Serological Testing: Why we did what we did!
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1 Serological Testing: Why we did what we did! W. John Judd, FIBMS, MIBiol Emeritus Professor University of Michigan
2 What we did Provided blood and blood products in a timely, cost-efficient manner. Implemented policies and procedures such that the product provided optimal clinical benefit to the recipient and did not cause adverse clinical effects or transmit disease Assisted in the prevention and management of HDFN Aided in the diagnosis of immune hemolysis
3 More specifically, what we did Performed blood group determination, and antibody detection/titration and identification as applied to: compatibility testing prenatal/perinatal testing immune hemolysis investigation
4 Why we did some of the things we did We had to (compliance) To improve patient care To save money To use staff better To eliminate redundant testing Because we had always done it that way! To stay in front
5 What we had to do Donor Recipient Donor/Recipient ABO/Rh requisition selection antibodies identity crossmatch disease sample issue ABO/Rh ABO/Rh bedside antibodies records
6 Donor Testing transfusion service ABO Rh Antibodies Infectious agents confirm RBC type on all RBC units direct tests with anti-d on RBC units labeled Rh- not required not required except for platelets
7 ABO/Rh on RBC Units Required by FDA and AABB Necessary for electronic crossmatch Done upon receipt from blood supplier Anti-A,B used to test units labeled group O IgM mab anti-d used to test units labeled Rh-negative About 100 mislabeled units/year reported to FDA
8 Protecting the Recipient REQUISITION right patient right reason right product IDENTIFICATION right patient SAMPLE right name right ID # right blood in tube TRANSFUSION right patient right reason right product
9 A1 B
10 Confirmation of Identity Old Way verbal verbal + visual ID bracelet patient unique SS number barcoded New Way digitalized thumbprint retinal scan voice recognition
11 Because We Must! Stoppered tube with firmly attached label first and last name identification number date (time) of collection Labeled at the bedside! matches requisition Obtained within 3 days of scheduled transfusion if patient transfused or pregnant in preceding 3 months
12 How did we do what we did? pretransfusion testing ABO/Rh Antibody detection Crossmatch
13 ABO/Rh Typing Requirements ABO Rh RBCs with anti-a and B Serum/plasma with A 1 and B RBCs Concordance between serum and RBCs Positive reactions must be >2+ Direct tests with anti-d Positive reactions must be >2+ Control to detect false-positives No test for weak D
14 Why didn t we? Because. Test patient RBCs with anti-a,b? Test apparent Rhpatient samples for weak D? Rare A/B subgroups given group O RBCs Weak D can result from partial D, requiring Rh- RBCs
15 Valid ABO/Rh Reactions tube tests anti-a anti-b Bioclone anti-d A 1 RBCs B RBCs 0 0 >2+/0 >2+ >2+ >2+ 0 >2+/0 0 >2+ 0 >2+ >2+/0 >2+ 0 >2+ >2+ 0* 0 0 * inert control required if positive
16 Weak Reactions why we care Less the expected 3+ or 4+ May represent a false-positive test Seen in newborns and in disease May result from non-abo-type specific transfusion May indicate partial D phenotype
17 Why did we automate? Increased workload from requirement to detect bacterial contamination in platelets Positive sample identification Standardized testing leading to better compliance with cgmp Increased costs of traditional reagents To stay in front
18 Valid ABO/Rh Reactions automated gel tests anti-a anti-b anti-d Control A 1 RBCs B RBCs 0 0 >3+/0 0 >1+ >1+ >2+ 0 >3+/0 0 0 >1+ 0 >2+ >3+/0 0 >1+ 0 >2+ >2+ >3+/
19 Why the change? Gel not optimal for detection of anti-a and B in plasma Discrepancies between tube and gel Rh types were associated in 3/13 cases with DAR form of partial D
20 Antibody Detection in the past Room temperature Albumin LIS 37 C Anti-IgG+C3 Autocontrol current 37 C Gel/LIS Anti-IgG
21 Approved Methods antibody detection serum:rbcs time AHG SAL >2:1, 3-4% PS/IgG ALB >2:1, 3-4% PS/IgG LIS 2:2, 2% PS/IgG GEL 1:2, 0.8% 15 IgG PEG 2:1, 3-4% IgG LIP 2:1, 1% 1 IgG SPA 1:1, 0.4% 15 IgG
22 Why Gel? LISA PEG GEL SPA sensitivity 91.2% 96.8% 95.9% 99.1% specificity 98.1% 97.8% 99.6% 90.1% Reilly et al. Transfusion 1997;37(S):64
23 Michigan Data GEL LISW sensitivity 98% 97% specificity 96% 90% PV+ 79% 57% efficiency 95% 89%
24 Gel in the RL Anti Found by: anti-c -E + -c LIS or Gel 32 39% -E + -c Ficin-Gel 21 26% -E alone LIS or Gel 29 35% total anti-c any method 53 65% 82 R 1 R 1 Patients with Anti-E
25 What we did most of all Eliminated testing that was not required.
26 Redundant Testing RT incubation Anti-C3 in AHG 3-cell-sample screen IAT-crossmatch (negative screen) DAT 37 C reading
27 No anti-c3 or RT? Unwanted Negatives Unwanted Positives LIS RT-37-IgG+C % 37-IgG+C % 37-IgG 5* 0.1% * All anti-jk
28 Reagent RBCs Two group O RBC samples that between them, carry C c D E e; K k; Fy a Fy b ; Jk a Jk b ; M N S s ; Le a Le b and P 1 R 1 R 1 and R 2 R 2, one Jk(a+b-)
29 Why Jk(a+b-) RBC? RBC Dosage Time AHG Double Single 10 IgG+C IgG+C IgG 34 23
30 Antibodies found at X-Match negative screen Year # Tests Method Wanted Unwanted ,674 ALB 8(1) ,639 LIS 10(1) ,444 LIS 17(4) ,730 35(6*) 400 * to low prevalence antigen; anti-i, -HI. M, etc
31 Seen only by X-Match C 3 E 11 e 1 c 2 K 3 Jk 7 C w 1 Fy 2 V 2 Js a 2 Wr a 1 Data used in support of IS-crossmatch and 2-cell-sample screen.
32 Why were antibodies only found at X-Match? Dosage? Note: A rr sample will not have afforded detection of 11 -E and 3 -C Better antigen expression on donor RBCs? More caution applied to reading X-match? Inconstant degree of agitation applied to tube tests
33 DAT/Autocontrol Study Samples 65,049 -ve serum IgG DAT Evaluated: transfusion reactive serum 43 0 diagnostic 15 7
34 Findings ELUATES negative 518 auto 192 drug 7 passive 9 allo 52 BY EVALUATION 3 Jk a eluate 2 K1 eluate 1 Lu a eluate 1 D serum-ficin 1 E serum-ficin 1 K serum K+k-RBCs PV+ = 0.29%
35 1992 Study on 37 C Reading 87,480 samples (tests) 3590 positives (4.1%) 475 positive only at 37 o C (37+IAT-) IAT- due to antibodies of potential significance (by specificity) latter in 72 patients (2 each in 3 patients) PV 37+IAT- = 21.7%; incidence = 0.12%
36 37 C Agglutinins significance n specificity harmless 196 I, HI, etc dubious 176 MN, Le, P 1, Lu a potential 103 E(63), D(4), C(1), ce(3) K(27), Jk(5) 87,480 samples
37 RBC Exposure patients (n = 75) transfusion E C ce D K Jk a < 4 months > 1 year none known 3 1 1
38 Risk Calculation How many patients/year will be exposed to how many incompatible units after test elimination? # cases x 0.34 (or 0.80) x 5 x % incompatible T&S patients transfused XM patients transfused average # units transfused
39 30,000 samples tested RISK 29,000 units transfused test eliminated PV+ cases per year patients at risk transfused # incompatible units transfused per patient DAT 0.29% IAT-XM 7.25% C 21.7% ,000 crossmatches
40 Because we could!
41 Electronic Crossmatch Replaces serological tests for ABO incompatibility Requires validated electronic record of patient and donor ABO/Rh types Not to be used if unexpected antibodies present
42 AABB/FDA Requirements On-site validation Only ABO incompatibility Verification of data entry
43 Method Selection IAT if unexpected antibodies Computer if two ABO/Rh IS if computer down
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54 Where I would like to have gone
55 e-match Computer Order Entry record of ordering physician algorithm to validate request Sample Collection electronic patient and phlebotomist ID label printed at bedside Testing centralized automated electronic operator ID results downloaded to LIS Remote Site validated computerized donor inventory data access via internet electronic XM on-site electronic patient and transfusionist ID
56 direct storage bar coded entry random compatment assignment single access retrieval automatic dispense of ABO matched unit storage optimization first in, first out benefits better staff utilization reduction of emergency requests reduced outdating
57 We Went Molecular! Because we could The antisera were running out and expensive! To stay in front To improve patient care
58 BeadChip TM Technology
59 Genotyping vs. Phenotyping Recently transfused patients Patients with a positive DAT Patients with more than two alloantibodies
60 Milestones at Michigan 1974 WJJ arrived 1986 IS-XM 1975 no anti-a,b 1987 anti-igg 1979 LISS 1992 computer XM 1980 IgG DAT 1996 no 37 C 1982 no RT 2003 gel automation 1985 no DAT 2007 molecular 2008 WJJ retired
61
62 No! No! PEG adsorptions Prewarmed tests Rh-negative, Du-positive
63 What s this? Anti- A Anti-B A1 RBCs B RBCs
64 What s this? Anti- A Anti-B A1 RBCs B RBCs by gel
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