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1 Presentation Handouts (9221-TC) Top Gun Serology October 13, :00 PM - 3:30 PM

2 Event Faculty List Event Title: 9221-TC: Top Gun Serology Event Date: Sunday, October 13, 2013 Event Time: 2:00 PM to 3:30 PM Director/Moderator/Speaker Susan Johnson, MSTM, MT(ASCP)SBB Director, Clinical Education BloodCenter of Wisconsin Disclosures: Yes Speaker Martha Rae Combs, MT(ASCP)SBB Analytical Specialist Duke University Medical Center Disclosures: No

3 Top Gun Serology Flight School Martha Rae Combs, MT(ASCP)SBB Duke University Medical Center Durham, NC Susan T Johnson, MSTM, MT(ASCP)SBB BloodCenter of Wisconsin Milwaukee, WI On March 3, 1969 (2006) the United States Navy (AABB) established an elite school for the top one percent of its pilots (blood bank technologists & physicians) Its purpose was to teach the lost art of aerial combat (antibody id) and to insure that the handful of men (and women) who graduated were the best fighter pilots (blood bankers) in the world They succeeded Today, the Navy calls it Fighter Weapons School (AABB calls it, Serology Flight School) The flyers (techs) call it: TOP GUN Top Gun Serology Flight School Martha Rae Combs, MT(ASCP)SBB aka Maverick Duke University Medical Center Durham, NC Susan T Johnson, MSTM, MT(ASCP)SBB aka Goose BloodCenter of Wisconsin Milwaukee, WI

4 Goose aka Sue Maverich aka Martha Rae Top Gun Serology Flight School

5 Let s find out about our flight school class What is your profession? 1 Medical Technologist / Laboratory Scientist 2 Physician 3 Other What is your position? 1 Medical Technologist /Laboratory Scientist 2 Supervisor/Manager 3 Medical Director 4 Educator 5 Student 6 Other

6 Where do you work? 1 Hospital Transfusion Service 2 Blood Center 3 Other These do not work!

7 Top Gun Case 1 Avoiding Gel And Solid Phase Junk Let s start with a question Gel users Do you use a different screening method when you get an unexpected positive gel screen or inconclusive gel panel? 1 Yes 2 No

8 Gel Users What is your alternative method? 1 LISS tube 2 PEG 3 Albumin 4 Saline Gel LISS tube Pro LISS-based Avoid anti-m Avoid anti-bg Avoid gel dependent autoantibody Avoid HTLA Con LISS tube less sensitive for significant antibodies?

9 Gel PEG tube Pro Equivalent or better sensitivity for significant antibodies Avoid HTLA type antibody Con Dysproteinemia May not avoid cold allo/auto antibody Possible increased sensitivity for warm autoantibody Let s find out what you do Solid phase users Do you use a different screening method when you get an unexpected positive solid phase screen or inconclusive/neg solid phase panel? 1 Yes 2 No

10 Solid phase users What is your alternative method? 1 Gel 2 LISS tube 3 PEG 4 Albumin 5 Saline Solid phase Gel Pro Avoid solid phase-only warm autoantibody Avoid solid phase junk Con Gel junk Gel-only anti-m, -Bg, HTLA type antibodies May detect IgM, rouleaux Less sensitive for Rh and Kidd?

11 Solid phase PEG Pro Avoid solid phase-only warm autoantibody and junk Con Less sensitive for Rh and Kidd May detect colds, IgM, HTLA type antibodies Possible interference due to dysproteinemia Solid phase LISS tube Pro LISS-based Avoid solid phase-only warm autoantibody Con Less sensitive for Rh, Kidd + others May detect IgM, HTLA type antibodies, rouleaux Avoiding the Junk AUS Antibody of undetermined specifity SPP Solid-phase

12 AUS Some find it exciting! Antibody of undetermined specificity: frequency, laboratory features, and natural history Liu and Grossman Transfusion 2013 May:53(5):931-8 July 1, 2009 Dec 31, antibodies in 6058 patients AUS 18% Anti-E 18% Anti-K 14% Liu and Grossman 1 st quarter 2012 AUS in 174 patients 78% reacted with 2 cells or less in gel 98% were 1+ or weaker 1 st time AUS also tested in PEG

13 Liu and Grossman 45 patients with AUS had repeat workups 31(69%) with persistent AUS 14(31%) AUS disappeared 7(16%) developed 10 new antibodies in 3-21 days (-E, -D, -C, -Jk b (2), -Le a, -s, warm auto) Liu and Grossman AUS is common Reactions heterogeneous May represent Antibody to low-prevalence antigens Antibody to Non-RBC antigens Developing clinically significant antibodies SP316 Ficin-treated cells in ID-MTS gel test identify clinically significant antibodies which may mimic non-specific reactivity Hill, Hanna, Adamski, Marques 352 cases 72 (20%) non-specific reactivity in gel 67 ficin-treated panel in gel 20 antibodies identified (17 clinically significant)

14 SP The Development of alloantibodies in patients that demonstrate Capture-R Solid Phase Phenomenon Meade, Armstrong, Sanford Panreactive SP antibody screens and panels negative in tube method, DAT-0 Subsequent samples evaluated PEG crossmatches SPP - Meade, Armstrong and Sanford 109 patients, June 2006 Dec % of workups 55 patients with follow-up testing 15 reactivity disappeared 34 remained the same 6 (11%) new alloantibody Conclusion: SPP is a good indicator that patients will develop alloantibody SP312 Early Antibody Detection using Solid Phase Technology Stein, Fischman, Heaton et al Previous FPA with new antibody ID 98% Previous negative ABSC with new antibody ID 35%

15 What is a bogey in Top Gun terms? 1 Unknown aircraft on radar 2 One stroke over par 3 Evil or mischievous spirit 4 Unexpected positive reactions Cautionary Tale 38 M, MVA Spinal injury, multiple fractures 6 RBC RBC Cautionary Tale, continued Positive SP screen, inconclusive SP panel, negative PEG screen 1 RBC , computer XM compatible Review of workup

16 Rh Kell Duffy Kidd Lewis P MN SP D C c E e K k Fy a Fy b Jk a Jk b Le a Le b P1 M N S s IAT Rh Kell Duffy Kidd Lewis P MN SP D C c E e K k Fy a Fy b Jk a Jk b Le a Le b P1 M N S s IAT Rh Kell Duffy Kidd Lewis P MN SP D C c E e K k Fy a Fy b Jk a Jk b Le a Le b P1 M N S s IAT

17 Kidd SP SP 1hr Jk a Jk b IAT IAT Was this SP-only anti-jk a significant? Pretransfusion sample Jk(a-) Unit received was Jk(a+) No hemolysis labs run Moral of Story When you have inconclusive results, closely evaluate reactive cells for a common phenotype!

18 Liu and Grossman 3 of 6 patients with AUS that developed alloantibody the six samples each reacted with one to four cells that express antigens corresponding to the alloantibodies identified later What would you do? If screen is negative by alternative method, what do you report? 1 Positive antibody screen 2 Negative antibody screen 3 Positive screen with comments

19 Due to false positive rate of antibody detection method, the antibody screen was repeated using a different method with increased specificity No alloantibodies were detected with the alternative method One more question! If screen is negative by alternative method, what type of crossmatch do you perform? 1 Computer or immediate spin 2 Alternative screening method 3 Original screening method (SP or Gel)

20 Duck Duck GOOSE! Top Gun Tactical Maneuver Crossmatch Methods What was Goose s real name in the movie? 1) John Carpenter 2) Felix Unger 3) Bryan Adams 4) Nick Bradshaw

21 Gel users, what type of crossmatch would you perform in your institution with a negative antibody detection test? 1 Computer XM 2 Immediate Spin in Gel 3 Immediate Spin in Tube 4 AHG in Gel 5 IS and AHG in Gel or tube 6 Computer XM and AHG in Gel or tube Gel users, what type of crossmatch would you perform in your institution with a positive antibody detection test? 1 AHG in Gel 2 IS and AHG in Gel or tube 3 Computer XM and AHG in Gel or tube SPRCA users, what type of crossmatch would you perform in your institution with a negative antibody detection test? 1 Computer XM 2 Immediate Spin in Tube 3 AHG using SPRCA 4 IS and AHG tube 5 Computer XM and AHG in tube

22 SPRCA users, what type of crossmatch would you perform in your institution with a positive antibody detection test? 1 Computer XM 2 Immediate Spin in Tube 3 AHG using SPRCA 4 IS and AHG tube 5 Computer XM and AHG in tube Test tube users, what type of crossmatch would you perform in your institution with a negative antibody detection test? 1 Computer XM 2 Immediate Spin in Tube 3 IS and AHG in tube 4 Computer XM and AHG in tube Test tube users, what type of crossmatch would you perform in your institution with a positive antibody detection test? 1 Computer XM 2 Immediate Spin in Tube 3 IS and AHG in tube 4 Computer XM and AHG in tube

23 BBTS Standards 28 th ed Serologic Crossmatch The crossmatch shall use methods that demonstrate ABO incompatibility and clinically significant antibodies to red cell antigens and include an antiglobulin test If no clinically significant antibodies were detected, at a minimum, detection of ABO incompatibility shall be performed History of Crossmatch December Report to BPAC If Type and Screen has been performed, should AHG crossmatch be eliminated? IS crossmatch may act as a doublecheck and should be retained The Sensitivity & Specificity of the IS Crossmatch EA Meyer and IA Schulman, Transfusion 1989, 29: Donor Recipient Berry-Dortch et al Schulman et al Sensitivity A O 200/ / % B O 100/ / % AB O 200/200 81/81 100% A B 200/200 81/81 100% B A 100/ / % AB A 200/200 52/ % AB B 181/200 24/ % A2B B 204/531 55/70 499% A2B A 100/100 NT 100% IS Crossmatch does not always detect incompatibility

24 Detection of ABO Incompatibility Using Gel EA Steiner & WJ Judd, Transfusion 1997 Vol 37 Supplement Does not always detect incompatibility A 2 B donor and B recipient 100 B Patients vs A 2 B donors % False Negative IS Tube 43 Buffered Gel 65 IgG Gel 46 Detection of ABO Incompatibility Automated Gel Crossmatch R Alrabeh et al, Transfusion 2012 Vol 52 Supplement Automated Gel Crossmatch vs IS XM? No Donor Recipient IS Negative Gel Negative 10 A B B A AB A AB B 7 9 Detection of ABO Incompatibility Limitations of Crossmatch Traves C et al Vox Sanguinis (2013) 105 (Suppl 1), P472 No Donor Recipient Gel Card Company 1 Negative Gel Card Company 2 Negative Tube IS Negative 4 A2 O A2B O B A A1B A A2 B A2B B No method detects all ABO incompatible crossmatches!

25 What do you think about the crossmatch test now? What do you think about any crossmatch test now? 1 There isn t any method that will detect all ABO incompatibility 2 Electronic crossmatch is Top Gun for detecting ABO incompatibility 3 Both 1 and 2 4 I m in a tailspin 5 Other Dave Bio Baranek Plays one at AABB Real Top Gun Instructor

26 Top Gun Case 2 Assessing Positive Reactivity of DATs & IATs Case 2 Patient s History 88 y/o Caucasian female 3 year history of chronic anemia Newly diagnosed with gastric cancer Multiple previous RBC transfusions Last transfusion 4 months ago Referred from community hospital to your academic medical center Case 2 Referring Hospital Results Pretransfusion Testing - Gel Anti-A Anti-B Anti-D Ctl A 1 cells B cells Interp A Pos Rh MNS P1 Lewis KEL Duffy Kidd Gel DCEc e M N S s P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT

27 Case 2 Antibody Identification Rh MNS P Lewis Kell Duffy Kidd GEL D C c E e M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b IAT s AC 2 Case 2 Referring Hospital Results Direct Antiglobulin Test Polyspecific AHG 2+ Anti-IgG 2+ Anti-C3 0 Control 0 What do you think?

28 What is the most likely reason for the positive DAT? 1 Delayed hemolytic transfusion reaction 2 Warm autoantibody 3 Cold autoantibody 4 Delayed serologic transfusion reaction 5 Drug-induced immune hemolytic anemia 6 Positive DAT not related to plasma antibody Assessing DAT Reactivity Test Tube < C Feldman & J O Conner Assessing DAT Reactivity - Gel <

29 Assessing DAT Reactivity Is there evidence to support hemolysis? Patient s diagnosis Recent transfusion Compare to serum/plasma reactivity WAIHA DAT is usually 3-4+ before antibody is in serum/plasma WARM AUTOANTIBODY IN PLASMA WARM AUTOANTIBODY COATING RBCs DAT 3-4+

30 WARM AUTOANTIBODY IN PLASMA DAT 3-4+ IAT 1+ Assessing DAT Reactivity Compare to serum/plasma reactivity DHTR/DSTR DAT is weak positive, sometimes mixed field Consider methods being used Tube vs SP or Gel May be unrelated to serum/plasma antibody Let s break for a question

31 Maverick buzzes the tower twice in the film What coincides with each of these stunts? 1 The tower radar is knocked off-line 2 The tower chief spills coffee on himself 3 Maverick is docked a rank 4 Maverick intones, "This one's for you, dad Back to our mission What test would you do next? 1 Eluate 2 Autologous Adsorption 3 Allogeneic Adsorption 4 Cold Screen/Panel 5 Saline IAT in Tubes 6 LISS IAT in Tubes 7 PEG IAT in Tubes 8 Send it to an IRL 9 Other

32 Case 2 Acid Eluate Rh MNS P Lewis Kell Duffy Kidd Eluate Last Wash D C c E e f M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b IAT IAT Case 2 IRL Results Test Tube Methods Rh MNS P1 Lewis KEL Duffy Kidd LISS PEG D C E c e M N S s P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT IAT I o II III Case 2 SPRCA Galileo Results Rh MNS P Lewis Kell Duffy Kidd SP D C c E e M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b IAT ?/

33 Case 2 SPRCA Galileo Results Rh MNS P Lewis Kell Duffy Kidd SP D C c E e M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b IAT / / / / / / / / / / / / / / / / / / 4 16 / / / / / / / / / / / / / / / / / / 0 Now what do you think? What do these results suggest? 1 Antibody to high prevalence antigen 2 Multiple antibodies 3 Warm autoantibody 4 Cold autoantibody 5 Drug-dependent antibody

34 Take my breath away Assessing Reactivity in IAT Consider Number of positive and negatives Phase or method antibody is reactive Agglutination strength of positive reactions (weak or 4+) Consistent or variable positive reactions Appearance of agglutination Mixed field, mixed cell (gel), loose, stringy Single Antibody LISS IS 37C IAT AC 0 0 0

35 Multiple Antibodies LISS IS 37C IAT AC Multiple Antibodies or Rh (-c or e) LISS IS 37C IAT AC Multiple Antibodies LISS IS 37C IAT AC 0 0 0

36 Antibody to High Prevalence Antigen LISS IS 37C IAT AC Warm Autoantibody LISS IS 37C IAT AC Antibody to High (Knops, JMH, Ch/Rg) LISS PEG IS 37C IAT IAT AC

37 Antibody to High (Knops, JMH, Ch/Rg etc) LISS Gel IS 37C IAT IAT AC Antibody to High (Knops, JMH, Ch/Rg etc) LISS SP IS 37C IAT IAT AC NT Let s review reactivity Rh KEL Duffy Kidd LISS PEG SP D C E c e K k Fy a Fy b Jk a Jk b IAT IAT IAT I II III ?/1 DAT is 2+ with Poly and Anti-IgG

38 Now, what do you think pilots? What do these results suggest? 1 Antibody to high prevalence antigen 2 Multiple antibodies 3 Warm autoantibody 4 Cold autoantibody 5 Drug-dependent antibody What additional testing would you perform? 1 Test selected e negative cells 2 Perform autologous adsorptions 3 Perform allogeneic adsorptions 4 Phenotype 5 Genotype 6 Other

39 Things to consider Selected e negative cells Method Auto or allo? Adsorptions Patient has been transfused Allogeneic adsorptions may be helpful if it is autoantibody More Things to consider Phenotype perform with caution as patient was transfused 4 months ago Patient has a positive DAT Use monoclonal reagents Genotype Recent transfusion is not a problem Turnaround time RBC Phenotype Results Mo Reagents Anti -C Anti -E Anti -c Anti -e Anti -K Anti -Jk a Anti -Jk b Anti -S + Ctl Ctl Pt mf 3 0

40 Selected Cells Rh Ss KEL Duffy Kidd LISS D C E c e S s K k Fy a Fy b Jk a Jk b 37C IAT 10 + w w Case Test Tube Methods Rh MNS P1 Lewis KEL Duffy Kidd LISS PEG D C E c e M N S s P1 Le a Le b K k Fy a Fy b Jk a Jk b IAT IAT I o II III Case 2 SPRCA Galileo Results Rh MNS P Lewis Kell Duffy Kidd SP D C c E e M N S s P 1 Le a Le b K k Fy a Fy b Jk a Jk b IAT ?/

41 We re almost there! What do you think about positive reactivity with cell 3 in SP? 1 Another alloantibody is present 2 Solid Phase junk 3 Probable warm autoantibody is present 4 Probable cold autoantibody is present 5 Probable panagglutinin is present Conclusions Anti-e and anti-fy a identified Probable panagglutinin reported Variable reactivity was key in identifying antibodies Be cautious of positive DATs assess reactivity in comparison to serum results What appears to be method junk can be alloantibody!

42 Goose is a RIO in the movie Top Gun What does RIO indicate? 1 Radio Intercept Officer 2 Refrigerator Intercept Officer 3 Radar Intercept Officer 4 Radio Instruction Officer 5 Really Ignorant Officer Keep up the great work! Not Everyone Gets to Be Top Gun!! Top Gun Tactical Maneuver QC

43 A movie question What was Maverick s real name in the movie? 1) Nick Bradshaw 2) Tony Mitchell 3) Pete Mitchell 4) Frank Lemon CAP TRM31400 Records document acceptable reactivity and specificity of typing sera and reagent cells on each day of use, including a check against known positive and negative cells or antisera, or manufacturer s directions for daily quality control are followed

44 What do you do in your hanger? Do You Test ABO Reagents Against Cells Lacking The Corresponding Antigen? 1 Yes 2 No Things to Consider Not required by some manufacturers May detect contaminated reagent Are you testing each vial or each lot?

45 A few more questions For ABO QC, What Are You Testing? 1 Each vial used? 2 One vial from lot in use? What Do You Choose as a D+ Control Cell? 1 R 2 R 2 2 R 1 R 1 3 R 1 r 4 rr 5 Random D+

46 CAP TRM31400 Unless manufacturer s instructions state otherwise, the following apply: Each cell used for antibody detection must be checked each day of use for reactivity of at least one antigen using antisera of 1+ or greater avidity Inquiring minds want to know How Do You Meet This Requirement (for tube tests)? 1 Each day of use, test each cell with antibody to high-prevalence antigen 2 Each day of use, test each cell with 2 different antibodies 3 Each day of use, test each cell with a single reagent containing a mixture of antibodies 4 Other

47 Manufacturer Suggestions Visual inspection Periodic testing for antigens likely to deteriorate, ie Le a Tested periodically with weak antibodies Do You Test Screening Cells with a Negative Control? 1 Yes 2 No How far are you taking this?

48 Do You Perform QC On All Antibody Detection Methods Used? 1 Only on routine method 2 All methods used are routinely tested each day 3 Only test methods on day of use What is a Bandit in Top Gun terms? 1 Identified enemy aircraft 2 The government 3 Dog in the TV show, Johnny Quest Do you perform any QC on in-date antibody panels? 1 Yes 2 No

49 Package Insert QC Recommendations Test a different red cell sample each day of use with a relatively weak IgGreactive antibody Test periodically with weak antibodies, ie anti-le a CAP TRM31400 Unless manufacturer s instructions state otherwise, the following apply: Each cell used for antibody detection must be checked each day of use for reactivity of at least one antigen using antisera of 1+ or greater avidity **No mention of antibody panels Another Couple of Questions

50 Do You Use Expired Panel Cells? 1 Yes, frequently 2 Yes, rarely 3 No Do You Perform QC on Expired Panels? 1 No QC - Use until hemolyzed 2 No QC use as long as SOP allows for post expiration 3 Test with a positive and negative control each day of use 4 Test cell for the antigen of the antibody being excluded 5 No - validated that antigens are stable post expiration TRM31250 Rare reagents may be used beyond expiration date if positive and negative controls are run each day This does NOT apply to reagents that are readily available

51 Things to Consider Panels are not rare reagents Expired panel cells should be used rarely and only as a last resort Develop a policy for the use of expired reagents - rare and not rare A result using an expired panel cell is a small part of the big picture This mission is almost complete! Top Gun Trivia The US Navy set up recruiting booths at theaters showing Top Gun Real Top Gun School members are fined $5 for mentioning the movie Sean Penn and Tom Hanks turned down the role of Maverick

52 Top Gun Tactical Maneuver Suspected hemolysis Top Gun Tactical Maneuver Dr Ice calls and tells you he thinks his patient (a Top Gun pilot) is hemolyzing While doing a practice flight the patient started falling asleep on the RIO Given the seriousness of even the tiniest error when flying a F-14 he is leaving no stone un turned to get to the bottom of this Time for an interrogation

53 What would you do after hanging up the phone? 1 Call your medical director and ask them to call Dr Ice 2 Ask Dr Ice for lab values 3 Look up lab results for patient 4 Ask Dr Ice to order a DAT/IAT 5 1 and 3 6 All of the above Which laboratory test results would NOT indicate hemolysis? 1 Hgb/HCT 2 LDH 3 Bilirubin Unconjugated (Indirect) 4 Bilirubin Conjugated (Direct) 5 Haptoglobin 6 Reticulocyte count 7 Peripheral Blood Smear Laboratory Tests for Hemolysis Things to Consider Hematology Hgb/HCT Reticulocyte Count usually marrow suppression

54 Laboratory Tests for Hemolysis Things to Consider Chemistry Tests LDH Total & Unconjugated Bilirubin Haptoglobin Hemoglobinemia intravascular hemolysis Hemoglobin Hemoglobin Macrophage in spleen Heme Biliverdin Bilirubin Unconjugated Plasma How would you routinely perform the initial DAT when ordered? 1 Anti-IgG Gel card 2 Anti-IgG & Anti-C3d Gel card 3 Test Tube with polyspecific AHG 4 Test Tube with Anti-IgG 5 Test Tube with Anti-IgG & Anti-C3 6 Solid Phase 7 Other

55 Last question!!! Would you do an eluate if the DAT is negative? 1 Yes 2 No 3 Call Medical Director and let them decide 4 Call results and let patient s physician decide 5 Defer decision to supervisor 6 Unsure Things to Consider Eluate with Negative DAT Newly forming antibody may only be detected in eluate Eluate concentrates antibody Antibody may have low affinity Difficult to detect in standard DAT Positive in eluate with cold wash

56 Top Gun the Musical Top Gun the Musical Crashes and Burns Is just plane bad Never takes off Won t take your breathe away Spins out of control If only the theater had ejector seats CONGRATULATIONS! You are now TOP GUN!!!