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Blood Donors-since it can be life threatening to give the wrong ABO group to the patient. Transfusion recipients-since we need to know the donor blood is ABO
compatible.
Transplant Candidates and Donors-ABO antigens are found in other tissues as well. Therefore the transplant candidates and donors must be compatible. Prenatal Patients-To determine whether the mothers may have babies who are suffering from ABO-HDN. It is also beneficial to know the ABO group should she start hemorrhaging. Newborns (sometimes) If the baby is demonstrating symptoms of Hemolytic Disease of the Newborn, the ABO group needs to be determined along with Rh and others. Paternity testing Since the inheritance of the ABO Blood Group System is very specific, this serves as one of the first methods to determine the likelihood that the accused father is the father or not.
O A B AB
45 40 11 4
49 27 20 4
ABO Typing
ABO typing involves both antigen typing and antibody detection. The antigen typing is referred to as the forward typing and the antibody detection is the reverse typing
The forward typing determines antigens on patient's or donor's cells a. Cells are tested with the antisera reagents anti-A, anti-B, (and in the case of donor cells anti-A,B) b. Reagents are either made from hyperimmunized human sources, or monoclonal antibodies. c. One advantages of the monoclonal antibodies are the antibody strength. d. Another advantage of monoclonals: human source reagents can transmit infectious disease (hepatitis). Reverse typing determines antibodies in patient's or donor's serum or plasma a. Serum tested with reagent A1 cells and B cells b. Reverse grouping is also known as backtyping or serum confirmation
ABO INHERITANCE
Inheritance Terminology:
gene: determines specific inherited trait (ex. blood type) chromosome: unit of inheritance. Carries genes. 23 pairs of chromosomes per person, carrying many genes. One chromosome inherited from mother, one from father locus:
site on chromosome where specific gene is located allele: alternate choice of genes at a locus (ex. A or B; C or c, Lewis a or Lewis b) homozygous: alleles are the same for any given trait on both chromosome (ex. A/A) heterozygous: alleles for a given trait are different on each chromosome (ex. A/B or A/O) phenotype: observed inherited trait (ex. group A or Rh positive) genotype: actual genetic information for a trait carried on each chromosome (ex. O/O or A/O) dominant: the expressed characteristic on one chromosome takes precedence over the characteristic determined on the other chromosome (ex. A/O types as A) co-dominant: the characteristics determined by the genes on both chromosomes are both expressed - neither is dominant over the other (ex. A/B types as AB) recessive: the characteristic determined by the allele will only be expressed if the same allele is on the other chromosome also (ex. can type as O only when genotype is O/O)
ABO Genes
The A and B genes found on chromosome #9. We inherit one gene (allele) from our father and one from our mother. The two co-dominant alleles are A or B. Anytime an individual inherits an A or B gene it will be expressed. The O gene signifies lack of A or B antigens. It is not expressed unless this gene is inherited from both parents (OO). Therefore the O gene is recessive. Below is the example of two individuals who are A. One inherited only one A gene along with an O gene and is therefore heterozygous. The other inherited 2 A genes and is homozygous for A.
1 = A/A 1 = Homozygous A Phenotype A Genotype A/A Can Contribute Only an A Gene to Offspring
Inheritance Patterns
We can't determine genotypes of A or B people unless family studies are done. Some basic rules of ABO inheritance are as follows: 1. A/A parent can only pass along A gene 2. A/O parent can pass along either A or O gene 3. B/B parent can only pass along B gene 4. B/O parent can pass along either B or O gene 5. O/O parent can only pass along O gene 6. AB parent can pass along either A or B gene
Offspring possibilities
Mother's Genes A O
Father's Genes B AB BO O AO OO
Mother's Genes A A
Father's Genes B AB AB B AB AB
Mother's Genes A A
Possibilities of an A/A mating with an O/O:
Father's Genes B AB AB O AO AO
Mother's Genes A A
Possibilities of an A/O mating with an O/O:
Father's Genes O AO AO O AO AO
Mother's Genes A O
Possibilities of an A/B mating with a O/O:
Father's Genes O AO OO O AO OO
Mother's Genes
Father's Genes O O
A B
AO BO
AO BO
2. H gene causes L-fucose to be added to the terminal sugar of precursor chain, producing H antigen (shown in this diagram of a Type 2 H antigen saccharide chaine).
3. Either A gene causes N-acetyl-galactosamine to be added to H substance, producing A antigen, (shown in this diagram) or
5. If both A and B genes present, some H-chains converted to A antigen, some converted to B antigen. 6. If H gene absent (extremely rare), no H substance can be formed, and therefore no A or B antigen. Result is Bombay blood group.
Subgroups of A and B
The subgroups of A and B are caused by decreased amounts of antigen on the red blood cells. They are inherited conditions. The most common are subgroups of A. Approximately 80% of the A's and AB's have a normal expression of A1. Most of the other 20% are either A2 or A2B. This subgroup has fewer H chains converted to A antigen result is more H chains on red cell, and
fewer A antigens.
There are other, weaker subgroups of A exist: A 3; Aint; Am, Ax; Ael. Each has a different pattern of reacting with anti-A, anti-A, and various antibody-like substances called lectins.
Lectins
Lectins are extracts of seeds of plants that react specifically with certain antigens. The two most common lectins used in Blood Bank are:
Ulex europaeus, or lectin H, which agglutinates cells that have H substance. Dolichos biflouros, or lectin A1, which agglutinates cells with A1.
Lectin-H reacts strongest with O cells, which has a high concentration of H antigen, and weakest with A1 cells, which have a low concentration of H. Lectin lectin-H O cells 4+ A2 cells 3+ A2B cells B cells 2-3+ 2+ negative A1 cells weak to negative positive A1B cells weak to negative positive Bombay cells negative negative
Differentiating Subgroups of A:
Use the following steps to help differentiate the subgroups of A: 1. Use lectin-A1 to differentiate A1 cells from all others - will agglutinate only A1 cells 2. Look for weaker or mixed field reactions 3. Look for anti-A1 in serum (serum reacts with A1 cells but not A2 cells) 4. Look at strength of reactions with anti-A,B or with lectin-H GROUP Reaction with anti-A Reaction with anti-A,B Reaction with LectinA1 Reaction with Lectin-H Presence of anti-A1 A1 4+ 4+ 4+ 0-w no A2 4+ 4+ 0 1-2+ may A3 mf mf 0 2+ may Ax 0 2+ 0 2-3+ often in serum
d.allele e.homozygous f.heterozygous g.phenotype h.genotype i.dominant j.co-dominant k.recessive 16. State the alleles in the ABO system. 17. State which alleles are co-dominant 18. State which allele is recessive 19. For each of the following phenotypes, give the possible genotypes: a. A b. B c. AB d. O 20. Predict all the possible phenotypes and genotypes from all blood type matings 21. Describe the sequence of events in the synthesis of the ABO antigens, beginning with the precursor substance. 22. State the sugars that are associated with each different blood group system 23. Describe the significant characteristics of the Bombay blood group. 24. Explain what lectins are. 25. Predict the reactions of each different blood group, including subgroups of A, with lectin-H. 26. Explain what reactions demonstrate a subgroup of A. Table of Contents Clinical Microbiology Syllabus
Methods: Forward Typing: The Forward Typing Area contains a separation membrane with one application zone and a detection area with parallel lines of antibody reagents specific
for blood groups A, B and D (principle see figure 1). Method for direct Forward Typing: 100 l of diluted blood or diluted erythrocyte sediment are transferred to the application zone, followed by 300 l of a diluent. Results can be read after 5 minutes. Reverse Typing: The Reverse Typing Area contains a separation membrane divided into four distinct application, migration and detection areas each (original principle see figure 2). Method: 25 l of a 5% suspension of reagent red cells for reverse grouping (Reverse Cyte A1, A2, B, O; Medion Diagnostics, Switzerland) are mixed in separate tubes with 100 l of plasma each. Suspensions are incubated for 2 minutes at room temperature, then, 50 l are transferred to the A1, A2, B, O application zones. Results can be read in the detection areas after another 3 minutes. Positive results in Forward Typing and Reverse Typing are seen as distinct red bands. Figure 1 Figure 2 Figure 1: Lateral Flow Forward Typing Assay Medion multicard with the configuration: A-B-D (VI-) -D (VI-) -K--C-C w
-c-E-e. Positives are recognized as distinct red bands. Results are valid when the val signal is positive (red spot) and the ctl is negative (no signal). Result: Blood group B Cc D. Ee kk Figure 2: Lateral Flow Reverse Typing: Principle Prototype strips used for testing a plasma from a donor with blood group O. Plasma is incubated with commercial A1, A2, B and O Reverse-Cyte cells, added to the application zone, and specific reactions are monitored in the detection zone. The presence of isoagglutinins is indicated by the red band in the detection area. Result: Blood group O plasma with anti-A1, anti-A2 and anti-B isoagglutinins. Figure 3 Figure 3: Lateral Flow Forward and Reverse Typing Assay Design of a possible combination device for the detection of blood group antigens and isoagglutinins. Results from a donor with blood group A (Rhesus positive) with anti-B isoagglutinin activity are shown. (Poster presentation at the 38. Jahreskongress der Deutschen Gesellschaft fr Transfusionsmedizin und Immunhmatologie (DGTI) 2