Immunohematology

Immunohematology introduces the theory of genetic and immunology of blood group antigens and antibodies. Medical Laboratory Technologists are exposed to the knowledge regarding the function of blood bank in transfusion medicine. Emphasis of Immunohematology is also on the acquiring of technical skill in testing ABO and Rh blood group and ensuring supply of safe and compatible blood to patients.

It introduces the Medical Laboratory Technologists with the knowledge and techniques to solve problem of incompatible cross-matching, to determine the causes of transfusion reactions as well as to perform investigations of hemolytic transfusion reactions by way of antibody identification. Medical Laboratory Technologists will also be exposed to the theory of blood bank management including quality control system. The role of aphaeresis in transfusion medicine is also included.

Main Reference Textbooks:
Textbook of Blood Banking and Tansfusion Medicine 2005 Sally V Rudman
Immunohematology: Principles and Practice, Quinley, Lippincott

Additional Reference Material:
Transfusion Medicine,2nd ed 2005 ISBN00443066485 Churchill Livingstone

The Gene H: It’s Role in Expression of A and B Genes

The expression of the A and B Genes appears to depend on the action of another gene, known as H. Most individual are homozygous for this gene (HH), though since its allele, h, is an amorphic gene, the heterozygote Hh cannot be recognized. The phenotype h is extremely rare. The genetic sequence leading to the expression of ABH genes on the red cell is believed to be as a precursor mucopolysaccharide substance is converted by the H gene to H substance. This “altered’ precursor substance (H substance) is partly converted by the A and B genes into A and B antigen. Some H substance remains unconverted. The O gene, being amorphic, effects no conversion of H substance. Since no conversion of H substance takes place by the action of the O gene, the H antigen is found in greatest concentration in group O individuals.

The addition of a sugar known as L-fucose to the terminal D-galactose of the basic precursor substance (Type 1 and Type 2 chains) gives the resultant molecule “H” specificity. The Fucose molecule is bound in alpha (1-2) linkage.

The presence of an A gene results in the attachment of the sugar N-acetyl-galactosamine to the substrate formed by the H gene. This gives the resultant molecule “A” specificity.

In much the same way, the presence of the B gene results in the attachment of D-galactose to the substrate formed by the H gene. In the absence of H gene, no L-fucose will be added to the terminal sugar. The A and B genes in this case will not be expressed. An individual of genotype hh will therefore group as “O” even when the A or B genes have been inherited. The order of reactivity usually follows the pattern O> A2 > A2B> B> A1> A1B

Chemical Characteristics of Blood Group Antigens

A great deal of work has been performed by various experts in attempts to understand the chemical characteristics of the red cell antigens. Apparently the facts remain sketchy. However, it is generally accepted that red cell antigens are glycolipids or proteins. The specificity of the antigens is determined by the sequential addition of sugar residues to a common “precursor substance”. This has proved to be the case in all blood group antigens studied so far. The precursor substance is composed of four sugar molecules in which two are known as D-galactose (GAL), one is N-acetyl-galactosamine (GAL NAc) and the last is N-acetyl-glucosamine (GNAc). Two types of precursor substance have been identified, known as Type 1 and Type 2 chains. These chains differ in the linkage of the terminal galactose molecule to the subterminal N-acetyl-glucosamine. In Type 1 chains the linkage is beta (1-3), whereas in Type 2 chains the linkage is beta (1-4) (Chase snd Morgan, 1991; Painter et al, 1963; Watkins, 1966).

The addition of another sugar to this basic precursor determines the specificity of the antigen with the rest of the polysaccharide chain remaining unchanged. Knowledge of the chemistry of blood group antigens has come mainly from work on the body secretions (semen, tears, saliva and cyst fluids), since blood group substances on the red cells are present in relatively small quantities and some of them are soluble only in alcohol. In secretions, these substances occur in much larger quantities and are soluble in water. It has been established that the same sugars are present on the red cells, though on the red cells they are bound through sphingosine to fatty acid moieties. (compounds of this type are known as glycolipids)

Blood Type

If an individual is exposed to a blood group antigen that is not recognized as self, the immune system will produce antibodies that can specifically bind to that particular blood group antigen, and an immunological memory against that antigen is formed. The individual will have become sensitized to that blood group antigen. These antibodies can bind to antigens on the surface of transfused red blood cells (or other tissue cells), often leading to destruction of the cells by recruitment of other components of the immune system.

When IgM antibodies bind to the transfused cells, the transfused cells can clump. It is vital that compatible blood is selected for transfusions and that compatible tissue is selected for organ transplantation. Transfusion reactions involving minor antigens or weak antibodies may lead to minor problems. However, more serious incompatibilities can lead to a more vigorous immune response with massive RBC destruction, low blood pressure, and even death.

A blood type (also called a blood group) is a classification of blood based on the presence or absence of inherited antigenic substances on the surface of red blood cells (RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood group system, and some of these antigens are also present on the surface of other types of cells of various tissues. Several of these red blood cell surface antigens that stem from one allele (or very closely linked genes), collectively form a blood group system.

Blood types are inherited and represent contributions from both parents. A total of 30 human blood group systems are now recognized by the International Society of Blood Transfusion (ISBT). Many pregnant women carry a fetus with a different blood type from their own, and the mother can form antibodies against fetal RBCs. Sometimes these maternal antibodies are IgG, a small immunoglobulin, which can cross the placenta and cause hemolysis of fetal RBCs, which in turn can lead to hemolytic disease of the newborn, an illness of low fetal blood counts which ranges from mild to severe.

Blood Group System - Scianna

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. This antigen was originally described by Schmidt et al. in 1962 and named Sm. Second is Sc2, described by Anderson et al. in 1963 and named Bua. Lewis et al. reported the original Sm- cells were Bu(a+) and suggested they be renamed Sc1 and Sc2 following conformation that they were the products of alleles. 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.

The Scianna antigens are encoded by a gene whose chromosomal location is on the short arm of chromosome 1 between 1p36.2-p22.1. The antigens are located on a glycoprotein containing disulfide bonds and an N-glycan, known as the Scianna glycoprotein (function unknown). To date, the antigens have only been found on erythroid cell lines. Individuals of the rare Sc:-1,-2 phenotype (the null phenotype ) do not appear to have any associated red cell membrane defect or anemia. The Sc:-1,-2 phenotype has been found most frequently (when found at all) amongst individuals native of the South Pacific Islands.

The antibodies against the Scianna antigens have been associated with mild to delayed transfusion reactions and mild hemolytic disease of the newborn. It is suspected that the Scianna blood group system could become as complex as some of the other blood groups. Some unique antibodies have been found that hint of an association with Scianna but lack the complete research necessary to qualify for blood group assignment. An example of possible expansion is the report of three Sc:1,-2 individuals that produced allo-antibodies that failed to react with Sc:-1,-2 red blood cells, and the antibodies were mutually incompatible. This is suggestive that there may be three more high incidence antigens within this system. In addition, the low incidence antigen Radin, may be a part of the Scianna system but is not an allele of Sc1 and Sc2.

Blood Group System - XG

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.

Biochemical analyses have shown that the Xga antigen is located on a 27,000 molecular weight sialoglycoprotein. This protein is encoded for by a gene on the X chromosome at position Xp22-32. The Xg locus is one of the few genes on the X chromosome that are not subject to Lyonization, ie. random "switching off" in females of one of the X genes. Goodfellow and Tippett have observed an interesting association between the Xga antigen and CD99. They found that the CD99 antigen also showed variable expression which appeared to be related to sex. Individuals who are high expressors of CD99 are Xg(a+). Although there are Xg(a-), CD99 low expressors, no "null phenotype" has yet to be found. Since these two proteins may associate with each other in the red cell membrane, the null would presumably lack both the Xg and CD99 proteins. No disease association has been found with a particular Xg phenotype.

Blood Group System - Diego

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.

The Diego system remained a two antigen system until the 1990s when Dia, Dib and the Wright antigens (Wra and Wrb) were determined to be amino acid substitutions on band 3, the anion exchange protein (AE1). The system quickly expanded when other antigens of low frequency were also identified on band 3 including: ELO, Redelberger, Traversu, Warrior, Wulfsberg, VanVugt, Waldner, Bishop, Hughes and Moen. No red cell lacking band 3 has been reported for this system which may indicate that its loss is lethal. There has been only one report of the Di(a-b-) phenotype. Antibodies to Wra are frequently found in patients having autoimmune hemolytic anemia or individuals having a positive direct antiglobulin test.

Blood Group System - Kidd

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.

The first example of the Jk(a-b-) phenotype was found in a woman who experienced a delayed transfusion reaction. She was a Filipino of some Chinese and Spanish ancestry. Another family of Filipino-Chinese ancestry was reported that contained three Jk(a-b-) members. Since these first reports, many such individuals of Asian or Polynesian extraction have been identified. One study found a total of 66 (0.9%) Jk(a-b-) donors among 7425 tested; all were of Polynesian backgrounds. Other populations reporting this phenotype include tribes from Mato Grasso, Brasil, Hindus from India and Japanese blood donors. The Jk(a-b-) phenotype is strikingly absent from Caucasians although rare cases have been found in a French, an Australian and a Finnish family. The molecular basis for Jknull has been shown to be splice-site and misense mutations, as well as a partial gene deletion. The Jka/Jkb polymorphism is a A8386 base pair change at amino acid 280, changing Asp to Asn.

Interestingly, the Jk(a-b-) red cells were shown to be resistant to lysis in high concentrations of urea as opposed to normal cells that completely lyze in ~1 minute. This observation led to biochemical studies which identified the Kidd antigens on the urea transport protein which is found not only in red blood cells but also in the kidney. One study of individuals with the Jk(a-b-) phenotype has reported that they have a decreased ability to concentrate urine but this does not appear to cause a health problem.

Blood Group System - Duffy

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.

The Duffy genes, located on chromosome one at position 1922-23, have recently been cloned and sequenced. The difference between Fya and Fyb is a change in the amino acid at position 43 from aspartic acid (Fya) to glycine (Fyb). Studies have shown that blacks whose erythrocytes express Fyb antigen also have the antigen on the cells of their kidney, heart, muscle, brain and placenta. The Duffy gene codes for a protein known as a chemokine receptor, which is important in the inflammatory process. Accordingly, the Fy protein is also known as DARC (Duffy Antigen Receptor for Chenokines).

Blood Group System - Lewis

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: one reacts only with Le(b+) cells of the A2 or O type (anti-LebH) while the other reacts with all Le(b+) cell regardless of ABO type.

The antigens of the Lewis system are carbohydrate (sugar) determinants carried either on proteins or lipids. Although they were first detected on red cells, the majority of the biochemical studies have been performed on Lewis substances isolated from plasma or saliva. Generally in both Caucasians and Blacks, the three major phenotypes are Le(a+b-), Le(a-b+) and Le(a-b-). These arise through the interaction of two genes- Lewis and secretor. If a Lewis gene is present the donor will be either Le(a+b-) or Le(a-b+); however, if there is no Lewis gene the red cells type as Le(a-b-). In Blacks the Le(a-b-) type occurs with a frequency of 20-25% as compared to 5% in Caucasians. Furthermore, red blood cells from newborns will type as Le(a-b-) regardless of their genetic makeup as the cells have not had time to absorb Lewis antigens from the plasma. Another type which is extremely rare in Caucasians and Blacks, ie. Le(a+b+), is found in the Oriental population and appears to be due to a weak secretor gene.

In 1993, Boren et al. reported that the bacteria Helicobacter pylori used the fucose sugar found in the Leb antigen as a receptor to establish infection. H. pylori has been implicated as the causative agent in gastritis, peptic ulcers and gastric carcinoma. Several studies suggest that transplant patients having the Le(a-b-) phenotype have shorter transplant survival times than those who have a Lewis gene. Interestingly, antibodies raised to cancer cells often have specificity within the Lewis blood group.

Blood Group System - Cartwright

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.

The two antigens of the Yt system, Yta and Ytb, are both expressed at birth, however in a slightly lower expression level than seen on adult cells. They were shown to be resistant to trypsin treatment, but sensitive to other protein cleaving enzymes. Antibodies to these antigens were implicated in cases of delayed transfusion reactions but were not reported to have caused hemolytic disease of the newborn. No examples of Yt(a-b-) individuals have been found despite numerous studies. From population studies, Ytb appeared to be lacking, or of very low incidence, from Orientals, Amer-Indians and southern Africans; Yta was found lacking in approximately one per thousand individuals of European origin.

This system remained unexciting until it was found that both of these antigens were weak or absent from paroxysmal nocturnal hemoglobinuria (PNH) III red blood cells. With a rush of activity in the early 1990's, it was found that the Yt blood group system antigens represented amino acid substitutions on the GPI-linked glycoprotein, acetylcholinesterase (AchE). This protein probably exists as a dimer (pair) in the red cell membrane. Yta and Ytb are equated to substitutions of a hisitidine and asparagine, respectively, at amino acid position 322 on the GPI-linked glycoprotein. AChE has been assigned a chromosome location at 7q22.

As of this date, there have been only two antigens associated with this system. Based on the present knowledge of the placement on AChE, it is far more likely that any new variants will be found investigating AChE peculiarities and their immune response than by standard serological methods.

Blood Group System - Kell

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.

The Kell antigens are encoded by a chromosomal location on the long arm of chromosome 7. They are located on a 93 kDa type II glycoprotein that makes a single pass through the membrane, is glycosolated at five sites and functions as a metalloprotease. The Kell antigen appears to be found on erythroid and nonerythroid tissue (primarily in testis). In nonerythroid tissues, as exemplified by skeletal muscle, Kell is disulfide-linked to XK.

This system, as with several other systems, has examples of "depressed phenotypes" and "null phenotypes". The McLeod phenotype (first detected during the investigation of the cells of a Dr. McLeod) have suppression of all inherited Kell blood group antigens, in addition to a unique red blood cell morphology; acanthocytes. Interestingly, the "null phenotype" cells, referred to as Ko, which by definition have no Kell blood group antigens, has normal discocytes.

The expression of the Kell genes is modified by epistatic effects, both from the Kell locus and from at least two regulatory genes, one of which is X-borne (see Kx).

Blood Group System - Lutheran

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).

The Lu(a-b-) phenotype or Lunull is very rare and may rise from one of the three distinct genetic circumstances. These individuals have mild acanthocytosis and poikilocytosis of their red cells. A dominant inhibitor gene, In(Lu), which is independent of the Lutheran locus, is responsible for the most common form of the Lu(a-b-) phenotype. The red cells appear to lack all Lutheran antigens using hemagglutination techniques; however, their presence can be demonstrated by absorption/elution methods. A second Lunull type is due to the homozygous inheritance of an amorph at the Lutheran locus. This is probably the true Lunull as no Lu antigens have been detected on the red cells even using absorption/elution techniques. Less than a half dozen individuals with this type are known. The final Lu(a-b-) type, due to an X-linked suppresser gene (XS2), also presents with weakened Lu antigens similar to the inhibitor type, however, only one such family has been found to date. The In(Lu) gene not only suppresses Lutheran but also the i, P1, Aua, Anton (An/Wj) and several other antigens defined by monoclonal antibodies.

The Lu protein has a molecular weight of 85 kD and is composed of 5 extracellular Ig- like domains. Lu and B-CAM (basel cell adhesion molecule) are different forms of the same protein/gene whose function is to bind/aminin.

Blood Group System - P

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, including anti-P, -Pk, -Tja, 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.

The biochemical nature of the antigens of the P blood group has been well defined. P system antigens are formed by the addition of carbohydrates to the fatty acid chain of sphingolipids. Because of the wide distribution of these antigens in nature, many of the antibodies to P system antigens result from immune response to other organisms. The Donath-Landsteiner antibody, found in cases of paroxysmal cold hemoglobinuria, has been thought to be such a response. Many cases of paroxysmal cold hemoglobinuria (PCH) in children are preceded by a flu-like illness or respiratory infection and are thought to be viral in nature. In adults, the Donath-Landsteiner antibody may appear in transient association with syphilis. It has been postulated that both the virus and spirochete carried a P-like carbohydrate structure that stimulates the autoantibody production. Recently, another virus, parvovirus B19, has been associated with the P blood group system. In healthy children, parvovirus B19 infection manifests itself with a malar rash while adult infection results in a mild flu-like illness. The persons at greatest risk of developing complications due to B19 are those with sickle cell disease and thalassemia.

The rare phenotype for this system is known as p [formerly Tj(a-)]. Soon after the discovery of this phenotype it was noted that the naturally occurring antibodies in p females were a cause of early abortion. Interestingly, the p phenotype is found more frequently in the Amish population.

Blood Group System - MNS

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.

The MNS antigens are located on either glycophorin A, glycophorin B, hybrid or mutant structures of both of these sialoglycoproteins which are encoded by two highly homologous and closely linked genes on the long arm of chromosome 4. This system was the first non-water soluble blood group system to be biochemically investigated. Many of the low incidence antigens associated with hybrid structures could only have been assigned to this system through biochemical and DNA investigation.

The MNS antigens are found predominately on the red cells with some found on the renal endothelium and epithelium. Antibodies against M are fairly common, being the most frequently found antibody in non-transfused children, however antibodies against N are exceedingly rare (undoubtedly because N can be encoded by some forms of glycophorin A when the N gene is present and the most common form of glycophorin B). Even though anti–M antibodies are found in multiply transfused individuals and multiparous females, it rarely if ever is associated with hemolysis of red cells. Antibodies against S, s and the majority of the remaining high and low incidence MNS antigens have been associated with hemolytic transfusion reactions and hemolytic disease of the newborn.

As with most blood group systems, this system has its "null" individuals. Those that lack both glycophorin A and B are referred to as MkMk (there are no detectable MNS antigens on these individuals' red cells). Those that lack glycophorin A but have a normal glycophorin B are En(a-) individuals and those that have glycophorin A but lack glycophorin B are S-s-U- individuals.

Interestingly, Landsteiner and Levine’s discovery prompted them to test apes with antisera prepared by immunizing rabbits with human M blood. These studies led them, conversely, to immunized rabbits with rhesus monkey blood in order to prepare anti-M reagents. Refer to the Rh blood group system and LW blood group system for their discovery.

Blood Group System - Indian

In 1973 a new antigen was reported that occurred in 5% of Indians from Bombay. Two years later, Dr. Giles reported the Salis serum which contained an antibody to a high frequency antigen that was antithetical to Ina, thus, it was named Inb. A higher frequency of Ina has been found in some Arabs (10-12%). Both of the Indian antigens can be suppressed by the In(Lu) gene (LU). In addition, the null phenotype, ie. "In(a-b-)", has been reported in a patient diagnosed with congenital dyserythropoietic anemia. In this case there appears to be a silent allele present (no protein is produced) but the exact molecular basis for this is unknown at this time.

The gene for Indian is located on chromosome 11p13 and is named CD44. CD44 is a 80,000 molecular weight glycoprotein that is found on many cells including white blood cells, brain, breast, heart, kidney, liver, lungs and skin to name a few. It is believed to be important in "lymphocyte homing". A point mutation at bp position 252 is responsible for the Indian antigens. The Ina antigen has proline at amino acid 26 while Inb has arginine. Preliminary data suggest that the antigen AnWj may also be carried on CD44 and that this antigen may soon be assigned to the IN system.

Although originally investigated as a serological problem, the IN/CD44 protein is of considerable importance in cancer. Alternative forms or sizes of the CD44 mRNA may be produced and are called isoforms. In certain types of cancer, eg. melanoma and colonic carcinoma, there is an over-expression of some CD44 isoforms. Thus, detection of these alternate forms is currently under investigation as a means for early detection of some cancers as well as new therapies for cancer.

Blood Group System - Rh

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. 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. That antibody produced in rabbits to rhesus monkey cells, and the similar human antibody specificities, have been named after the two original investigators, Landsteiner and Wiener (refer to LW blood group system).

Blood Group System - ABO

In 1900, Landsteiner observed that the red cells of some individuals were agglutinated by the serum of others and his detailed report a year later heralded the discovery of the first human blood groups. His limited experiments on laboratory colleagues demonstrated three distinct groups; his pupils Von Decastello and Sturli discovered a fourth group in 1902. It was 25 years before these groups were shown to be inherited as Mendelian characters by means of three allelomorphic genes A, B and O and were, in fact, entities of one blood group system. For most of medicine this was considered a meaningless curiosity, but for those involved with the early development of transfusions it was quickly noted that transfusion of blood only of the same group could mean the difference between a successful outcome or a fatality.

While various nomenclatures have been used to describe these factors, it was internationally decided in the mid-1940's that the characteristics would be individually identified as A, B, AB and O and the blood group system know as ABO. Since that time there have been billions of tests preformed and almost as many publications describing various aspects and associations of this blood group systems or its allelic products.

The genes controlling the ABO system are located on the long arm of chromosome 9 and encode for either an a (1-3) N-acetylgalactosaminyltransferase (for A) or a (1,3) galactosyltransferase (for B). These transferases incorporate immunodominant sugars (GalNAc for A; Gal for B) on to one of four different types of oligosaccharide chains (type 2 is predominant on red cells, type 1 is found in secretions, plasma and some tissue) carried on glycosphingolipid or glycoprotein molecules. The placement of the A and B antigens is dependent upon the existence of a substrate produced by the Hh blood group system (ISBT # 018). Some individuals have weakened or variant expression of A and/or B which can be attributed to inheritance of variant forms of transferases. Those individuals that have neither transferases, inheritors of two amorph genes, are group O. To date, 14 A alleles, 14 B alleles and 8 O alleles have been identified at the molecular level and more remain to be found.

The ABO antigens, as stated above are not restricted to the red blood cell membrane but can be found in saliva and all body fluids except spinal fluid if the individual has inherited a secretor gene. The antigens are also found on most epithelial and endothelial cells. It also appears on lymphocytes and platelets as it is adsorbed from the plasma. Alterations of ABH expression have been found in various forms of cancer. Furthermore, antigens of the ABO system may play a role in resistance to bacteria or viruses. There is tremendous population diversity in the percentage of the ABO groups throughout the world. The ease of testing for the ABO blood groups contribute to the broad knowledge base of this blood group system.

Blood Group

Blood groups are individual differences in proteins and/or carbohydrates that make up part of the red blood cell membrane. Most of them are inherited just like you inherit eye or hair color. At one time we could count all of the known blood group antigens on two hands. The first blood group was very simple- ABO. The designation of O was suppose to mean that no antigen , ie. A or B was present on the red blood cells. Later it was discovered that the type O cells actually had another antigen which was named H (for "human"). In the 1920s, animal experiments led to the discovery of additional blood group factors which were given the letters M, N and P. There seemed to be lots of room for expansion in the early 1940s when the Rh blood group was found and the antigens were assigned the letters C/c, E/e and D (there is no d).

However, by the late 1940s the single letter blood group antigens became in short supply and a new approach was taken. The last name of the first person to make a new antibody was now used to name the system, eg. Lewis, and the first two letters of the last name were used for the abbreviation, eg. Le. But even this caused problems when Mrs. Duffy and Mrs. Kidd were found. The D and K antigens had already been assigned and were too similar, so the new blood groups became Duffy (Fy) and Kidd (Jk). The blood group antigen list continued to grow in this manner until over 240 types were known.

None of these terms were easily adaptable to the new computer age and so another system was designed which used numbers. However, this became cumbersome and finally in 1980 the International Society of Blood Transfusion established a Working Party for Nomenclature to consider blood group terminology. Under the direction of Dr. Fred Allen, they devised a genetically based numerical terminology. The original report published in 1990 has been periodically updated as new systems have been established and antigens properly assigned. However, the ISBT Working Party recognized the fact that "popular" terms might still be in use and has tried to incorporate that into their recommendations. In presenting the following information on blood groups we have utilized the ISBT system for both the system name and number.

Blood Bank

This was my last place during industrial training. Blood bank is an essential unit in Pathology, and the heart of every hospital. Its function is to supply matched and screed blood and blood products to the wards. Recruiting potential donors and the flow of regular donors is done to maintain the commitment of blood bank to supply blood to the patient effectively. The blood bank must have adequate number of personnel. These individuals must be properly trained and have adequate experience to enable them to carry out their work properly. The test that been done after working hours are grouping and cross matching and Coomb’s test.

All specimens for routine tests must be sent to blood bank before 3.15pm, all the specimens must be sent to the blood bank immediately either on working hours or after working hours. A request form is required when requesting blood. In order to maintain the safeness of the blood, eight service health goals are required which are wellness focus, person focus, informed person/Personalized information, self care, care close to home, seamless care and high quality care. Blood bank will sent the blood to IMR, Pathology Department, HKL, and Pusat Darah Negara for immunophenotyping for leukemia and lymphoma cases, cytogeneticts cases in Haematology Department, IMR and DNA analysis for thalassemia cases.

Working procedure in blood bank are compatibility test (Major and Minor Crossmatching), anti human globulin tests (Coomb’s Test), preparation of Platelet/Fresh Frozen Plasma, preparation of Cyroprecipitate/Cytosuspension, blood donor selection, ABO and Rhesus (D) Grouping by Tile Method, Rhesus (D) Grouping Du Antigen Typing by Tube Method, haemoglobin estimation by Copper Sulphate Method, quality assurance guideline for manufacture component, preventive and Corrective Action on Blood Refrigerator or Freezer, taking Temperature with Temperature Logger, released Blood and Blood Components from Unscreened (refrigerator) Freezer/Platelet Agitators to Screened and washing of Glass Tube. During my practical in blood bank, the tasks that I was assigned are compatibility test (Major and Minor Crossmatching), anti Human Globulin Tests (Coomb’s Test).Preparation of Platelet/Fresh Frozen Plasma and Haemoglobin Estimation by Copper Sulphate Method.

Reference Laboratory

Reference laboratories are the department of pathology Hospital Teluk Intan in a secondary lab. Confirmatory and specialized test that are not carried out here are send to its reference laboratories. The main and designated reference laboratory is Hospital Ipoh. Other reference laboratories are Institute for medical research (IMR), Hospital Kuala Lumpur (HKL), National Blood Transfusion Centre and other places. Therefore, the specimens are dispatched by specific time to reference laboratory and the test result normally will be obtained from the reference hospital. Example of the test is leptospirosis test which is sent to IMR for the result.