COMPATIBILITY TESTING

The purpose of pre-transfusion compatibility testing is to PREVENT hemolytic transfusion reaction

DIFFERENT LEVELS OF COMPATIBILITY TESTING

Landsteiner is noted for his development in 1902 of the modern system of classification of blood groups from his identification of the presence of agglutinins in the blood

Landsteiner, 1899

ABO TYPING
ABO typing has two stages: one direct red blood cells -RBCstyping using commercial antisera (anti-A, anti-B) and one indirect typing utilizing plasma/serum and known - A and B RBCs suspensions. 1. Direct ABO blood typing (or front grouping) This test detects the presence of A and B antigens on RBCs surface by means of agglutination utilizing commercial antisera. It is a basic and simple test, it can be performed on few drops of venous or capillary blood (i.e. obtained by finger pricks) . The RBCs are then examined for clumping (gross observation, gel suspension)
Antisera- serum containing antibodies

Anti A

Anti B

Anti A

Anti B

Anti A

Anti B

Anti A

Anti B

AB

False positives
Are rarely seen and they are essentially caused by the plasma proteins when whole blood is used as specimen. These plasma-related agglutinations may be false or they may be real agglutinations caused by auto-antibodies. These apparently positive results turn generally negative when saline-suspended RBCs are tested instead of whole blood. Remember that auto-antibodies usually cause agglutination of all tested blood drops so beware of AB positive results ! In these cases the reverse typing will help to clarify the situation .

False negative
In theory it is possible if antisera are no longer working. So antisera should be checked regularly with RBCs samples of known A and B type. If reagents are working properly the only false negatives may be due to the presence of very rare group A or B variants. These may be also suspected because of discrepancies between the direct typing and the reverse or indirect serum grouping.

The H antigen is an essential precursor to the ABO blood group antigens

The H locus is located on chromosome 19. It contains 3 exons that span more than 5 kb of genomic DNA, and it encodes a fucosyltransferase that produces the H antigen on RBCs.

This H substance is present in all RBCs, except those of hh (i.e., Bombay blood group) and also produce antibody to both A and B antigens, and are therefore compatible only with other hh donors. So, in the Bombay blood in serum or in reverse grouping there is anti-A, anti-B, and anti-H.

Reverse serum grouping

This test is used to confirm and must not substitute the front grouping that remains the pillar of ABO typing. Reverse grouping helps to identify the rare cases of false negative and false positive results. The test puts in evidence the presence of naturally occurring Anti-A and Anti-B that are present after 6 months of life in all subjects that lack the reciprocal A/B antigen. In order to perform the reverse test we need a saline 3-5% suspension of fresh RBCs of group A, B and O. The O type RBCs suspension represents a negative control. Technique: 1) Mix 2 drops of serum or plasma under investigation with one drop of each 3-5% RBCs suspension (group A, B and O); and 2) read for agglutination

ABO TYPING
Back or reverse type with A and B cells
Commercially available A and B cells are added to two tubes of plasma
AB
A B A

B
B A

A
B A

O
B

Note: another sample of blood is usually added for Rh-D typing

How do we know whether or not the host (or recipient) has antibodies to minor blood group antigens?

Add commercial RBCs with known important minor antigens on their surface to host (or recipient) plasma and centrifuge. Then incubate at body temperature for 15-30 minutes Then add antiglobulin

Ex. of minor blood group [MNSs, Duffy (Fy) and Lewis (Le)].

Antiglobulin reagent

Purification, concentrate and specific test

Composition of antiglobulin reagent

1. Anti human globulins ( 1:32) 2. Anti-complement (C3d, low titer agglutinins to prevent false positive from cold autoantibody

polyspecific

These natural cold autoantibodies occur at low titers, less than 1:64 measured at 4C, and have no activity at higher temperatures

Classification of antiglobulin test

1. Direct antiglobulin test: To detect IgG bound on red cell 2. Indirect antiglobulin test: To detect free IgG in serum

Direct antiglobulin test

Antihuman globulin (AHG)

Antiglobulin test

Indirect antiglobulin test

If recipient antibodies have coated commercial RBC surfaces

Rabbit antiglobulin will bind to the Antibodies and the RBCs will clump

Method of antiglobulin test

Tube test
Gel test

Application of Direct Antiglobulin Test (DAT)

1. Hemolytic disease of the new born (HDN) 2. Autoimmune hemolytic anemia (AIHA) 3. Drug-induced hemolytic anemia

4. Transfusion reaction hemolytic anemia

Application of indirect antiglobulin test

1. Antibody identification 2. Crossmatch / compatibility test

3. Red cell typing 4. Investigate for HTR

Hemolytic transfusion reaction (HTR)

Factors influence in antiglobulin test

Sensitization phase - Temperature - Reaction medium - Antigen-antibody ratio - Time Washing phase: wash out unbound IgG

False negative in antiglobulin test 1. Expired or out date serum, cells, reagents 2. Labile complement in aged plasma 3. Improper time and temperature 4. Washing: - Inadequate - longer time - low speed - non-continuous
5. Contamination in reagent serum

False positive in antiglobulin test

1. 2. 3. 4. 5. Auto-aggregation of red cell Contamination of colloidal silica in saline Contamination of metallic ion in saline Contamination of detergent in container Longer time in centrifugation

Hemolytic disease of the new born

Rh incompatibility occurs when the mother's blood type is Rh negative and her fetus' blood type is Rh positive (inherited the Rh-positive antigen from the father)

If some of the fetus' blood passes into the mother's blood stream, her body will produce antibodies in response

These antibodies could pass back through the placenta and harm the fetus' red blood cells, causing mild to serious anemia in the fetus

Before birth, options for treatment include intrauterine transfusion or early induction of labor when pulmonary maturity has been attained, fetal distress is present, or 35 to 37 weeks of gestation have passed. The mother may also undergo plasma exchange to reduce the circulating levels of antibody by as much as 75%. After birth, treatment depends on the severity of the condition, but could include temperature stabilization and monitoring, phototherapy, transfusion with compatible packed red blood, exchange transfusion with a blood type compatible with both the infant and the mother, sodium bicarbonate for correction of acidosis and/or assisted ventilation. Rhesus-negative mothers who have had a pregnancy with/are pregnant with a rhesus-positive infant are given Rh immune globulin (RhIG) at 28 weeks during pregnancy and within 72 hours after delivery to prevent sensitization to the D antigen. It works by binding any fetal red cells with the D antigen before the mother is able to produce an immune response and form anti-D IgG.

How to treat the HDN

Whats new?
2007 Nature Biotechnology USA, Denmark, France, Sweden Convert blood types A, B, and AB to O, using bacterial glycosidase enzymes to cleave the antigens from the RBC surface. Need D negative cells

Red blood cell compatibility table

Recipient
O-

Donor O- O+ A- A+ B- B+ AB- AB+

O+
AA+

BB+ AB-

AB+

Plasma compatibility table

Recipient Donor

O A B A B
AB A

B
O