Use and Abuse of Reagents and Techniques. Joann Moulds PhD, MT(ASCP)SBB Director, Scientific Support Services LifeShare Blood Centers Shreveport, LA.

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1 Use and Abuse of Reagents and Techniques Joann Moulds PhD, MT(ASCP)SBB Director, Scientific Support Services LifeShare Blood Centers Shreveport, LA.

2 Antibody Screening & Identification Blood Grouping Reference Laboratories 1. Speed is not the major issue (limited sample, history, transfusion requirements) 2. Necessary methods may be complex; staff should be very skilled 3. Should detect causative antibody, and any other relative to the testing methods used 4. Method must be equal to but should be better (more sensitive) than the referring institution

3 Quote from John Moulds Would you: 1. Take your car to a mechanic that promised to work on it as fast as he could? 2. Would you trust the thoroughness of a physical exam if it only took three minutes? If it takes you longer to look up the history or to fill out paper work than it does to do an incubation phase then your priorities are in the wrong place. Time spent on the proper incubation will eliminate repeat testing because of nebulous results.

4 Let s Use the Right Temperature If weak reactivity observed at AHG, do a room temperature phase If clinical signs of hemolysis but no AHG reactivity, do a room temperature Anti-Vel, anti-pr, anti-en a Bring back reading at 37 C Hemolytic Lewis antibodies

5 Antigen-Antibody Interaction 100% Saturation Antigen Stability Antibody Stability Time Antibody uptake is time & temperature dependent; proportional to contact between antibody/antigen t

6 Antigen-Antibody Interaction 100% Saturation Antigen Stability Antibody Stability Lowering Ionic strength of solution Rising Ionic strength of solution Time

7 Use & Abuse of Potentiators Adding Lo-Ion to enzyme treated RBCs Originally reported as a settling technique Jorgensen J, et al. Vox Sang 179;37: Control for false positives Performing an antibody titration in LISS (Lo-Ion) by diluting in the sample in normal saline

8 Using the Proper Saline Bacterial or fungal growth Leached heavy metals ph changes during processing/storage 26 sources of saline; ph ranged from Anti-S (titer 32) by ICT was 0 using ph 4.8 saline Also depressed MNSs, Rh, K, Fy and Jk Suggested PBS ph (minimum >6) Bruce M. et. al. Transfusion 1986:26; Rolih S. et. al. Immunohematology 1993:9;15-18

9 Using Saline to Your Advantage Saline containing azide destroys Knops system antibody reactivity Solid phase membrane contain azide= Knops non-reactive by solid phase

10 Use and Abuse of Enzymes Trypsin- removes Kn, Do, Lu antigens 4 vols of 2.5 mg/ml (Sigma), 30 37C Daniels G. Immunohematology 1992:8;53 Enzyme controls Satisfactory enzyme treatment should produce RBCs that are directly agglutinated by an antibody that reacts only by IAT with untreated RBCs. AABB Technical Manual (17 th ed.), eg. anti-d Same enzyme treated cell without antibody should be negative Use of enzyme treated RBCs with monoclonal reagents is an inappropriate control No microscopic reading of enzyme treated RBCs

11 Use and Abuse of Chemicals Chemicals that disrupt disulfide bonds can be used to treat RBCs Remove bound immunoglobulin Destroy antigens: Kell, Yt, Do, Kn Can also be used to treat serum Determine IgM vs. IgG AET>DTT>2-ME (use PBS ph=8) 2-ME must be dialyzed prior to use Store in dark container 2-3 4, < 6

12 Seldom Used Techniques Instead of titrating weak AHG antibodies Ficin= Neg (Ch/Rg, JMH) Trypsin= Neg (Knops, Dombrock, Yt) C4d coated RBCs Plasma inhibition DTT Treat RBCs

13 Using C4d Coated RBCs Use sucrose method and fresh serum to add more C4 antigen to RBCs Group O, can be stored frozen in LN 2 Kraemer K, Moulds JJ, Judd WJ. Transfusion 1981:21; Ideally use known source with high levels of C4 representing different C4 allotypes Rg- Ch-

14 Using Plasma Inhibition Ideally use known source with high levels of C4 representing different C4 allotypes can have single source Ch+ or Rg+ Some donors will be partial inhibitors more frequent with anti-rg due to presence of null genes Blacks frequently have duplicated C4B* (Ch+)

15 Use of Adsorptions To investigate ABH subgroups Do not use monoclonal reagents Better resolved by genotyping To remove antibody from a serum Alloantibodies Multiple specificities- don t need selected cells High incidence antigen- adsorb onto matched cells Autoantibodies Cold or warm antibodies

16 Use and Abuse of Adsorptions To remove auto antibody from a serum Cold auto antibody (I, IH) enzyme treat RBCs rabbit RBCs or stroma may adsorb other IgMs Warm auto antibody Autologous either enzyme or ZZAP treated Heterologous with R 1 R 1, R 2 R 2 and rr cells* *Just because the patient has a positive DAT and the antibody is removed doesn t make it an auto

17 Techniques to Remove Antibody from RBCs Gentle heat elution Chloroquine diphosphate Edwards J, Moulds JJ, Judd WJ. Transfusion 1982;22:59-61 Use at 24 C not 37 C with IAT reagents Removes IgG and Bg antigens Does not remove complement EDTA Glycine-Acid (EGA) McKelvey-Kindblade J, Edwards-Moulds J. Lab Med 1984;1: Destroys Kell system, Bg and Er a Strict 2 min. incubation

18 Case Study- Positive DAT Patient HA is a 40 y/o Hispanic male with diagnosis of hemolytic anemia Sample submitted to regional reference lab for serological investigation

19 Case cont. Initial panel: all cells positive (RT- AHG) 2+ to 3+ by tube LISS Auto control: 3+ DAT: Poly= 3+, IgG= 3+, C3=- 2+ Eluate : all cells react 3+ at AHG

20 Case cont. Antigen typing with monoclonal reagents R 2 R 2, K+, M+N- Antigen typing post EGA treated cells Fy(a+b+), Jk(a+b+), S+s+ IRL suggested DNA typing be performed with next admission to determine k type since chloroquine treatment failed to remove all antibody from RBCs

21 Genotyping by Microarray

22 Case cont. What may have caused discrepant results? o o o Was there a sample identification error on either of the two samples? Was there incomplete removal of bound antibody despite negative DAT? Was polyspecific AHG used instead of anti- IgG? Complement would remain on cells post EGA treatment

23 Did we use the techniques correctly? Or did we abuse the techniques?

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