Unit 4 Principles of Serological Testing in Immunohematology

1. Unit 4 Principles of Serological Testing in Immunohematology Terry Kotrla, MS, MT(ASCP)BB

2. Blood Group Antigens and Antibodies • Antigens are embedded structures in or protruding from RBCs, WBCs or platelets. • Exposure to blood group antigens through transfusion or pregnancy. • Individual may form antibodies to antigens they lack. • Most concerned with IgG and IgM class antibodies. • IgM naturally occurring, react at RT, easily detected. • IgG are “immune” requires exposure to the actual antigen and requires special serological test (Coomb’s) to detect. • ALWAYS testing for antibodies in serum/plasma and antigens on cells.

3. Detection Tests • Detection of antigens routinely done to determine individuals’ ABO and D type. • Antibody detection tests used to • Confirm ABO antigen typing. • Detect unexpected antibodies in serum/plasma which may cause destruction of transfused cells containing antigen.

4. Dynamics of Antigen-Antibody Reactions • Union of antigen with antibody depends on structure and charge of molecule. • Lock and key • Opposite charges on antigen and antibody create attraction forces. • Reactions is REVERSIBLE .

5. Dynamics of Antigen-Antibody Reactions • Physical forces hold antigen and antibody together. • Weak • Attractive forces vary in strength with changes in pH, ionic strength, temperature and nature of solven. • Blood group antigens and antibodies bind until dynamic equilibrium is reached. • Represented by bell shaped curve based on concentration. • Prozone • Postzone • Zone of equivalence.

6. Antigen Antibody Reactions

7. Detection of Antigen-Antibody Reactions • Agglutination • RBCs have antigens which combine with antibodies present in serum (either patient or reagent). • Antibody bridges form with antigens on adjacent cells resulting in agglutination. • Hemolysis caused by antibody binding, activation of complement which results in destruction of RBC membrane. • Solid Phase to detect antigens or antibodies • Primarily done in donor centers. • Microplates coated with antigen or antibody. • Positive, cells adhere to sides of well. • Negative, cells fall to bottom and form a button

8. Agglutination – Principle and Variables • TWO stages involved • First stage is sensitization, attachment of an antibody to corresponding antigen on RBC membrane. • Second stage is lattice formation, bridges form between sensitized cells. • IgG have 2 antigen binding sites, one binds to antigen on one cell, the other binds to antigen on adjacent cell. • IgM has 5 antigen binding sites and can bind to several antigen sites on different cells. • Lattice formation will result in hemagglutination.

9. Agglutination • IgG agglutinating RBCs • IgM agglutination RBCs.

10. Variables Affecting First Stage • Antigen-antibody ratio – CRITICAL • Optimal amount of serum/plasma must be determined. • Increase serum/plasma increase amount of antibody – more sensitive. • pH of 6.5 to 7.5, for routine procedures 7.0 used. • Ionic strength of suspending medium • Saline partially neutralizes oppositely charged sites on antigen and antibody molecules which hinders association. • Low ionic strength saline (LISS) with lower salt concentration enhances uptake of antibody onto cells. • Albumin, unless used under low ionic conditions, does little to affect antibody uptake onto cell, usually affects second stage.

11. Variables Affecting First Stage • Temperature requirements depend on antibody class detected. • IgG reacts optimally with antigens at 37C. • IgM reacts optimally at RT (20-24C) or below (4C) • Incubation time • Choose incubation time which favors maximal binding of antibody to antigen. • Enhancement agents increase antibody uptake onto cells thereby decreasing incubation time.

12. Variables Affecting Second Stage • Immunoglobulin Class • IgG is a small monomer with the ability to sensitize RBCs but NOT agglutinate. • IgM is huge pentamer, very easily agglutinates RBCs • Antigen sites may be present in low numbers on surface resulting in no agglutination. • Electrostatic Repulsion Forces • Lattice formation depends on overcoming electrical charges (repulsion forces) of RBCs. • Zeta potential is the term used to describe the electrical forces which keep RBCs separated, use methods to decrease.

13. Zeta Potential • IgG unable to overcome zeta potential to cause direct agglutination. • I could not copy the original picture. • Visit http://tinyurl.com/4f9d24y , EXCELLENT materials • IgG unable to overcome zeta potential to cause direct agglutination. • I could not copy the original picture. • Visit http://tinyurl.com/4f9d24y , EXCELLENT materials

14. Principle of the Antiglobulin Test • Most important test in the blood bank after ABO/D typing. • Small IgG antibodies can sensitize but rarely cause agglutination. • Cannot overcome zeta potential • If undetected (false negative) will cause destruction of RBCs. • 1945 Coombs, Mourant and Race described test for non-agglutinating coating antibodies now called the anti-human globulin (AHG or Coomb’s) test. • You MUST know the principle of the test backwards, forwards and sideways.

15. Principle of the Antiglobulin Test • A reagent antibody to IgG (anti-IgG) and/or complement is produced (AHG serum) and is added to washed cells after incubation with serum/plasma or antisera. • If cells are sensitized (coated) with IgG during incubation the anti-IgG will attach to the Fc portion of the IgG coating the cell causing agglutination. • You must MEMORIZE the principle of this test and be able to draw a diagram for future exams.

16. Coomb’s Test

17. Importance of Coomb’s Test • Prior to 1945 could only detect IgM class antibodies, only a few blood groups had been identified. • ABO antibodies and antigens were detected but hemolysis of ABO compatible transfused cells continued to occur. • This test provided the explanation that IgG class antibodies were responsible, could sensitize but not be detected in direct agglutination tests. • Determined that IgG was capable of destroying transfused RBCs and also crossed the placenta to destroy fetal RBCs. • IT IS NOW A REQUIRED TEST

18. Preparation of AHG Serum • Two methods. • Animal Inoculation • Purified human IgG injected into suitable animal, usually rabbits, and rabbit would make anti-IgG. • Animal bled, unwanted antibodies removed. • AHG will react with IgG bound to cells or free in serum/plasma.

19. Preparation of AHG Serum • Monoclonal Reagent Preparation • Animal (mouse most popular) injected with appropriate antigen (IgG) and produces anti-IgG • Make hybridoma • Fuse antibody secreting cell (mouse spleen cell) with myeloma cell which produces massive quantities of antibody. • The myeloma cell produces antibody without specificity. • Fusion with mouse spleen cell “programs” the myeloma cell to produce antibody with one specificity. • Cell is immortal. • Grow in culture. • Harvest the antibody containing fluid from hybridoma.

21. Monoclonal Antibody Production

22. Types of AHG • THREE types • Polyspecific • Monospecific anti-IgG • Monospecific anti-Ce

23. Polyspecific AHG • Consists of a POOL of anti-human IgG, anti-C3b and anti-C3d. • May be produced by hybridomas, rabbits or a mixture. • May be used for routine compatibility testing, antibody identification and DAT. • Most important function is to detect IgG antibodies coating the cells. • The importance of the presence of anti-complement in AHG serum is very controversial for routine compatibility testing. • Antibodies which are detectable only by their ability to bind complement are rare, so it's not as useful for compatibility testing as many false positive reactions occur.

24. Anti-IgG (monospecific) • Major component is antibody to human gamma chains (anti-IgG) contains no anti-complement, is heavy chain specific. • Used as alternative to polyspecific AHG in routine blood bank procedures. • Advantage of utilizing anti-IgG is that it eliminates the false positives frequently obtained when using polyspecific AHG due to complement and non-specific cold reacting antibodies yet will detect clinically significant alloantibodies.

25. Anti-Complement (Monospecific) • Contains anti-complement antibodies to detect complement coating the cells. • Used for classifying the coating proteins on RBCs to determine if IgG, complement, or both are present. • Primary importance is for classification of autoimmune hemolytic anemias and drug induced hemolysis of RBCs.

26. Antiglobulin Techniques • TWO applications of the antiglobulin test • Direct antiglobulin test (DAT) • Indirect antiglobulin test (IAT)

27. Direct Antiglobulin Test (DAT) • Used to demonstrate or detect in-vivo coating of RBCs with globulin, particularly IgG and/or C3. • One drop of a patient's cell suspension is washed 3 times to remove all contaminating proteins. • AHG is added directly to the washed cells, centrifuged and observed for agglutination, which is indicative of IgG antibodies coating the cells, the AHG serum will bind to the IgG on the cells forming lattices = agglutination. • Primary use is detecting maternal antibody coating fetal cells. • Also used in detecting autoantibody coating on patient cells.

28. Direct Antiglobulin Test

29. Indirect Antiglobulin Test (IAT) • Detects in-vitro sensitization with antibody complement or both. • Patient serum (or special types of anti-serums) are added to RBCs and incubated at 37 C (body temperature) for a specified time. • May be read after incubation because some IgG antibodies may coat the RBCs heavily enough after incubation to cause agglutination. • The RBCs are washed free of contaminating protein and AHG serum is added, if the cells have been coated with IgG during the incubation phase the anti-antibody in the AHG will form lattices which resulting in agglutination.

30. Indirect Antiglobulin Test

31. Antiglobulin Techniques • The crossmatch procedure may require the IAT, • patient serum is incubated with donor cells, washed and tested with AHG serum. • Agglutination indicates that the patient has antibody to an antigen on the donor cells • The unit cannot be given to the patient (it is incompatible). • When a patient has an antibody identified in their serum it is critical to find donor units which lack the antigen, typing the donor may require the IAT.

32. False Negative Results in the DAT and IAT.MEMORIZE! • Failure to wash red blood cells adequately. • Testing interrupted or delayed during the washing procedure. • Loss of activity in AHG reagents • Failure to add AHG. • Improper centrifugation • Under centrifugation • Over centrifugation • Incorrect concentration of red blood cells in test system. • Prozone reactions

33. False Negative Results in the DAT and IAT.MEMORIZE! • Negative DAT may not necessarily mean absence of coating globulins. • Poly and anti-IgG will detect approximately 200 molecules of IgG per cell. • Presence of weak antibody may go undetected. • If a negative reaction is obtained, IgG coated reagent RBCs (check cells), are added to the test system, agglutination ensures that the AHG serum is active. • In the DAT complement coating may not be apparent as agglutination upon immediate reading.

34. False Negatives in the IAT • IAT and DAT are different. • Red cells and serum lose reactivity if improperly stored. • Occasional examples of anti-Jka and anti-Jkb may be detected only in the presence of active complement. • Temperature and incubation time affect attachment of antibody or complement to the red blood cells. • Optimal proportion of serum to cells should be achieved.

35. False Positive Resultsin the DAT and IAT. MEMORIZE! • Red cells may be agglutinated before they are washed. • Saline stored in glass bottles may contain colloidal silica particles that can leach from the container into the saline. • Improperly cleansed glassware. • Overcentrifugation • Improperly prepared AHG reagents.

36. DAT - additional considerations • Complement components, primarily C4, may nonspecifically bind to red blood cells from clotted blood samples kept at 4C. • False positive DAT results may occur with blood collected into tubes containing silicone gel. • Blood samples collected from intravenous fluid lines used to administer solutions. • Septicemia in a patient or bacterial contamination of stored specimens.

37. IAT - Additional Considerations • Red cells coated with IgG cannot be tested with antisera that react only by IAT, such as weak D or anti-Fya. • Procedures are available for removing IgG from in vivo coated red blood cells

38. Role of Complement in the Antiglobulin Reactions • Complement-binding antibodies attach complement to the cell surface when they react with red cell antigens. • Some blood group antibodies bind complement to the red cell membrane. • Clinical significance of complement binding antibodies is variable. • Immune complexes present in plasma activate complement components that may adsorb to red blood cells in a non-specific manner. • Complement coated red cells may or may not proceed to hemolysis.

39. References • Indian Immunohematology Initiative http://www.indianinitiative.org/lectures/ • Life’s Blood – EXCELLENT http://tinyurl.com/4ar2q56 • Merck Manual http://www.merckmanuals.com/professional/sec11/ch131/ch131a.html • Monocolonal Antibody Productionshttp://www.accessexcellence.org/RC/VL/GG/monoclonal.php • Monoclonal Cell Culture Picturehttp://tinyurl.com/4mnyrpa

40. End of Unit 4