Life's Blood 

 

Table of ContentsCLASS NOTES

by M. Schroeder and M. Jensen

ABO DISCREPANCIES DEFINITION:

Any deviation from the expected pattern of antigen on the cell and the opposite antibody in the serum . ANTI-A ANTI-B A1 CELL B CELL

4+ 0 2+ 4+  

ANTI-A ANTI-B A1 CELL B CELL 4+ 0 0 0

 

ANTI-A ANTI-B A1 CELL B CELL 4+ 4+ 4+ 4+

What are their ABO types ? ? ? ? ? ? ?

ABO Discrepancies MUST BE RESOLVED

In RECIPIENTS the discrepancies must be resolved before any blood component is transfused. If not resolved before blood is needed, transfuse Group O (O NEGATIVE if there is a discrepancy in the Rh type also).

In DONORS the discrepancies must be resolved before any blood is labeled with a blood t;ype..

GENERAL RULES TO RESOLVE:

1. Always re-test first.  

2. Check for clerical/technical errors  

3. Weakest reaction is usually the one in doubt.  

4. Check results of the screening cells.  

5. Check the patient’s age.  

6. Check the diagnosis  

7. Check the transfusion history.

KINDS OF DISCREPANCIES:

CLERICAL ERRORS (TRANSCRIPTION ERRORS)

 

Life's BloodTable of Contents

CLASS NOTES

LABORATORY TECHNIQUES: REAGENTS, REACTION GRADING; CELL WASHING; SAMPLE COLLECTION

BLOOD BANKING REAGENTS

The techniques used in Blood Bank involves mixing antigens, usually on red blood cells with antibodies.  The environment where this reaction occurs can range in temperature from 4oC to 37oC.  With the most common being room temperature for ABO and the initial Rh(D) testing and 37oC when screening and identifying other clinically significant antigen-antibody reactions.  In a number of situations we are looking for particular antigens on the red cell such as looking for A or B antigens to determine a patient's ABO type.  Other times we may be looking for particular antibodies that may cause transfusion reactions or hemolytic disease of the newborn.  Depending on whether we are looking for a particular antigen or antibody will determine what reagents we are going to use.  If we are looking for an A antigen on a patient's red cells, we will use known anti-A reagent that will cause agglutination of the A antigens on the red cells.  If the patient has on B antigens or no ABO antigens, as in the case of an O individual, their cells will not agglutinate with anti-A reagent.

Sources of Antigen Testing:

In almost all blood bank techniques we have red cells with antigens present.  These red cells may either reagent red cells with known antigens, patient red cells, or donor red cells.  The reagent red cells are commercially prepared  and have all the red cell antigens identified. 

When we use red cells where the antigens have already been determined, we can identify the possible antibodies present.  For example:  Anti-A and Anti-B are expected antibodies in patient's who lack that particular antigen.  Therefore if a patient or donor has only the A antigen on their red cells, then they should have anti-B in their serum.  When we test their serum with A1 and B cells, agglutination will occur with the B cells and not with the A1cells since they have an anti-B.  The reagent cells used for blood banking include the following:

A1 and B cells for confirmation of the ABO type in all patients and donors other than newborn babies

Antibody screening cells are O cells that have been studied to determine the presence of a number of antigens for specific antibodies that are known to cause transfusion reactions and hemolytic disease of the newborn.  The antibody screening technique is part of all compatibility tests done before blood is transfused.  Some of the more common antibodies detected are anti-D, anti-E, anti-K.

Antibody identification cell panel are again O cells with the specific antigens known.  Usually there are between 8 and 12 different cells in a cell panel.  The pattern of positive and negative reactions help identify the antibody.

Sources of Antibody for Testing

Antibody is found in serum.  If it is the patient's serum that is being tested, we do not know what antibody may be present so we are using one of the 3 types of reagent

 

Life's Blood

CLASS NOTES (Under construction)

QUALITY ASSURANCE IN BLOOD BANKING

Quality Programs 

When discussing Quality Programs  it includes:

Quality Control Quality Assurance  Quality Improvement 

For Blood Banking  Quality Programs are essential requirements of 2 Federal Agencies:

1. Centers for Medicare and Medicaid Systems (CMS), formerly HCFA, under CLIA-88 which covers all Clinical Laboratory activities and related federal payments

2. Food and Drug Administration that has the following concerns: a. Responsibilities of the blood product requirements (anticoagulants and

preservatives, shelf life etc.)  b. Specific requirements related to independent quality control and quality

assurance for overall quality of blood products and the processes related to dispersion of those products.

A number of accrediting agents have quality requirements as well: 

1. American Association of Blood Banks (Blood Banks and Transfusion Services)

2. Joint Commission on the Accreditation of Healthcare Organizations  3. College of American Pathologists

AABB Quality System Essentials & FDA Guidelines for Quality Assurance in Blood Establishments 

Organization: active support of quality systems must be place for the following procedures;

SOP's - Standard Operating Procedures Training plans and development of procedures Approval of lot release of reagents and quality control reagents

Life's Blood Table of Contents

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.

 

 

 

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 ChromosomeABO A, B, O 9

MNSsU M, N, S,s,U 4P P1 22

Rh D, C, E, c, e 1Kell K, k, Kpa, Jsa,Kpb, Jsb 7

Lewis Le, le 19Duffy Fya, Fyb, Fy3 1Kidd Jka, Jkb 18Xg 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 AOO 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 frequencyObserved frequency

MS = 0.53 (M) X 0.33 (S) = 0.17 0.24Ms = 0.53 (M) X 0.67 (s) = 0.36 0.28NS = 0.47 (N) X 0.33 (S) = 0.16 0.08Ns =  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 Phenotypeh ABO Oh

or Bombay

=r

Rh Rhnull

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, 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

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:-1k1+, K:1+, K(1), K:(1), K1-, K:1-, K1-negative

Phenotypes A Rh+, B Rh-A+ (means positive for A antigen)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?

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.

 

Life's BloodTable of Contents

CLASS NOTES 

by M. Schroeder and M. Jensen

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. 

 

 

 

 

Incidence (%)  of

ABO Bloo

d Groups in the US

PopulationABO Group

Whites

Blacks

O45

49

A40

27

B11

20

AB

4 4

ABO Typing

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

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

Red Cell ABO Group

Reaction of Serum Tested Against

Reverse ABO 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)

Mother's GenesFather's Genes

B O

A AB AO

O BO OO

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

Mother's GenesFather'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)

Mother's Genes

Father's GenesB O

A AB AO

A AB AO

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

Mother's GenesFather's Genes

O O

A AO AO

A AO AO

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

Mother's Genes

Father's GenesO O

A AO AO

O OO OO

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

Mother's Genes

Father's GenesO O

A AO AO

B BO 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 cellsBombay cells

lectin-H 4+ 3+ 2-3+ 2+weak to negative

weak to negative

negative

Lectin-A1 negative negative negative negative positive positive negative

 

Differentiating Subgroups of A:

Use the following steps to help differentiate the subgroups of A:

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

GROUP A1 A2 A3 Ax

Reaction with anti-A 4+ 4+ mf 0

Reaction with anti-A,B 4+ 4+ mf 2+

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

lectin-H. 26. Explain what reactions demonstrate a subgroup of A.

Table of Contents  

 

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 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 PresentPhenotype

Percentage

R1 RHD RHCe D,C,e R1 42%  r RHce dce   r 37% 

R2  RHD RHcE DcE  R2  14% 

Ro RHD RHce (more common in Blacks)

Dce  Ro  4% 

r' RHCe dCe  r' 2%r"  RHcE dcE  r"  1%Rz  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. R always refers to D whether it is Ro, R1, R2, or the very rare Rz. 2. r always refers to  the lack of D

3. o refers to having no C or E 4. 1 or ' always refers to C 5. 2 or " always refers to E 6. 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 Rz 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

Antigens Present

Genotype Incidence(%)

DCEWeiner

HaplotypesWhites Blacks

1 D, C, c, e DCe/ce R1r 31.1 8.8    DCe/Dce R1Ro 3.4 15

    Dce/Ce Ror 0.2 1.82 D, C, e DCe/DCe R1R1 17.6 2.9    DCe/Ce R1r' 1.7 0.73 ce ce/ce rr 15 74 DCcEe DCe/DcE R1R2 11.8 3.7    DCe/cE R1r" 0.8 <0.1    DcE/Ce R2r' 0.6 0.45 DcEe DcE/ce R2r 10.4 5.7    DcE/Dce R2Ro 1.1 9.76 Dce Dce/ce Ror 3.0 22.9    Dce/Dce RoRo 0.2 19.47 DcE DcE/DcE R2R2 2.0 1.3    DcE/cE R2r" 0.3 <<0.1

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 Rho 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 AGT Interpretation

Anti-D Rh Co Anti-D Rh Co Anti-D Rh Co

+ 0         D positive0 0 0 0 + 0 Weak D0 0 0 0 0 0 True D negative

0 0 0 0 + +

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 SYSTEM1. 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. Correctly perform, interpret, and record the Rh type of any given sample 2. Recognize when chemically modified or saline Rh reagents must be used. 3. Correctly perform, interpret, and record a weak D (Du)test. 4. Correctly perform, interpret, and record the Rh phenotype of any given

sample, and state its most likely genotype.

 

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-Phenotype

Adult Phenotype Incidence in %Lea Leb Whites Blacks+ 0 Le(a+b-) 22 23

 

Life's BloodTable of Contents

CLASS NOTES 

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 anti-U 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

PhenotypePhenotype Frequency %

Whites Blacks

M+N- 28 26

M+N+ 50 44

M-N+ 22 30

S+s-U+ 11 3

S+s+U+ 44 28

S-s+U+ 45 69

S-s-U- 0 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

 

Life's BloodTable of Contents

CLASS NOTES 

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

 

 

 

 

Antibody ScreeningPRINCIPLE 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 #, IIPatient 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.

6.  Add one drop of Screening Cell I to tube I; add one drop Screening Cell II to the II tube; add one drop