red cell suspension may trap non-agglutinated cells presenting a fine pink line on top of the gel while most of the cells are on the bottom of the microtube after centrifugation. • Strict adherence to the procedures and recommended equipment is essential. The equipment should be checked regularly according to GLP procedures. • Use of suspension solutions other than the specified ID-Diluent may modify the reactions. • Too heavy or too light red cell suspension can cause aberrant results.

2.2.3.3 ID-Card “Liss/Coombs”

Polyspecific anti-human globulin (AHG) reagents are used for routine detection and identification, compatibility tests and the direct anti-human globulin test (DAT).

The most important function of the polyspecific AHG reagent is to detect the presence of IgG. The importance of anticomplement in the AHG reagent is debatable since antibodies detectable only by their ability to bind compliment are rather rare. However, anti-C3d activity is important for the DAT in the investigation of autoimmune haemolytic anaemia (AIHA). A positive DAT generally indicates that the red cells are coated in vivo with immunoglobulin and/or compliment.

The ID-Card “Liss/Coombs” is siutable for the DAT, for the compatibility test, for antibody screening and identification with “ID-DiaCell” and “ID-DiaPanel”.

2.2.3.3.1 Card reagent content

The microtubes of the ID-Card “Liss/Coombs” contain plolyspecific AHG, to be used for antibody screening, anti-body identification, crossmatch and the DAT. For the indirect antihuman globulin test (IAT), labour intensive washing procedures are eliminated, due to the fact that the red cell suspension is added to the microtubes before the plasma/serum, creating a barrier over the gel suspension, thus avoiding neutralization of the AHG by serum IgG proteins.

Store at room temperature (18-25°C) and do not store near any heat, air conditioning sources or ventilation outlets. For stability, see expiratory date.

2.2.3.3.2 Sample material

The determination should be with a blood sample not older than 24 hours after sampling, max 1 week storage at 2-8°C. Draw blood samples using acceptable phlebotomy techniques. Preferably, blood samples should be drawn into Citrate, EDTA, Heparin or CPD-A.

When the use of serum instead of plasma is required, the serum must be well cleared, by centrifugation at 1500g for 10 minutes, before use to avoid fibrin residues, which may interfere with the reaction pattern.

Where samples are not for immediate testing they should be stored at 2-8°C after separation for a maximum of 48hours, thereafter at –20°C.

2.2.3.3.3 Controls

Controls should be included in accordance with the relevant guidelines of quality assurance.

2.2.3.3.4 Remarks

Do not use ID-Cards which show signs of drying, have bubbles or damaged seals. Allow the test cell reagents and samples to reach room temperature before use and resuspend gently.

As for all products covered by GLP rules, the sensitivity of the above procedures should be validated using antibodies of known potency.

2.2.3.3.5 Observations on reaction

2.2.3.3.5.1 Antibody screening reactions

• A negative reaction indicates the absence of detectable irregular antibodies in the patient’s or donor’s serum or plasma. • A positive reaction indicates the presence of irregular antibodies. Enter the reactions obtained on an antigram. Verify that the lot number of the test cell reagents “ID-DiaCell I-II-III” or “ID-DiaCell I-II-III P” corresponds to the lot number indicated on the antigram. • Following the reaction pattern ant the antigen configuration, the type of antibody present may be indicated. Perform the usual further tests to identify the antibody. • A positive reaction with one or more test cells and a negative autocontrol suggest the presence of a specific antibody. • A positive reaction with all test cells and a positive autocontrol may be due to a non-specific reaction. • A positive reaction with all test cells and a positive autocontrol but one or more test cells showing a stronger positive reaction than the autocontrol, the patient sample should be submitted for further testing to investigate the possibility of an underlying allo-antibody.

2.2.3.3.5.2 Antibody Identification reactions

• A positive reaction indicates the presence of irregular antibodies. Enter the reactions obtained on an antigram. Verify that the lot number of the test cell reagents “ID-DiaPanel” corresponds to the lot number indicated on the antigram. • Following the reaction pattern and the antigen configuration, the type of antibody present can, in the most cases, be identified (autocontrol must be negative). • A positive reaction with all “ID-DiaPanel” test cells and a negative autocontrol suggest the presence of non-specific reactions or may indicate the presence of an alloantibody directed against a high frequency antigen. • A positive reaction with all test cells and a positive autocontrol may be due to a non-specific reaction.

• A positive reaction with all “ID-DiaPanel” test cells and the autocontrol but with one or more test cells showing a stronger positive reaction than the autocontrol, the patient sample should be submitted for further testing to investigate the possibility of an underlying alloantibody and further investigation should be undertaken.

2.2.3.3.5.3 Compatibility testing

• A negative reaction indicates compatibility of the donor blood with the recipient. • A positive reaction indicates incompatibility of the donor blood with the reception, due to presence of antibodies directed against antigens on the donor red cells. Further investigation to identify the antibody specificity should be performed.

2.2.3.3.6 Limitations

• Certain drugs are known to cause a positive reaction in anti-himan globulin procedures. • Some pathological conditions are also reported as causing positive reactions in anti-human globulin procedures. • Cells that have become polyagglutinable, due to cryptantigen exposure e.g. T antigen, either in vivo or in vitro may react with all human sera. Further investigation of such reactions is required. • Bacterial or other contamination of materials used can cause false positive or false negative results. • Fibrin residues in the serum or red cell aggregates in the red cell suspension may trap non-agglutinated cells presenting a fine pink line on top of the gel while most of the cells are on the bottom of the microtube after centrifugation. • Srict adherence to the procedures and recommended equipment is essential. The equipment should be checked regularly according to GLP procedures. • Use of suspension solutions other than ID-Diluent1 may modify the reactions. • Too heavy or too light red cell suspension can cause aberrant results.

2.2.3.4 ID-Card “NaCl, enzyme test and cold agglutinins”

Saline techniques are used to detect antibodies that react predominantly at 4°C or at 18-25°C (room temperature), such as anti-M, -N, -P1, -Lea, -Leb, -I. They are also used to define haemolytic anaemia associated with cold antibodies.

Enzyme techniques are useful when increased sensitivity in antibody screening is desired. It enhances the reactions of certain antibodies, notably in Rh, Kell and Kidd systems. Since photolytic enzymes usually destroy some antigens such as M (MNS1), N (MNS2), S (MNS3), Fya (FY1), Fyb (FY2), an enzyme technique should never be the only method employed.

It is not considered necessary to include the room temperature saline test in routine antibody screening procedures, but the saline test at 4°C can be used to detect cold agglutinin.

The ID-Card “NaCl, enzyme test and cold agglutinins” is suitable for antibody screening and identification procedures, compatibility testing and reverse grouping.

2.2.3.4.1 Card reagent content

ID-Card “NaCl, enzyme test and cold agglutinins” with 6 microtubes containg neutral gel suspension and as a presevative < 0.1% NaN3.

Store at room temperature (18-25°C) and do not store near any heat, air conditioning sources or ventilation outlets. For stability, see expiratory date.

2.2.3.4.2 Sample material

The determination should be with a blood sample not older than 24 hours after sampling, max 1 week storage at 2-8C. Draw blood samples using acceptable phlebotomy techniques. Preferably, blood samples should be drawn into Citrate, EDTA, Heparin or CPD-A.

When the use of serum instead of plasma is required, the serum must be well cleared, by centrifugation at 1500g for 10 minutes, before use to avoid fibrin residues, which may interfere with the reaction pattern.

2.2.3.4.3 Controls

Controls should be included in accordance with the relevant guidelines of quality assurance.

2.2.3.4.4 Remarks

Do not use ID-Cards which show signs of drying, have bubbles or damaged seals. Allow the test cell reagents and samples to reach room temperature before use and resuspend gently.

As for all products covered by GLP rules, the sensitivity of the above procedures should be validated using antibodies of known potency.

If hemolysis is observed in the upper part of the microtube in the absence of a hemolysed sample it should be interpreted as positive.

2.2.3.4.5 Observation on reaction

• One or more positive reaction indicates the presence of irregular antibodies. Enter the reactions obtained on an antigram. Verify that the lot number of the test cell reagents corresponds to the lot number indicated on the antigram. • Following the reaction pattern and the antigen configuration, the type of antibody present can, in the most cases, be identified (autocontrol must be negative). • A positive reaction with all “ID-DiaPanel P” test cells and a negative autocontrol suggest the presence of non-specific reactions or may indicate the presence of an alloantibody directed against a high frequency antigen. • A positive reaction with all test cells and a positive autocontrol may be due to a non-specific reaction. • A positive reaction with all “ID-DiaPanel P” test cells and the autocontrol but with one or more test cells showing a stronger positive reaction than the autocontrol, the patient sample should be submitted for further testing to investigate the possibility of an underlying alloantibody and further investigation should be undertaken. • It should be noted that enzyme auto-antibodies may react less strongly with the patient’s cells in the one-stage technique than against “ID-DiaCell I-II-III P” (two stage technique). • Strong cold antibodies can b the cause of positive reactions, especially in the enzyme test procedure. Complete antibody screening procedures should be undertaken. • Anti-I is a common specificity in cold agglutinin disease.

2.2.3.4.6 Limitations

• Bacterial or other contamination of materials used can cause false positive or false negative results. • Fibrin residues in the serum or red cell aggregates in the red cell suspension may trap non-agglutinated cells presenting a fine pink line on top of the gel while most of the cells are on the bottom of the microtube after centrifugation.

• Strict adherence to the procedures and recommended equipment is essential. Use of suspension solutions other than ID-Diluent1 may modify the reactions. • Too heavy or too light red cell suspension can cause aberrant results.

2.3 Methods

2.3.1 ABO and Rh D typing

2.3.1.1 Principle

The reverse group uses cells with known ABO blood group antigens on their surface. These are reacted with the individual's serum. An individual's serum possesses naturally occurring antibodies to the A, B, H antigens that their cells lack. If the naturally occurring antibodies recognize the corresponding antigens on the cells with the known ABO antigen they will agglutinate these cells.

Antibodies that react with red cell antigens coat the red cell and sometimes agglutination results. When antibody-coated red cells are added to an anti-human globulin solution the antibodies in this solution react with the antibody on the red cells and agglutination results. When agglutinates are centrifuged through gel, the rate of travel through the column is proportional to the size of the agglutinates. In the DiaMed system for typing, red cells are spun into a column containing antiserum to the red cell antigens. For screening, the patient's serum and reagent red cells are placed in the reaction chamber above the column. Upon centrifugation, the red cells are exposed to the antiglobulin reagent and agglutinated cells are trapped. Non-agglutinated cells that are not impeded form a button at the base of the column. The difference in specific gravity between the red cells and serum results in the red cells passing into the column while the serum is excluded (less dense). This eliminates the wash step traditionally required when the antiglobulin test is carried out in tubes. The polymers in the diluent, in addition to excluding the serum, also enhance agglutination (http://www.palmslab.com.au/Education/transfus/grouping.shtml).

2.3.1.2 Sample and Materials required

1. Blood sample in EDTA. 2. ID-Diluent 1 at room temperature.

3. 0.8% suspension of reagent red cells (ID-DiaCell A1 and ID-DiaCell B). 4. Micropipette. 5. DiaMed system centrifuge. 6. Disposable pipette tips. 7. ID-Reader M.

2.3.1.3 Procedure

1. Sample preparation Prepare a 5% red cell suspension in ID-Diluent 1 as follows: • Dispense 0.5ml of ID-Diluent 1 into a clean test tube. • Add either 50µl of whole blood or 25µl of packed cells. • Mix gently. • Allow to stand for 10 minutes at room temperature.

2. Method • Label the cassette with the patient’s donation number and remove aluminium foil.

• Add 50µl ID-DiaCell A1 and ID-DiaCell B to the respective reaction chamber of a microtube that contains neutral gel, followed by 50µl of serum or plasma to be tested. Incubate for 10 minutes at 18-25°C. • Add 10µl of patient red cell suspension to the cassette containing specific typing reagents. • Centrifuge the card using the DiaMed centrifuge for 10 minutes. • Read the front and back of the individual columns for agglutination. • Read and record the results by means of the ID-Reader M machine.

3. Interpretation of results

Figure 2.8: ABD-ctl-A1B Gel Card (Serology SOP NBTC Labs – Malta)

A positive reaction {score 3 (1+w); 5 (1+); 8 (2+); 10 (3+); 12 (4+)} is recorded when red cells are retained in or above the gel column after centrifugation.

A negative reaction is recorded (as 0) when a distinct button of cells sediment to the bottom of the column after centrifugation.

Anti-A Anti-B Anti-D Ctl A1 B Blood Group ++++ Negative ++++ to + Negative Negative ++++ to + A Positive ++++ Negative Negative Negative Negative ++++ to + A Negative Negative ++++ ++++ to + Negative Negative Negative B Positive Negative ++++ Negative Negative Negative Negative B Negative Negative Negative ++++ to + Negative ++++ to + ++++ to + O Positive Negative Negative Negative Negative ++++ to + ++++ to + O Negative ++++ ++++ ++++ to + Negative ++++ to + Negative AB Positive ++++ ++++ Negative Negative ++++ to + Negative AB Negative Table2.1: Blood Group interpretation (Serology SOP NBTC Labs – Malta)

2.3.2 Antibody Screening

2.3.2.1 Principle

Over 500 antigens can be detected on human RBCs. Some of these are found on other tissues. Antigens that are carried by a particular cell line of almost all persons are known as high-incidence or public antigens. Others, of low incidence, are sometimes called private antigens.

Human blood is classified, or typed, according to the presence or absence of certain markers (called antigens) on the surface of red blood cells. Blood typing tests are carried out before a person receives a blood transfusion or to check a pregnant woman's blood type. Blood typing may also be done to determine whether two people are likely to be blood relatives (for instance, to help establish paternity).

Antibody screening for unexpected anti-RBC antibodies is routinely done on pretransfusion specimens from prospective recipients and prenatally, on maternal specimens. Unexpected antibodies are specific for RBC blood group antigens other than a A and B, eg, Rh0(D), Kell (K), or Duffy (Fy ). Early detection is important because such antibodies can cause hemolytic disease of the newborn and serious transfusion reactions, and they may greatly complicate and delay compatibility testing and procurement of compatible blood.

The polyspecific antibody screening cassettes contain rabbit antibody to human IgG, C3b/c and C3d. The reagent contains a buffer solution with bovine albumin, sodium azide and macromolecules. The reagent is coloured blue. One cassette will accommodate two separate patient antibody screens. It is acceptable to only use one half of the cassette provided that the remaining half remains totally sealed. (http://www.palmslab.com.au/Education/transfus/screenin.shtml)

2.3.2.2 Sample and Materials required

1. Blood sample in EDTA. 2. ID-Diluent 2 at room temperature. 3. 0.8% suspension of reagent red cells (ID-DiaCell I-II-III and ID-DiaCell I-II- IIIPapainized). 4. Micropipette. 5. DiaMed system centrifuge. 6. DiaMed system incubator. 7. Disposable pipette tips. 8. ID-Reader M.

2.3.2.3 Procedure

1. Method • Label the cassette with the patient’s donation number and remove aluminium foil. • Add 50µl ID-DiaCell I-II-IIIP to the first three reaction tubes (orange/red) of the card. • Add 50µl ID-DiaCell I-II-III to the first three reaction tubes (green) of the card. • Add 25µl of patient’s plasma to each microtube. • Incubate the cards for 15 minutes at 37°C in the DiaMed incubator. • Centrifuge the card using the DiaMed centrifuge for 10 minutes. • Read the front and back of the individual columns for agglutination. • Read and record the results by means of the ID-Reader M machine.

2. Interpretation of results

Figure 2.9: Reverse Grouping and Antibody Screen Gel Card (Serology SOP NBTC Labs – Malta)

A positive reaction {score 3 (1+w); 5 (1+); 8 (2+); 10 (3+); 12 (4+)} is recorded when red cells are retained in or above the gel column after centrifugation.

A negative reaction is recorded (as 0) when a distinct button of cells sediment to the bottom of the column after centrifugation.

I-II-IIIP I-II-III Result ++++ to + Negative Antibodies Present (possibility of Cold Antibodies – most likely Anti-I) Negative ++++ to + Antibodies Present ++++ to + ++++ to + Antibodies Present (possibility of Cold Antibodies)

Negative Negative Antibodies Absent

Table2.2: Antibody screen interpretation (Serology SOP NBTC Labs – Malta)

Enter the reactions obtained in the antigen table (Appendix 2) and following the reaction pattern the particular antibody or antibodies can be suspected.

2.3.3 Antibody identification

2.3.3.1 Principle

Following a positive screen the serum is reacted with a panel of cells. It is essential that antibodies are quickly and accurately identified in order that donor blood may be provided (http://www.palmslab.com.au/Education/transfus/abid.shtml).

When an antibody is detected in the patient's serum this is identified using panels of red cells of known phenotype. There is a defined procedure for the identification of antibodies. • Read the results of the gel cards and record this on the panel sheet on which the donor's phenotype is shown. • When no positive reaction is observed, exclude antigens with a cross for homozygotes and a single line for heterozygotes. • Examine the antigens that have not been excluded to see if any of them account for all the reactions observed and that the reaction is in the phases that are usual for an antibody of that specificity. • Further investigation with cells of known phenotype (negative for an antigen where there is an identified reaction, but positive for an antigen to be excluded), with composite panels or by typing the patient, may be required.

The pattern of reactivity seen with a panel could arise by chance and there is a requirement that the result be significant to at least the 95% confidence level. Three antigen positive reactive cells and three antigen negative unreactive cells are required.

2.3.3.1.1. Coombs antibody identification

2.3.3.1.1.1 Sample and Materials required

• Blood sample in EDTA. • ID-Diluent 2 at room temperature. • 0.8% suspension of reagent red cells (ID-DiaPanel I-II-III). • 0.8% suspension of patient’s red cells. • Micropipette. • DiaMed system centrifuge. • DiaMed system incubator. • Disposable pipette tips. • ID-Reader M.

2.3.3.1.1.2 Procedure

1. Sample preparation for ABO/D determination Prepare a 8% red cell suspension in ID-Diluent 2 as follows: • Dispense 1.0ml of ID-Diluent 2 into a clean test tube. • Add 10µl of blood • Mix gently.

2. Method • Label two cassettes with the patient’s donation number and remove aluminium foil. • Add 50µl ID- ID-DiaPanel I-II-III to the appropriate microtubes. • Add 50µl of the patient’s red cell suspension to the appropriate tube of the card (auto control). • Add 25µl of patient’s plasma to all microtubes. • Incubate the cards for 15 minutes at 37°C in the DiaMed incubator. • Centrifuge the card using the DiaMed centrifuge for 10 minutes. • Read the front and back of the individual columns for agglutination.

• Read and record the results by means of the ID-Reader M machine.

3. Interpretation of results

Figure 2.10: Liss/Coombs identification card (Serology SOP NBTC Labs – Malta)

A positive reaction {score 3 (1+w); 5 (1+); 8 (2+); 10 (3+); 12 (4+)} is recorded when red cells are retained in or above the gel column after centrifugation.

A positive reaction indicates the presence of irregular antibodies. Enter the reactions obtained in the antigen table (Appendix 3) and following the reaction pattern the type of antibody present, in most cases, can be identified. Auto control must be negative.

Positive reaction with all ID-DiaPanel test cells but a negative control may indicate the presence of an alloantibody. If the auto control is also positive, this may be due to unbound autoantibodies or non-specific reactions.

A negative reaction is recorded (as 0) when a distinct button of cells sediment to the bottom of the column after centrifugation.

A negative reaction indicates the absence of any irregular antibodies tested for.

2.3.3.1.2 Enzyme antibody identification

2.3.3.1.2.1 Sample and Materials required

1. Blood sample in EDTA. 2. ID-Diluent 2 at room temperature. 3. 0.8% suspension of reagent red cells (ID-DiaPanel I-II-IIIPapainized). 4. ID-Papain. 5. 0.8% suspension of patient’s red cells. 6. Micropipette. 7. DiaMed system centrifuge. 8. DiaMed system incubator. 9. Disposable pipette tips. 10. ID-Reader M.

2.3.3.1.2.2 Procedure

1. Sample preparation for ABO/D determination Prepare a 8% red cell suspension in ID-Diluent 2 as follows: • Dispense 1.0ml of ID-Diluent 2 into a clean test tube. • Add 10µl of blood. • Mix gently.

2. Method • Label two cassettes with the patient’s donation number and remove aluminium foil. • Add 50µl ID- ID-DiaPanel I-II-IIIP to the appropriate microtubes. • Add 50µl of the patient’s red cell suspension to the appropriate tube of the card (auto control). • Add 25µl of patient’s plasma to each microtube except the one containing the auto control. • Add 25µl of ID-Papain to the microtube containing the auto control.

• Incubate the cards for 15 minutes at 37°C in the DiaMed incubator. • Centrifuge the card using the DiaMed centrifuge for 10 minutes. • Read the front and back of the individual columns for agglutination. • Read and record the results by means of the ID-Reader M machine.

3. Interpretation of results

Figure 2.11: NaCl Enzyme identification card (Serology SOP NBTC Labs – Malta)

A positive reaction {score 3 (1+w); 5 (1+); 8 (2+); 10 (3+); 12 (4+)} is recorded when red cells are retained in or above the gel column after centrifugation.

A positive reaction indicates the presence of irregular antibodies. Enter the reactions obtained in the antigen table (Appendix 3) and following the reaction pattern the type of antibody present, in most cases can be identified.

Positive reaction with ID-DiaPanel test cells may indicate the presence of an alloantibody.

A negative reaction is recorded (as 0) when a distinct button of cells sediment to the bottom of the column after centrifugation.

A negative reaction indicates the absence of any irregular antibodies tested for.

Alloantibodies do not react with antigens present on the RBCs of the antibody producer. Unexpected alloantibodies are antibodies other than naturally occurring anti-A or-B. Such antibodies may be found in some 0.3-3.8% of the population, depending upon the selected group of patients or donors studied and the sensitivity of the test methods. Immunisation to “foreign” RBC antigens may result from pregnancy or transfusion, or following injection with immunogenic material.

Once an unexpected antibody is detected, its specificity should be determined and its clinical significance assessed. A clinical significant antibody is one that shortens the anticipated survival of transfused RBCs or has been associated with haemolytic disease of the new born.

The serum under investigation should be tested by the desired techniques with a panel of eight or more group O reagent RBC samples of known blood group phenotype. To be functional, a reagent RBC panel must make it possible to identify with confidence those clinically significant alloantibodies that are most frequently encountered such as anti-D, -E, -K and –Fya. When a serum contains only one of these antibodies, the reagent RBC phenotypes should be such that the presence of most other common alloantibodies can be at least tentatively excluded.

It is important to know how the serum investigation reacts with the autologous RBCs. This helps determine whether alloantibody, autoantibody or both are present.

Chapter 3

Results and Conclusions

3.1 Results

For this test analysis a batch of 511 randomly selected samples was considered. The batch size cannot be calculated statistically since the rate of incompatible crossmatches is low and an accurate percentage is not even available. Hence, it is not guarantied that in a given amount of crossmatches there would be a specified amount of incompatibilities.

The samples were tested using the DiaMed microtyping system. For each sample a blood group determination consisting of a forward and reverse grouping, including testing for the rhesus D antigen, was performed. Furthermore, the sample was screened for the presence of antibodies. Necessary controls for blood typing were also included. These consisted of AB POS and O NEG blood groups, so as to check the positivity and negativity of the cards and reagents.

Controls for antibody screen were carried out using anti-E and anti-Fya. So as to ensure the stability of the reagents, these known antibodies were freshly prepared by the Q. C. staff and run every 34 samples. These antibodies, i.e., anti-E and anti-Fya, were chosen because of their particular properties. Anti-E reacts in both antihuman globulin and enzyme, whilst anti-Fya reacts only in the antihuman globulin. Thus the two possible types of antibody reactions were covered.

After appropriate pipetting, incubation and centrifugation the cassettes where inserted into the ID-ReaderM for confirmation of the reactivity grading. All those samples that had a positive antibody screen had to undergo identification testing to identify the antibody present. The sera of these positive samples were frozen at -40C for batching. Once all available samples were typed and screened, the positive samples were thawed and the identification performed. Anti-E and anti-Fya were again used as controls and the grading noted.

Both the screen and identification were compared with the appropriate antigrams and the antibodies identified.

Samples that gave a positive reaction in all wells could not have their antibodies identified. Such a situation probably arises due to cold reacting antibodies and would require further investigation to ensure a safe transfusion.

Mixed field results were also obtained, probably due to either the deterioration of the media used or the sample itself. In such cases repeated testing is suggested.

The actual crossmatch was carried out routinely and the screening was carried out blindly, without knowing the result of the compatibility test. The two were later compared (Appendix 4).

Taking the number of incompatible crossmatches as 100%, we can calculate the percentage of the number of incompatible crossmatches in which an antibody was detected. This calculates the percentage safety of the type and screen as compared to AHG compatibility testing.

No of incompatible X-match = 100% No of incompatible X-match with an antibody detected = 22/23 x 100% = 95.65%

The type and screen procedure gave a safety of 95.65%. Ideally the percentage safety of a type and screen procedure should be 99%

If we consider the number of tests performed to be 100%, excluding those results that had a mixed field, the percentage of samples in which unexpected antibodies were found can be calculated.

No of individuals tested (excluding mixed fields) = 100% No of individuals with unexpected antibodies = 23/503 x 100 = 4.57%

3.2 Conclusion

In this study, one result in particular gave a negative screen with an incompatible crossmatch. There are various reasons why such a result might be obtained: • A blood group error, on the donor blood or the recipient. This should also be picked up by an immediate spin crossmatch, which is also part of the type and screen procedure. • The crossmatch was incorrectly performed or read as incompatible. In such a case no transfusion reaction would occur. • The antibody screen was incorrectly performed or read as negative. A transfusion reaction would have occurred. • The patient had a rare antibody, which was not present on the panel but present on the donor. A transfusion reaction would have occurred. • The patient had an antibody, which showed dosage that reacted with a homozygous antigen on the donor’s blood but not with a heterozygous antigen on the panel. A transfusion reaction would have occurred. • The patient had a weak antibody, which deteriorated during storage of samples. This should not occur during clinical practice.

This study did not show the type and screen to reach the expected safety level 99%, taking that of the full antiglobulin crossmatch to be 100%. A type and screen with an immediate spin crossmatch is thought to give a safety of 99.9%. Hence even if only 99% of unexpected antibodies are detected the likelihood of a rare antibody meeting a rare antigen in transfused blood is very low, thereby adding to increased safety of 99.9%.

A more extensive study should be considered. The usefulness of the type and screen was shown through the detection of unexpected antibodies in 4.57% of cases. The detection and identification of these antibodies would help select blood in advance for patients undergoing surgery and the results should be retained in the patients’ records in case these weaken with time but still give rise to haemolytic transfusion reactions.

Appendix I

1.1 The Rhesus system

Rh is the most complex of the blood groups systems, embracing over 45 distinct antigens, the absence or presence of which combine to exhibit an individual's Rh blood group type. The most clinical important antigen, D or Rho, was the first discovered in 1940 and has been generally referred to as the Rh antigen, being present in over 85% of the random population. Those individuals that lack the D antigen are considered to be Rh negative. Natural antibodies against the Rh antigens do not occur. Rhesus antigens are unique nonglycosylated, very hydrophobic cell surface proteins of 32 kDa. They are structurally related to the band 3 and band 4.5 glycoproteins, which suggests that they too may be transporter proteins (http://ntri.tamuk.edu/immunology/blood.html)

Figure I.1: Chemistry of the rhesus antigen

The Rh antigens are encoded by two highly homologous and closely linked genes on the short arm of chromosome 1. The RHD gene producing the D antigen, or most of its components; RhCE gene producing the Cc and Ee antigens or their variants. The majority of the antigens within this system represent products of gene cross-over, point mutations or deletions within one or both genes.

The Rh antigens appear to be red cell specific, appearing early during development of red blood cells, and have not been found on other body tissues. Antibodies against the Rh antigens have caused severe and fatal transfusion reactions and hemolytic disease of the newborn. The importance of the Rh antigens in the erythroid membrane is exemplified by the fact that in many examples of auto-immune hemolytic anemia, auto-Rh antibodies are frequently found.

Moreover, in hematological testing the extremely rare (only 32 known throughout the world) individuals who have no detectable Rh antigens, Rhnull individuals, a shortened red cell survival is quite common. Rhnull cells exhibit stomatocytosis and spherocytosis, and have increased permeability to potassium suggesting that they lack a crucial membrane component. A current model suggests that Rh assembles in the membrane as a complex with CD47, LW, RhAG and glycophorin B. Mutations of the

RhAG gene accounts for most examples of Rhnull

It is truly ironic that this blood group system received this name because it was originally thought to be similar to an antibody produced in rabbits that had been immunized with rhesus monkey cells. By the time it was scientifically proven that they were two distinct antibody specificities there were too many publications referring to the Rh factor as the product of the D gene and the symbol Rh was well entrenched for this blood group system. Hence, the rhesus association to the system name had been made, but in fact, there is no association with rhesus monkeys what so ever.

1.2 The MNS system

MNS was the second blood group system to be discovered (1927). In a deliberate attempt to discover more blood group antigens, Landsteiner and Levine immunized rabbits with human red blood cells. The discovery and elucidation of inheritance was one of the most brilliant achievements in this field of biology; out of forty-one sera four were found to have a distinctive agglutinin that reacted independent of the then know ABO blood group types. By selective immunization and absorption, the serological specificities and inheritance of M and N were described. It was twenty years before the third antigen of the group, S, was identified; followed shortly by the discovery of the product of its antithetical allele, s. Because of this system's usefulness in testing inheritance within pedigrees, several newly discovered blood group antigens were found to be associated with this system; some being high incidence antigens (i.e. U, Ena) or, more frequently, low incidence antigens (i.e. Mg, He, Mta, etc). To date there have been over 43 antigens associated with this blood group system (http://jove.prohosting.com/~scarfex/blood/2.html).

Rahuel et al. (1988) characterized 2 cDNA clones encoding glycophorin A from human fetal cDNA libraries. They used these clones to locate the structural gene to 4q28-q32. They concluded further, by Southern blot analysis of genomic DNA from normal En(a+) and rare En(a-) persons, that the glycophorin A gene has a complex organization and is largely deleted in persons of the En(a-) phenotype (Finnish type), who lack glycophorin A on their red cells. Rahuel et al. (1988) concluded that the Finnish variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C.

In the genome of the UK variant of En(a-), Rahuel et al. (1988) identified several abnormalities of the glycophorin A and B genes, leading them to conclude that both are largely deleted, being replaced by a gene fusion product composed of the N-terminal portion of a blood group M-type glycophorin A and of the C-terminal portion of glycophorin B. Okubo et al. (1988) described 2 Japanese sisters with consanguineous parents who were apparently homozygous for M(k). Total absence of sialoglycoproteins α and β from red cell membranes was demonstrated in 1 of the sisters. This is the third

reported family; one of the other families was also Japanese. All affected individuals had been healthy except for the proposita in the present study who had Hodgkin disease.

Huang et al. (1988) studied a family in which 3 different glycophorin mutations were present in 2 individuals of a 16-member family. The variant Dantu glycophorin showed properties consistent with a delta-alpha (GPB/GPA) hybrid glycophorin. This gene was linked to a gene coding for the M-specific alpha glycophorin. Another variant glycophorin, Mi-III glycophorin, was transmitted as an autosomal dominant trait and was associated with N blood group activity. The inheritance pattern indicated that it could be a variant of delta glycophorin (glycophorin B). In the persons with both Dantu and Mi-III glycophorins, a delta glycophorin deficiency was observed, suggesting that a deletion or alteration of the delta gene may exist on the same chromosome as the Dantu gene. Huang et al. (1989) showed that the St(a) (Stone) antigen is likewise determined by a fusion hybrid of the glycophorin A and B genes.

1.3 The Lutheran system

The Lutheran blood group was initially described in 1945 when the first example of anti-Lua was discovered in the serum of a patient following transfusion of a unit of blood carrying the corresponding low frequency antigen. The new antibody was named Lutheran, a misinterpretation of the patient's name, Luteran. In 1956, Cutbush and Chanarin described anti-Lub, which defined the high frequency antithetical partner. The Lutheran blood group system, now consists of 18 antigens, including four allelic pairs: Lua (Lu1) and Lub (Lu2); Lu6 and Lu9; Lu8 and Lu14; Aua (Lu18) and Aub (Lu19) (http://jove.prohosting.com/~scarfex/blood/5.html).

The protein carrying the antigens for the Lutheran blood group system is a product of a single gene LU, which consists of 15 exons distributed over ~12 kb. The gene encodes a type I integral membrane glycoprotein (Lu gp)present as two 85kD and 78kD isoforms differing at the C terminus, due to alternative splicing. The 85kD isoform is the predominant species. and the 78kD form is also known as B-CAM , the basal cell adhesion molecule antigen. The Lu glycoprotein is a member of the Ig superfamily., that share a similar extracellular domain composed of five Ig-like domains. Lu gps thus belong to a subset of adhesion molecules and, in particular, function as laminin receptor (Parsons et al.2001).

1.4 The Kell sytem

The first Kell system antibody was described in 1946, shortly after the implementation of the use of the then recently described rabbit anti-human globulin reagent. The system's clinical importance was obvious from the first case: an example of hemolytic disease of the newborn. As with most systems, over the years, more antigens have been found that were proven by inheritance to be of the Kell blood group system. At present it is a system comprised of 22 blood group antigens, several having been shown to be products of allelic genes. Some of the antigens have also shown a distinct racial prevalence (K antigen is more frequently found in Northern European, the Jsa antigen is most frequently found in those of African descent and the Kpc antigen has been more frequently found in Japanese). All of this was very suggestive of a chromosome location that might have three or more regions with mutation points (http://jove.prohosting.com/~scarfex/blood/6.html).

Monospecific Kell blood group antibodies, of either human alloimmune or mouse monoclonal origin, react with a single surface-exposed protein of 93,000 daltons. Chymotryptic peptide maps of the 93,000-dalton protein isolated by antibodies of two different specificities (anti-K7 or anti-K14) indicate that Kell epitopes reside on the same protein. Kell protein is similar in size to band 3 protein but differs markedly in its tryptic and chymotryptic peptide maps, indicating that they are different proteins. In addition, sheep antibody to human band 3 does not react with Kell protein. Rabbit antibody to Kell protein reacts, by Western immunoblotting, with membrane proteins from Kell antigen positive red blood cells but not from those of a Ko (Kell null) cell. In intact red cells only a small portion of the Kell protein is available to lactoperoxidase-catalyzed iodination. Under nonreducing conditions Kell antigen is isolated not only as a 93,000-dalton protein but also as larger protein complexes ranging in size from above 200,000 to 115,000 daltons. Treatment of red cells with iodoacetamide, prior to isolation of Kell protein, reduces the amount of the very large complexes, but Kell protein occurs both as 115,000- and 93,000-dalton proteins (Readman et al. 1986).

1.5 The Lewis system

The first description of an antibody in the Lewis system was published in 1946 by Mourant. Lewis system antibodies are some of the most frequently encountered in pre- transfusion or pre-natal screening. Anti-Lea is the most frequent antibody in the Lewis system, is often naturally occurring and is of the IgM class. Anti-Leb exists in two forms: bH one reacts only with Le(b+) cells of the A2 or O type (anti-Le ) while the other reacts with all Le(b+) cell regardless of ABO type (http://jove.prohosting.com/~scarfex/blood/7.html).

The Lewis system involves genetically variable antigens in the body fluids and only secondarily are the antigens absorbed to red cells. Grollman et al. (1969) showed that Lewis-negative women lack a specific fucosyltransferase which is present in the milk of Lewis-positive women. The enzyme is apparently required for synthesis of the structural determinants of both Lewis (a) and Lewis (b) specificity. The same enzyme is involved in the synthesis of milk oligosaccharides, because 2 oligosaccharides containing the relevant linkage were absent from the milk of Lewis-negative women.

Grubb (1953) provided the ingenious interpretation of the interactions between the Les locus determining presence/absence of Lewis substance in the saliva and on red cells and the Se locus determining secretion of ABH blood group substances in the saliva and Le(a) or Le(b) expression in red cells.

The Lewis determinants are structurally related to determinants of the ABO and the H/h blood group systems. They are made by sequential addition of specific monosaccharides onto terminal saccharide precursor chains on glycolipids or glycoproteins. On the erythrocyte surface they reside on glycolipids. In contrast to the other blood group antigens the synthesis of these glycolipids does not occur in erythroid tissues and they are acquired by the erythrocyte membranes form other tissues through the circulating soluble forms bound to lipoproteins.

1.6 The Duffy System

In 1950, the Duffy blood group was named for the multiply transfused hemophiliac whose serum contained the first example of anti-Fya. In 1951, the antibody to the antithetical antigen, Fyb, was discovered in the serum of a woman who had been pregnant three times. Using these antibodies three common phenotypes were defined: Fy(a+b+), Fy(a+b-), and Fy(a-b+). Differences in the racial distribution of the Duffy antigens were discovered four years later when it was reported that the majority of Blacks had the erythrocyte phenotype Fy(a-b-). This phenotype is exceedingly rare in Whites. The frequency of the Fy(a-b-) phenotype is 68 percent in American Blacks and 88-100 percent in African Blacks. The molecular basis for the Fy(c-b-) phenotype is the result of a point mutation in the erythroid specific promoter. The absence of Duffy antigens on erythrocytes results in their resistance to invasion by two malaria parasites, Plasmodium vivax and Plasmodium knowlesi. This racial variation in distribution of the Duffy system antigens provides one of the few known examples of selective advantage conferred by a blood group phenotype (http://jove.prohosting.com/~scarfex/blood/8.html).

The Duffy glycoprotein is expressed along postcapillary venules throughout the body, except in the liver. Erythroid cells and postcapillary venule endothelium are the principle tissues expressing the Duffy transcripts. The Fy(a-b-) individuals do not produce Duffy mRNA in the bone marrow, in accordance with the absence of Duffy glycoprotein on their erythrocytes. However, in organs other than bone marrow of Duffy negative individuals, mRNA of the same size but less quantity than those of Duffy positive individuals is expressed.

Chaudhuri et al. (1995) demonstrated the Duffy glycoprotein on the endothelial cells of Fy(a-b-) individuals. Iwamoto et al. (1996) identified a novel first exon and spliced form mRNA that was the predominant Duffy transcript in both erythroid and postcapillary venule endothelium. The novel exon started at nucleotide position -332 in erythroid cells and -380 in endothelial cells. The 5-prime flanking region of the novel first exon was regarded as a transcription controlling unit for both tissues. The tissue-specific lack of expression in Fy(a-b-) indicated that the transcriptional control of the Duffy gene is under tight tissue-specific regulation.

Iwamoto et al. (1996) characterized a base substitution in the promoter of Duffy negative individuals: a 1-bp substitution (-365T-to-C) was found in the proximal GATA motif from 3 black Fy(a-b-) individuals. Iwamoto et al. (1996) found that the black-type mutation abolished chloramphenicol acetyltransferase transcription in human erythroleukemia cells but not in human microvascular endothelial cells. Deletion mutagenesis studies revealed that the proximal GATA motif represents the erythroid regulatory core region for the Duffy gene. Gel shift assay showed that the proximal GATA motif is the target sequence of GATA1. These studies indicated that the black- type mutation abolishes Duffy gene expression in erythroid but not in postcapillary venule endothelium, which is compatible with the Northern blot and immunohistochemical observation in black Fy(a-b-) individuals.

1.7 The Kidd sytem

Shortly after the development of the antiglobulin test for the detection of red cell antibodies, the first example of a Kidd antibody was reported in 1951. A patient, Mrs. Kidd, was described who produced an antibody that caused hemolytic disease in her newborn son. After determining that the new antigen was independent of the other then- known blood groups, it was given the name Jka. Soon afterwards, the allele was found by Plaut and designated Jkb. In 1959, the first example of the null phenotype, i.e., Jk (a- b-), was founnd in a woman who had produced an antibody that appeared to be anti-Jka plus anti-Jkb. Since the specificities were inseparable, the antibody was renamed anti- Jk3 which recognizes an antigen found whenever Jka or Jkb is present. To date, no low frequency antigens have been associated with the Kidd blood group (http://jove.prohosting.com/~scarfex/blood/9.html).

Leppert et al. (1987) found a linkage of blood group Kidd to 2 DNA markers on chromosome 18; the maximum lod scores were 3.61 at theta = 0.168 and 4.18 at theta = 0.218. This is, of course, inconsistent with linkage of Jk to Km (147200).

Pausch and Mayr (1987) presented additional data supporting linkage of Jk and IGK. Together with the data of Field et al. (1985), the maximum lod score reached 3.0 for theta = 0.32. However, the evidence from linkage studies using DNA markers is overwhelming; HGM9 concluded provisionally that the Jk locus is at 18q11-q12 (Geitvik et al., 1987). The L2.7 probe used in the assignment to chromosome 18 was thought to

lie on the short arm, close to the centromere. The maximum lod score was 8.53 at recombination fraction of 0.03 (upper probability limit 0.11). In these data also, linkage of Jk to IGK was found (total lods = 4.12 at theta = 0.30). No obvious explanation for the conflicting gene mapping data could be found. Geitvik et al. (1987) quoted deletion data excluding Jk from a considerable part of chromosome 18 and contributing to the assignment of 18q11-q12.

1.8 The Bombay (Hh) system

The history and biochemical nature of the H antigen is entangled with that of the ABO and Lewis blood group systems. The recognition of the Hh blood group probably begins with the discovery by Bhende et al. (1952) of the first three individuals who completely lacked A and B antigens but were not group O. This null phenotype was named "Bombay" after the city where it was found. Although the Bombay phenotype is extremely rare in most populations, a number of examples have been found in India over the years. Early suggestions were that this type was due to a new allele at the ABO locus, but others suggested an inhibitor gene and finally the possibility of a genetically independent but related gene. We now know that the Bombay phenotype is due to a recessive gene at the H locus; ie. h and is therefore referred to as Oh. Subsequently, intermediate form of the H gene have been found and these individuals are called "para- Bombays" (http://jove.prohosting.com/~scarfex/blood/18.html).

Larsen et al. (1990) cloned and sequenced a gene which they showed encodes the H blood group antigen. When expressed in COS-1 cells, the cDNA directed expression of cell surface H structures and a cognate alpha-(1,2)FT activity with properties analogous to the human H blood group alpha-(1,2)FT. The cDNA predicted a 365-amino acid polypeptide. Southern blot analysis showed that this cDNA identifies DNA sequences syntenic to the human H locus on chromosome 19.

A study of null alleles isolated from individuals with the Bombay phenotype will be of interest. Studies along that line were reported by Kelly et al. (1994). Inactivating point mutations were identified within the coding portions of the FUT1 gene in a Bombay pedigree.

They were also found in a para-Bombay pedigree. (According to a 2-locus model, the H blood group locus determines expression of the H antigen (as well as A and/or B antigens) in the erythroid lineage, whereas the SE locus controls H expression (and thus A or B antigen expression) in a variety of secretory epithelia and in saliva.) Nonsecretors are homozygous for null alleles at the SE locus, whereas individuals with the Bombay phenotype lack H determinants in all tissues and appear to be homozygous for null alleles at both the H and the SE loci. Individuals of the para-Bombay phenotype synthesize H determinants in their secretory epithelia but not in the erythroid lineage. These persons are believed to be homozygous for null alleles at the H locus, but apparently have at least one functional SE allele.

The H and secretor loci are alternatively known as FUT1 and FUT2, respectively. Since the para-Bombay individuals studied by Kelly et al. (1994) maintained a functional SE-determined fucosyltransferase, but not an H-encoded fucosyltransferase, it could be concluded that the SE locus must correspond to a fucosyltransferase gene distinct from the one defective in the Bombay phenotype. Thus, additional support was provided for the conclusion that the human H and SE blood group loci correspond to distinct fucosyltransferase genes.

1.9 The Catwright sytem

The first Yt (also known as Cartwright) blood group system antigen, ie. Yta, was described 1956 by Eaton et al. This blood group antigen was proven to be inherited as a dominant character and independent from the other know systems at that time. In 1964, Giles and Metaxas reported the first example of an antibody that detected the product of the expected antithetical allele, Ytb. This latter discovery raised the stature of the Yt system, which then became a chromosome marker of about the same potential usefulness as the Lutheran system (http://jove.prohosting.com/~scarfex/blood/11.html).

1.10 The P system

The P blood group was identified by Landsteiner and Levine after they deliberately inoculated rabbits with human red cells in order to find new blood group factors. Since the resulting discovery of anti-P1, a number of related antibodies have been identified, k a including anti-P, -P , -Tj , and -Luke (LKE). Some other antibodies, such as anti-IP1, -iP1, and -iP, require the specific Ii antigens, in combination with P antigens. Most of these antibodies are cold-reactive and thus are of little significance in transfusion. The Donath- Landsteiner antibody, a biphasic hemolysin, has been shown to have P specificity (http://jove.prohosting.com/~scarfex/blood/3.html).

1.11 The Diego System

The first example of anti-Dia was discovered in Venezuela in 1956, as a cause of hemolytic disease of the newborn. The family was Caucasian but there appeared to be Native American admixture. This led to the recognition that Dia was a useful marker for persons of Mongolian descent while being of very low frequency in other populations. The frequency in Native Americans ranges from 2-36% while 3-10% of Orientals are positive. Anti-Dib was not recognized until 1967 when two examples were reported in two Mexican women that were being transfused (http://jove.prohosting.com/~scarfex/blood/10.html).

The Diego blood group system is controlled by 2 allelic genes: Di(a) and Di(b). The Di(a) antigen was first described in Venezuela on the basis of an antibody that had been the cause of hemolytic disease of the newborn (Levine et al., 1956). A second example of anti-Di(a) was found in Buffalo in the serum of a Polish mother, whose child also suffered from hemolytic disease of the newborn (Tatarsky et al., 1959). The Diego system shows polymorphism mainly in Mongolian peoples, e.g., Chinese and American Indians. In a family of Polish origin, Kusnierz-Alejska and Bochenek (1992) found anti- Di(a) antibody in the serum of a mother who gave birth to a newborn with severe hemolytic anemia. They identified the Di(a) antigen in 45 of 9,661 donor blood samples from different regions of Poland (0.46%). All 45 were of Polish ancestry.

1.12 The Scianna sytsem

There are three antigens within the Scianna blood group system recognized by the International Society of Blood Transfusion (ISBT) Working Party on Terminology for Red Cell Surface Antigens. The first is Sc1, a high frequency antigen found in greater than 99 % of most populations. The frequency of Sc2 is about 1% of Northern Europeans but the frequency is much lower in other populations. The incidence of Sc: 1,2 is more common in Mennonites, as a selected population. The third antigen is Sc3, a high frequency antigen found on all cells except the extremely rare individuals that type Sc: -1, -2. Reported by McCreary in 1973, it was found while working with a sample of a patient from the Marshall Islands (http://jove.prohosting.com/~scarfex/blood/13.html).

1.13 The Colton system

The Colton blood group system, despite the knowledge known about its genetics and biochemistry, has remained a relatively uncomplicated system. Antibodies to the Colton antigens have caused both mild and moderate hemolytic transfusion reactions and mild hemolytic disease of the newborn, except in one severe case due to anti-Co3. In this case the antibody titer was greater than 32,000 and the baby was supported by intrauterine and post-delivery maternal transfusions. Investigation of blood from three unrelated Co (a-b-) individuals revealed that the serological aberration was due to three separate causes: a frame shift after Gly104, a deletion of exon 1 and a Leu38 (instead of a Pro38) causing a stop. These three individuals lacked or had a very low amount of CHIP- 1. The CHIP-1 protein (hence the Colton blood group antigens) is located on a variety of surfaces including epithelia, endothelium, descending tubules and apical surfaces of proximal tubules in addition to red blood cells. However, the Co(a-b-) individuals do not appear to have any health related problems related to the loss of this protein. (http://jove.prohosting.com/~scarfex/blood/15.html).

Smith et al. (1994) demonstrated that the Colton blood group antigens result from an ala-val polymorphism at residue 45, located on the first extracellular loop of the aquaporin-1 protein. In red cells from 3 individuals who lacked Colton antigens, i.e., were Co(a-b-), Preston et al. (1994) found mutations in the AQP1 gene that resulted in a

nonfunctioning CHIP molecule. Surprisingly, none of the 3 suffered any apparent clinical consequences

1.14 The XG system

In 1962, Mann reported an antibody which he found in the serum of a multiply transfused Caucasian male (Mr. And) that seemed to be associated with the sex of the donor red cells. In other words, the antigen frequency differed between males (XY) and females (XX) of the same race. Thus, this new antigen was named Xga as it appeared to be controlled by the X (sex) chromosome. The Xg antigen is well developed at birth although cord blood cells may give weaker reactions than adult cells. The Xg system, however, is unusual in that no other antigens have been identified to date. Curiously, most of the antibody producers have been males (http://jove.prohosting.com/~scarfex/blood/12.html).

1.15 The Landsteiner and Wiener

Landsteiner and Wiener used blood from a monkey (Macacus rhesus) to immunize rabbits and guinea pigs in order to define new antibody specificities. One of the antibodies they produced appeared to have the same specificity as a human antibody found in several woman who had stillborn fetuses. Consequently, the antibodies were named anti-Rh for rhesus. However, as early as 1942 it was known that the rabbit/guinea pig and human antibodies could not be the same. When the guinea pig antibody was used to test Rh+ and Rh negative cord blood cells (as defined by the human antibody) all samples were reactive. The guinea pig antibody was given the name "D-like". Then Race and Sanger found two women whose antibodies could be absorbed by Rh negative red cells and which appeared to be similar to the "D-like" antibody from animals. Following further studies by Levine the "D-like" name was changed to LW in honor of Landsteiner and Wiener. (http://jove.prohosting.com/~scarfex/blood/16.html).

The LW blood group antigens reside on a 42-kD erythrocyte membrane glycoprotein. Bailly et al. (1994) isolated 2 forms of LW cDNA. The predicted LW protein

was found to exhibit sequence similarities, with approximately 30% identity, with intercellular adhesion molecules ICAM1, ICAM2, and ICAM3, which are the counterreceptors for the lymphocyte function-associated antigen LFA1. The extracellular domain of LW consists, like that of ICAM2, of 2 immunoglobulin-like domains, and the critical residues involved in the binding of LFA1 to ICAMs were partially conserved in LW. Hermand et al. (1996) characterized the LW gene, which is organized into 3 exons spanning approximately 2.65 kb of DNA.

1.16 The Dombrock sytem

The first example of anti-Doa, reactive with 64% of the Caucasian population, was reported by Swanson et al. in 1965. It was not until 1972 that Molthan et al. reported the antithetical antibody anti-Dob. Thus, the Dombrock blood group system was defined and was estimated to be the fifth most useful blood group marker in Caucasians. However, limited examples of both antibodies greatly restricted broad investigations. Dombrock system antigens Doa and Dob appear to be poor stimulis and most examples of both antibodies rarely are found as a single specificity in a serum; and when detected, are notorious for disappearing in vivo. Antibodies within this system have been associated with weak to moderate transfusion reactions, but not with clinically defined cases of hemolytic disease of the newborn (http://jove.prohosting.com/~scarfex/blood/14.html).

Appendix II

Appendix III

Appendix IV

No Typing Screen Crossmacth No Type Screen Crossmacth 1 A Pos Negative Compatible 43 O Pos Negative Compatible 2 O Pos Negative Compatible 44 A Pos Negative Compatible 3 O Pos Negative Compatible 45 A Pos Negative Compatible 4 A Pos Negative Compatible 46 O Pos Negative Compatible 5 O Pos Negative Compatible 47 O Pos Unidentified Incompatible 6 O Pos Negative Compatible 48 A Pos Negative Compatible 7 A Pos Negative Compatible 49 O Pos Negative Compatible 8 O Pos Negative Compatible 50 O Pos Negative Compatible 9 O Pos Negative Compatible 51 A Pos Anti-Lea Incompatible 10 B Pos Negative Compatible 52 O Pos Negative Compatible 11 A Pos Negative Compatible 53 O Pos Negative Compatible 12 A Pos Mixed Field Compatible 54 B Pos Negative Compatible 13 A Neg Negative Compatible 55 A Pos Negative Compatible 14 O Pos Negative Compatible 56 A Pos Negative Compatible 15 B Pos Negative Compatible 57 A Neg Negative Compatible 16 A Pos Negative Compatible 58 O Pos Negative Compatible 17 O Pos Negative Compatible 59 B Pos Negative Compatible 18 A Pos Negative Compatible 60 A Pos Negative Compatible 19 O Neg Negative Compatible 61 O Pos Negative Compatible 20 A Pos Negative Compatible 62 A Pos Negative Compatible 21 O Pos Negative Compatible 63 O Neg Negative Compatible 22 A Pos Negative Compatible 64 A Pos Negative Compatible 23 A Pos Negative Compatible 65 O Pos Negative Compatible 24 O Pos Negative Compatible 66 A Pos Negative Compatible 25 O Pos Negative Compatible 67 A Pos Negative Compatible 26 A Pos Negative Compatible 68 O Pos Negative Compatible 27 O Pos Negative Compatible 69 O Pos Negative Compatible 28 O Pos Negative Compatible 70 A Pos Negative Compatible 29 A Pos Negative Compatible 71 O Pos Negative Compatible 30 O Pos Negative Compatible 72 O Pos Negative Compatible 31 O Pos Negative Compatible 73 A Pos Negative Compatible 32 B Pos Negative Compatible 74 O Pos Negative Compatible 33 A Pos Negative Compatible 75 O Pos Negative Compatible 34 A Pos Negative Compatible 76 B Pos Negative Compatible 35 A Neg Negative Compatible 77 A Pos Negative Compatible 36 O Pos Anti-K Compatible 78 A Pos Negative Compatible 37 B Pos Negative Compatible 79 A Neg Negative Compatible 38 A Pos Negative Compatible 80 O Pos Negative Compatible 39 O Pos Negative Compatible 81 B Pos Negative Compatible 40 A Pos Negative Compatible 82 A Pos Negative Compatible 41 O Neg Negative Compatible 83 O Pos Negative Compatible 42 A Pos Negative Compatible 84 A Pos Anti-E Compatible

No Typing Screen Crossmacth No Type Screen Crossmacth 85 O Neg Negative Compatible 127 O Pos Negative Compatible 86 A Pos Negative Compatible 128 A Pos Negative Compatible 87 O Pos Negative Compatible 129 O Neg Negative Compatible 88 A Pos Negative Compatible 130 A Pos Negative Compatible 90 O Pos Anti-K Incompatible 131 O Pos Negative Compatible 91 O Pos Negative Compatible 132 A Pos Negative Compatible 92 A Pos Anti-e Incompatible 133 A Pos Negative Compatible 93 O Pos Negative Compatible 134 A Neg Negative Compatible 94 O Pos Negative Compatible 135 O Pos Negative Compatible 95 A Pos Negative Compatible 136 B Pos Negative Compatible 96 O Pos Negative Compatible 137 A Pos Mixed Field Compatible 97 O Pos Negative Compatible 138 O Pos Negative Compatible 98 B Pos Negative Compatible 139 A Pos Negative Compatible 99 A Pos Negative Compatible 140 O Neg Negative Compatible 100 A Pos Negative Compatible 141 A Pos Negative Compatible 101 A Neg Negative Compatible 142 O Pos Negative Compatible 102 O Pos Negative Compatible 143 A Pos Negative Compatible 103 B Pos Negative Compatible 144 A Pos Negative Compatible 104 A Pos Negative Compatible 145 O Pos Negative Compatible 105 O Pos Negative Compatible 146 O Neg Negative Compatible 106 A Pos Negative Compatible 147 A Pos Negative Compatible 107 O Neg Negative Compatible 148 O Pos Negative Compatible 108 A Pos Negative Compatible 149 B Pos Negative Compatible 109 O Pos Negative Compatible 150 A Neg Negative Compatible 110 A Pos Negative Compatible 151 A Pos Negative Compatible 111 A Pos Negative Compatible 152 A Pos Negative Compatible 112 O Pos Negative Compatible 153 A Pos Negative Compatible 113 O Pos Negative Compatible 154 A Neg Negative Compatible 114 A Pos Anti-c Incompatible 155 AB Pos Negative Compatible 115 O Pos Negative Compatible 156 O Pos Negative Compatible 116 O Pos Negative Compatible 157 O Pos Negative Compatible 117 A Pos Negative Compatible 158 A Pos Negative Compatible 118 O Pos Negative Compatible 159 O Pos Negative Compatible 119 O Pos Negative Compatible 160 A Pos Negative Compatible 120 B Pos Negative Compatible 161 B Pos Negative Compatible 121 A Pos Negative Compatible 162 O Pos Negative Compatible 122 A Pos Negative Compatible 163 A Pos Negative Compatible 123 A Neg Negative Compatible 164 A Pos Negative Compatible 124 O Pos Negative Compatible 165 A Pos Negative Compatible 125 B Pos Negative Compatible 166 A Pos Anti-Cw Incompatible 126 A Pos Negative Compatible 167 B Pos Negative Compatible

No Type Screen Crossmacth No Type Screen Crossmacth 168 O Pos Negative Compatible 208 A Pos Negative Compatible 169 A Pos Negative Compatible 209 AB Pos Negative Compatible 170 A Pos Negative Compatible 210 AB Pos Negative Compatible 171 O Pos Negative Compatible 211 A Neg Negative Compatible 172 A Pos Negative Compatible 212 O Pos Negative Compatible 173 A Pos Negative Compatible 213 O Pos Anti-K Compatible 174 AB Pos Negative Compatible 214 O Pos Negative Compatible 175 B Pos Negative Compatible 215 O Pos Negative Compatible 176 O Pos Negative Compatible 216 O Pos Negative Compatible 177 O Pos Negative Compatible 217 A Pos Negative Compatible 178 A Pos Negative Compatible 218 A Pos Negative Compatible 179 O Pos Negative Compatible 219 A Pos Negative Compatible 180 O Pos Negative Compatible 220 A Pos Negative Compatible 181 AB Pos Negative Compatible 221 O Pos Negative Compatible 182 O Pos Unidentified Incompatible 222 A Pos Negative Compatible 183 A Pos Negative Compatible 223 AB Pos Negative Compatible 184 B Pos Negative Compatible 224 O Pos Negative Compatible 185 A Pos Negative Compatible 225 A Pos Negative Compatible 186 O Pos Negative Compatible 226 B Pos Negative Compatible 187 O Pos Negative Compatible 227 O Pos Negative Compatible 188 O Pos Negative Compatible 228 O Pos Negative Compatible 189 A Pos Negative Compatible 229 A Pos Negative Compatible 190 B Pos Negative Compatible 230 A Pos Negative Compatible 191 A Pos Negative Compatible 231 O Pos Negative Compatible 192 O Pos Negative Compatible 232 O Pos Negative Compatible 193 O Pos Negative Compatible 233 AB Pos Negative Compatible 194 O Pos Negative Compatible 234 O Pos Negative Compatible 195 A Pos Mixed Field Compatible 235 O Pos Negative Compatible 196 A Neg Negative Compatible 236 B Pos Negative Compatible 197 AB Pos Negative Compatible 237 A Pos Negative Compatible 198 O Neg Negative Compatible 238 O Pos Negative Compatible 199 A Neg Negative Compatible 239 AB Pos Negative Incompatible 200 O Pos Negative Compatible 240 O Pos Negative Compatible 201 O Pos Negative Compatible 241 A Pos Negative Compatible 202 A Pos Negative Compatible 242 O Pos Negative Compatible 203 A Pos Unidentified Compatible 243 O Pos Negative Compatible 204 O Pos Negative Compatible 244 O Pos Negative Compatible 205 A Pos Negative Compatible 245 A Pos Negative Compatible 206 O Pos Negative Compatible 246 A Pos Negative Compatible 207 O Pos Negative Compatible 247 AB Pos Negative Compatible

No Type Screen Crossmacth No Type Screen Crossmacth 248 A Pos Negative Compatible 288 A Pos Unidentified Incompatible 249 A Pos Negative Compatible 289 A Pos Negative Compatible 250 A Pos Negative Compatible 290 O Pos Negative Compatible 251 A Pos Negative Compatible 291 A Pos Negative Compatible 252 O Neg Negative Compatible 292 AB Pos Negative Compatible 253 A Pos Negative Compatible 293 A Neg Negative Compatible 254 O Pos Mixed Field Compatible 294 A Pos Negative Compatible 255 A Pos Negative Compatible 295 O Pos Negative Compatible 256 A Pos Negative Compatible 296 O Pos Negative Compatible 257 A Pos Negative Compatible 297 A Pos Negative Compatible 258 A Pos Negative Compatible 298 O Pos Negative Compatible 259 A Pos Negative Compatible 299 O Pos Negative Compatible 260 A Pos Negative Compatible 300 A Pos Negative Compatible 261 A Pos Negative Compatible 301 O Pos Negative Compatible 262 A Pos Negative Compatible 302 A Pos Negative Compatible 263 A Pos Negative Compatible 303 A Pos Negative Compatible 264 B Pos Negative Compatible 304 A Pos Negative Compatible 265 B Neg Negative Compatible 305 A Pos Negative Compatible 266 O Pos Negative Compatible 306 O Pos Negative Compatible 267 A Pos Negative Compatible 307 O Pos Negative Compatible 268 O Neg Negative Compatible 308 A Pos Negative Compatible 269 O Pos Negative Compatible 309 A Pos Negative Compatible 270 O Pos Negative Compatible 310 O Pos Negative Compatible 271 A Pos Anti-e Incompatible 311 O Pos Negative Compatible 272 A Pos Negative Compatible 312 A Pos Negative Compatible 273 O Pos Negative Compatible 313 O Pos Negative Compatible 274 O Pos Negative Compatible 314 A Pos Negative Compatible 275 B Neg Negative Compatible 315 A Pos Negative Compatible 276 A Pos Negative Compatible 316 A Neg Negative Compatible 277 A Pos Negative Compatible 317 O Pos Negative Compatible 278 A Pos Negative Compatible 318 A Neg Anti-K Incompatible 279 A Pos Negative Compatible 319 O Pos Negative Compatible 280 A Pos Negative Compatible 330 A Pos Negative Compatible 281 O Pos Negative Compatible 331 O Pos Negative Compatible 282 A Pos Negative Compatible 322 O Pos Negative Compatible 283 O Pos Negative Compatible 333 O Pos Negative Compatible 284 A Pos Negative Compatible 334 A Pos Negative Compatible 285 A Neg Negative Compatible 335 O Neg Anti-Jsb Incompatible 286 O Pos Negative Compatible 336 A Pos Negative Compatible 287 O Pos Negative Compatible 337 A Pos Negative Compatible

No Type Screen Crossmacth No Type Screen Crossmacth 338 A Neg Negative Compatible 368 A Pos Negative Compatible 339 A Pos Negative Compatible 369 O Pos Negative Compatible 330 A Pos Negative Compatible 370 O Pos Negative Compatible 331 A Pos Negative Compatible 371 B Pos Negative Compatible 332 A Pos Negative Compatible 372 O Pos Negative Compatible 333 O Pos Negative Compatible 373 A Pos Negative Compatible 334 O Neg Negative Compatible 374 O Pos Negative Compatible 335 AB Pos Negative Compatible 375 A Pos Negative Compatible 336 O Pos Negative Compatible 376 O Pos Negative Compatible 337 A Pos Negative Compatible 377 A Pos Mixed Field Compatible 338 A Neg Negative Compatible 378 A Pos Negative Compatible 339 A Pos Negative Compatible 379 A Pos Negative Compatible 340 B Pos Negative Compatible 380 O Pos Negative Compatible 341 A Pos Negative Compatible 381 B Pos Negative Compatible 342 O Pos Negative Compatible 382 B Pos Negative Compatible 343 A Pos Negative Compatible 383 A Pos Negative Compatible 344 O Pos Negative Compatible 384 A Pos Negative Compatible 345 O Pos Negative Compatible 385 A Pos Negative Compatible 346 A Pos Negative Compatible 386 A Pos Negative Compatible 347 O Neg Negative Compatible 387 A Pos Negative Compatible 348 O Neg Mixed Field Incompatible 388 O Pos Negative Compatible 349 A Pos Negative Compatible 389 A Neg Negative Compatible 350 B Pos Negative Compatible 390 A Pos Negative Compatible 351 O Pos Unidentified Incompatible 391 O Pos Negative Compatible 352 O Pos Negative Compatible 392 AB Neg Negative Compatible 353 O Pos Negative Compatible 393 A Neg Negative Compatible 354 O Pos Negative Compatible 394 A Pos Negative Compatible 355 O Pos Negative Compatible 395 A Neg Negative Compatible 356 A Pos Negative Compatible 396 A Pos Negative Compatible 357 B Pos Negative Compatible 397 O Pos Negative Compatible 358 A Pos Negative Compatible 398 O Pos Negative Compatible 359 A Pos Negative Compatible 399 A Pos Negative Compatible 360 O Pos Negative Compatible 400 O Pos Negative Compatible 361 O Pos Negative Compatible 401 O Pos Negative Compatible 362 A Pos Negative Compatible 402 A Pos Negative Compatible 363 O Pos Negative Compatible 403 A Pos Negative Compatible 364 A Neg Negative Compatible 404 O Pos Negative Compatible 365 A Pos Negative Compatible 405 O Pos Negative Compatible 366 A Pos Negative Compatible 406 A Pos Negative Compatible 367 A Pos Negative Compatible 407 A Pos Negative Compatible

No Type Screen Crossmacth No Type Screen Crossmacth 408 O Pos Anti-Lua Incompatible 448 A Pos Negative Compatible 409 B Pos Negative Compatible 449 A Pos Anti-e Incompatible 410 A Pos Negative Compatible 450 A Pos Negative Compatible 411 B Pos Negative Compatible 451 A Pos Negative Compatible 412 O Pos Negative Compatible 452 A Pos Negative Compatible 413 A Pos Negative Compatible 453 O Pos Negative Compatible 414 O Neg Negative Compatible 454 O Pos Unidentified Incompatible 415 O Pos Negative Compatible 455 A Neg Negative Compatible 416 A Pos Negative Compatible 456 B Pos Negative Compatible 417 O Neg Negative Compatible 457 A Pos Negative Compatible 418 B Pos Mixed Field Compatible 458 A Pos Negative Compatible 419 O Pos Negative Compatible 459 A Pos Negative Compatible 420 A Pos Negative Compatible 460 A Pos Negative Compatible 421 A Pos Negative Compatible 461 O Pos Negative Compatible 422 O Neg Negative Compatible 462 A Pos Negative Compatible 423 O Pos Negative Compatible 463 O Pos Mixed Field Compatible 424 O Pos Negative Compatible 464 A Pos Negative Compatible 425 O Pos Negative Compatible 465 A Pos Negative Compatible 426 O Pos Negative Compatible 166 A Pos Negative Compatible 427 B Pos Negative Compatible 467 O Pos Negative Compatible 428 AB Pos Negative Compatible 468 O Pos Negative Compatible 429 O Pos Negative Compatible 469 O Pos Negative Compatible 430 A Pos Negative Compatible 470 A Pos Negative Compatible 431 O Pos Negative Compatible 471 O Pos Negative Compatible 432 O Pos Negative Compatible 472 A Pos Negative Compatible 433 A Pos Negative Compatible 473 B Pos Negative Compatible 434 A Pos Negative Compatible 474 O Pos Negative Compatible 435 A Pos Anti-Lea Incompatible 475 A Pos Negative Compatible 436 A Pos Negative Compatible 476 O Pos Negative Compatible 437 A Pos Negative Compatible 477 A Pos Negative Compatible 438 O Pos Negative Compatible 478 A Neg Anti-Lea Incompatible 439 O Pos Negative Compatible 479 O Pos Negative Compatible 440 O Pos Negative Compatible 480 O Pos Negative Compatible 441 O Pos Negative Compatible 481 A Pos Negative Compatible 442 O Pos Negative Compatible 482 A Pos Negative Compatible 443 A Pos Negative Compatible 483 A Pos Negative Compatible 444 O Pos Negative Compatible 484 AB Pos Anti-Lea Compatible 445 O Pos Negative Compatible 485 O Neg Negative Compatible 446 A Pos Negative Compatible 486 O Pos Negative Compatible 447 O Pos Negative Compatible 487 A Pos Negative Compatible

No Type Screen Crossmacth No Type Screen Crossmacth 488 A Pos Negative Compatible 500 A Pos Negative Compatible 489 B Pos Anti-Lua Incompatible 501 A Pos Negative Compatible 490 A Neg Negative Compatible 502 O Neg Negative Compatible 491 O Pos Negative Compatible 503 AB Pos Anti-K Incompatible 492 A Pos Negative Compatible 504 O Pos Negative Compatible 493 B Pos Negative Compatible 505 B Neg Unidentified Incompatible 494 A Pos Negative Compatible 506 O Pos Negative Compatible 495 A Neg Negative Compatible 507 O Pos Negative Compatible 496 A Pos Negative Compatible 508 O Pos Unidentified Incompatible 497 A Pos Negative Compatible 509 A Pos Negative Compatible 498 A Pos Negative Compatible 510 A Pos Negative Compatible 499 AB Pos Negative Compatible 511 A Pos Negative Compatible

References

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