Serological Testing: Why we did what we did!

W. John Judd, FIBMS, MIBiol

Emeritus Professor

University of Michigan

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

More specifically, what we did…

compatibility testing

prenatal/perinatal testing

immune hemolysis investigation

Performed blood group determination, and antibody detection/titration and identification as applied to:

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

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

Donor Testingtransfusion service

ABO confirm RBC type on all RBC units

Rh direct tests with anti-D on RBC units labeled Rh-

Antibodies not required

Infectious agents

not required except for platelets

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

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

A1 B

Confirmation of Identity

Old Way verbal

verbal + visual

ID bracelet– patient unique

– SS number

– barcoded

New Way digitalized thumbprint

retinal scan

voice recognition

Because We Must!

Stoppered tube with firmly attached label– first and last name

– identification number

– date (time) of collection

Labeled at the bedside!

Obtained within 3 days of scheduled transfusion if patient transfused or pregnant in preceding 3 months

matches requisition

How did we do what we did?pretransfusion testing

ABO/Rh

Antibody detection

Crossmatch

ABO/Rh Typing Requirements

ABO RBCs with anti-A and –B

Serum/plasma with A1 and B RBCs

Concordance between serum and RBCs

Positive reactions must be >2+

Rh Direct tests with anti-D

Positive reactions must be >2+

Control to detect false-positives

No test for weak D

Why didn’t we…? Because….

Test patient RBCs with anti-A,B?

Rare A/B subgroups given group O RBCs

Test apparent Rh-

patient samples for weak D?

Weak D can result from partial D, requiring Rh- RBCs

Valid ABO/Rh Reactionstube tests

anti-A anti-BBiocloneanti-D

A1 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

Weak Reactionswhy 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

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

Why did we automate?

Valid ABO/Rh Reactionsautomated gel tests

anti-A anti-B anti-D Control A1 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+/0 0 0 0

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

Antibody Detection

Room temperature

AlbuminLIS

37 C

Anti-IgG+C3

Autocontrol

37 C

Gel/LIS

Anti-IgG

in the past current

Approved Methodsantibody detection

serum:RBCs time AHG

SAL >2:1, 3-4% 30-60’ PS/IgG

ALB >2:1, 3-4% 15-30’ PS/IgG

LIS 2:2, 2% 10’-15’ PS/IgG

GEL 1:2, 0.8% 15’ IgG

PEG 2:1, 3-4% 15-30’ IgG

LIP 2:1, 1% 1’ IgG

SPA 1:1, 0.4% 15’ IgG

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

Michigan Data

GEL LISW

sensitivity 98% 97%

specificity 96% 90%

PV+ 79% 57%

efficiency 95% 89%

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 R1R1 Patients with Anti-E

Gel in the RL

Eliminated testing that was not required.

What we did most of all…

Redundant Testing

RT incubation

Anti-C3 in AHG

3-cell-sample screen

IAT-crossmatch (negative screen)

DAT

37 C reading

No anti-C3 or RT?

Unwanted

Negatives

Unwanted

Positives

LIS RT-37-IgG+C3 0 1.41%

37-IgG+C3 0 0.61%

37-IgG 5* 0.1%

* All anti-Jk

Reagent RBCs

Two group O RBC samples that between them, carry C c D E e; K k; Fya Fyb; Jka Jkb; M N S s ; Lea

Leb and P1

R1R1 and R2R2, one Jk(a+b-)

Why Jk(a+b-) RBC?

RBC Dosage

Time AHG Double Single

10 IgG+C3 37 29

15 IgG+C3 37 33

10 IgG 34 23

Antibodies found at X-Matchnegative screen

Year # Tests Method Wanted Unwanted†

1975-76 82,674 ALB 8(1) 201

1979-80 58,639 LIS 10(1) 85

1983-84 81,444 LIS 17(4) 114

222,730 35(6*) 400

* to low prevalence antigen; † anti-I, -HI. –M, etc

Seen only by X-Match

C 3 E 11 e 1 c 2

K 3 Jk 7 Cw 1 Fy 2

V 2 Jsa 2 Wra 1

Data used in support of IS-crossmatch and 2-cell-sample screen.

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

DAT/Autocontrol Study

Samples 65,049 -ve serum

IgG DAT+ 3570 3133

Evaluated: 778 489

transfusion 720 482

reactive serum 43 0

diagnostic 15 7

Findings

ELUATESnegative 518

auto 192

drug 7

passive 9

allo 52

BY EVALUATION3 Jka eluate

2 K1 eluate

1 Lua eluate

1 D serum-ficin

1 E serum-ficin

1 K serum K+k-RBCs

PV+ = 0.29%

1992 Study on 37 C Reading

87,480 samples (tests)

3590 positives (4.1%)

475 positive only at 37oC (37+IAT-)

103 37+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%

37 C Agglutinins

significance n specificity

harmless 196 I, HI, etc

dubious 176 MN, Le, P1, Lua

potential 103 E(63), D(4), C(1),

cE(3) K(27), Jk(5)

87,480 samples

RBC Exposurepatients (n = 75)

transfusion E C cE D K Jka

< 4 months 32 2 1 20 2

> 1 year 9 1 1 1 1

none known 3 1 1

Risk Calculation

# cases x 0.34 (or 0.80) x 5 x % incompatible

How many patients/year will beexposed to how many “incompatible”units after test elimination?

T&S patientstransfused

XM patientstransfused

average # unitstransfused

RISK

test

eliminated

PV+

cases per

year

patients at

risk

transfused

# incompatible

units transfused

per patient

DAT 0.29% 3.25 1.1 2.05

IAT-XM 7.25% 9.7 7.75 1.85

37 C 21.7% 25 8.5 1.45

30,000samplestested

29,000units

transfused

46,000 crossmatches

Because we could!

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

AABB/FDA Requirements

On-site validation

Only ABO incompatibility

Verification of data entry

Method Selection

IAT if unexpected antibodies

Computer if two ABO/Rh

IS if computer down

Where I would like tohave gone…

Computer Order Entry• record of ordering physician• algorithm to validate request

Sample Collection• electronic patient andphlebotomist ID

• label printed at bedside

Testing• centralized• automated• electronic operator ID• results downloaded to LIS

Remote Site• validated computerizeddonor inventory

• data access via internet• electronic XM on-site• electronic patient andtransfusionist ID

e-match

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

We Went Molecular!

Because we could

The antisera were running out and expensive!

To stay in front

To improve patient care

BeadChipTM Technology

Genotyping vs. Phenotyping

Recently transfused patients

Patients with a positive DAT

Patients with more than two alloantibodies

Milestones at Michigan

1974 WJJ arrived

1975 no anti-A,B

1979 LISS

1980 IgG DAT

1982 no RT

1985 no DAT

1986 IS-XM

1987 anti-IgG

1992 computer XM

1996 no 37 C

2003 gel automation

2007 molecular

2008 WJJ retired

No! No!

PEG adsorptions

Prewarmed tests

Rh-negative, Du-positive

What’s this?

Anti- A Anti-B A1 RBCs B RBCs

0 0 0 4+

What’s this?

Anti- A Anti-B A1 RBCs B RBCs

4+ 0 3+ 3+

by gel