COVER STORY

Approaches to Problem Solving in Antibody Identification

By Cora Jean Apollon

A 35-year-old Caucasian female is admitted to the hospital for delivery of her third child. A crossmatch for 2 units of blood is ordered because the patient has a history of bleeding episodes. An antibody screening test and panel are positive. The patient states she received 3 units of blood during post-partum hemorrhage at her last delivery. Her hospital record shows no previous antibody detected.

Based on the information provided in the panel, what is the specificity of this antibody? What is the most probable Rh phenotype of the patient? What percent of blood will be compatible with this patient? Can this antibody cause HDN?

Questions of this kind are standard in the blood bank, where performance of serological procedures for identification of unexpected blood group antibodies detected during routine testing is the responsibility of the blood bank medical technology staff.

Most blood group antibodies encountered can be identified if antibody identification procedures are established and followed, technologists have a basic understanding of the serologic characteristics of antibodies, and careful serologic technique is employed.

Only qualified individuals by training and/or experience who have demonstrated their competence may perform these procedures.¹ Unified Operation Instructions (OIs) should be developed and followed by all technical personnel to fulfill these requirements.

Blood group antibody screening tests are performed as part of processing donor units, pretransfusion testing, resolving transfusion reactions, prenatal evaluations, evaluating hemolytic disease of the newborn (HDN), and evaluating some disease processes. Unexpected blood group antibodies may be detected during the testing.

Once an unexpected antibody is detected, determining its specificity, clinical significance and importance is necessary. The clinical significance and importance are not determined by its specificity but by its ability to bind red cells at 37 degrees Celsius. However, knowing its specificity may indicate its clinical significance or importance.

Antibody Identification

Antibody identification is necessary for the selection of appropriate blood for transfusion, and in the investigation of HDN and immune hemolytic anemia (IHA).

Methods of Identification: Using a panel of group O reagent red blood cells (rbcs) of known blood group phenotype, the serum under investigation should be tested by the desired techniques at all phases at which antibody activity was initially detected. It must be noted that the reactivity of some antibodies may be increased by: extending incubation periods, increasing the serum-cell ratio, decreasing the pH, lowering temperatures, and using enzyme or other enhancement techniques. The saline, albumin, low-ionic strength saline (LISS), polyethylene glycol (PEG), low-ionic Polybrene^R (LIP) and enzyme techniques are all suitable methods for antibody identification. Additional procedures such as adsorption and elution techniques may be necessary to identify some antibodies.

Reagent Red Cells (Panel): To be functional, a reagent rbc panel must be able to identify most commonly encountered antibodies and rule out antibodies that are not present.² The antigram which is part of the panel states which antigens are present and which are not present on the panel cells. The antigram indicates whether each donor cell tests positive or negative for the Rh antigens: D, C, E, c, e, f, C^w, and usually V and VS; the M, N, S, and s antigens; the P₁ antigen; the Le^a and Le^b antigens; the Lu^a and Lu^b antigens; Kell system antigens (K, k, Kp^a and Js^a);, the Fy^a and Fy^b antigens; the Jk^a and Jk^b antigens, and the Xg^a antigen. The antigram will usually indicate the effects that temperature and enzymes have on certain antigen-antibody reactions.

For conclusive antibody identification, there must be sufficient rbc samples tested that lack, and that carry, the antigen to which an antibody appears to display specificity. A p value of 0.05 is the accepted minimum value at which an interpretation is considered statistically valid. To obtain this level of statistical confidence, at least six cells must be tested. Three cells must be positive and three cells negative for the corresponding antigen.

Autocontrol: The autocontrol is not required as a part of the antibody detection test. However, it may provide valuable information if an antibody is detected. A positive autocontrol is an abnormal finding and usually means that the patient has a positive direct antiglobulin test (DAT). Positive autocontrols are associated with autoimmune hemolytic anemia (AIHA), and with a variety of benign conditions. The autocontrol may be positive in recently transfused patients because alloantibodies may mimic autoantibodies in these patients. Therefore consider elution studies in recently transfused patients when the autocontrol is positive.

Interpreting Serologic Results: The specificity of an antibody is determined by comparing the pattern of positive and negative reactions as indicated on the antigram. It is important to look for this pattern and to examine the phenotypes of both reactive and nonreactive rbc samples in interpreting the results.³ Record the reactions: positive-H (hemolysis), 4+, 3+, 2+, 1+, + -, and = (negative) on the antigram sheet as you read them. This should be done in accordance with an OI to ensure proper identification and avoid missing antibodies that may be hidden.²

The following observations will assist in making a tentative simplifying assumption:

* Is the autocontrol negative or positive?

* In what phase(s) and strength did the positive reactions occur?

* What are the effects of temperature, suspending medium or proteolytic enzymes on the reaction with a particular rbc sample?

* Is there any variation in the strength of agglutination observed among reactive rbcs which suggests the effect of zygosity (dosage)?

* Did more than one screening cell react and, if so, did they react at the same strength and phase?

* Is hemolysis or mixed-field agglutination present? Certain antibodies (anti-Le^a, -Le^b, -P+ P₁ + P^k and -Vel) are known to cause in vitro hemolysis; mixed-field agglutination is associated with anti-Sd^a and -Lu^a.

* Are the reactions with cord cells negative or positive?

* What antibodies can be ruled out as possibilities? It is essential to "cross out" in identifying an antibody. Use an (X) to "cross out" when the cell is homozygous (EE) for the antigen and use an (/) when the cell is heterozygous (Ee).

* Does the serum reactivity match any of the remaining specificities?

* Are all commonly encountered rbc antibodies ruled out?

* Is there sufficient evidence to prove the suspected antibody?

* Is the patient lacking the antigen corresponding to the antibody? (Type the patient's rbcs for the antigen corresponding to the antibody identified. The results should be negative for the antigen.)

Problems in Identification

Problems may occur in the identification process, as indicated in the following situations:

Single Alloantibodies: If the antibody reacts in an unexpected manner (i.e., anti-S which usually reacts in the AHG phase may react in the saline, room temperature immediate spin reading). Also, depending on the antigen dosage, antibodies in the MNSs, Kidd and Duffy systems may react preferentially with cells from homozygotes carrying a double dose of the antigen. Another potential problem is that some antibodies such as anti-Bg may exhibit no discernible specificity.

Multiple Antibodies: Suspect when

1. more than one screening cell may be positive;

2. cells react at different phases and strengths;

3. observed pattern of reactivity does not fit a single antibody;

4. variation in strength of reactivity cannot be explained based on antigen dosage;

5. panel cells show reactivity at different phases or the effect of enzyme treatment of the panel cells is variable;

6. unexpected reactions are obtained when attempts are made to confirm the specificity of a single antibody.

Antibodies to High-Incidence Antigens: may be present when all cells are reactive and autocontrol is negative; may react at room temperature and may cause lysis of panel cells at 37 degrees Celsius; may have reduced or absent reactivity in enzyme tests; or may exhibit weak nebulous reactions in the AHG phase.

Antibodies to Low-Incidence Antigens: should be suspected when only one cell in the panel or one donor cell reacts with the serum. These antibodies usually cause no difficulty in compatibility testing because the antigens are rare.

Anomalous Serologic Reactions: antibodies to a variety of drugs, suspending media, additives, dyes and other substances can cause positive results with reagent rbcs.

To ensure that all personnel use the same procedures in resolving alloantibody problems they must follow the established OIs as directed. Basic knowledge, close attention and deductive reasoning are all assets essential to antibody identification.

* About the author: Cora Jean Apollon, a bone marrow transplant support specialist at Wilford Hall Medical Center, Lackland Air Force Base, San Antonio, has 43 years' experience in laboratory medicine. A biology major in college, she received her laboratory training as a member of the U.S. Air Force. At the end of her active tour, she continued her career as a civil servant.

References

1. Standards for Blood Banks and Transfusion Services: 16th Edition. AABB. 1994.

2. Harmening, D., Modern Blood Banking and Transfusion Practices: 2nd Edition. 1989.

3. Technical Manual: 11th Edition. AABB, 1993.

4. Blood Group Antigens and Antibodies. Ortho Diagnostics, 1982.

Other Resources

Anstall, Blaylock and Craven, Managing Hazards in the Transfusion Service, ASCP, 1993.

Berte, L.M., "Antibody Identification," Problem Solving in Immunohematology, ASCP Press, 1992.

Issitt, Peter D., Applied Blood Group Serology: 3d Edition. Montgomery Scientific Publications, 1985.

Judd, W., "Resolving Urgent Blood Bank Problems," Blood Banking in a Changing Environment. AABB Workshop, 1994.

Larison, J., "The Decision Making Process," Clinical Laboratory Science, Vol. 5, No. 4 Jul/Aug 1992.

Mollison, P.L., et al., Blood Transfusion in Clinical Medicine: 9th Edition. Blackwell Scientific Pub., 1993.

Challenges for the 90s. AABB Workshop, 1989.

Emerging Trends in Technology: A Workshop. AABB, 1992.

Review of Current Methods in Blood Banking and Transfusion Medicine: Conference. ASCP, 1993.

CHARACTERISTICS OF SOME UNEXPECTED ANTIBODIES:

1. Rh system antibodies are usually immune. They usually react at 37 degrees Celsius, and with antihuman globulin. They may cause transfusion reactions and may cause HDN.

2. Kell system antibodies are usually immune. They usually react at 37 degrees Celsius, and with antihuman globulin. They may cause transfusion reactions and may cause HDN.

3. Duffy system antibodies are usually immune. Enzymes inhibit the reactions of anti-Fy^a and anti-Fy^b.

4. Kidd system antibodies are usually immune. Enzymes enhance the reactions of anti-Jk^a and anti-Jk^b.

5. Lewis system antibodies are usually naturally occurring antibodies. They may cause in vitro hemolysis. They are usually not clinically significant.

6. MNSs system antibodies: anti-M and anti-N are usually naturally occurring. Anti-S and anti-s are usually immune. Anti-M and anti-N are unlikely to cause transfusion reactions and do not usually cause HDN. Anti-S and anti-s may cause transfusion reactions and may cause HDN.

7. Anti-P is usually naturally occurring, usually not clinically significant. There are no published reports of this antibody causing HDN.

8. Lutheran system antibodies: anti-Lu^a may be natural and may be immune. They may cause transfusion reactions and may cause HDN. Anti-Lu^b is probably immune, and may cause transfusion reactions and may cause HDN.

Additional and Alternative Procedures Useful in Resolving Antibody Identification Problems:

1. Adsorption kits

* Human Platelet Concentrate (HPC)--for use in reduction or removal of unwanted HLA antibodies (e.g., Bg, etc.).

* Rabbit Erythrocyte Stroma (RESt)--for removal of unwanted cold agglutinins (such as anti-I, -H or -IH).

* Warm Autoantibody Removal Medium (WARM)--for use in removal of warm autoantibodies from Direct Antiglobulin Test (DAT) red cell specimens, freeing antigen sites for adsorption of serum autoantibody.

2. Enhancement media

* Gamma PeG^® (Polyethylene Glycol Additive)--for antibody detection tests to provide a supplemental technique for antibody detection or identification.

* EM-X (Enhancement medium--incubation 10 minutes)--low ionic strength bovine albumin medium for human red cell antigen-antibody testing; to be used in the indirect antiglobulin test as a potentiating and enhancement medium.

3. Additional advances

* The gel test.

* The Galvanic Immunosensor Assay.

* Use of Thiol (Sulfhydryl) reagents to distinguish IgM from IgG antibodies.

(table/courtesy Cora Jean Apollon)