Serological Testing:
Why we did what we did!
W. John Judd, FIBMS, MIBiol
Emeritus Professor
University of Michigan
What we did…

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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…
Performed blood group determination,
and antibody detection/titration and
identification as applied to:
compatibility testing
 prenatal/perinatal testing
 immune hemolysis investigation

Why we did some of the things
we did…
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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 Testing
transfusion service
ABO
Antibodies
confirm RBC type on all RBC
units
direct tests with anti-D on
RBC units labeled Rhnot required
Infectious
agents
not required except for
platelets
Rh
ABO/Rh on RBC Units


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

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


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

REQUISITION
right patient
right reason
right product
SAMPLE
right name
right ID #
right blood in tube
IDENTIFICATION
 right patient
TRANSFUSION
 right patient
 right reason
 right product
A1
B
Confirmation of Identity
Old Way
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
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verbal
verbal + visual
ID bracelet
– patient unique
– SS number
– barcoded
New Way

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
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


matches
requisition
Labeled at the bedside!
Obtained within 3 days of scheduled
transfusion if patient transfused or
pregnant in preceding 3 months
How did we do what we did?
pretransfusion testing
ABO/Rh
 Antibody detection
 Crossmatch

ABO/Rh Typing Requirements
ABO
Rh
RBCs with anti-A and –B
Serum/plasma with A1 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
Why didn’t we…?
Because….
Test patient RBCs with Rare A/B subgroups
anti-A,B?
given group O RBCs
Test apparent Rhpatient samples for
weak D?
Weak D can result
from partial D,
requiring Rh- RBCs
Valid ABO/Rh Reactions
tube tests
Bioclone
anti-A anti-B
A1 RBCs B RBCs
anti-D
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 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

Why did we automate?
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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
Valid ABO/Rh Reactions
automated 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
in the past
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Room temperature
AlbuminLIS
37 C
Anti-IgG+C3
Autocontrol
current
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37 C
Gel/LIS
Anti-IgG
Approved Methods
antibody detection
SAL
ALB
LIS
GEL
PEG
LIP
SPA
serum:RBCs
time
AHG
>2:1, 3-4% 30-60’ PS/IgG
>2:1, 3-4% 15-30’ PS/IgG
2:2, 2%
10’-15’ PS/IgG
1:2, 0.8%
15’
IgG
2:1, 3-4% 15-30’
IgG
2:1, 1%
1’
IgG
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%
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 R1R1 Patients with Anti-E
What we did most of all…
Eliminated testing that
was not required.
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?
LIS
Unwanted
Negatives
RT-37-IgG+C3
0
37-IgG+C3
0
37-IgG
5*
* All anti-Jk
Unwanted
Positives
1.41%
0.61%
0.1%
Reagent RBCs
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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-Match
negative 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
35(6*)
400
222,730
* 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?
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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
720
482
reactive serum
43
0
diagnostic
15
7
transfusion
Findings
ELUATES
negative
auto
drug
passive
allo
518
192
7
9
52
BY EVALUATION
3
2
1
1
1
1
Jka
K1
Lua
D
E
K
eluate
eluate
eluate
serum-ficin
serum-ficin
serum K+k-RBCs
PV+ = 0.29%
1992 Study on 37 C Reading

87,480 samples (tests)
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3590 positives (4.1%)
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475 positive only at 37oC (37+IAT-)
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103 37+IAT- due to antibodies of potential
significance (by specificity)
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latter in 72 patients (2 each in 3 patients)
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PV 37+IAT- = 21.7%; incidence = 0.12%
37 C Agglutinins
significance
harmless
n
specificity
196 I, HI, etc
a
dubious
176 MN, Le, P1, Lu
potential
103 E(63), D(4), C(1),
cE(3) K(27), Jk(5)
87,480 samples
RBC Exposure
patients (n = 75)
transfusion
E
C cE D
< 4 months 32
> 1 year
9
none known
3
1
K
a
Jk
2
1 20
2
1
1
1
1
1
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
30,000
samples
tested
29,000
units
transfused
RISK
patients at
risk
transfused
# incompatible
units transfused
per patient
test
eliminated
PV+
cases per
year
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
46,000 crossmatches
Because we could!
Electronic Crossmatch
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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 to
have gone…
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

direct storage
• bar coded entry
• random compatment
assignment
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single access retrieval
• automatic dispense of ABO
matched unit
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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
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Milestones at Michigan
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1974
1975
1979
1980
1982
1985
WJJ arrived
no anti-A,B
LISS
IgG DAT
no RT
no DAT
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1986
1987
1992
1996
2003
2007
2008
IS-XM
anti-IgG
computer XM
no 37 C
gel automation
molecular
WJJ retired
No! No!
PEG adsorptions
 Prewarmed tests
 Rh-negative, Du-positive

What’s this?
Anti- A
Anti-B
0
0
A1 RBCs B RBCs
0
4+
What’s this?
Anti- A
Anti-B
4+
0
A1 RBCs B RBCs
by gel
3+
3+