CLS 3311 Advanced Clinical Immunohematology

1. CLS 3311 Advanced Clinical Immunohematology Identification Of Unexpected Antibodies

2. Antibody Identification • When an unexpected antibody has been detected using an IAT or a previous record indicates its presence then… • We need to perform an Antibody Identification to discover the unexpected antibody’s specificity. • We react a PANEL of reagent red blood cells (usually 11) with known phenotypes with the patients serum or plasma, including an Auto Control Tube to rule in or out any auto antibodies. • The next slide is an example of a panel reporting sheet for a Gamma Biologics panel.

4. Antibody Identification • The next hope is that the antibody will actually react with the Panel cells. • There are ways to enhance antibody reactivity that we discussed in the Ag/Ab reactions lecture. Remember? • How about increasing the serum to cell ratio? • Using enhancement media? • Enzyme treatment of red blood cells? Etc. • The following slides are a set of questions and approaches to basic antibody identification. These need to be mastered before we move on to enzyme treatment, absorptions, elutions, etc.

5. What is the ABO group of the patient? What is the patient’s Rh type? What is the gender of the patient? Isthere a previous record on this patient? A1B person may have anti-H in their serum. Is this significant? Why? Rh negative may be producing anti-D, anti-C, anti-E, etc. Females may be multiparous If so, was an antibody identified? You also cannot assume that it is currently the only one present. General Questions that need to be answered prior toAb Id

6. What is the diagnosis or suspected diagnosis of your patient? What is the patient’s transfusion record? 5.Need to communicate with other members of the healthcare team - certain hemolytic anemia’s can result from an autoimmune process or medication. 6. Has the patient been transfused with packed rbc’s within the last three months? If so, accurate antigen typing of their red cells for an antigen to a corresponding antibody will not be possible. Why?

7. If you are recording weak reactions with many or all cells in the panel but NOT the auto-control, you may be seeing: A patient who has hypergammaglobulinemia or has recently received a volume expander. Rouleaux formation! Panel 1 High titer low avidity (HTLA) antibody. Panel 2 A patient who has an antibody to a high frequency antigen. i.e. anti-Jsb, anti-k, etc. Panel 3 Panel 1 represents what may be seen in situation A above. Notice that the AHG phase is completely negative. Why?

8. Panel #1 With Rouleaux the reaction strength may vary more than indicated and the auto control is positive. Confirmation of rouleaux involves performing a saline replacement test.

9. Panel #2 This represents the reactivity of a High Titer Low Avidity (HTLA) antibody. Weak (Microscopic to 1+) reactions with all cells. Weak reactions that persist through many dilutions (titer: at least to 1:64).

10. Panel #3 This represents an antibody to a high incidence antigen (99% of the population).

11. Are the Positive reactions of equal Strength with each cell? This would suggest that there is only one antibody specificity causing the reactions.

12. Does patient serum react with the same panel cells at 37oC and AHG? This, too would suggest that only ONE unexpected antibody is causing the observed reactions.

13. Variation in Agglutination Strength on Ab Panels Multiple Antibodies present • Can cause variation in agglutination strength in a single phase. i.e. 1+ to 3+ reactions at 37oC phase • Can cause variation in agglutination strength between phases: i.e. 1+ at 37oC and 3+ at AHG Dosage • Can cause variation in agglutination strength in a single phase. i.e. 1+ to 3+ reactions at 37oC phase

14. Look at the reaction strengths and how they correlate to the zygosity of the Anti-Fya antibody. How strong is the agglutination with the Homozygous cells compared to the Heterozygous cells? DOSAGE can account for this.

15. This panel appears to be demonstrating the presence of Multiple Antibody specificities. There is variation in reaction strength within each phase and between phases.

16. RULING OUT!! • Look at each Panel Cell that has a NEGATIVE reaction with the serum being tested. • Rule out by drawing 1 line through the Antigen specificity at the top of the sheet. • Repeat the process for each negative cell in the panel. • At the end of the process count the number of mark through lines in each specificity. • Each specificity that has 2 or more strikes are considered RULED OUT! They are statistically eliminated as the antibody causing those reactions. Circle all specificity's which have NOT been RULED OUT!

17. General Rule • For some antibody specificity’s which are known to show dosage you cannot Rule Out the antibody as being absent unless 2 Homozygous Cells fail to react with the patient serum. Antibody specificity’s that are included under this rule are: Duffy(Fya, Fyb), Kidd (Jka, Jkb), M, N, S,ands.

18. Ruling out using Homozygous cells: Panel cells 1-4 have no reactivity at any phase. These can be used to rule out. Each highlighted box represents a homozygous cell enabling us to rule out one of the blood groups that meet that requirement: Kidd, Duffy and MNSs.

19. RULING OUT!! • The next slide is a panel that has animated the ruling out process. As you click the mouse button lines will indicate a strike against a blood group specificity. Two strike through’s statistically eliminate that antibody specificity from causing the observed panel reactions. • Each Circled Specificity indicates an antibody that could possibly be causing the reactions observed on the panel.

20. Rule out by drawing 1 line through the Ag specificity at the top of the sheet for each negative cell with a + in the box. For example Cell #4 is the first negative cell. Moving from left to right beginning with the D antigen is a + on cell #4, this is where we begin by drawing a single line through the D. It now has one strike.

21. RULING OUT!! • The next slide is the same panel without the rule out strikes or circles. Go through the rule out process by yourself and compare results with the ruled out panel. • Remember, begin with a negative cell, homozygous cells are required for Kidd, Duffy and MNSs to rule out. Circle all specificities that cannot be ruled out.

23. Panel Write-Up #1. List all of the circled specificities: D, C, V, VS, Cw, S, Lea, Lua, K, Kpa, Jsa,Fyb, Jkb • These represent all of POSSIBILITIES of antibodies that could be causing the reactions on this panel. These are the ones that have not been ruled out. #2. List those antibody specificity’s that cannot be ruled out but which were so RARE that either no cell on the panel was positive for them or only one cell was positive and does not appear to be the cause of the reactions: V, VS, Cw,Lua, Kpa, Jsa.

24. Panel Write-Up #3.List the antibodies that are not the probable cause because their typical PHASE OF REACTIVITY does not match the observed phase of reactivity: Lea, Lua • On this panel all the reactions occurred at AHG phase (using Monospecific -IgG AHG). • What is the optimal phase of reactivity for Lea and Lua? Both are cold reacting antibodies. They should demonstrate reactivity at IS or Room Temp.

25. Panel Write-Up #4. List the MOST LIKELY ANTIBODY based on pattern of reactivity. Anti-C • Look at the pattern of the agglutination reactions and compare them to each remaining antibody specificity. Begin at the left with Anti-D. Is there a reaction between the serum and the antigen specificity EACH TIME the antigen is present on the panel cells? • Is there NO reaction between the serum and the panel cells each time the antigen is NOT present on the panel cells? • Does this antibody normally react at this Phase? • Is the Auto Control Negative?

26. Panel Write-Up #5.Listthe selected cells that you could use to rule out any remaining possibilities from #1 above and indicate how many cells you would need of each selected cell type. For example: • Remaining specificities: D, S, K, Fyb and Jkb The most likely antibody is anti-C so to eliminate the remaining specificities I need a cell that is NEGATIVE for C antigen and positive for the remaining specificities: C =,D+, SS+ (2), K+, FybFyb +, JkbJkb +

27. Panel Write Up #5 Continued… • In your blood bank you will go to another panel (either another Lot # or Manufacturer) and look for cells that are negative for the offending antibody and positive for the remaining specificities that have not been ruled out. You will select cells that fit this description and set up another Panel, called a Selected Cell Panel. • Using the Selected Cell Panel you will test the patients serum to rule out the remaining antibody specificities.

28. Follow up Testing • Once an antibody has been identified, testing the patients red blood cell for the corresponding antigen is supporting evidence. i.e. if an anti-E is identified then we would test the patients RBCs for the E antigen. If anti-E is the offending antibody then the patient’s RBCs should be negative for the E antigen. • The patient cannot have been transfused within the last three months! Why?

29. Panel Write Up • The preceding Panel Write Up format will be used every time a panel is performed in this course. • When a panel is assigned, like the last two slides of this lecture, you will email and post only the write up: steps 1-5. Or if a panel is on an exam, your answer will have steps 1-5 completed for full credit. • This way I don’t have to see your actual rule out sheet but can get a pretty good idea of your thought process.

30. Additional Panels • The next two slides are practice panels. • I will post the write ups on the discussion board when I have all student write ups emailed to me. • These are practice and more will be posted as soon as I see how everybody is doing.