GENETICS IN BLOOD BANKING

Mendelian Inheritance and Significance Terms

Basic Principles: 1. Each parent contributes 1/2 of the genetic information. 2. The genetic information is contained on chromosomes composed of DNA 3. Humans have 23 pairs of chromosomes a. 22 matched (autosomal) chromosomes and b. 1pair of sex chromosomes (females have 2 X chromosomes and males a X and a Y chromosome). Examples of Chromosome locations for common Blood Groups are as follows: System ABO MNSsU P Rh Kell Lewis Duffy Kidd Xg Common Genes A, B, O M, N, S,s,U P1 D, C, E, c, e K, k, Kpa, Jsa,Kpb, Jsb Le, le Fya, Fyb, Fy3 Jka, Jkb Xga Located on Chromosome 9 4 22 1 7 19 1 18 X

4. Genes are the units of inheritance within the chromosomes. 5. At each location, or loci, on the chromosomes there are possibilities of different forms of the genes, these different forms are called alleles. (For example the ABO Blood Group System, there are A1, A2, B, and O as common alleles. or allelic genes) 6. When the inherited alleles are the same the person is homozygous such as OO, when the individual inherits 2 different alleles such as AO, they are heterozygous for both the A and O genes. 7. On occasion we will see examples of dosage where some antibodies will react more strongly with homozygous cells than with heterozygous cells. For example, an anti-E that reacts as a 3+ with EE cells and only 1+ with Ee cells. 8. A Punnett Square is used to determine the inheritance possibilities for a particular mating. For example if the mother's genotype (genes) are AO and the father's genotype (genes) are BO, you would have the following Punnet square possibilities. In this example there three heterozygous possibilities AB, AO, and BO and one homozygous possibility OO Dad B O

Mom

A O

AB BO

AO OO

9. In the above Punnett Square, the AB genotype will have both A and B antigens, therefore the phenotype is AB since both are expressed. AO and BO genotypes will demonstrate only the A and the B antigens respectively and therefore the phenotypes are A and B respectively. The individual that is OO will have the O phenotype. 10. A and B genes are dominant, or co-dominant, and the O gene is recessive. The dominant genes will be expressed if present. Recessive genes will only be expressed if they are homozygous. 11. Most Blood Group genes are co-dominant and therefore will be expressed if present.

Mitosis and Meiosis

Two kinds of cell division: Mitosis is cell division that leads to two identical cells that has the same number of paired chromosomes. (In humans there are 23 pairs or 46 chromosomes) Meiosis is the cell division that occurs when gametes (sperm and eggs) are formed and will not have pairs of chromosomes. (In humans there will be 23 chromosomes in the sperm that will match up with the 23 chromosomes in the egg when fertilization occurs to form the gametocyte.). The sex of the child is determined by the X and Y chromosomes. Males provide either X or Y chromosome and females provide only provide X chromosomes. Genes that are found only on the X chromosome are said to be sex-linked. Genes found on the other 22 pairs of chromosomes are autosomal.

Genotypes, Phenotypes, Amorphs, and Pedigree Charts

Here is a pedigree chart for three generations. The ABO phenotypes are listed for the known blood types.

The mother in the first generation has the AB genes since her phenotype is AB. In the mating for the second generation, the genotype for the father could either be BB or BO since his father's phenotype is unknown. It would appear the mother is AA since both her parents are A, but..... Look at their children's blood types. What is the mother's genotype now since both children are B?

There are no O individuals in the above example but O is considered an amorph since it has no detectible traits. The lack of D antigen is considered another example of an amorph since no reaction with anti-D indicates the individual is D negative (Rh negative). These two examples are recessive genes that need to be homozygous for it to be demonstrated.

Other Concepts Relating to Blood Group Genetics

Contributions of Blood Genetics to the Field of Human Genetics

Certain characteristics that make Blood Genetics useful for the field of human genetics 1. 2. 3. 4. Simple and unquestionable pattern of inheritance Can test or determine the phenotypes readily More than 1 allele occurring fairly frequently Environment does not affect the expression of the genes.

Some discoveries that were found in blood genetics:

Multiple alleles seen in ABO system Linkage between the secretor genes with the Lutheran genes on the same chromosome

Population Genetics Linkage

Linkage between the secretor genes with the Lutheran genes on the same chromosome was already noted. 1. We now know that the D gene is closely linked to the Cc and Ee genes. The most frequently inherited Rh positive set of genes is CDe and the most frequent Rh negative gene is cde or ce since d is an amorph. 2. The MNSs genes are also linked, MS, NS, Ms, Ns leading to a difference between the expected frequency and the observed frequency. Expected frequency MS = 0.53 (M) X 0.33 (S) = 0.17 Ms = 0.53 (M) X 0.67 (s) = 0.36 NS = 0.47 (N) X 0.33 (S) = 0.16 Ns = 0.47 (N) X 0.67 (S) = 0.31 Observed frequency 0.24 0.28 0.08 0.39

Silent Genes
As indicated already there are some amorph blood group genes that exist and lead to none expression of a blood antigen. The following are some examples of silent genes. Blood Group Gene h = r Ko Lu Jk Fy Blood Group System ABO Rh Kell Lutheran Kidd Duffy Homozygous Phenotype Oh or Bombay Rhnull Knull Lu(a-b-) Jk(a-b-) Fy(a-b-)

Blood Group Nomenclature

Accepted terminology according to AABB Technical Manual - 50th Anniversary 1953 2003, 14th edition, 2003, p221. 1. Genes encoding the expression of blood group antigens are written in italics (or underlined if italics are not available). If the antigen name includes a subscript (A1), the encoding gene is expressed with a superscript (A1) 2. Antigen names designated by a superscript or a number (eg, Fya, Fy:1) are written in normal (Roman)script....Superscript letters are lowercase.... 3. When antigen phenotypes are expressed using single letter designation, results are usually written as + or -, set on the same line as the letter(s) of the antigen: K+ k-. 4. To express phenotypes of antigens designated with a superscript letter, that letter is placed in parentheses on the same line as the symbol defining the antigen: Fy(a+) and Fy(-). 5. For antigens designated by numbers, the symbol defining the system is notated in capital letters followed by a colon, followed by the number representing the antigen tested. Plus signs do no appear when test results are positive (K:1), but a minus sign is placed before negative test results: K:1, K:-1. If tests for several antigens in one blood group have been done, the phenotypes is designated by the letter(s) of the locus or blood group system followed by a colon, followed by antigen numbers separated by commas: K: -1, 2, -3, 4. Only antigens tested are listed;...

Table 10-4. Examples of Correct and Incorrect Terminology

(AABB Technical Manual, p.222

Term Description Phenotype Phenotype Antibody Antigen Antibody Phenotype Phenotypes Phenotype Phenotype

Correct Terminology Fy(a+) Fy(a+b-) Anti-Fya K anti-k K:1, K:-1 A Rh+, B RhM+NRh:-1, -2, -3, 4,5

Incorrect Terminology Fya+, Fy(a+), Fya(+), Fya+, Fya(+), Duffya+ Fya+b-, Fy(a+b-), Fya(+)b(-), Fya(+)b(-) Anti Fya, Anti-Duffy Kell (name of system) Anti-Cellano k1+, K:1+, K(1), K:(1), K1-, K:1-, K1negative A+ (means positive for A antigen) B- (means negative for B antigen) M(+), MM (unproven genotypes) Rh: -1, -2, -3, +4, +5, Rh: 1-,2-,3-, 4+,5+

Public versus Private Genes

Public Genes are found in most of the population. In the Kell Blood Group System, the Kpb is found in close to 100% of the population

Genes that are very rare are referred to private genes. Kpa is very rarely found (2.3% in whites and almost never in African Americans) and therefore close to being a private gene.

Paternity Testing
Today most paternity testing is done using the following technology:

Red Cell Testing for the following Blood Group System: ABO, MNSs, Rh, Duffy, Kidd, Kell, White Cell Testing using HLA antigens DNA testing

1. Can a mother who types A and the alleged father who types O have a child who types B? 2. Can a mother who types CC and the alleged father who types cc have a child who types CC?

OBJECTIVES - Genetics in Blood Banking

1. Describe the importance of blood group genetics as it relates to the overall field of genetics. 2. Demonstrate the basics of inheritance of blood group traits relating to chromosomes, dominant and recessive genes, alleles, genotypes, phenotypes, heterozygous and homozygous inheritance, autosomal and sex-linked inheritance. 3. Relate DNA and RNA roles in inheritance. 4. Identify what are inheritance patterns and pedigree charts. 5. Distinguish between mitosis and meiosis. 6. Explain the inheritance of dominant versus recessive versus codominant traits. 7. Differentiate between phenotypes and genotypes. 8. Identify the role of population genetics in calculating gene frequencies. 9. Explain crossing over and linkage. 10. Differentiate between public and private genes. 11. Explain the use of blood group genes as genetic markers.

ABO BLOOD GROUP SYSTEM

ANTIGENS AND ANTIBODIES
Definition:
Blood group system

A series of antigens exhibiting similar serological and physiological characteristics, and inherited according to a specific pattern.

Importance of the ABO system:

Most important (clinically significant) Blood Group System for transfusion practice Why? This is the only blood group system in which antibodies are consistently, predictably, and naturally present in the serum of people who lack the antigen. Therefore ABO compatibility between donor and recipient is crucial since these strong, naturally occurring A and B antibodies are IgM and can readily activate complement and cause agglutination. If ABO antibodies react with antigens in vivo, result is acute hemolysis and possibly death.

Indications for ABO grouping:

ABO grouping is required for all of the following individuals:

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.

Discovery of the ABO system:

In 1900 Karl Landsteiner reported a series of tests, which identified the ABO Blood Group System. In 1910 he won Nobel prize for medicine for this discovery. He mixed the serum and cells of all the researchers in his lab and found four different patterns of agglutination. From those studies he developed what we now know as Landsteiner's rules for the ABO Blood Group:
1. A person does not have antibody to his own antigens 2. Each person has antibody to the antigen he lacks (only in the ABO system) 3. Below are the four blood groups and the antigens and the expected, naturally-occurring antibodies present.

BLOOD GROUP A B AB O

ANTIGEN A B A and B neither anti-A or anti-B

ANTIBODY anti-B anti-A neither anti-A,B

Incidence (%) of ABO Blood Groups in the US Population

ABO Group O A B AB Whites 45 40 11 4 Blacks 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

Routine ABO Typing

Reaction of Cells Tested With
Anti-A 0 + Anti-B 0 0 O A Red Cell ABO Group

Reaction of Serum Tested Against

A1 Cells + 0 B Cells + +

Reverse ABO Group

O A

0 +

+ +

B AB

+ 0

0 0

B AB

Discrepancies in ABO typing

1. Results of forward and reverse typing must agree before reporting out blood type as seen in the about table. 2. If forward and reverse do not agree, must identify cause of discrepancy. 3. If cannot resolve discrepancy, must report out blood type as UNKNOWN and give group O blood

Characteristics of ABO antigens:

ABO antigens are glycolipid in nature, meaning they are oligosaccharides attached directly to lipids on red cell membrane. These antigens stick out from red cell membrane and there are many antigen sites per red blood cell (approximately 800,000) Besides their presence on red blood cells, soluble antigens can be present in plasma, saliva, and other secretions. These antigens are also expressed on tissues other than red cells. This last fact is important to consider in organ transplantation. ABO antigens are only moderately well developed at birth. Therefore ABO-HDN not as severe as other kinds of Hemolytic Disease of the Newborn. .

Characteristics of ABO antibodies:

1. These are expected naturally occurring antibodies that occur without exposure to red cells containing the antigen. (There is some evidence that similar antigens found in certain bacteria, like E.coli, stimulate antibody production in individuals who lack the specific A and B antigens.) 2. Immunoglobulin M antibodies, predominantly 3. They react in saline and readily agglutinate. Due to the position of the antigen and the IgM antibodies it is not necessary to overcome the zeta potential. 4. Their optimum temperature is less than 30oC, but reactions do take place at body temperature 5. Not only are these antibodies expected and naturally occurring, they are also commonly present in high titer, 1/128 or 1/256. 6. They are absent at birth and start to appear around 3-6 months as result of stimulus by bacterial polysaccharides. (For this reason, newborn blood is only forward typed.)

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

2 = A/O
2 = Heterozygous A Phenotype A Genotype A/0 Can Contribute A or O 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. 2. 3. 4. 5. 6. A/A parent can only pass along A gene A/O parent can pass along either A or O gene B/B parent can only pass along B gene B/O parent can pass along either B or O gene O/O parent can only pass along O gene AB parent can pass along either A or B gene

ABO phenotypes and genotypes

1. Group A phenotype = A/A or A/O genotype 2. Group B phenotype = B/B or B/O genotype 3. Group O phenotype = O/O genotype 4. Group AB phenotype = A/B genotype

Offspring possibilities
Possibilities of an A/O mating with a B/O: (Children's genotypes in purple) Father's Genes Mother's Genes B A O AB BO

O AO OO

Possibilities of AA mating with BB: (Children's genotypes in purple) Father's Genes Mother's Genes B A A AB AB

B AB AB

Possibilities of an A/A mating with a B/O: (Children's genotypes in purple)

Father's Genes Mother's Genes

A A Possibilities of an A/A mating with an O/O: Father's Genes Mother's Genes O A A Possibilities of an A/O mating with an O/O: AO AO O AO AO B AB AB O AO AO

Father's Genes Mother's Genes

A O Possibilities of an A/B mating with a O/O: O AO OO O AO OO

Mother's Genes

Father's Genes

O A B AO BO

O AO BO

BIOCHEMISTRY OF THE ABO SYSTEM

The ABO antigens are terminal sugars found at the end of long sugar chains (oligosaccharides) that are attached to lipids on the red cell membrane. The A and B antigens are the last sugar added to the chain. The "O" antigen is the lack of A or B antigens but it does have the most amount of next to last terminal sugar that is called the H antigen.

Production of A, B, and H antigens

The production of A, B and H antigens are controlled by the action of transferases. These transferases are enzymes that catalyze (or control) addition of specific sugars to the oligosaccharide chain. The H, A, or B genes each produce a different transferase, which adds a different specific sugar to the oligosaccharide chain. To understand the process let's look at the sequence of events:
1. Precursor chain of sugars is formed most frequently as either Type 1 or Type 2 depending on the linkage site between the N-acetylglucosamine (G1cNAc) and

Galactose (Gal).

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

4. B gene causes D-galactose to be added to H substance, producing B antigen.

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.

Bombay blood group:

The Bombay blood group lacks H gene and therefore cannot make H antigen (H substance). Since the H substance is the precursor for the A and B antigens, these antigens also are not made. The cells type as O and the serum has anti-A, anti-B, and anti-H since the individual lacks all of these antigens. Anti-H agglutinates O cells. The only cells Bombay individuals do not agglutinate are from other Bombay blood people since they lack the H antigen,

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.

A small percentage of the individuals

There are other, weaker subgroups of A exist: A3; 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 O cells A2 cells A2B cells B cells A1 cells A1B cells Bombay

cells lectin-H Lectin-A1 4+ negative 3+ negative 2-3+ negative 2+ negative weak to negative positive weak to negative positive negative negative

Differentiating Subgroups of A:
Use the following steps to help differentiate the subgroups of A:
1. 2. 3. 4. Use lectin-A1 to differentiate A1 cells from all others - will agglutinate only A1 cells Look for weaker or mixed field reactions Look for anti-A1 in serum (serum reacts with A1 cells but not A2 cells) Look at strength of reactions with anti-A,B or with lectin-H 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

GROUP Reaction with anti-A Reaction with anti-A,B Reaction with Lectin-A1 Reaction with Lectin-H Presence of anti-A1

Problems with Ax:

Because Ax cells initially type as O and serum usually has anti-A1, (along with anti-B), patient forwards and reverses as O. Unfortunately when Ax is transfused into an O individual, the naturally occurring anti-A,B will react with the donor cells causing a transfusion reaction. Therefore: To prevent Ax from being erroneously typed as O, confirm all group O donors with anti-A,B.

OBJECTIVES ABO SYSTEM

1. 2. 3. 4. 5. 6. Explain why the ABO system is the most important for blood transfusion practice. List the situations in which an ABO type would be required. Describe 6 significant characteristics of ABO antigens. Describe 6 characteristics of ABO antibodies. Explain how the ABO system was discovered. State Landsteiner's rules.

7. List the blood groups in the ABO system, the antigen(s) present on the e cell in each blood group and the antibody(ies) in the serum for each, for adults. 8. State the differences in ABO antigens and antibodies in newborns. 9. State which ABO groups could safely receive a red cell transfusion from donors of each of the following blood types: A, B, AB, O 10. State which ABO groups could safely receive a plasma transfusion from donors of each of the following blood types: A, B, AB, O 11. Explain how ABO blood types are determined. 12. Explain what is meant by forward and reverse grouping, backtyping, and serum confirmation. 13. Explain what an ABO discrepancy is, and what must be done if the discrepancy cannot be resolved 14. State the incidence of each ABO blood group in the Caucasian population, and how the percentages differ in the Black and Oriental populations. 15. Define each of the following and give an example of each within the ABO system: a.gene b.chromosome c.locus 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 lectinH. 26. Explain what reactions demonstrate a subgroup of A.

Rh SYSTEM
History
In 1939, Hemolytic Disease of the Newborn was first described by Levine and Stetson. The cause of hemolytic disease of the cause was not specifically identified but maternal

antibody suspected. A year later (1940) Karl Landsteiner and Alexander Wiener injected animals with Rhesus monkey cells to produce an antibody which reacted with 85% of human red cells, which they named anti-Rh. Within a year Levine made connection between maternal antibody causing HDN and anti-Rh. Between 1943-45 the other common antigens of the Rh system were identified. For many years the exact inheritance of the Rh factors were debated Weiner promoting Rh and hr terminology and Fisher-Race utilizing DCcEe for the various Rh antigens. In 1993, Tippett discovered true mode of Rh inheritance using molecular diagnostics

Rh Antigens
D (Rho) is the most important antigen after A and B antigens. Unlike the anti-A and antiB antibodies, anti-D antibodies are only seen if a patient lacking D antigen is exposed to D + cells. The exposure of D+ cells usually occurs through pregnancy or transfusion.

There are 5 principle antigens that may be found in most individuals. They are:

D found in 85% of the population C found in 70% of the population E found in 30% of the population c found in 80% of the population e found in 98% of the population (d) which has never been identified but refers to the 15% of the population who has no D antigen

There are at over 50 Rh antigens that have been identified including those that are either combinations of these antigens or weak expressions of the above antigens, but most Rh problems are due to D, C, E, c or e.

Alleles:
The common alleles are:

C and c are alleles with Cw occasionally seen as a weaker expression of C. E and e are alleles although E is seen only a third as often as e. The e antigen is referred to as a high incidence antigen since it is found in 98% of the population. D and the lack of D (or d) are alleles.

Characteristics of Rh antigens
The Rh antigens together are proteins of 417 amino acids. These proteins cross the red cell membrane 12 times. There are only small loops of the protein on the exterior of the cell membrane.

Therefore the Rh antigens are not as available to react with their specific antibodies and there are fewer antigen sites than ABO. Unlike the ABO system the Rh antigens are not soluble and are not expressed on the tissues. They are well developed at birth and therefore can easily cause hemolytic disease of the newborn if the baby has a Rh antigen that the mother lacks. Besides the antigens being well-developed at birth, they are very good immunogens. This is especially true to D, which if the most immunogenic after A and B antigens.

Rh Antibodies
Unlike the ABO antibodies that are mainly IgM, the Rh antibodies are commonly IgG. They are NOT naturally occurring and therefore are formed by immune stimulus due to transfusions or baby's red blood cells during pregnancy. The most common antibody to form is anti-D in Rh negative individuals. Since Rh antibodies are IgG they bind best at 37oC and their reactions will be observed with the indirect antiglobulin technique. Agglutination reactions are enhanced by high protein (albumin), low-ionic strength saline (LISS), proteolytic enzymes (ficin) and polytheylene glycol (PEG). Rh antibodies will react more strongly with homozygous cells than with heterozygous cells. For example, an anti-E will react with strongly with E+E+ cells and more weakly with E+e+ cells. This is called dosage. Both Hemolytic Disease of the Newborn and Hemolytic Transfusion Reactions can occur due the various Rh antibodies. Anti-D has been the biggest concern since it was recognized in the 1940's as being the most common cause of hemolytic disease of the newborn. Since the D antigen is so immunogenic we screen all donor units for the D antigen. Therefore if an individual is A+, it means both the A and the D antigens are present. On the other hand, if an individual is A-, the A antigen is present and the D antigen is absent. To prevent problems due to anti-D:

we try to always give Rh-negative individuals Rh-negative blood and we give Rhoimmune globulin to Rh-negative mothers to prevent the formation of anti-D during pregnancy.

The incidence of Rh antibodies

Anti-D most common antibody seen in Rh(D) negative people

Anti-E most common antibody seen in Rh pos people since only 30% of the population have the antigen Anti-C or Anti-c less common - most people have the antigen Anti-e often seen as autoantibody and will make it difficult to find compatible blood since 98% of the population have the e antigen Anti-C,e or Anti-c,E often seen in combination. If a patient lacks both a C and e and has made an anti-C, then enhancement techniques should be done to make sure that an anti-e is not also present.

Rh System Inheritance
From the 1940's to the 1990's the mechanism for inheritance of the Rh Blood Group System was in question. The terminology that is part of the Fisher-Race Theory is most commonly used even today.

Fisher-Race Theory
The Fisher-Race theory involved the presence of 3 separate genes D, C, and E and their alleles c and e and the absence of D since an anti-d has never been found. These three genes are closely linked on the same chromosome and are inherited as a group of 3. The most common group of 3 genes inherited is CDe and ce (D negative) is the second most common.

Weiner Theory
Weiner believed there was one gene complex with a number of alleles resulting in the presence of various Rh antigens. According to Weiner there were 8 alleles, Ro, R1, R2, Rz, r, r', r", ry , which ended up with different antigens on the red cells that he called Rho, rh', rh", hr', hr". Weiner terminology is not use as often today, but you will often see Rho(D) when a person considered to be Rh-positive. At times the gene terms are easier to use than Fisher-Race. If a person has the Fisher-Race genotype of DCe/DCe, it is easier to refer to that type as R1R1 2. Made up of combinations of genetic products

Tippett Theory
In 1986, Tippett predicted that there are two closely-linked genes - RHD and RHCE. The RHD gene determines whether the D antigen that spans the membrane is present. Caucasians who are D negative have no gene at that gene loci. In the Japanese, Chinese, and Blacks of African descent have an inactive or partial gene at this site. The RHCE gene determines C, c, E, e antigens produced from the alleles:

RHCe RHCE RHcE

RHce

Rh Gene Complexes, Antigens, Possible Combinations and Percentages Haplotypes Genes Present Antigens Present Phenotype Percentage 1 R RHD RHCe D,C,e R1 42% r RHce dce r 37% R2 RHD RHcE DcE R2 14% RHD RHce Ro (more common in Dce Ro 4% Blacks) r' RHCe dCe r' 2% r" RHcE dcE r" 1% z R RHD RHCE DCE Rz <1% ry RHCE dCE ry <1%

Translating From Wiener To Fisher-Race

There are times when you will need to convert Weiner to Fisher-Race or vice versa. It will be easier to do these conversions if you remember the following: 1. 2. 3. 4. 5. 6. R always refers to D whether it is Ro, R1, R2, or the very rare Rz. r always refers to the lack of D o refers to having no C or E 1 or ' always refers to C 2 or " always refers to E The very rare haplotypes that have both a C and E are given letters from the end of the alphabet z and y.

Determining Genotypes From Phenotypes

The following steps will be helpful in determining from the individual's phenotype. These rules are based on probability so the least likely genotypes will involve R z or ry. 1. Type patient for the five Rh antigens: D, C, c, E, e 2. Start with D: is it positive or negative? 1. If negative, the individual will be homozygous d. 2. If positive for D, you can't tell yet whether the individual is homozygous or heterozygous for D. Therefore, put D on just one chromosome. 3. Look at C: is it positive or negative? 1. If negative, put c on each chromosome. 2. If positive, look at c result to determine if the C is homozygous or heterozygous. If there is no c present, there would be two C and it would be homozygous. If a c is present as well as C, they are heterozygous. 3. If homozygous, then put C on each chromosome. 4. If heterozygous, put C on same chromosome as D; put c on other. 4. Look at E: is it positive or negative? 1. If negative, put e on each chromosome.

2. If positive, look at e result to determine if homozygous or heterozygous. 3. If homozygous, put E on each chromosome. 4. If heterozygous, put E on same chromosome as the D unless the D already has a C; put e on other chromosome. DCe is more common than DcE and DCE is extremely rare. 5. Put C and E together on same chromosome only if no other possible combinations

Most Common Genotypes

The following genotypes are listed as the most common with 1 being the most common in Whites and 7 the least common. Rz and ry are so rare they are not included in the following table. Incidence of the most common genotypes Genotype Incidence(%) Weiner DCE Whites Blacks Haplotypes DCe/ce R1r 31.1 8.8 1 o DCe/Dce RR 3.4 15 Dce/Ce Ror 0.2 1.8 DCe/DCe R1R1 17.6 2.9 1 DCe/Ce R r' 1.7 0.7 ce/ce rr 15 7 1 2 DCe/DcE RR 11.8 3.7 1 DCe/cE R r" 0.8 <0.1 DcE/Ce R2r' 0.6 0.4 2 DcE/ce Rr 10.4 5.7 DcE/Dce R2Ro 1.1 9.7 o Dce/ce R r 3.0 22.9 o o Dce/Dce RR 0.2 19.4 DcE/DcE R2R2 2.0 1.3 2 DcE/cE R r" 0.3 <<0.1

Antigens Present 1 D, C, c, e

2 3 4

D, C, e ce DCcEe

5 6 7

DcEe Dce DcE

Applications of Rh genotyping
Paternity testing of the blood group antigens is based on a process of exclusion. Since the RHD and RHCE are closely linked and Ce, ce, cE are produced by a single gene, there are limited combinations that the father can provide. HDN predictability by testing the father's Rh genotype. This helps predict likelihood of HDN due to D when mom has anti-D. The most common Rh genotype of the father will indicate whether the baby has O%, 50%, or 100% probability of being D positive.

If the father is also D negative (ce/ce), the baby will be D negative as well and there is a 0% probability of the baby suffering from Rho HDN.

If the father's Rh genotype appears to be either, R1r, R2r or Ror, the baby has a 50% probability of being D positive and suffering from Rh o HDN. On the other hand if a father's Rh genotype appears to be any of the following, R1R1, R2R2, R1R2, RoRo, R1Ro, or R2Ro, the baby has a 100% probably of getting a D gene from his father and therefore being D positive and suffering from Rho HDN.

Variants
Weak D (Du)
Weak D is a weakly expressed D antigen that will only be demonstrated after incubation at 35-37oC followed with antiglobulin testing. (ie being demonstrated only by Coombs technique). An Rh control must always be run along with the weak D test. Always consult the product insert to determine if Rh Control needs to be run when performing the immediate spin D testing. The following results could be obtained when performing the D testing: Immediate Spin 37oC Anti-D Anti-D Rh Co Anti-D Rh Co + 0 0 0 0 0 0 0 0 0 0 0 0 0 AGT Anti-D Rh Co + 0 + 0 0 + Interpretation D positive Weak D True D negative Or any time the Rh control is positive, you can not interpret the results and need to perform further testing

Testing for Weak D

AABB requires that all donor blood that originally fails to react with anti-D at immediate spin must be tested for weak D. Units that test weak D positive would be labeled D positive and would be transfused only to D positive individuals. Hospitals may or may not test all Rh negative recipients for weak D. The cost of time and reagents is minimized if only the immediate spin. This may create some confusion with the recipient if their donor card indicates they are Rh positive but they type Rh negative when they are the recipient. Recipients that type D negative at immediate spin would be given D negative blood, which not create a problem for the patient. When performing testing prenatal and postnatal mothers, D-negative blood at immediate spin would be tested for weak D as well to determine if they are eligible for Rho Immune Globulin. Since Rho Immune Globulin is actually anti-D it is safe for a true D negative, but not for a weak D positive mother. Why do weak D's exist?

There are three explanations for weak D's.

Quantitative Weak D There are individuals that quantitatively produce fewer D antigen sites. This is more common in Blacks and is often seen with the Dce haplotype. On rare occasions among Whites an unusual DCe or DcE may also produce a quantitatively decrease weak D. Position Effect Weak D In this case the D is weakened by the position of a C on the opposite haplotype which is called the trans position. The two Rh genotype combinations where this type of weak D is seen are: Dce/Ce and DcE/Ce. Today this type of weak D would type as a regular D due to the improvement of reagents. Partial D antigen (Mosaic D) It has been found that some D-positive individuals make an alloanti-D that reacts with other D positive cells but not their own. Many of these will demonstrate a weak D type of reaction. In this type of weak D, the individuals are lack some of the components of the D antigen and therefore are able to make allantibodies to those specific components if they are transfused with D positive blood.

Other Rh System Variants

There are presently 46 Rh antigens identified and named. The following are the most common of those variants

Cw is a low frequency antigen found in approximately 2% of Whites and 1% of Blacks. It is not an allele of C and c. Its allele is MAR, which is found in 99.9% of the population. V and VS are low frequency alleles found in 1% or less of the Whites, but are more common in Blacks. V is found in 30% of the Blacks and VS in 32%. G is present when D or C present due to the present of serine at the 103 position of the Rh polypeptide. Anti-G will react with both D+ and C+ cells. f is present when c and e together on same chromosome: Dce or ce. This is the most common of what are called cis product antigens. Rhnull has no Rh antigens on their red cells but these individual can transmit normal Rh antigens to their offspring. In the most common type the core Rh polypeptide is missing. A less common type has the regulator gene that turns off the expression of Rh. There have been at least 43 individuals in 14 families that are Rhnull. In these individuals the red blood cell membrane is abnormal and some of these have been identified when it was observed that they had hemolytic anemia and abnormal red cell morphology. If these individuals develop an Rh antibody following a transfusion or pregnancy, it is considered a anti-total Rh antibody.

Rh Typing
False Positives
False positive D's occur:

1. When following through to AGT for weak D and will be identified as false positive by a positive Rh control. This is seen when a patient/donor has strong positive DAT. The cells are coated with antibody (not necessarily Rh antibody) in vivo. Albumin is necessary in reagent Anti-D to overcome the zeta potential allowing cells coated with IgG Anti-D to get close enough together to agglutinate, but cells coated in vivo with any IgG antibody will also agglutinate in albumin. These false positives are corrected by using form of Anti-D that does not require albumin. There are two types of alternative types of anti-D:

Monoclonal (IgM) anti-D will cause agglutination of D positive cells without the presence of albumin at room temperature. A number of facilities normally use this type of anti-D and therefore do not routinely use Rh control. Chemically modified anti-D has been modified by breaking the disulfide bonds closest to the hinge region so antibody can reach cells that are farther apart.

2. False positive can also be caused by rouleaux formation, which will look like agglutination macroscopically. Rouleaux would be identified microscopically due to the "coin-stacking" appearance of the red cells. This false positive would be corrected by washing cells 3 to 4 times and then retesting.

False Negatives
False negatives are not readily identifiable, but can occur in the following instances:

The most common is the result of too heavy cell suspension due to too many cells for the amount of antibody in the antisera. They may also rarely be caused by extremely strong positive DAT. In this case a the patient's D antigen sites are coated in vivo and there are no sites left for commercial anti-D to attach to. This can be fixed by heating cells gently to elute off antibody without damaging cells, then re-test.

OBJECTIVES - Rh SYSTEM
1. Briefly describe how and when the Rh system was discovered 2. List the major Rh antigens and state the frequency each is seen in the Caucasian population. 3. Describe the characteristics common to the major Rh antigens and compare them to the ABO system. 4. Explain the Tippett theory of inheritance. 5. For any given Rh phenotype, predict the most likely genotype in both the Wiener and Fisher-Race nomenclatures. 6. For any given Wiener genotype, list the Rh antigens present. 7. Explain why Rh genotyping is important. 8. Give three explanations for the weak D phenotype. 9. Discuss how the weak D phenotype applies to donors, recipients, and obstetrical patients. 10. State the relative frequencies of the Cw, V and VS antigens.

11. Explain the G, f, and Rh null phenotypes. 12. Describe characteristics common to antibodies in the Rh system. 13. List the more common antibodies seen in the Rh system. 14. Discuss the use of albumin and enzymes in identifying Rh antibodies. 15. Explain how false positives can occur when testing for the Rh antigens, and describe how the problem may be overcome. 16. Explain how false negatives can occur when testing for the Rh antigens, and describe how the problem may be overcome. 17. Differentiate between high-protein anti-D, chemically modified anti-D, and saline anti-D.

Performance objectives:
1. 2. 3. 4. Correctly perform, interpret, and record the Rh type of any given sample Recognize when chemically modified or saline Rh reagents must be used. Correctly perform, interpret, and record a weak D (Du)test. Correctly perform, interpret, and record the Rh phenotype of any given sample, and state its most likely genotype.

LEWIS BLOOD GROUP SYSTEM

There are distinct differences in regards to the Lewis Blood Group System

Manufactured by the tissues Lewis antigens are secreted into body fluids Absorbed onto red cells from the plasma

The Lewis Blood Group System is also similar to the ABO system

The antigens are part of the same oligiosaccharides that are part of the ABH antigens The Lewis antigen (fucose) is added onto the N-acetyl-glucosamine that is just before the galactose where the fucose is added for the H antigen in the secretions.

Lewis Antigens
Alleles
The development of the Lewis antigens is controlled by two alleles of the Lewis blood group system.

Le is dominant and results in the presence of Lewis antigen. The recessive le (absence of Lewis gene) is recessive and therefore 2 le/le needs to be inherited

Genotypes

Both Le/Le or Le/le result Lewis positive antigen. Lewis antigen exists as either Lewis a (Lea) or Lewis b (Leb). Lewis negative results from le/le.

Phenotypes: Lewis System Phenotypes and Their Incidence

(Modified from AABB Technical Manual, 2002, p. 287) Adult Phenotype Incidence in % Reactions with AntiPhenotype Lea Leb Whites Blacks + 0 0 + 0 + 0 + Le(a+b-) Le(a-b+) Le(a-b-) Le(a+b+) 22 72 6 Rare 23 55 22 Rare

Formation of Lewis Antigens

What Lewis antigens are formed depends on interaction between Lewis (Le/le), Secretor (Se) and H genes in the tissues to produce Lewis antigens in the secretions. Lewis antigens have a similar structure to ABO antigens: 3. Formed at terminal sugars of Type I precursor substance made by tissue cell in plasma a. If person has the Lewis gene, it adds fucose to second sugar from the end = Lea b. If person is also a secretor, H gene adds fucose to terminal sugar of precursor substance = Leb antigen c. If person NOT a secretor, no H added to precursor substance made by tissue cell, so it remains as Lea

4. NOT part of rbc membrane - synthesized by tissue cells, carried by plasma, adsorb onto rbc surface 5. Not present on newborn red cells 6. Can disappear during pregnancy

Lewis Antibodies

Types of antibodies 1. Anti-Lewisa 2. Anti-Lewisb 3. Anti-Lewisa and Lewisb B. Almost always produced by Lewis a negative; Lewis b negative people C. Naturally occurring but not regularly occurring (unexpected antibodies) D. Frequently seen in pregnancy (due to loss of Lewis antigen during pregnancy) E. IgM, therefore usually not clinically significant 1. Does not cross placenta 2. Reacts best at RT, but some MAY react at 37C 3. If anti-Lea present at 37C, may cause hemolytic transfusion reaction 4. Anti-Leb is usually clinically insignificant III. Secretor Status A. Secretor status controlled by Secretor (Se) gene 1. Secretor = Se/Se or Se/se (80%) 2. Non-secretor = se/se (20%) B. Secretors have soluble A, B and H antigens in body fluids - plasma, tears, saliva C. Can test saliva for presence of ABH antigens D. Practical application of saliva testing: 1. Determine ABO type in patients with ABO discrepancies 2. Determine ABO type in patients massively transfused with another blood type 3. Can also use Lewis types to determine secretor status: E. Lewis a positive, Lewis b negative = non-secretor F. Lewis a negative, Lewis b positive = secretor G. Lewis a negative, Lewis b negative = can't tell secretor status from Lewis types

H. Can only use this method if patient has not been heavily transfused recently - need to type patient red cells, not donor IV. Testing for Secretor Status A. Utilizes principle of AGGLUTINATION INHIBITION 1. Patient's saliva boiled and cleared 2. Cooled saliva mixed with reagent anti-A, anti-B and anti-H 3. If soluble A, B, or H antigens present in saliva, these will react with antibodies in reagent antiserum, and neutralize it, so no antibody available to agglutinate test cells 4. if no soluble A, B or H antigens in saliva, antibodies in reagent antiserum will not be neutralized, and will be free to react with test cells B. Group A Secretor: 1. A antigens in saliva 2. Addition of reagent anti-A: antibodies tied up by soluble A antigens 3. Addition of known A cells results in no agglutination (no free A antibody to cause agglutination)

NO AGGLUTINATION = POSITIVE TEST FOR SECRETOR STATUS

C. Group A Non-Secretor: 1. NO ABO antigens in saliva

2. Addition of reagent Anti-A leaves free antibody in serum

3. Addition of known A cells causes agglutination. AGGLUTINATION = NEGATIVE TEST FOR SECRETOR STATUS

OBJECTIVES - LEWIS BLOOD GROUP SYSTEM 1. State the alleles in the Lewis system 2. Explain how Lewis antigens are produced 3. Explain the difference between Lewis a and Lewis b 4. Explain the relationship between the secretor gene and Lewis antigen production 5. Describe the general characteristics of Lewis antigens, including presence at birth and effect of pregnancy 6. State the possible Lewis phenotypes, and their approximate percentages in the Caucasian population 7. State which Lewis phenotype is most likely to make antibodies in the Lewis system 8. Describe the typical characteristics of Lewis antibodies 9. State which of the Lewis antibodies is more clinically significant, and explain why. 10. Explain what is meant by the term "secretor" 11. Explain the principle of agglutination inhibition, and how it applies to saliva testing 12. Given the results of any saliva agglutination inhibition test, state whether or not the patient is a secretor, and determine his/her ABO type 13. Explain how secretor status can be determined from the Lewis phenotype 14. State limitations to the above system 15. Give two practical applications of saliva testing Performance objectives: 1. Correctly perform and interpret the saliva test for agglutination inhibition, to determine your own secretor status 2. Correctly perform and interpret the antigen typing for Lewis a and Lewis b on your own red cells 3. Correlate the results of your Lewis typings with the results of you saliva testing

OTHER BLOOD GROUP SYSTEMS

MNS SYSTEM
Antigens and Their Inheritance
The antigens M and N are co-dominant alleles that are closely linked to the S and s antigens, which are also co-dominant. Chromosome 4 contains these linked genes. These antigens are inherited by a complex pattern similar to the Rh system. The Ms and Ns linkage is more common than the MS and NS linkages. All these antigens, however are fairly frequent in the population with the following overall frequencies:

M = 78% N = 72% S = 55% s = 89% U = Greater than 99%

U antigen is a high incident antigen NOT seen in individuals who lack both S and s antigens. Individuals who lack this antigen (<1%) have a high likelihood of forming antiU as well as anti-S and anti-s.
AABB Technical Manual, 13th ed.: Modified Table 15-3 Phenotypes and Frequencies in the MNS System p.318 Phenotype Frequency % Phenotype Whites M+NM+N+ M-N+ S+s-U+ S+s+U+ S-s+U+ S-s-U28 50 22 11 44 45 0 Blacks 26 44 30 3 28 69 Less than 1

Biochemistry of the MNS antigens

The M and N antigens are glycoproteins containing sialic acid that cross the cell membrane. Carboxyl terminus extends into the red cells interior, a hydrophobic segment as part cell membrane and an amino terminal segment on the external environment of the red cell. The external components of the antigens are destroyed by the enzymes like fiacin, typsin and papain.

The antigens of the MNSU blood group system are well developed in newborns. Therefore a mother who is negative for one of these antigens could be stimulated to make antibodies thatmay cause HDN. The antibody would have to be an IgG immunoglobulin that reacts at 37oC)

MNS System Antibodies Anti-M is frequently seen as a saline agglutinin if testing is done at room temperature.

It is predominantly IgM and can be naturally occurring. It will show dosage and therefore M homozygous cells will react with antibody more strongly than heterozygous cells. Therefore the predominant form of the antibody is not clinically significant. There are instances where some or all of the antibody is IgG in nature. If you have an anti-M that strongly at 37oC and/or AHG, it should be considered to be potentially clinically significant. Mild to severe cases of hemolytic disease of the new born have be reported. Since the M antigen can be removed by enzyme, the reactivity of anti-M can be destroyed by enzyme. When performing a crossmatch on a recipient's specimen that contains anti-M, a prewarmed crossmatch should be preformed.

Anti-N is very rare and has similar reactivity as anti-M. Most often seen in kidney dialysis patients as cross-Reacting antibody to formaldehyde. Formaldehyde is used to sterilize Dialysis equipment. Anti-S anti-s and anti-U usually form following red cell immunization due to transfusions and pregnancies.

They are usually IgG and react best after 37oC with AGT (Coombs) technique. All are capable of causing Hemolytic Transfusion Reactions (HTR) (delayed) and Hemolytic Disease of the Newborn (HDN) S is usually destroyed by enzyme but s is variable and U is not destroyed by enzyme treatment. Anti-U is rare but should be considered if a previously transfused or pregnant Black patient has an antibody to a high-incident antigen.

Summary of Antibody Characteristics

Reactivity
Antibody < RT* 37 AHG

% Bind In Vitro Compatible HTR** HDN*** Comments Enzymes Complement Hemolysis in US Population Destroy (Rare) Destroy (Rare) No No Few MildSevere 22% Usually Clinically Insignificant

M N

Most Few Most Few

Few Few

Rare Moderate 28%

Some Some Most

Variable Some Effect Variable Few Effect No (Rare) Change

No

Yes

Mild MildSevere MildSevere

45% W 69% B 11% W 3% B <1%

Few Few

Most

No

Yes

Rare Some Most

No

Yes

*Room Temperature **Hemolytic Transfusion Reaction ***Hemolytic Disease of the Newborn

KELL SYSTEM
Antigens and Their Inheritance
In Dr. Mourant's article on the discovery of antiglobulin test, he recounts that they tested the serum of a number of mothers, who were believed to have babies with hemolytic disease of the newborn. One of the reactions was not due to Anti-D. "The non-Rh antigen involved in this case was that subsequently known as Kell. Thus at the very outset the test had detected a previously unknown blood group system which has since proved to be of some clinical importance." ("The Discovery of the Anti-Globulin Test") There are three pairs of alleles within the Kell system. Each pair has a high frequency and low frequency gene that are co-dominate if present. The three pairs are:

K (Kell), or K1, and k (Cellano), K2 Kpa (K3) and Kpb (K4)(Penney) Jsa (K6)and Jsb (K7)(Sutter)

K, Kpa and Jsa are low frequency antigens and k, Kpb and Jsb are high frequency antigens. There is a Kell phenotype, K null (Ko or K5), is very rare and K, k, Kpa, Kpb, Jsa, Jsb antigens are not expressed. The Kell Systems antigens are found in only small amounts on the red cell carried on a single protein. K has approximately 3500 sites and k has between 2000-5000. The function of this protein is unknown.
AABB Technical Manual, 13th ed.: Modified Table 15-5 Phenotypes and Frequencies in the Kell System p.322 Phenotype Frequency % Phenotype Whites Blacks

K+kK+k+ K-k+ Kp(a+b-) Kp(a+b+) Kp(a-b+) Js(a+b-) Js(a+b+) Js(a-b+) Ko [K-,k-,Kp(a-b-),Js(a-b-)]

0.2 8.8 91 Rare 2.3 97.7 0.0 Rare 100.0 Extremely rare

Rare 2 98 0 Rare 100 1 19 80

Kell System Antibodies

Anti-Kell is the most clinically significant antibody within this system. The Kell antigen is considered the next most antigenic after the D antigen of the Rh system. Individuals lacking the K antigen can make anti-Kell after only two exposures to Kell-positive blood. Because over 90% of the population are Kell negative it is not difficult to find donor blood that is compatible with the recipient. Antibodies to other antigens in Kell system are very rare. Antibody Characteristics of the antibodies to the Kell System are:

IgG Cause HDN and HTR (delayed) React best in Coombs after 37oC incubation Does not show dosage (homozygous KK and heterozygous Kk cells react with the same strength) Enzyme has no effect

Summary of Antibody Characteristics

Reactivity
Antibody < RT* 37 K AHG

% Bind In Vitro Compatible HTR** HDN*** Comments in US Complement Hemolysis Enzymes Population No Some No Yes Mild91

Some Some Most

change k Few Few Most No (Some) change No (Some) change No (Some) change No (Some) change No (Some) change No Yes

Severe Mild 0.2

Kpa

Some Some Most

No

Yes

Mild

99.7

Kpb

Few

Few

Most

No

Yes

Mild

<0.1 100 W 80 B

Jsa

Few

Few

Most

No

Yes

Moderate

Jsb

(No)

(No)

Most

No

Yes

Mild1B Moderate

*Room Temperature **Hemolytic Transfusion Reaction ***Hemolytic Disease of the Newborn

DUFFY SYSTEM
Antigens and Their Inheritance
The Duffy antigens Fya and Fyb are a pair of co-dominant alleles found on chromosome 1. The phenotypes Fy(a-b+), Fy(a+b+), Fy(a-b+) are very common among the US white population. Fy(a-b-) is very rare in the white population but makes up 68% of the black population of the United States. Biochemically the Duffy antigens are glycoproteins that has an external loop. This external loop can be destroyed by enzymes such as ficin, papain, and trypsin. The Fya and Fyb antigens are receptors for the malarial parasite, Plasmodium vivax. Therefore individuals that are phenotypically Fy(a-b-) have a resistance to malaria. This particular phenotype is found up to 100% of western Africa and of course 68% of the American Blacks.
AABB Technical Manual, 13th ed.: Modified Table 15-6 Phenotypes and Frequencies in the Duffy System p.325 Phenotype Frequency % Phenotype Whites Fy(a+b-) 17 Blacks 9

Fy(a+b+) Fy(a-b+)

49 34 Very rare

1 22 68

Fy(a-b-)

Duffy System Antibodies

Duffy antibodies frequently seen in multiply-transfused Blacks. Anti-Fya much more common than anti-Fyb and is more likely to cause HTR and HDN. 3. Characteristics

IgG Anti-Fya can cause HDN and HTR (delayed) and anti-Fyb is milder and no HDN cases have been reported but could possibly be a cause. React best in Coombs after 37oC incubation Reactions destroyed by enzyme of the red cells

Summary of Antibody Characteristics

Reactivity
Antibody < RT* 37 AHG

% Bind In Vitro Compatible HTR** HDN*** Comments Enzymes Complement Hemolysis in US Population Destroy Some No Yes MildSever (Yes) 34 W 17 W 77 B

Fya

Rare

Rare

Most

Fyb

Rare

Rare

Most

Destroy Some

No

Yes

*Room Temperature **Hemolytic Transfusion Reaction ***Hemolytic Disease of the Newborn

KIDD SYSTEM
Antigens and Their Inheritance
Jka and Jkb antigens are inherited on chromosome 18 where urea transport mechanisms are located. Cells that are Jk(a-b-) are less likely to to lyse in the presence of high concentration of urea. These antigens are inherited by the co-dominant alleles Jka and Jkb that are high frequency antigens. The Kidd antigens are thought to be

grouped very close together in clusters on the red cell membrane. Due to the close proximity of the antigens when the antibodies are attached complement can be activated. The activation of complement can cause intravascular transfusion reactions. Approximate frequencies among the white US population are the following:

Jka = 75% Jkb = 75% Approximately 50% of the white population is Jka and Jkb positive

The more specific frequencies are listed in the table below for both whites and blacks.
AABB Technical Manual, 13th ed.: Modified Table 15-7 Phenotypes and Frequencies in the Kidd System p.326 Phenotype Frequency % Phenotype Whites Jk(a+b-) Jk(a+b+) Jk(a-b+) Jk(a-b-) 28 49 23 Extremely rare Blacks 57 34 9

Kidd System Antibodies

Both anti-JKa and anti-Jkb are hard to detect and identify since they are very weak and are detected primarily at the antiglobulin phase of testing. These antibodies are usually low titer as well as being weak reactions. The antibodies disappear rapidly from circulation and also in stored serum since their recognition is enhanced if complement is present. These antibodies will often show dosage. Enzyme can enhance the reaction as well as PEG enhancement solution. Characteristics of anti-Jka and anti-Jkb are as follows:

gG React best after 37oC with Coombs technique Can cause HDN Can cause Hemolytic Transfusion reactions that are acute intravascular reactions, or they may be delayed transfusion reactions that show up after the patient's immune system is reexposed and the memory cells quickly produce antibodies to the antigens. While most of the antibodies discussed in newsletter are more likely to cause extravascular hemolytic transfusion reactions, the Kidd antibodies can activate complement and therefore cause intravascular hemolytic transfusion reactions.

Summary of Antibody Characteristics

Reactivity
Antibody < RT* 37 AHG

% Bind In Vitro Compatible HTR** HDN*** Comments Enzymes Complement Hemolysis in US Population Enhance All Some Yes Mild 23 Associated with Severe Delayed HTR

Jka

Few

Few

Most

Jkb

Few

Few

Most

Enhance All

Some

Yes

Mild

28 W 57 B

*Room Temperature **Hemolytic Transfusion Reaction ***Hemolytic Disease of the Newborn

Bg Antibodies
The Bg antibodies were first described by Bennett and Goodspeed. They are not considered clinically significant, but may mask clinically significant antibodies. They are antibodies to white cell antigen remnants on red cells

Anti-Bga reacts with Human Leukocyte Antigen (HLA)-B7 Anti-Bgb reacts with Human Leukocyte Antigen (HLA)-B17 Anti-Bgc reacts with Human Leukocyte Antigen (HLA)-A28

Reactions relating to these antibodies are weak, react possibly at room temperature or more commonly in the antiglobulin (Coombs) phase. They react with only a few cells in a cell panel. They do not cause hemolytic disease of the newborn or hemolytic transfusion reactions. They are seen frequently seen in multiply transfused patients or in multiparous women (multiple pregnancies)

HTLA ANTIBODIES = High Titer Low Avidity

A summary of these antibodies are as follows

Not clinically significant, but serological reactions make them look like they are High Titer if the antibodies are titered. The titers are usually at least 1:64 and often will be over 1:1000 Reactions are very weak and will break apart very readily due to the weak attraction between the antigens and antibodies (low avidity).

These antibodies basically have a high titer but a very weak reaction. Some institutions actually score the strength of the antigen reaction in points which will allow them to differentiate some of the differences seen in various reactions.

Score = strength of reaction given points or score

4+ = 12 points 3+ = 10 points 2+ = 8 points 1+ = 5 points neg = 0 points

Specific serologic characteristics of the HTLA antibodies are:

IgG React best in Coombs after 37oC incubation W+ to 1+ reactions React with most cells (antibodies to high-frequency antigens) Not clinically significant since they are not known to cause hemolytic disease of the newborn or hemolytic transfusion reactions. Since they are antibodies to high-frequency antigens they may mask clinically significant antibodies that are also in the serum.

OBJECTIVES - BLOOD GROUPS OTHER THAN ABO/RH

For each of the blood groups listed below:

MNS Kell Duffy Kidd

Ii Lewis P

1. Recognize and translate the symbols used to denote antigens and antibodies 2. List the major alleles and the relative frequency of each in the Caucasian population. (High-incidence or low-incidence) 3. Describe the serological characteristics of the antibodies:

class temperature of reaction effect of enzyme complement binding dosage effect

4. Discuss the clinical significance of each antibody regarding transfusion reactions and hemolytic disease of the newborn 5. Describe the relationship between the Duffy system and malaria. 6. Describe the typical serological characteristics common to all HTLA antibodies.

7. Explain the difference between an antibody's titer and an antibody's score. 8. Describe the typical serological characteristics of the Bg antibodies 9. Describe the clinical significance of HTLA and Bg antibodies. 10. Explain the relationship between white cells and the Bg antibodies.

I AND P BLOOD GROUP SYSTEMS

I Blood Group System
I/i Antigens
The I antigen is found on almost all adults and is part of the precursor component of the oligosaccharide that forms the A, B, and H antigens. There are two types of oligosaccharides: Type 1 and Type 2. These chains that are part of the ABH antigens form the i antigen found in infants.

By the time the child reaches 2 years of age a number of b1-->6 linkages are added between the residual galactose components. In adults these precursor components are become branched with a 1-->6 binding between 2 galactose molecules forming a branched oligosaccharides that make up the I antigen seen in most adults.
Therefore I (almost all adults) are formed from branched sugar chains on precursor substance.

All cord bloods form i antigen from straight-line sugar chains on precursor substance of the A, B, and H antigens.

As indicated: in most children i is converted to I by the age of 2 years.

Anti-I, Anti-IH, and Anti-i Antibodies

Anti-I antibodies are the most common antibodies found if antibody screenings are done at immediate spin, room temperature. Their characteristics are:

IgM immunoglobulins and therefore are saline agglutinins with their optimal temperature at 4oC. Since they are IgM they also do not cross the placenta. Anti-I is naturally occurring often due to a Mycoplasma pneumoniae infection or some lymphomas and leukemias. These antibodies are USUALLY not clinically significant Anti-I reacts with all adult cells (including patients own, all reagent cells, all donor cells) Anti-I does not react with cord cells Auto-anti-I is a common cold agglutinin

High titers of auto-anti-I can interfere with serological testing: In ABO typing, the forward grouping may have patient cells coated with IgM autoantibody, so all cells agglutinate. When this happens all results are positive and the group looks like AB+. On the reverse grouping anti-I in serum reacts with all adult cells, so A1 and B cells always agglutinate. All results are positive and the group looks like an O. Cells + anti-A 4+ Cells + anti-B 4+ Cells + Serum + Serum + B anti-A,B A1 cells cells 4+ 4+ 4+ Cells + anti-D 4+ Cells + Rh Interpretation Cont Unknown 4+ ABO Group

Method to resolve these Problems

Correct forward typing by washing cells in warm saline and re-testing. Correct reverse by warming the serum before testing, or adsorbing the cold autoantibody out of the serum.

Crossmatch results will result in all donors being incompatible. Correct by:

warming the donor cells and patients serum before crossmatching, omitting potentiators like LISS and using monospecific anti-IgG.

Anti-IH antibodies are directed against both I antigens and H antigens. Therefore they react most strongly with group O adult cells, least with cord cells or A1 cells.

Antibody Anti-I Anti-IH Anti-H Anti-i

Comparison of Anti-I, Anti-IH, and Anti-H Reactions Adult A Cells Adult O Cells Cord A Cells Cord O Cells 2+ 2+ 0 0 2+ 4+ 0 2+ 0 2+ 0 2+ 0 0 2+ 2+

Anti-IH is seen most often in A1 adults and is not clinically significant but may cause problems with the ABO grouping and crossmatch results just as anti-I does. Follow the same steps for resolving the problem as indicated for anti-I. Diseases 1. Anti-I and anti-IH are only clinically significant when they cause Cold Hemagglutinin Disease (CHD). In this disease a strong autoanti-I is present. The blood in patient toes, fingertips, earlobes is cooler than than 37 oC and the antibody coats cooled cells, binds complement, and causes hemolysis. This disease is treated by keeping the patient's extremities warm so the blood is not allowed to cool. 2. High-titer anti-I also commonly associated with Mycoplasma. pneumoniae infections. Treatment would be giving the patient appropriate antibiotics. 3. Anti-i, which is fairly uncommon and transient, is associated with infectious mononucleosis.

P System
Antigens of the P system are Pk, P, P1 .

Basic structure is the precursor substance, but is called globoside (P) or paragloboside (p) in the P system as the diagram below shows:
from the AABB Technical Manual, 13th, 1999, p.291

P antigens poorly developed at birth and therefore do not usually cause Hemolytic Disease of the newborn. They are present in variable amounts on different red cells and seems to diminish in storage. Like ABO and Ii antigens, they are composed of sugar chains as seen in the accompanying figure. Like A and B antigens, they are present on tissue cells as well as red cells. Similar antigens found in nature (bird droppings, pigeon eggs, hydatid cyst fluid) and these substances can be used to neutralize anti-P in a patient's serum. These similar antigen solutions can be useful when the antibody is interfering with ABO and crossmatch testing. The Pk antigen occurs when the Pk antigen does not convert to P

P Phenotypes
80% of people with P antigen are P1 phenotype (have P and P1 antigens) and 20% of people with P antigen are P2 (P antigen only - no P1). The P2 phenotype merely signifies absence of P1 antigen in people with P antigen (globoside). Individuals who are Pk consistantly have an alloanti-P that is an IgM antibody. The absence of the P antigen is very rare and is designated as p.

P Antibodies
Alloanti-P1 is the most common of the antibodies in the P system. This antibody is commonly seen in P2 people. IAlloanti-P1'ss characteristics are:

IgM class and therefore is saline reactive, enhanced by cold incubation, may bind complement. Not clinically significant Naturally occurring but not regularly occurring. It is formed with no known red blood cell stimulus - either pregnancy or transfusion. If individuals are P1 negative, they do not automatically have anti-P1 i, but it is fairly common Antigen-antibody reactions in P system enhanced by adding albumin to system, or by treating red cells with enzymes

Auto anti-P is an IgG antibody seen in P1 or P2 persons and causes clinically significant Paroxysmal Cold Hemoglobinuria (PCH). The Donath-Landsteiner Test helps diagnose PCH. It is designed to detect biphasic anti-P. A biphasic antibody reacts with the antigen on the red blood cell at colder temperature, binding complement . When antigen-antibody-complement complex warms up, the red blood cells are hemolyzed. Donath-Lansteiner test is performed by incubating patient's serum with his own cells first at 4oC, then at 37oC, and then looking for hemolysis.

A positive Donath-Landsteiner = no hemolysis at 4oC, no hemolysis at 37oC, hemolysis only in the tube that was incubated at both temperatures

A negative Donath-Landsteiner = neg at both temps, OR hemolysis at 4oCas well as 37oC Donath-Landsteiner Test

4oC incubation:

37oC incubation:

no hemolysis = NEGATIVE no hemolysis = NEGATIVE

4oC + 37oC incubation: hemolysis: = POSITIVE

OBJECTIVES - I AND P BLOOD GROUP SYSTEMS

1. Differentiate between I and i antigens regarding structure and age when developed. 2. State the optimal temperature of reaction of anti-I. 3. Explain how anti-I interferes with ABO typing and crossmatch results. 4. Explain how the above problems may be overcome. 5. Explain why anti-I is always an autoantibody. 6. Explain why anti-I or anti IH is not generally clinically significant. 7. Explain the difference in reactions between anti-I and anti-IH 8. Describe the cause, symptoms and treatment of Cold Hemagglutinin Disease. 9. State what other disease auto-anti-I may be associated with. 10. State what disease anti-i may be associated with. 11. State the major antigens of the P system, and how they are distributed in the population. 12. State which antigens make up the following phenotypes in the P system: P1, P2, p 13. Explain what paragloboside or globoside is. 14. List three other sources of the P antigen in nature, and explain when this knowledge may become useful. 15. State the most common allo-antibody in the P system. 16. Explain how antigen-antibody reactions in the P system may be enhanced. 17. State the cause of paroxysmal cold hemoglobinuria (PCH). 18. Describe the Donath-Landsteiner test and explain what it detects.

Antibody Screening

PRINCIPLE AND APPLICATIONS

The patient's serum is tested for the presence of clinically significant antibodies using an indirect antiglobulin method. The serum is tested against unpooled Group O cells selected to possess the relevant blood group antigens. The antibody screen is routinely performed as part of compatibility testing; it is also performed upon request on prenatal patients, on Rh immune globulin candidates, as part of an organ transplant workup, or on other patients for whom an "indirect Coombs" is requested.

SAMPLE
Since the patient's serum is tested, a 10 ml clotted (red top) tube is preferred. A 4 or 5 ml red top is acceptable; a 2 ml red top is acceptable from a newborn. The sample should be tested when fresh; old or improperly stored samples may lose complement activity and lead to false negatives.

REAGENTS, EQUIPMENT, AND SUPPLIES

12 x 75 mm test tubes Lighted agglutination viewer Plastic test tube holder Reagent Screening Cells I & II, and III Indelible marking pen PEG Large bore dispo pipettes Anti-Human Globulin (Coombs serum) 37oC waterbath or heat block Coombs Control Cells Serofuge Wash bottle with physiologic saline

PROCEDURE
1. Verify that patient information on the sample matches information on the worksheet. 2. Centrifuge the sample and separate the serum to a labeled tube. 3. Prepare a washed 3% cell suspension from the patient's cells. See WASHED 3% CELL SUSPENSIONS. 4. Label 3 tubes Patient last name/or #, I Patient last name/or #, II Patient last name/or #, III 5. Using a large bore pipette, add 2 drops of patient serum to all tubes. Hold the dropper at a consistent 45-degree angle.

Add one drop of Screening Cell I to tube I; add one drop Screening Cell II to the II tube; add one drop Screening Cell III to the III tube. 7. Shake all tubes to mix and centrifuge at high speed the time appropriate for the saline spin calibration in the serofuge. 8. Gently resuspend and examine macroscopically for agglutination using the lighted agglutination viewer. 9. Record all negative reactions as ; grade positive reactions on a scale of w+ to 4+. 10. Again holding the dropper at a consistent angle, add 2 drops of PEG to all tubes. 11. Shake to mix and incubate at 37oC at least 15 but no more than 30 minutes. (NOTE: IF A WATERBATH IS USED FOR THE 37oC INCUBATION, THE INCUBATION TIME MAY BE REDUCED TO 10 MINUTES). 12. Wash all tubes 4 times, decanting well after each wash, mixing the cell button well between washes, and blotting the last drop of saline after the final wash. 13. Immediately add one drop AHG to each tube, shake to mix, and centrifuge the time appropriate for the Coombs spin calibration in the serofuge (usually the same as the saline calibration). 14. Immediately resuspend gently and check macroscopically for agglutination using the lighted agglutination viewer. 15. Read and record results as described above, along with your initials and the date, if not already recorded on the worksheet. 16. Add one drop Coombs Control Cells to all negative reactions and centrifuge 1530 seconds in the serofuge. 17. Resuspend and macroscopically examine for agglutination. There must be agglutination in this step or the test is invalid. Record results. 6.

INTERPRETATION

The lack of agglutination indicates the absence of antibodies to antigens on the reagent test cells, and the test is reported negative. Agglutination in either Screening Cell tube prior to the addition of Coombs Control Cells indicates the presence of unexpected alloantibodies. The test is reported positive and further testing is necessary to identify the antibody(ies). See ANTIBODY IDENTIFICATION. Agglutination in the autocontrol (AUTO) indicates coating of cells circulating in the patient. This may be due to an antibody to medications, autoantibody, passively transfused alloantibody, or alloantibody coating transfused cells in the patient. Perform a DAT and obtain the patient's medication list, diagnosis and transfusion history.

NOTES AND PRECAUTIONS

1. Use of PEG requires a strict serum/PEG ratio. Also, add no more than 2 drops of serum when using PEG. 2. AHG must be added to the cells immediately following washing. Antibodies may elute from the cells if they are allowed to sit in saline without the addition of AHG. 3. The Coombs Control Cells must be positive at the end of the procedure. Reasons for negative results here include:

Inadequate washing of the cells in the Coombs phase. Residual serum neutralizes AHG. A small fibrin clot has formed in the tube, neutralizing AHG. Be sure sample has thoroughly clotted. Coombs reagent was omitted or is inactive.

4. Weak or questionable reactions may be enhanced by:

Increasing the amount of serum used to 3-4 drops. PEG must be omitted, and the incubation time extended to 30 minutes. Omitting PEG and adding albumin to the test mixture. Extend the incubation time to at least 20 minutes. Using enzyme treated cells. See manufacturer's directions. Repeat testing using PEG to the test mixture. See manufacturer's directions.

5. Occasionally you may get a moderately strong reaction at room temperature that persists weakly in Coombs. This may not be clinically significant if it is due to complement binding at room temperature from a cold-reactive antibody. To determine if the reaction is due to complement binding, do a pre-warmed screen (SEE PREWARMED CROSSMATCH TECHNIQUE).