Life's Blood

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 Common Genes Located on Chromosome

ABO A, B, O 9
MNSsU M, N, S,s,U 4
P P1 22
Rh D, C, E, c, e 1
Kell K, k, Kpa, Jsa,Kpb, Jsb 7
Lewis Le, le 19
Duffy Fya, Fyb, Fy3 1
Kidd Jka, Jkb 18
Xg Xga 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 AB AO
O BO 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. Simple and unquestionable pattern of inheritance

2. Can test or determine the phenotypes readily

3. More than 1 allele occurring fairly frequently

4. 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 Observed frequency

MS = 0.53 (M) X 0.33 (S) = 0.17 0.24

Ms = 0.53 (M) X 0.67 (s) = 0.36 0.28
NS = 0.47 (N) X 0.33 (S) = 0.16 0.08
Ns = 0.47 (N) X 0.67 (S) = 0.31 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 Blood Group System Homozygous Phenotype

h ABO Oh or Bombay
=
Rh Rhnull
r
Ko Kell Knull
Lu Lutheran Lu(a-b-)
Jk Kidd Jk(a-b-)
Fy Duffy 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, Fy a, 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 Correct Terminology Incorrect Terminology

Phenotype Fy(a+) Fya+, Fy(a+), Fya(+), Fya+, Fya(+), Duffya+

Phenotype Fy(a+b-) Fya+b-, Fy(a+b-), Fya(+)b(-), Fya(+)b(-)
Antibody Anti-Fya Anti Fya, Anti-Duffy
Antigen K Kell (name of system)
Antibody anti-k Anti-Cellano
Phenotype K:1, K:-1 k1+, K:1+, K(1), K:(1), K1-, K:1-, K1-negative
A+ (means positive for A antigen)
Phenotypes A Rh+, B Rh-
B- (means negative for B antigen)
Phenotype M+N- M(+), MM (unproven genotypes)
Phenotype Rh:-1, -2, -3, 4,5 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?

Life's Blood

Class Notes

ANTIGLOBULIN TESTING

The antiglobulin test, which is also referred to as the anti-human globulin test (AHG) or the Coombs test, is the
cornerstone of detecting clinically significant unexpected antibodies that have coated cells either in vivo or in vitro. For a
historical perspective, see "The Discovery of the Anti-Globulin Test" written by A. E. Mourant pages 180 to 183 Vox Sang. 45: 180-
83 (1983)

Principle of Antiglobulin Test

Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged. These cells are said to
be sensitized with IgG or complement.

IgG-coated red blood cells

Complement-coated red blood cells

In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the
C3b or C3d component of complement, must be added to the system.

This will form a "bridge" between the antibodies or complement coating the red cells, causing agglutination.

The light-colored antibody molecule represents the anti-globulin reagent that binds
with the Fc portion of the IgG antibody attached to the red blood cells.

The light-colored antibody molecule represents the anti-globulin reagent

that binds with the complement attached to the red blood cells.
Traditionally rabbits were immunized with human gamma globulin to make this antibody to IgG or C3d.

Types of Antiglobulin Tests

The original work done by Coombs and Mourant was detecting those antibodies, especially in the Rh system, that would
cause hemolytic disease of the newborn, which we now classify as the Indirect Antiglobulin Test.

There are two types of antiglobulin tests:

Direct Antiglobulin Test (DAT) - Detects antibodies or complement coating patient's cells in vivo.

Indirect Antiglobulin Test (IAT) - Uses a 37oC incubation step so antibodies in serum can react with antigens on
cells in vitro, After washing the cells antiglobulin reagent is used to detect antibody coating of cells.

Reagents

Production Methods of Anti-Human globulin (AHG or Coombs) Reagent

May be made by injecting rabbits with purified human IgG or C3, then harvesting the antibodies produced by the
rabbit.

Monoclonal technology may be used to make monoclonal antiglobulin reagent

Specificity types

Polyspecific Anti-human Globulin: blend of Anti-IgG & Anti-C3b, -C3d

Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone

Note: Reagent does not contain antibodies to IgM. Information about IgM coating of cells comes from the presence of C3
coating the cells since IgM is a strong complement activator.

Interpretation of Antiglobulin Tests

Whether the cells have been coated, or sensitized, in vivo or in vitro the final interpretation is based on the following

Positive Antiglobulin Test

(Wash Bottle Image)

Wash cells three times to

 remove unbound antibody

Only antibody attached to the

cells remain

Add Anti-Human Globulin

(Anti-globulin Image)

Visible Agglutination in the test tube:

Grade the reaction strength

Summary of the reaction:

1. Antigen-antibody reaction, which can take place either in vivo or in vitro

2. Cells coated with IgG antibody and/or complement

3. Cells washed 3-4X to remove unbound or free antibody or complement

4. The only antibody or complement left is attached to red cells

5. AHG (Coombs serum) added

6. Antibodies in Coombs serum react with antibodies or complement on red cells, causing agglutination

7. If no agglutination add Coombs control reagent cells* (CCC).

*Coombs control reagent cells will be discussed under False Negative Reactions.

Negative Antiglobulin Test

 Antibodies are not attached to the
antigens during incubation.

Wash the cells 3 times to remove any unattached antibodies.

Add Anti-human globulin

No visible agglutination and therefore a negative test

Add Coombs Control Check Cells

Check cells agglutinated and original test

cells remain unagglutinated.

Coombs Control Agglutinated by Anti-Human Globulin

Coombs Control Check Cells tell you if you did the test properly when you have a negative test.

1. NO antigen-antibody reaction occurred.

2. No attachment of antibody or complement to red cells

3. Cells washed three to four times = all plasma or serum antibodies was washed away.

4. Anti-human globulin, Coombs, serum added, which would react with antibody-coated cells if present.

5. But no agglutination, because no antibodies or complement on red cells for the anti-human globulin, Coombs,
serum to react with

6. Must add Coombs Control Check Cells to negative reactions

CCC are cells coated with IgG antibody

Will react with antibodies in Coombs serum still "floating around" in the tube.
 Agglutination will now result

Agglutination following addition of CCC verifies negative result

False Positives and Negatives

False-Negative Reactions

False-negative reactions can occur when antigen-antibody reactions have occurred but WASHING IS INADEQUATE and
free antibody remains when the anti-human globulin is added.

Anti-human globulin (Coombs) antibody prefers to react first with free antibody and then with antibody-coated
cells

If the free antibody has already reacted with the anti-human globulin, no free Coombs serum to react with
Coombs Control Check Cells (CCC)

False negatives that are detected by negative Coombs control cells includes

inadequate cell washing

delay in adding antiglobulin reagent after the washing step

presence of small fibrin clots among the cells

inactive, or forgotten, antiglobulin reagent

Inadequate cell washing will lead to unbound antibody remaining in the red cell suspension that are available to neutralize
the AHG (Coombs serum) so it will not react with red cells bound with antibody.

Delay in adding Coombs serum after washing step will lead to antibody eluting off, detaching from, cell while cells are
sitting in saline. Now free antibody present in the saline neutralizes the AHG, Coombs, serum so it will not be able to
react with the cells bound with antibody.

Small fibrin clot among the cells that were not washed away will have immunoglobulins and complement present. The
antibodies and complement in the fibrin clot neutralizes AHG, Coombs, serum leading to a negative test.

Inactive AHG (Coombs serum) or the failure to add AHG (Coombs serum) will also be detected by a negative reaction
when adding Coombs Control Check Cells.

There are also false negatives NOT detected by negative Coombs Control Cells that include:

Too heavy cell suspension

Delay during cell washing procedure, which can lead to antibody eluting off cells while they are sitting in saline
and then the antibody is washed away during the remaining washes

Improper centrifugation can either lead to lost of cells during the washing or the need to shake too hard during
resuspension.

False positives

False positive reactions can also occurred when performing this test. These would not be detected by the use of Coombs
Control Check Cells. Reasons for a false positive reaction could be the following:

Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test, DAT)

Spontaneous agglutination (cells heavily coated with IgM)

Non-specific agglutination ("sticky cells")

All of these reactions would be the result of cells appearing to agglutinate, or actually agglutinating. Using a clotted tube
for the DAT may allow complement to become activated in the test tube since calcium ions are free to be part of the
complement cascade.

Direct Antiglobulin Testing

Principle

The Direct Antiglobulin Test detects in vivo coating of patient cells - either IgG antibodies, complement, or both. Within the
patient's blood stream antibodies attach to their specific antigens on the red blood cells. This happens in Hemolytic
Disease of the Newborn (HDN), in transfusion reactions, and in autoimmune hemolytic anemia. Certain drugs are also
known to activate complement and it can also coat the cells in vivo.

When the blood is drawn the antibodies and/or complement have already attached to the red cells. Those red cells from
the EDTA tube will be washed 3 or more times and a 3% cell suspension is made. A drop of cell suspension and the anti-
human globulin are mixed in a tube and then centrifuged. If agglutination occurs, it indicates the patient has a positive
Direct Antiglobulin Test due to antibody coating the cells in vivo. If IgM antibodies involved, DAT will be identified by
complement binding since the polyspecific antisera has both anti-IgG and anti-C3. The meaning of a positive DAT is
found under Clinical Causes of a Positive DAT.

Technique

1. Add 1 drop of patient cells from EDTA tube to tube

2. Wash these drops of blood 3-4X to remove plasma antibodies and make a 3% cell suspension.

3. Add a drop of 3% cell suspension to a clean, labeled tube.

4. Add drop of Polyspecific AHG (Coombs serum) to the tube.

5. If test is positive with polyspecific reagent, set up again using monospecific reagents to see if it is antibody or
complement or both coating the cells.

6. We want to make the test as sensitive as possible, so allow all negatives to incubate 5 minutes to enhance
complement coating.

7. Read all negatives microscopically to detect weak coating.

8. False pos. possible if red top tube used to collect sample.

In-vitro complement coating frequently happens when sample clots or cools down due to weak cold-acting auto-
antibodies like anti-I

Prevent by using lavender top tube to tie up Ca+ and Mg+ ions and prevent complement activation in vitro.

8. Whenever positive DAT is obtained, obtain the following information on the patient:
 Diagnosis (particularly autoimmune hemolytic anemia, hemolytic disease of the newborn and transfusion
reactions)

Medications

Recent transfusion history of both red cell and plasma components

Other lab values that may indicate red cell destruction (hematocrit, bilirubin, LDH)

Clinical Causes of Positive DAT

1. Normal patient with unexplainable reasons for a positive DAT

2. Transfusion reaction work-ups require that a DAT be performed on the post-transfusion specimen since the
patient's antibodies and/or complement may coat the transfused donor cells. These reactions are usually a weak
positive or mixed field agglutination since you are testing a mixed population of patient and donor cells.

3. Warm-acting Autoimmune disease, can lead to patient antibodies coating their own cells. This results in a strong
positive result. A cold-acting autoimmune hemolytic anemia would be due to IgM antibodies that in turn activate
complement. The complement-coated cells would then be detected by the antiglobulin reagent.

4. Hemolytic disease of the newborn is due to the mother's IgG antibodies crossing the placenta and coating the
antigens on the fetal red blood cells. Cord blood collected at the time of birth would be tested, but may need to
followed up by a heel stick of EDTA blood. The reaction is usually a strong positive.

5. Complement on the red cells may be the result of antigen-antibody reactions which may t involve red cells.
Complement can also be activated if immune complexes are present in the plasma and the activated complement
attaches to the red cells. Complement can also become activated by the C3 by-pass mechanism and the lectin
activation process. Again once the activation of complement occurs in the blood stream, it can become attached
to the red cells.

6. Passive transfer of antibody from donor units of plasma or platelets may attach to the patient's red cells since
recipients are given ABO compatible blood but other unexpected red cell antibodies may not have been
detected. These antibodies in donor plasma can coat antigens on patient cells when group AB, A, or B receive
group O plasma products (and possibly platelets)

7. ABO mismatched transplants of particularly bone marrow can occur if an universal "O" donor bone marrow is
given to an A, B, or AB recipient. "Passenger lymphocytes" from group O donor organ make antibody to group
AB, A, or B recipient cells and these in turn can activate complement. It is also more common for "O" individuals
to make an IgG anti-A,B, which would also contribute to a positive DAT.

8. Sensitization of red cells due to medications like penicillin and cephalosporins that usually involves non-specific
coating of red cells. Other drugs like tetracyclines, antihistamines and sulphonamides cause the development of
immune complexes that are capable of activating complement. Some drugs, like ibuproten, levodopa and
methyldopa, are also known to cause autoimmunity. If a patient has a positive DAT, drug-induced problems
should be considered.

Indirect Antiglobulin Testing

The indirect antiglobulin test is one of the most important and commonly used techniques in immunohematology. It is
used to commonly for the detection of:
 Weak D's in donor bloods and pregnant females of individuals who type D (-) at room temperature when doing
ABO and Rh typing.

The presence or absence of antigens on a person cells from particularly the Kell, Kidd, and Duffy Blood Group
systems.

Unexpected, clinically significant antibodies in the patient's serum during the antibody screening procedure and
the antibody identification procedure..

Principle

The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells. This is done when sensitization
does not lead to direct agglutination. This occurs when there are too few antigens on the red cell, too few antibodies in
the serum and those antibodies are in the IgG class.

Summary of the Indirect Antiglobulin Technique

1. Incubate cells with serum at 37oC for the recommended time. (Usually 15 to 30 minutes.)

2. After incubation wash the cells three to four times.

3. Add AHG, Coombs reagent, centrifuge and read for agglutination.

4. If the test is negative, add Coombs Control Check Cells to check for false negatives.

Uses:

Screening Serum for Unexpected Antibodies Procedure

1. Involves patient serum plus reagent red cells (Screening Cells) Duet I and II (attach photo of screening cells)

The patient's serum potentially has unknown antibody.

Screening Cells have known antigens for the common clinically significant antibodies. (attach screening cell
sheet)

2. If there is agglutination after Coombs step with either (or both) Screening Cells, patient has an unexpected
antibody.

3. If antibody screen positive, must do additional tests to specifically identify antibody

The uses for antibody screen are:

Testing donor plasma to make sure no unexpected antibodies will be transfused to the recipient.

Testing recipient serum before transfusion to make sure patient has no unexpected antibodies to react with donor
cells.

Testing maternal serum to make sure pregnant mother has no antibodies to react with fetal cells causing
hemolytic disease of the newborn.

Red Cell Antigen Typing

Red cell antigen typing involves patient cells plus reagent antiserum. The patient's cells are the unknown antigen and the
reagent antiserum is the known antibody. The antiglobulin technique is used for antigen typing for a weak D and a
number of other clinically significant antibodies like the Kell, Kidd, and Duffy antibodies. If there is agglutination after the
addition of anti-human globulin, or Coombs step, patient cells had that specific antigen.

The specific procedure varies depending on what antigen is being tested for, and what brand of antiserum is being used.
Remember you must always read and follow directions in product insert carefully

Uses for red cell antigen typing are:

Typing donors for antigen if patient has antibody. You would want units that are negative for that antigen.

Verifying that patient is negative for antigen if he/she has made the antibody.

Typing patient to see what antigens he/she lacks so can predict what antibodies he/she is capable of making if
they seem to be particularly likely to make additional antibodies.

Controls

When performing red cell antigen testing always run known positive and negative controls. This will verify that antiserum
is acting properly and helps you interpret your test results. The positive control should be heterozygous for the antigen to
ensure antiserum is capable of detecting weaker antigens. For example, when performing antigen typing for K, you would
want a cell that is K+ and k+.

POSITIVE CONTROL NEGATIVE CONTROL PATIENT CONTROL PATIENT TEST

Heterozygous Positive cells Cells without Antigen Patient Cells Patient cells

+ + + +

Reagent Antiserum Reagent Antiserum Rh control Reagent. Antiserum

May be Positive (2+)
Should be at least 2+ Should be Negative Should be Negative Negative or
Mixed Field

OBJECTIVES - ANTIGLOBULIN TESTING

1. Explain the principle of the antiglobulin test.

2. Explain the difference between the direct antiglobulin test and the indirect antiglobulin test.
 3. Explain how Coombs reagents are produced.

4. Explain the role of Coombs control cells in antiglobulin testing.

5. Explain the mode of action of the Coombs control cells.

6. Name five reasons for a false negative antiglobulin test.

7. Name two reasons for a false positive antiglobulin test

8. Explain why a lavender top tube is best for DAT testing.

9. Describe the differences in the procedure between testing for IgG on the cells and testing for complement on the
cells.

10. Name two reasons for a false positive DAT, and explain why each would produce a false positive result.

11. List six clinical situations in which the DAT would be positive, and explain why each would cause the positive DAT

12. List the patient information that should be obtained if a positive DAT result is obtained on his or her sample

13. Describe the basic procedure for indirect antiglobulin testing

14. List three applications of the indirect antiglobulin test to detect red cell antigens

15. List three applications of the indirect antiglobulin test to detect serum antibodies

16. State the controls used in antigen typing and explain the purpose of each

Performance objectives:

1. Correctly perform and interpret the DAT, using good washing technique.

2. Use monospecific reagents appropriately, and correctly interpret results.

3. Use Coombs control cells appropriately.

4. Correctly perform and interpret serum antibody screens.

5. Correctly perform and interpret red cell antigen typings.

6. Correctly select and use controls when performing red cell antigen typing.

Table of Contents

Life's Blood
Table of Contents

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 ANTIGEN ANTIBODY
A A anti-B
B B anti-A
AB A and B neither
O neither anti-A or anti-B anti-A,B

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

ABO Group Whites Blacks

O 45 49
A 40 27
B 11 20
AB 4 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

Red Cell ABO Reverse ABO

Reaction of Cells Tested With Reaction of Serum Tested Against
Group Group

Anti-A Anti-B A1 Cells B Cells

0 0 O + + O
+ 0 A 0 + A
0 + B + 0 B
+ + AB 0 0 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 2 = A/O

1 = Homozygous A 2 = Heterozygous A
Phenotype A Phenotype A
Genotype A/A Genotype A/0
Can Contribute Only an A Gene to Offspring 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. 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

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 O
A AB AO
O BO OO

Possibilities of AA mating with BB: (Children's genotypes in purple)

Father's Genes
Mother's Genes
B B
A AB AB
A AB AB

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

Father's Genes
Mother's Genes

B O
A AB AO
A AB AO

Possibilities of an A/A mating with an O/O:

Father's Genes
Mother's Genes
O O
A AO AO
A AO AO

Possibilities of an A/O mating with an O/O:

Father's Genes
Mother's Genes

O O
A AO AO
O OO OO

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

Mother's Genes Father's Genes

 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

Life's Blood

CLASS NOTES

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 anti-B antibodies, anti-D antibodies
 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

Clinical Microbiology Syllabus

Life's Blood

CLASS NOTES

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)

Reactions with Anti- Adult Phenotype Incidence in %

Phenotype

Lea Leb Whites Blacks