9 - ANTIBODY DETECTION AND IDENTIFICATION

Clinically unexpected antibodies

occur in less than 3% of the population

• Women ; pregnancy

• 60 years old ; transfusion

Multiple antibodies are more frequently seen in:

• ______ than in men because of possible sensitization during ________

• Among patients older than __________ who have undergone __________ multiple times

ANTIBODY SCREEN

Involves the reaction between patient serum or plasma with 2 or 3 reagent phenotyped for multiple antigens; called antihuman globulin test

ANTIBODY SCREEN

Purpose: detect red cell antibodies other than expected anti-A and anti-B antibodies; these are called "unexpected antibodies" since they are present only in 0.3 to 2% of the general population

ANTIBODY SCREEN

Importance:

• Selection of appropriate blood for transfusion

• Investigation of HDN, immune hemolytic anemia, and transfusion reaction

ANTIBODY IDENTIFICATION

If the antibody screen is positive, and antibodies are present in the serum, a more definitive test called antibody identification or panel is performed

direct antiglobulin test (DAT)

Another test to detect and identify antibodies

Direct antiglobulin test (DAT)

A test to detect antibodies coating the surface of red cells in vivo. An antibody detection method or panel also identifies these antibodies

elution

Ab ID:

These antibodies may be removed from red cells by a process known as ______ and then identified by a panel

Ab ID

Select cell panel, Antigen typing, Enzyme panel

Antibody Screen → Positive rxn in 1 or more cells → Perform __________________ →(3)

DAT

Elution and Antibody Identification

Antibody Screen → Positive rxn in autocontrol → Perform ____ →POSITIVE RESULT →_________________________________

No further testing required

No further testing required

Antibody Screen → Negative with all cells → _________________________ → NEGATIVE RESULT → _________________________

Reagent Red cells (antibody screening cells)

commercially prepared Group O red cell suspensions obtained from individual donors that are phenotyped for the most commonly encountered and clinically important RBC antigens

Group O cells

are used so that "naturally occurring" expected anti-A or Anti-B will not interfere with detection of unexpected antibodies

Screening cells

are detected so that the following antigens are present on at least one of the RBC samples: D,C,E,c, e, M, N,S,S, P1, Lea, Leb, K, k, Fya, Fyb Jka, Jkb

antigram

The reagent red cells are provided in either two or three vials. Each vial contains cells from a single, different donor. The vials are provided with a description of the antigen content of each of the cell. This description is provided in a chart known as an?

Enhancement reagents

Are solutions added to serum and cell mixtures to promote antigen-antibody binding or agglutination

1. Low lonic Strength Saline (LISS)

2. Polyethylene Glycol (PEG)

3. Bovine albumin

Examples of Enhancement reagents (3)

Antihuman globulin reagents (AHG)

Used to promote agglutination of RBC sensitized with immunoglobulin G (IgG) or complement molecules

Coomb's Control Red Blood Cells (Check Cells)

are RBC coated with human IgG; prepared by incubating D positive RBC with potent anti-D

Coomb's Control Red Blood Cells (Check Cells)

Are used to ensure that AHG tests with negative results are not false negatives because of the inactivation of the AHG reagent

Coomb's Control Red Blood Cells (Check Cells)

In a negative AHG test, there is free AHG reagent in the test tube; when this reagent is added, the free AHG in the test should cause agglutination of the sensitized RBC

• Omission in the addition of AHG

• Neutralization of the AHG reagent

Antibody Screening

Principle: When the test is performed, each vial of reagent red cells is tested with the serum of the patient or donor

Antibody Screening

• Media of reactivity include the following:

  • Saline (immediate spin at room temperature)

• Enhancement medium at 37°C

  • Albumin

  • Low ionic strength solution (LISS)

  • Polyethylene glycol (PEG)

  • AHG sera (after incubated cells are washed with AHG)

Antibody Screening

Results from each phase are recorded and evaluated at the end of testing; by using the antigrams and knowing temperature and media reactivity, the potential antibodies may be narrowed and a more definitive diagnosis is provided by the use of the antibody detection panel

immediate spin

M, N, Lea, Leb and P1

lgM antibodies usually react on ______________ and include (5)

AHG phase

Kell, Kidd, Rh, Duffy

IgG antibodies usually react in the _________ and include (4)

negative autocontrol

A __________________ (using patient cells and patient serum) effectively rules out the presence of an autoantibody

Antibody Identification

Principle: The patient's serum constitutes the unknown with the potential for the presence of antibody; whereas the cells represent known antigens; most commonly, there are between 8 and 16 different cells included in the panel

Rh and Kidd

Enzyme treatment with ficin, papain, trypsin or bromelin enhance the reaction of some antibodies: (2)

M, N, S, Duffy

Enzyme treatment with ficin, papain, trypsin or bromelin denature other antibodies: (4)

ABO, P, Le, Jk, Kell systems

Hemolysis should be noted because some antibodies can cause hemolysis: (5)

1. Cold reacting antibodies, such as anti-I

2. Rouleaux

lf all panel cells and autocontrol agglutinate strongly at room temperature but less strongly at 37°C and in the AHG phase; the causes are (2)

warm antibody

If panel cells and autocontrol agglutinate only in the AHG phase, the cause maybe a?

1. Multiple antibodies

2. Antibody against a high frequency antigen found on all panel cells

If the panel cells agglutinate but autocontrol is negative, the causes are (2)

warm AlHA

If all panel cells are negative or give variable positive reactions and autocontrol is positive in AHG, the cause may be?

1. Absorption of antibodies

2. Elution of antibodies

3. Titration of antibodies

4. Typing of immunoglobulin

5. Enzyme testing

OTHER BLOOD BANKING TECHNIQUES (5)

ABSORPTION OF ANTIBODIES

It is the process of removing antibody from the serum

ABSORPTION OF ANTIBODIES

Non-specific antibodies can be removed by absorbing them from the serum by allowing clotting at the optimum temperature of the antibody

Absorption techniques

are helpful in the following situations:

• Remove non-specific antibodies from the serum

• Separate mixtures of antibodies to aid in their identification

ABSORPTION OF ANTIBODIES

Determine the presence of specific isoantibody as well as a non-specific auto antibody in the serum of a patient with acquired hemolytic anemia

ABSORPTION OF ANTIBODIES

Cold antibodies (lgM) are absorbed at 4°C, while warm antibodies (lgG) at 37°C

• anti-A ; anti-A1 ; A2 ; anti-A ; anti-A1

• A2 ; anti-A1

Application:

• The serum of Group B individuals contain _____ and _____; ____ red cells are added to the serum of Group B individuals; ______ antibodies react with A2 cells, while _______ do not react with A2 cells

• After centrifugation, the _____ cells absorbed by anti-A1 are removed, while ________ antibodies are left in the serum. The latter is called absorbed anti-A1 typing sera or anti-A1 typing sera

ELUTION OF ANTIBODIES

Removal of antibodies from the cells

• heat elution ; ABO

• ether elution ; non-ABO

ELUTION OF ANTIBODIES

Techniques:

• Heating the washed cell suspension at 56°C water bath for 10 minutes and agitating the mixture frequently (__________); this is done to elute ______ antibodies from RBC

• By the addition of ether (_________), low pH acid, digitonin-acid, cold acid, chloroform, xylene, or methylene chloride; this is done to elute ________ antibodies

ELUTION OF ANTIBODIES

Application

• Demonstrate and identify the antibody on the RBC of infants or cord blood in case of HDN

• Identify the antibody absorbed on the RBC in acquired hemolytic anemia

• ldentify the antibody absorbed on the RBC of recipient in transfusion reactions

• Separate and identify antibodies in a mixture of antibodies

TITRATION OF ANTIBODIES

done to determine the relative amount of antibody in the serum

TITRATION OF ANTIBODIES

Determined by making serial two-fold dilutions of the unknown serum and testing against saline and albumin suspended cells of the appropriate type; amount these cells added to each tube is constant

TITRATION OF ANTIBODIES

The titer is expressed as the reciprocal of the HIGHEST dilution in which agglutination is OBSERVED MACROSCOPICALLY (e.g., saline agglutination end at 1:256, so this is reported as a saline agglutination titer of 256)

TYPING OF IMMUNOGLOBULIN

Accomplished by the use of dithiothreitol (DTT) and 2-mercaptoethanol (2-ME)

TYPING OF IMMUNOGLOBULIN

DTT and 2-ME are used to remove or inactivate IgM, leaving the IgG intact; they are sometimes useful to remove or break up red cell agglutination by strong IgM cold autoantibodies

DTT and 2-ME

These 2 reagents are used to remove or inactivate IgM, leaving the IgG intact; they are sometimes useful to remove or break up red cell agglutination by strong IgM cold autoantibodies

ENZYME TESTING

Done to facilitate the aqglutination of red cells in the presence of certain antibodies

ENZYME TESTING

In the procedure, RBCs are expressed to proteolytic (or protein-digesting enzymes either before the antiserum is added or at the same time it is added

ENZYME TESTING

The enzymes used in blood bank are trypsin, papain, bromelin and ficin; papain is obtained from papaya, while bromelin from pineapple

Enzymes

These digest away some of the RBC surface, thus making RBC more readily accessible to agglutination by certain antibodies

Excessive enzyme treatment

must be avoided since it causes red cells to agglutinate spontaneously in the absence of antibodies

MN and Duffy

Ss, K, Jka, U

Enzyme treatment destroys (2) and may lower activity of (4) antigens