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Blood Grouping Systems and Typing Techniques

 

Introduction

 

A blood group system consists of antigens produced, either directly or indirectly, by alleles located at a single genetic focus or at loci so closely linked that crossing-over between the loci rarely occurs.(1) Inheritance of an allele usually, but not always, leads to the appearance its corresponding antigen on RBC membranes or in body secretions. Variant forms of a given allele can be inherited that produce increased or depressed amounts of the corresponding antigen. Antigen expression can also be influenced by genes inherited independently other blood group loci. For example, the expression of A or B antigens is affected by the action of the I gene or by alleles at the H blood group locus.

ABO, H, P, I or Lewis blood group antigens are constructed on structurally related carbohydrate molecules through the activity of gene-encoded glycosyltransferase enzymes. Since it is possible for the carbohydrate molecules to carry the determinants of more than one of these blood groups, the activities of the genes of one of these blood groups may affect the expression of the antigens of another. The antigens appear when specific sugars are added by the transferases to the ends of carbohydrate chains called oligosaccharides, The sugars added to the precursor chains by the gene transferases are referred to as immunodominant sugars since the sugars confer specific antigenic activity to the terminal portions of the converted oligosaccharide chains .(2)

Oligosaccharides consist of chains of sugars that can be attached to either glycoprotein or glycosphingolipid carrier molecules. In glycoproteins, oligosaccharides are linked via galactosamine (GalNAc) to polypeptide backbones. Structurally similar oligosaccharides are attached via glucose (Glc) to ceramide residues in glycosphingolipids. Glycosphingolipids form part of RBC membranes and also the membranes of most endothelial cells. Soluble forms are present in plasma, but are not secreted in body fluids. Soluble glycoproteins with blood group antigen activity exist in the body's serous and mucous secretions. Membrane-associated glycoproteins are present on RBCs and other body cells. (3)

 

The ABO System

 

A series of tests performed by Karl Landsteiner in 1900 led to the discovery of the ABO blood groups and to the development of routine blood grouping procedures. Landsteiner tested blood samples from his colleagues by mixing each person's serum with suspensions of RBCs from the others. Noting agglutination in some mixtures, but not in others, he was able to classify the blood samples into one of three groups, now named A, B and 0. (The fourth group, AB, was discovered in 1902 by Landsteiner's pupils von Decastello and Sturh.) Landsteiner recognized that the presence or absence of only two antigens, A and B, was sufficient to explain the three blood groups he saw. He also showed the serum of each person contained an antibody directed against the antigen absent from that person's RBCs. The first blood group system to be discovered, the ABO blood groups remain the most significant for transfusion practice. It is the only system in which the reciprocal antibodies are consistently and predictably present in the sera of normal people whose RBCs lack the corresponding antigen(s)"Landsteiner's Rule".  ABO incompatibility between a recipient and the donor is the foundation on which all other pretransfusion testing rests.(5.6)

 

 

Biochemical and Genetic Considerations

Glycosphingolipids carrying A or B oligosaccharides are integral parts of the membranes of RBCs, epithelial and endothelial cells; they are also present in soluble form in plasma. Glycoproteins that carry identical oligosaccharides are responsible for the A and B activity of secreted body fluids such as saliva. A and B oligosaccharides that lack carrier protein or lipid molecules are found in milk and urine.

Genes at three separate loci (ABO, Hh, and Sese) control the occurrence and the location of the A and B antigens. Three common alleles --A, B and O-are located at the ABO locus on chromosome 9. The A and B genes encode glycosyltransferases that produce the A and B antigens, respectively.(7) The O gene is considered to be amorphic since no detectable blood group antigen results from its action. The RBCs of group O persons lack A and B, but carry an abundant amount of H antigen because this antigen is the precursor material on which A and B antigens are built.

Family studies have shown that the genes at the remaining two loci, Hh and Sese (secretor), are closely linked. The chromosome on which they are located has not yet been identified. It is suggested that one of these loci may have arisen through gene duplication of the other. (8) Two recognized alleles reside at each locus. Of the two alleles at the H locus, one of these, H, produces an enzyme that acts at the cellular level to construct the antigen on which A or B are built. The other allele at this locus, h, is very rare. No antigenic product has been linked to h, so this gene is also considered an amorph. The possibility exists that other alleles occur at the Hh locus that differ from H in that they cause the production of only very small amounts of H antigen. (9)

The Se gene is directly responsible for the expression of H (and indirectly responsible for the expression of A and B) on the glycoproteins in epithelial secretions such as saliva. Eighty percent of the population are secretors because they have inherited the Se gene and produce H in their secretions that can be converted to A and/or B (depending on the genetic background of the secretor). The se gene, having no demonstrable product, is an amorph.

Oligosaccharide chains on which the A and B antigens are built can exist as simple structures of a few sugar molecules linked together in linear fashion. They can also exist as more complex structures that are composed of many sugar residues connected together in branching chains. It has been proposed that the differences in cellular A, B and H activity seen between specimens from infants and adults may be related to the number of branched structures carried on the cellular membranes of each group.(10) The RBCs of infants are thought to carry A, B and H antigens built predominantly on linear oligosaccharides. Linear oligosaccharides have only one terminus to which the H, then A and B, sugars can be added. In contrast, the RBCs of adults appear to carry a high proportion of branched oligosaccharides. Branching creates additional portions on the oligosaccharide that can be converted to H and then to A and B antigens.

A and B genes do not produce antigens directly but instead produce enzymes called glycosyltransferases that add specific sugars to oligosaccharide chains that have been converted to H by the action of the H gene. H antigens are constructed on precursor oligosaccharide chain endings called Type 1 and Type 2. (2.7)  The number 1 carbon of the terminal 6-carbon sugar b-D-galactose (Gal) is linked to the number 3 carbon of subterminal N-acetyl-glucosamine (GluNAc) in Type 1 chains and to the number 4 carbon of GluNAc in Type 2 chains. Blood group-active glycoproteins present on cell surfaces or in body fluids carry either Type 1 or Type 2 chains. Glycosphingolipids present in the plasma and those on the membranes of most glandular and parenchymal cells also have either Type 1 or Type 2 chain endings. In contrast, the glycolipid antigens produced by the RBCs; appear to be formed exclusively of Type 2 chains. These chains are carried on a class of glycosphingolipids called paraglobosides.

At the cellular level, the H gene transferase produces a fucosyltransferase that adds fucose (Fuc) in alpha (1-2) linkage to the terminal Gal of Type 2 chains. The A and B gene transferases can only attach their immunodominant sugars when the Type 2 (or Type 1) chains have been substituted with Fuc (ie, changed to H) thus, the A and B antigens are constructed at the expense of H. The A gene-specified N-acetyl-galactosaminyl-transferase and the B gene-specified galactosaminyl-transferase add GalNAc and Gal, respectively in alpha (1-3) linkages to the same Gal acted on by the H gene transferase.

The alleles at the ABO locus that result in subgroups (phenotypes of A and B that differ from each other with respect to the amount of A or B carried on the RBCs) produce transferases that differ from one another in their ability to convert H antigen .(7,11)  The O gene is thought to produce a protein that can be detected immunologically but has no detectable transferase activity. As a consequence, the RBCs of group O persons carry readily detectable, unconverted H antigen. The secretion of Sese persons contain Type I and Type 2 chains with no H, A or B activity. It has been suggested that the H and Se genes each encode a different  fucosyltransferase. (8,11) The enzyme produced by H acts primarily on Type 2 chains and in RBC membranes. That produced by Se prefers (but does not limit its action to) Type 1 chains and acts primarily in the secretory. Studies performed on the secretions of persons with the rare Oh phenotype support the concept that two types of H antigen exist.(9)  Persons of this  phenotype, who are genetically Hh and Sese, have no H and therefore, no A or B antigens on their RBCs or in their secretions. However, H, A and B antigens are found in the secretions of genetically hh persons, who, through family studies, appear to possess at least one Se gene.

 

A and B antigens are detected in direct agglutination tests with anti-A and anti-B reagents. ABO reagents frequently produce weaker reactions with the RBCs of newborns than with RBCs from adults. Weaker reactions are encountered because A and B antigens are not fully developed at birth, even though they can be detected on the RBCs of embryos 5-6 weeks old. (5-13)  By the time a person is 2-4 years old, RBC A and/or B antigen expression is fully developed. Antigenic expression remains fairly constant throughout life, although decreases have been seen in old age.

 

 

Subgroups of A

 Subgroups of A are phenotypes that differ from others of the same ABO group with respect to the amount of A antigen carried on RBCs, and, in  secretors, present in the saliva.  The two principal subgroups of A are A1, and A2. RBCs of both react strongly with anti­ A reagents in direct agglutination tests. The serologic distinction between A1, and A2 is based on results obtained in tests with reagent anti-A1, prepared from group B human serum or the lectin of Dolichos biflorus seeds. Under prescribed testing conditions, anti-A1, reagents agglutinate A1, but not A2 RBCs. The RBCs of approxi­mately 80% of group A or group AB persons are agglutinated by anti-A1, and, thus are classified as A1, or A1B. The remaining 20% whose RBCs are agglutin­ated by anti-A, but not by anti-A1, are called A2, or A2B. (16)

Anti-A1 occurs in the serum of 1% to 8% of A2 persons and 22% to 35% of A2B persons.(13)  Anti-A1 can cause discrepancies in ABO testing and incompatibilities in crossmatches with A1, or A1B RBCs. It is considered to be clinically insignificant unless it reacts at 37 C. It is not necessary to test group A RBCs with anti A1, to confirm their subgroup status except when working with samples from people whose sera contain anti-A1.

Subgroups weaker than A2 occur infrequently and, in general, are characterized by decreasing numbers of A antigen sites on the red cells and a reciprocal increase in H antigen activity. The genes responsible constitute less than 1% of the total pool of A genes. Classification of weak subgroups is generally based on the:

 

  • Degree of RBC agglutination by anti-A and anti-A1.

  • Degree of RBC agglutination by anti­ A, B.

  • Degree of H reactivity of the RBCs.

  • Presence or absence of anti-A1 in the serum.

  • Presence of A and H substances in the saliva of secretors.

 

RBCs of the Ax Ael, Aint or A3 subgroups are seen only infrequently in transfusion practice. Ax and Ael RBCs are readily recognized as subgroups of A by the discrepancies they produce between RBC and serum grouping tests. Ax RBCs are, in general, agglutinated by human anti-A,B but not by human anti-A. However, Ax RBCs react with some murine monoclonal anti-A reagents.(15) Ael RBCs fail to react with anti-A or anti-A,B of any origin. Adsorption and elution studies are necessary to show that these RBCs carry the A antigen. RBCs of the Aint phenotype can be identified only if tests with anti-A1 are performed. Aint RBCs react more weakly than A1 RBCs with anti-A1, yet more strongly with anti-H than do A2 RBCs. A3 RBCs produce a characteristic mixed-field pattern of small agglutinates among many free RBCs in tests with anti-A and anti-A,B. Weak subgroups of A such as Ax, Ael, and Aint, cannot be identified on the basis of blood grouping tests alone. Saliva studies and adsorption/elution studies must be performed.

Subgroups of B are even less common than subgroups of A.

 

Antibodies to A and B

Ordinarily, people possess antibodies directed toward the A or B antigen absent from their own RBCs.  This predictable complimentary relationship permits serum grouping in addition to RBC ABO grouping tests . The immunoreactive configurations that confer A and B specificities to molecules of the RBC membrane also exist in other biologic entities, notably bacterial cell walls. Bacteria are widespread in the environment and it appears that their presence in intestinal flora, dust, food and other widely distributed agents ensure a constant exposure of all persons to A-like and B-like antigens. Immunocompetent persons react to the environmental antigens by producing antibodies to those that are absent in their own systems. Thus, anti-A occurs in the sera of group O and group B persons and anti-B occurs in the sera of group O and group A persons. Group AB people, having both antigens, make neither antibody.

Time of Appearance

Anti-A and anti-B production generally begins after the first few months of life. Occasionally infants can be found that are already producing these antibodies at the time of birth. (5) Antibody production remains fairly constant until late in adult life. In elderly people, anti-A and anti-B levels may be lower than those seen in young adults. (1,16)  Since antibody production normally begins after birth, results that are obtained with the sera of newborns or infants to about 4-6 months cannot be considered valid because the antibodies may have been acquired through the placental transfer of maternal IgG anti-A and anti-B.

 

Reactivity of Anti-A and Anti-B Anti-A produced by group B persons and anti-B produced by A people are composed predominantly of IgM molecules.(5) Small quantifies of IgG molecules are also present in the sera of these two groups. IgG is the dominant form of anti-A and anti-B of group O serum. The IgG forms readily cross the placenta and can cause ABO hemolytic disease of the newborn (HDN). Because of the predominance of IgM antibodies in the sera of group A or B persons, ABO hemolytic disease is rarely seen in ABO-incompatible infants born of group A or B mothers. (5,13)

The distinguishing features of IgM and IgG anti-A and anti-B are given in Table 10-3. Both immunoglobulin types preferentially agglutinate red cells at room temperature (20-25 C) or below. Both are efficient activators of complement at 37 C. The complement-mediated lytic capabilities of these antibodies are most apparent when an incubation phase at 37 C is added to serum grouping tests. Occasionally, patients or donors can be found whose sera cause the hemolysis of ABO-incompatible red cells at temperatures below 37 C. Hemolysis by ABO antibodies in serum grouping tests should be suspected when a pink to red discoloration appears in the supernates or when the buttons of reagent ABO grouping RBCs are reduced in size or are missing. Hemolysis must be interpreted as a positive result. Hemolysis of RBCs will not occur if reagent RBCs are suspended in solutions that contain EDTA or other anticoagulants that prohibit complement activation.

 

Agglutinin development and cause

 “It is difficult to understand how agglutinins are produced in individuals who do not have the respective antigenic substances in their red blood cells. However, small amounts of group A and B antigens are believed to enter the body in the food, in bacteria, or by other means, and these substances presumably initiate the development of anti-A or anti-B agglutinins.”

-Guyton, Textbook of Medical Physiology

 

Anti-A,B (Group O Serum)

 Serum from group O persons contains an antibody designated as anti-A,B. It reacts with A and B RBCs and activities for both RBC groups cannot be separated by differential adsorption. Eluates prepared from group A RBCs that have been used to adsorb group O serum contain anti-A and an antibody that reacts with both A and B RBCs. Similar findings are obtained when B RBCs are used for adsorption. Saliva from A or B secretors inhibits the activity of this antibody with either A or B RBCs.

Anti-A1

The anti-A of group B serum appears, from simple studies, to contain separable anti-A and anti-A1. In direct tests, group B serum agglutinates A1 and A2 RBCs, yet following adsorption with A2 RBCs, group B serum reacts only with A1 RBCs. If further tests are performed, the differences between A antigen expression on A1 and A2 RBCs appears to be quantitative rather than qualitative. Further adsorption of group B serum with A2 RBCs will remove all serum activity for A1 RBCs. The apparent anti-A1 made by adsorption of group B serum can be thought of as a weakened form of anti-A. It reacts with A1 RBCs because they have more A antigen than do A2 RBCs. The sera of persons of certain weak subgroups of A may contain anti-A1 that is serologically similar to the anti-A1 of group B adsorbed serum.

Adsorbed group B serum can be used at the practical level to differentiate the two common A subgroups. More frequently, however, anti-A, reagents are employed that are manufactured from the lectin of Dolichos biflorus. The lectin will react with A, and A2RBCs unless it has been diluted appropriately. Reagent anti-A, lectins have been diluted by the manufacturer to react with & but not A2, RBCs.

 

Routine Testing for ABO

RBC typing tests, using anti-A and anti-B to determine the presence or absence of the antigens, are often referred to as direct or forward grouping tests. Serum grouping tests, using reagent A, and B RBCs to detect serum anti-A and anti-B, are sometimes called reverse grouping tests. Routine grouping of donors and patients must include both RBC and serum tests, each serving as a check on the other. It is permissible to test RBCs only when ABO grouping is performed to confirm the group of donor blood that has already been labeled with a blood group designation or when testing is performed on samples from infants less than 6 months of age.

Some ABO RBC grouping reagents are prepared from pools of sera from persons who have been stimulated with A or B blood group substances to produce antibodies of high titer. Other ABO grouping reagents are manufactured from monoclonal antibodies derived from cultured cell lines. Both types of reagents are potent and agglutinate most antigen positive RBCs on direct contact without centrifugation. Serum testing is most reliably performed by tube or microplate methods. Anti-A and anti-B occurring in the sera of patients and donors are frequently too weak to agglutinate RBCs without centrifugation. Therefore, it is not recommended that serum grouping tests be performed on slides.

On occasion, other reagents are incorporated into ABO grouping procedures. These include anti-AB (RBC grouping) and reagent A2 and O RBCs (serum grouping). Anti-A,B reagents, such as anti-A and anti-A,B, are derived either from human sera or monoclonal cell lines. Some workers elect to use anti-A,B routinely in grouping tests to avoid mistakenly classifying weakly reactive A or B RBCs as group O. Unfortunately, there exists a misconception that anti-A,B is more potent than either anti-A or anti-B and thus, will detect most weak subgroups of A or B. With the exception of Ax, anti-A,B (particularly that of human origin) does not agglutinate the RBCs of less common subgroups that fail to react with anti-A or anti-B. (13) Human-source anti-A,B does not react well with Ax RBCs in immediate-spin tests. The reagent and RBCs must be incubated together for 1060 minutes at room temperature for reactions to occur. If the manufacturer's directions recommend using anti-A,B for the detection of weak subgroups, it means that its reactivity against A. RBCs has been demonstrated. AABB Standards  does not require the use of anti-A,B to detect weak A or B subgroups since such bloods often distinguish themselves from O by failing to produce the expected serum grouping results. Moreover, the adverse consequences associated with the transfusion of weak subgroups of A and B to group O recipients are minimal.

Some commercially prepared reverse grouping reagents contain A2 and O RBCs in addition to A, and B RBCs. The sole purpose of A2 RBCs in these reagents is to facilitate the recognition of anti-A, in subgroups of A  Since the majority of A blood does not contain anti-A2, many workers employ this reagent only when discrepancies between RBC and serum tests are encountered. Group O RBCs of reverse grouping sets can be used to identify those sera that contain cold-reactive agglutinins that may interfere with serum grouping tests. Generally, such RBCs cannot be used for the detection or identification of unexpected antibodies since they have not been manufactured to meet the requirements of the FDA for these purposes.

Manufacturers of ABO reagents provide, with each reagent package, detailed instructions for the use of the reagent. Instructions may vary from one manufacturer to another in testing requirements. Therefore, it is important to follow the directions supplied with the specific reagent in use.

 

Discrepancies

Some discrepancies can be traced to problems arising in RBC grouping tests.

 

  • Samples obtained from patients who have received transfusions recently or who have received a bone marrow transplant may produce unexpected reactions if the samples contain a mixture of RBCs that differ from each other in their ABO group (transfusion or transplantation chimera).

  • Blood samples from persons who have inherited variant A or B genes may carry poorly expressed antigens. Weak antigens are also found on the red cells of some people with diseases such as leukemia. (1,5) Samples from these people may fail to produce the expected reactions in direct agglutination tests with anti-A and anti-B.

  • Abnormalities of an inherited or acquired nature, leading to what is referred to as polyagglutinable states, can result in RBCs with modified membranes. The modified RBCs can be unexpectedly agglutinated by reagent anti-A, anti-B or both.

  • Abnormal concentrations of serum proteins, the presence of macromolecules (or in cord blood samples, the presence of Wharton's jelly) may cause nonspecific aggregation that simulates agglutination if RBCs are suspended in their own serurn.

  • High concentrations of A or B blood group substances in the serum have been found, on rare occasions, to inhibit the activities of reagent antibodies to such an extent that unexpected negative reactions are obtained when serum- or plasma-suspended RBCs are used. (13,23)

  • The sera of some persons contain antibodies to the dyes used to color anti-A and anti-B reagents. These antibodies can causefalsely positive agglutination reactions if serum- or plasma-suspended RBCs are used in testing. (13)

   

Acquired B Phenotype

 

Acquired B state should be considered when the serum of a patient contains anti-B and the patient's RBC appears to be group AB with a weak B antigen. The acquired B phenotype arises through the modification of the A antigen by microbial enzymes called deacetylases. The enzymes modify cellular A immunodominant sugars (GalNAc) so they become more like the B sugar (Gal). A, RBCs are the only group that exhibits acquired B activity in vivo.' When present in sufficient numbers, acquired B antigens react with human anti-B in direct agglutination tests. While many examples of RBCs with acquired B antigens react weakly with anti-B, some examples can be found that are agglutinated quite strongly.

 

To confirm that group A1 RBCs carry the acquired B structure:

 

  • Check the patient's diagnosis. Acquired B antigens tend to be associated with carcinoma of the colon or rectum, infection with gram-negative organisms and intestinal obstructions.

  • Test the patient's serum against his or her own RBCs. The anti-B in the patient's serum will not agglutinate his or her own RBCs when they carry the acquired B determinant.

  • Test the RBCs with monoclonal anti-B. Some monoclonal reagents, unlike human-source antibodies, do not react with the acquired B phenotype - Such information may be carried in the instructions that accompany the monoclonal reagent. Test the RBCs with human anti-B serum. acidified to pH 6.0. Acidified anti-B sera do not react with the acquired B receptor.

  • If the patient is a secretor, test saliva for the presence of A and B substances. Patients whose RBCs carry acquired B structures will have A, but not B, substance in their saliva.

 

Acquired A-Like Antigens

 

ABO discrepancies are sometimes associated with Tn polyagglutination. (1,13) Tn activated RBCs have glycoproteins that carry improperly formed oligosaccharides. Such structures appear when there is a genetic dysfunction in a hematopoietic stem cell resulting in a deficiency of a particular glycosyltransferase. When group  O ,Tn or group B, Tn RBCs are tested, they may behave as if they have acquired an A-like antigen reacting with human or monoclonal anti-A reagents. The A like antigen of Tn RBCs can be differentiated from A arising through the action of an A gene transferase if RBCs are treated with proteolytic enzymes before testing. A-like antigens of Tn RBCs are destroyed by enzymes.

 

Mixed-Field Agglutination

In some cases, samples are encountered that contain two distinct, separable populations of RBCs. Usually a mixture occurs because group O RBCs were transfused to a group A (or group B) patient. RBC mixtures also occur in a condition called chimerism, resulting either from the intrauterine exchange of erythropoietic tissue by fraternal twins or from mosaicism arising through dispermy. Less frequently, it occurs when a patient has received a transplant of bone marrow that is of an ABO group different from the patient's own.

Mixed-field agglutination is characteristically seen when A3 RBCs are tested with anti-A. If the agglutinated RBCs are removed and the remaining RBCs again tested with anti-A, mixed-field agglutination occurs in the residual population as well. Mixed-field agglutination may also be seen with RBCs carrying A antigens weakened by diseases such as leukemia or with Tn RBCs.

 

Antibody-Coated Red Blood Cells

RBCs from infants with HDN, or from adults suffering from AIHA or HTRs may be heavily coated with IgG antibody molecules. Such RBCs often agglutinate spontaneously in the presence of high protein reagents such as anti-D. In some cases, sensitization is such that the RBCs also agglutinate in low-protein ABO reagents. RBCs coated heavily with IgM cold reactive autoagglutinins will agglutinate spontaneously in saline tests. If the RBCs are washed several times with saline warmed to 37 C, the antibodies can be eluted from the RBC membranes.

 

 

Unexpected alloantibodies

 

Unexpected alloantibodies, such as anti-P, or anti-M, react at room temperature and may agglutinate the reagent RBCs used in serum grouping that have the corresponding antigen. In general, reagent RBCs used for antibody detection will also be agglutinated at room temperature. (Rarely, the serum may react with an antigen on the RBCs other than A and B that is not present on the antibody detection RBCs.) To determine the correct ABO group of sera containing other cold-reactive alloantibodies:

 

  • Identify the alloantibody, as described in Chapter 15.

  • Test the reagent A, and B RBC to determine which reagent, if either, carries the corresponding antigen.

  • Test the serum against examples of A, and B RBCs that lack the corresponding antigen. For instance, if anti-M is identified, test the serum against A,, M - and B, M RBCs to resolve the discrepancy. If the antibody detection test is negative, repeat serum ABO tests with several examples of A, and B RBCs. Since the antibody is directed against an antigen of low frequency, most randomly selected A, and B RBCs will lack the corresponding antigen.

 

Rouleaux

Serum from patients with abnormally high concentrations of serum proteins, who have altered serum protein ratios or who have received plasma expanders of high molecular weight can cause reagent RBCs to appear agglutinated. Some of these samples cause rouleaux to occur. Rouleaux formation can be easily recognized microscopically if the RBCs aggregate in what have been described as "stacks of coins." More often, such sera cause aggregates that appear as irregularly shaped clumps that closely resemble antibody mediated agglutinates.

 

The H System

As mentioned previously, the genes of the H blood groups are H and h. H leads to the production of the H antigen that serves as the precursor molecule on which A and B antigens are built. The amount of H antigen is, in order of diminishing quantity, O>A2>B>A2B>A1>A1B. H like antigens are found in nature, and persons of the rare Oh phenotype, whose RBCs lack H, have (in addition to anti-A and anti-B) potent anti-H in their serum that is considered to be clinically significant .(21) Occasionally, group & A,B or (less commonly) B persons have so little unconverted H antigen on their RBCs that they may produce anti-H. In such situations the antibody is relatively weak and virtually always reacts at room temperature or below. In contrast, the anti-H of Oh persons reacts well over a wide thermal range (from 4-37 C)  with all RBCs except those of other Oh people. Oh patients must be transfused with only Oh blood because their anti-H rapidly destroys the H + RBCs of the other ABO groups. (28)

 

Oh Phenotype

The term "Bombay" has been used for the Oh phenotype because examples of such RBCs were first discovered in Bombay, India. The symbol Oh has been selected to denote the phenotype bemuse results obtained in routine ABO grouping tests mimic those of group O persons. Oh RBCs are not agglutinated by anti-A, anti-B or anti-A,B. That a sample is Oh (and not group O) is generally not recognized until serum from the Oh person is tested against group 0 RBCs. Group 0 RBCs are agglutinated by Oh sera as strongly as A and B RBCs. The Oh phenotype can be proven if the RBCs are tested with the anti-H lectin of Ulex europeaus. Anti-H lectin fails to agglutinate Oh RBCs, although it agglutinates group O RBCs quite strongly. Further confirmation testing can be performed if other examples Of Oh RBCs are available. The serum of a suspected Oh person will be compatible only with the RBCs of other Oh, people.

 

Para-Bombay Phenotypes, & and Bh

Ah and Bh RBCs lack serologically detectable H antigen, but carry small amounts of A or B, depending on the genotype of the donor. Weak reactions are obtained in grouping tests with anti-A or anti-B.(6,11,13) The RBCs are nonreactive with anti-H lectin or with the anti-H sera of Oh Persons. The para-Bombay phenotype is thought to result from the inheritance of variant H genes that produce only minute amounts of H antigen. All of the H is converted to A or B by the products of the A and B genes, respectively. The sera of Ah and Bh people contain anti-H in addition to the expected anti-A or anti-B.

 

The Lewis Antigens

The common Lewis antigens, Lea and Leb, are not intrinsic to RBCs, but are carried on plasma glycosphingolipids that are adsorbed from plasma to the RBC membranes. Their presence or absence in plasma and on RBCs is dependent, in part, on whether a person has inherited one Le or two le genes. The Le gene encodes a fucosyltransferase that adds Fuc in alpha(1-4) linkage to the subterminal GlcNAc of Type I oligosaccharides. (11)  The resulting structure has Lea activity. Persons who have inherited the dominant Se(H) gene in addition to Le produce an antigen called Leb. When Leb is produced it is adsorbed preferentially over Lea to RBC membranes. Leb is made when Type I chains are first modified into H by the Se(H) gene transferase. The Le gene transferase then adds Fuc to this structure to form Leb. The le gene is an amorph. Persons who are lele produce no Lea and no Leb antigens. Table 10-4 shows the Lewis phenotypes, together with their frequencies in the population. RBCs that type as Le(a+b+) are only rarely found when human antisera are used in typing. Such RBCs are seen more frequently when more potent monoclonal anti-Lea and anti-Leb reagents are used.

 

Lewis Antibodies

Lewis antibodies occur almost exclusively in the sera of Le(a-b-) people, and usually without known RBC stimulus. People whose RBC phenotype is Le(a- b+) do not make anti-Lea because small amounts of unconverted Lea are present in their saliva and plasma. It is unusual to find anti-Leb in the serum of a Le(a+ b-) person. Anti-Lea and anti-Leb may occur together in sera. They are almost always IgM and do not cross the placenta. Because of this, and because Lewis antigens are poorly developed at birth, the antibodies have not been implicated in HDN. Lewis antibodies may bind complement. Fresh sera that contain anti-Lea (or infrequently anti- Leb) may cause the in vitro hemolysis of incompatible RBCs. In vitro hemolysis is more often seen with enzyme-treated RBCs than with untreated RBCs.

Most Lewis antibodies agglutinate saline-suspended RBCs of the appropriate phenotype. The resulting agglutinates are often fragile and are easily dispersed if RBC buttons are not resuspended gently after centrifugation. Agglutination sometimes is seen after incubation at 37 C, but rarely of the strength seen in tests incubated at room temperature. Some examples of anti-Lea, and less commonly  anti-Leb, produce positive indirect antiglobulin reactions, providing complement is present in the reaction mixture and polyspecific antiserum is used.

Sera with anti-Leb activity can be divided into two categories. The most common examples react best with RBCs of group O and A2 These have been designated as anti-Lebh. Those that react equally well with the Leb antigen on RBCs of all ABO phenotypes are called anti-LebL . Anti-Lebh , but not anti-Le bL is neutralized by saliva from group O, Le(a- b-) persons who are secretors of H substance. Table 10-5 lists the serological behavior of the common Lewis system antibodies.

Two additional antibodies have been given names in the Lewis system although the determinants with which they react are not determined by Lewis genes. Anti-Lec has been reported in one human subject as a cold-reactive agglufinin.  This antibody agglutinated the RBCs; of Le(a-b-) people who are sese and are therefore nonsecretors, of H substance. The antibody called anti-Led agglutinates the RBCs of Le(a- b-) secretors. The product defined by anti-Led has been identified as the Type I oligosaccharide to which Fuc has been added at the H-active site. Anti-Led should more correctly be called anti-Type I H. The material that reacts with anti-Lec seems to be the Type 1 chain with no added Fuc molecules. No examples of anti-Le d have been found in humans but both anti-Lec and anti-Led have been successfully produced in goats injected with saliva from Le(a- b-) nonsecretors and Le(a- b-) secretors of H, respectively.

 

 

Lewis Antigens in Children

RBCs from newborn infants usually fail to react with both human anti-Lea and anti-Leb and, thus are considered to be Le(a- b-). Some can be shown to carry small amounts of Lea when tested with potent monoclonal or goat anti-Le a reagents. Reliable Lewis grouping of young children may not be possible, as test reactions may not reflect the correct phenotype until 6 years of age. Among children, the incidence of Le(a+) RBCs is high and that of Le(b+) RBCs low. The phenotype Le(a+ b+) may be observed as a transient phase in children whose phenotypes as adults will be Le(a- b+).

Cord RBCs are agglutinated by certain sera that agglutinate the Le(a+ b- ) and Le(a-b+), but not Le(a-b-), RBCs of adults.33 In serological. tests, these sera appear to contain inseparable forms of anti-Lea and Le b. They define a determinant that has been called Lex, and which is present on the majority of cord RBCs and on the Le(a +) or Le(b +) RBCs of adults. Many serologists, have suggested that anti-Lex may represent a more potent or more avid form of anti-Lea.

 


 

References

 

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2.     Watkins WM, Morgan WTJ. Possible genetical pathway for the biosynthesis of blood group mucopolysaccharides. Vox Sang 1959;4:97-119.

3.     Steane S. Proteins, lipids, carbohydrates and nucleic acids. In: Pierce S, Steane 5, eds. Biochemistry for blood bankers: selected topics. Arlington, VA: American Association of Blood Banks, 1983:43-66.

4.     Mourant AE, Kopec AC, DomaniewskaScoczak K. The distribution of the human blood groups and other polymorphisms. 2nd ed. London: Oxford University Press, 1976.

5.     Mollison PL. Blood transfusion in clinical medicine. 7th ed. Oxford: Blackwell Scientific Publications, 1983.

6.     Grundbacher Fj. Changes in the human A antigen of erythrocytes with the individual's age. Nature 1964;204:192-4.

7.     Watkins WM. The glycosyltransferase products of the A, B, H and Le genes and their relationship to the structure of the blood group antigens. In: Mohn JF, Plunkett RW, Cunningham RK, Lambert RM, eds. Human blood groups. Basel: S Karger, 1977:134-42.

8.     Oriol R, Danilovs J, Hawkins BR. A new genetic model proposing that the Se gene is

9.     Hakomori SI. Blood group ABH and Ii antigens of human erythrocytes: chemistry, polymorphism and their developmental change. Sernin Hematol 1981;18:39-47.

10. Salmon C, Cartron JP, Rouger P. The human blood groups. New York: Masson Publishing, USA, 1984.

11. Yoshida A. Identification of genotypes of blood group A and B. Blood 1980;55:119-23.

12. Beattie KM. Discrepancies in ABO grouping. In: A seminar on problems encountered in pretranfusion tests. Washington DC: American Association of Blood Banks, 1972;12965.

13. Race RR, Sanger R. Blood groups in man. 6th ed. Oxford: Blackwell Scientific Publications, 1975.

14. Rolih SD. New frontiers in serologic testing. In: Wallas CH, McCarthy LJ, eds. New frontiers in blood banking. Arlington, VA: American Association of Blood Banks, 1986;12733.

15. Toivanen P, Hirvonen T. Iso- and heteroagglutinins in human fetal and neonatal sera. Scand J Haematol 1969;6:42-8.

16. Code of federal regulations. Current edition. Title 21 CFR, part 660.26. Washington, DC: US Government Printing Office.

17. Holland PV, ed. Standards for blood banks and transfusion services. 13th ed. Arlington, VA: American Association of Blood Banks, 1989.

18. Judd WJ, Butch SH. Streamlining serological testing. In: Smith DM, Judd WJ, eds. Blood banking in a changing environment. Arlington, VA: American Association of Blood Banks, 1984:15-39.

19. Moulds JM. Polyagglutination: overview and resolution. In: Beck ML, Judd WJ, eds. Polyagglutination. Washington, DC: AmerIcan Association of Blood Banks, 1980:1-22.

20. Beck ML: Blood group antigens acquired de novo. In: Garratty G, ed. Blood group antigens and disease. Arlington, VA: American Association of Blood Banks, 1983:45-66.

21. Judd WJ. Microbial-associated forms of polyagglutination (T, Tk and acquired B). In: Beck ML, Judd WJ, eds. Polyagglutination. Washington, DC: American Association of Blood Banks, 1980:23-54.

22. Barber M, Dunsford 1. Excess blood-group substance A in serum of patient dying with carcinoma of stomach. Br Med J 1959;1:607.

23. Pierce S. Anomalous blood bank results. In: Trouble-shooting the crossmatch. Washington, DC: American Association of Blood Banks, 1977:85-114.

24. Reid M. Autoagglutination dispersal utilizing sulphhydryl compounds. Transfusion 1978;18:353-5.

25. Judd WJ, Steiner EA, Oberman HJ. Reverse and typing errors due to prozone: how safe is the immediate spin crossmatch? (abstract). Transfusion 1987;27:527.

26. Bhatia HM, Sathe MS. Incidence of "Bombay" (0,,) phenotype and weaker variants of A and B antigens in Bombay (India). Vox Sang 1974;27:524-32.

27. Davey RJ, Touralt MA, Holland PV. The clinical significance of anti-H in an individual with the 0, (Bombay) phenotype. Transfusion 1978;18:738-42.

28. Gunson HH, Latham V. An agglutinin in human serum reacting with cells from Le(a - b -) nonsecretor individuals. Vox Sang 1972;22:344-53.

29. Potapov MI. Production of immune anti-Lewis sera in goats. Vox Sang 1972;30:211-3.

30. Waheed A, Kennedy MS, Gerhan S. Transfusion significance of Lewis system antibodies: report on a nationwide survey. Transfusion 1981;21:542-5.

31. Issitt PD. Antibodies reactive at 30 C, room temperature and below. In: Clinically significant and insignificant antibodies. Washington DC: American Association of Blood Banks, 1979:13-28.

32. Arcilla MC, Sturgeon P. Lel, the spurned antigen of the Lewis blood group system. Vox Sang 1974;26:425-38.

33. Levene C, Sela R, Rudolphson Y et al. Hemolytic disease of the newborn due to anfi_PP~Pk (anti-Tia). Transfusion 1977;17:569-72.

34. Levine P. Comments on hemolytic disease of the newborn due to anti-PP,Pk (anti-Tjl). Transfusion 1977:17:573.

 

 



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