Transcript RBC Serology: Rh Discrepancies in Prenatal Patients

Rh Discrepancies in
Prenatal Patients
-A Practical Approach to
Management
CBS TM Resident Session
September 15th, 2009
Dr. Judith Hannon
Dr. Gwen Clarke
Rh genetics… a brief review
Rh genetics
RHD and RHCE
• 2 RH Genes RHCE and RHD on Chromosome
1; 10 exons each
• D and CE vary at only 32 -35 amino acid
positions (or are 97% identical)
• C/c and E/e encoded by RHCE
Rh Genetics
www.uni-ulm.de/~wflegel/RH/
D negative
• Deletion of RHD due to unequal crossover
between upstream and downstream genetic
elements (15 – 17% in caucasians)
• RHD with 37bp internal duplication and
premature stop codon(66%) or hybrid RHD-CED with no D expression (15%) of Africans
• 10 – 30% of Asians with D negative serology
are DEL and do express very low levels of D
antigen; most arise from deletion or mutation in
exon 9
Rh D and Rh CcEe
Science.uwe.ac.uk
D positive but…
Weak and partial D
• More than 100 D alleles identified by
sequencing studies
• Weak D characterized by single or few aa
changes primarily in transmembrane or
cytoplasmic part of D protein; 0.2 – 1% of
Caucasians; may react weakly or not at all in
direct agglutination assays
• Partial D characterized by aa changes in the
extracellular portions of D polypeptide; type as
D positive with some antisera
Weak and partial D
Blood, Vol. 93, Issue 1, 385-393, January 1, 1999
Wagner, Gassner, Muller, et al
How often does this happen?
• Weak D:
– 0.2 – 1% of Caucasian population
– Types 1, 2 and 3 represent >90% of these
• These types are NOT immunized to make anti D
• Partial D:
– Most react strongly with most anti D reagents
• May present when they make anti D
• Most that make anti D are DVI representing 0.02 – 0.05% of
population
How do we detect the D
antigen?
Anti D anti Sera
• Monoclonal anti D
– Antibody directed against a single epitope of the D
antigen
– Produced in vitro from a cell line (recombinant)
expressing a particular immunoglobulin gene
sequence
– Several monoclonals may be “blended”
• Polyclonal anti D
– A group of anti D antibodies directed against a
variety of epitopes on the protein; naturally
occurring following an immune response to D
immunization.
Anti Sera reactivity
• Varies with combination and number of
epitopes detected by a particular reagent
• Blended monoclonals have broad
reactivity with most weak and partial D and
produce strong reactions.
• Limited specificity monoclonals react with
fewer epitopes and react with only some
examples of partial D
Rh D and Rh CcEe
Science.uwe.ac.uk
Reaction strength and phase
• In general, D positive individuals have
strongly agglutinated cells in the presence
of anti D (3 – 4+)
– Repeated 1 – 2+ reactivity may indicate weak
or partial D status
• Weak D testing – or testing at the IAT
phase will result in almost all weak and
partial D’s typing as D positive – including
DVI individuals.
Commercially available anti D
antisera in Canada
• Monoclonal
–
–
–
–
–
Novaclone IgM IgG
Bioclone
ANTI D Monoclonal blend
ANTI D Monoclonal IgM
Immucor Series 4
Monoclonal Blend
– Immucor Series 5
Monoclonal Blend
• Polyclonal
–
–
–
–
Olympus (for PK7300)
Immucor
Seraclone
Biotest Anti D
Development of an Algorithm to
Resolve Serological RhD
Typing Discrepancies Post-Galileo
Implementation in the Prenatal
Laboratory
Background
• March 2006- CBS discontinued weak D testing of
prenatal patients (moms) in keeping with international
practises.
• May 2006 – Immucor GalileoTM replaced Olympus
PK7200 in CBS Prenatal Testing Laboratory, Edmonton.
• Required reagent change from Novaclone to Immucor
Series 4 (S4) and Series 5 (S5) anti-D.
• Validation included parallel testing for 3 days (673
samples) with 100% concordance for RhD results.
RhD Discrepancies
• May 31, 2006, 1st patient identified who had previously tested RhD
POS (Olympus PK7200 – Jan 2005) and was now testing RhD NEG
on Galileo (Immucor S4 and S5 anti-D) - Rh Inconclusive.
Manual Tube (IS):
Immucor S4
Immucor S5
DBL Novaclone
Gammaclone
w+ micro
NEG
2+
2+
• Sample sent to Mount Sinai Hospital in Toronto for molecular
testing- wk D Type 1 allele- known not to form anti-D- report
amended to RhD positive.
• Cause of discrepant results? Reagent specificity? Method?
(incubation time or temperature)
Molecular Testing
•
May 2006 to Dec 31, 2006 – discrepant samples retested with manual tube
technique using antisera panel (S4, S5, Novaclone, Gammaclone). RhD
status assigned based on serological testing.
•
Hospitals were receiving discrepant results and CBS was finding
discrepancies with previous test records, both prenatal and XM.
•
Reporting results as Rh inconclusive compromised the ability of hospitals to
use an electronic X-match which is in widespread use in Alberta.
•
Jan 1, 2007 to Apr 14, 2008 all RhD discrepant samples sent to Mount Sinai
Hospital Molecular Testing Laboratory on a study basis (Dr. Greg
Denomme)
•
Total samples screened on Galileo = 88,972. Total discrepant samples sent
for DNA analysis = 209 (0.23% of total)
Molecular Testing of Prenatal RhD Typing
Discrepancies Following Galileo
Total Tested = 88 972
Total discrepancies sent for DNA typing=209 (0.23% of total)
DNA Typing Results
# of
Patients
Rh Status
Assigned
RHIG
Recommended
% of DNA
Results
Received
Weak D Type 1
60
Pos
No
28.7
Weak D Type 2
19
Pos
No
9.1
Weak D Type 3
38
Pos
No
18.2
Weak D Type 4*
16
Neg/Pos
Yes/No
7.7
DAR
3
Neg
Yes
1.4
Partial DVI Type I
3
Neg
Yes
1.4
Partial DVI Type II
1
Neg
Yes
0.5
DVI Type II
2
Neg
Yes
1.0
DVa partial
1
Neg
Yes
0.5
Partial DVa like allele
1
Neg
Yes
0.5
65
Neg
Yes
31.0
Unclassified
TOTAL
*Pre May 2007 Rh Neg
Post May 2007 Rh Pos
209
100
63.7%
5.3%
31%
Based on data from molecular testing an
algorithm was proposed for assigning RhD
status to patients with discrepant results on
serological testing
• At least 1 Galileo anti-D score ≤ 1+ prompted
standardized tube tests with S4 and S5
– If tube tests agreed & < 2+ →Rh NEG
– If tube tests agreed & ≥ 2+ →Rh POS
– If tube tests did not agree (with ≥ 2+ difference
between reagents)
→Molecular testing
Examples of Discrepant Results –
WD Types 1 & 2
(NOT AT RISK)
MOLECULAR
MANUAL TUBE
Algorithm
Rh Assignment
GALILEO
Novaclone
Gamma
Series 4 Series 5 Galileo S4 Galileo S5
2+
3+
2+
3+
0
2+
0
1+
0
1+
0
?
Rh NEG
Rh NEG
1+
2+
1+
2+
0
0
0
0
1+
0
0
0
Rh NEG
Rh NEG
Weak D Type 1 n=52
Weak D Type 2 n+18
At least 1 Galileo Score ≤ 1 → Tube Tests (S4, S5)
Tube Tests Agree ≥ 2+ Rh POS
Tube Tests Agree < 2+ Rh NEG
Tube Tests Disagree (2+ diff) MOLECULAR Testing
Examples of Discrepant Results –
WD Types 3 & 4
(NOT AT RISK)
MOLECULAR
MANUAL TUBE
Algorithm
Rh Assignment
GALILEO
Novaclone Gamma Series 4 Series 5 Galileo S4 Galileo S5
Weak D Type 3 n=30
n=17
n=11
n=2
Weak D Type 4 n=13
n= 10
n=2
n=1
2+
4+
3+
2+
3+
3+
1+
3+
4+
1+
2+
1+
0
1+
?
0
?
?
Rh NEG
Rh POS
Molecular
3+
3+
3+
2+
3+
4+
1+
3+
3+
1+
2+
1+
1+
1+/?
2+
0
0/0
1+
Rh NEG
Rh POS
Molecular
At least 1 Galileo Score ≤ 1 → Tube Tests (S4, S5)
Tube Tests Agree ≥ 2+ Rh POS
Tube Tests Agree < 2+ Rh NEG
Tube Tests Disagree (2+ diff) MOLECULAR Testing
Examples of Discrepant Test Results
Unclassified, DAR, DVI
(AT RISK)
n = 56
Unclassified
n=51
n=41
n=7
n=3
DAR n=2
n=1
n=1
DVI Types
n=3
Novaclone
Gamma
Series 4
Series 5
Galileo S4
Galileo S5
Algorithm Rh
Assignment
3+
3+
3+
3+
3+
3+
2+
3+
4+
1+
2+
1+
2+
?
3+
?
0
1+
Rh NEG
Rh POS
Molecular
1+
0
?
0
0
0
Rh NEG
Rh NEG
0
0
0
Rh NEG
3+
3+
1+
1+
1+
1+
All correctly reported as Rh NEG
0
0
0
At least 1 Galileo Score ≤ 1 → Tube Tests (S4, S5)
Tube Tests Agree ≥ 2+ Rh POS
Tube Tests Agree < 2+ Rh NEG
Tube Tests Disagree (2+ diff) MOLECULAR Testing
Using the Proposed Algorithm Results of
Previous Molecular Testing were
Analyzed
(Unknown, DVI, DAR, etc)
AT RISK
56
Tube Tests Agree & ≥ 2+ →
Call Rh POS → RHIG withheld
inappropriately
Tube Tests do not Agree
(≥ 2+ difference) → Send for
Molecular Testing
Tube Tests agree and <2+ →
Call Rh NEG
NOT AT RISK
n=169
113
8/56 (14.2%) †
13/113 (11.5%)
3/48*
3/100*
45/56 (80.4%)
(Weak D Type 1, 2, 3, and 4)
→ Called Rh Pos → RHIG
withheld appropriately
→
97/100 (97%)**
RHIG given appropriately
→ RHIG given needlessly
† Included 7 unclassified and 1 DAR
*6/169 (3.6%) Would be sent for molecular testing
3 remained Rh NEG, unclassified
3 reported Rh POS (2 WDT3, 1 WDT4)
** 52/52 WDT1, 18/18 WDT2; 17/30 WDT3, 10/13 WDT4
Problem!
• 97% of individuals classified as Rh NEG using algorithm were WDT 1,
2, 3 and 4 and did not require RHIG. They would receive an
unnecessary blood product exposure.
• 14.2% of individuals (unclassified, DAR) would be classified as Rh
POS. Current evidence suggests that with-holding RHIG in such cases
may result in RhD alloimmunization but whether this translates into
a risk for HDFN is uncertain.
• How to decrease the number of NOT AT RISK individuals (WDT
1,2,3,4,) classified as Rh NEG while correctly identifying
individuals AT RISK for RHD alloimmunization?
Enhancement Techniques
• Extended incubation: 5’ incubation added to tube
testing with S4 and S5 to standardize technique - 10/12
samples reclassified to Rh POS but 3/10 shown to have
‘weak expression of D, unclassified’ on molecular
analysis.
• Conclusion: Extended incubation may assist in
minimizing the number of individuals typed
inappropriately as Rh NEG. However, some of the
individuals whose Rh type changed from NEG to POS
may be at risk for D alloimmunization.
Reanalyzed Data to Determine Implications
of Adding Novaclone to Algorithm
(Novaclone ≥ 2+ = Rh POS)
•
59% (57/97) of NOT AT RISK patients would continue
to have RHIG recommended unnecessarily (versus
97% without Novaclone) – mainly WDT1 and 2
•
19% (10/53) (9 ‘unclassified’ & 1 DAR) AT RISK
patients would be designated Rh NEG & RHIG
recommended appropriately
•
81% (43/53) (42 ‘unclassified’ & 1 DAR) would be
designated Rh POS & ineligible for RHIG. DAR is
known to be at risk for anti-D formation; whether the
‘unclassified’ cases are at risk is uncertain.
CBS Edmonton Rh Discrepancy
Algorithm for Prenatal Testing
Rh Discrepancy Algorithm
*Tube Test – Direct Agglutination (DA) is 5 min RT
incubation for S4 & S5.
Canadian Blood Services
Galileo Prenatal Testing
Galileo Results
S4 and S5 both ≥ 1+
Galileo Results
S4 and S5 both negative
Galileo Results
Series 4 and Series 5 are
inconclusive (?/0, 0/1+)
Comment 1 – This patient appears to have a weak
expression of the D antigen and may be capable of
forming Anti-D. Patient could be reported as RhD
positive by another laboratory that uses different
RhD antisera.
* Perform DA with S4
and S5, Novaclone
& monoclonal control
Yes
Pos ≥ 2+ (with
Novaclone &
S4 &/or S5
No
Neg (with all
reagents)
Genotyping
available
Comment 2 – Rh Immune Globulin to be give at
discretion of physician.
Comment 3 – Patient may have been previously
reported as Rh negative. This patient probably has
a weak or variant D antigen and may be capable of
forming anti-D.
No
Comment 1, 2
Yes
Report Rh Positive
**Previously reported as Rh Neg Comment 3
Report Rh Indeterminate
Comment 4, 5
Send for Genotyping
**Rh Discrepancy with previous testing – review
historical results to ensure there isn’t a collection
error.
Report Rh Negative
Comment 4 – Unable to determine Rh typing at this
time. Current testing shows variable results with
different anti-D typing sera. Until Rh(D) type is
determined consider patient as Rh(D) negative and
eligible for antenatal Rh Immune Globulin.
Comment 5 – Sample has been forwarded for RHD
genotyping to______________________ .
Further Investigation of
Unclassified Cases
Initial Testing: RhD multiplex PCR for exons 3-7
PCR-RFLP for weak D types 1, 2, 3, 4
Direct sequencing of exons 5 and 7
28 unresolved samples further investigated using a Qiagen DNA
extraction kit (Hilden, Germany) and BAGene DNA-SSP Partial D-Type kit
(Lich, Germany). This kit is able to identify D categories II, III, IV, V, VI, VII
as well as partial D DAU, DBT, DFR, DHMi, DHMii, DNB, DHAR and DEL.
Results: Allelic assignment was resolved in 16/28 (57%) samples.
13
2
1
12
D category VII
DHMi
D category IIIa/c/IV (further testing b/c phenotype not
concordant with DNA findings)
Unresolved (pattern consistent with standard D allele)
Reference: Pavenski K, Denomme G, Hannaford K, Appados A, Hannon J. Identification of Partial D
Variants in Prenatal Specimens with Serologically Weak or Undetermined D. Transfusion 2008; 48 (Suppl):
185A
Current Developments
•
•
•
•
•
•
•
Molecular testing not widely available in Canada and expensive approach to
resolving RhD discrepancies.
Slight variations in algorithms in use in CBS Perinatal Laboratories.
CBS Perinatal Committee is working towards a standardized algorithm
which is agreeable to all provincial prenatal laboratories and which will
facilitate implementation of the new CBS LIS system in June 2010.
Efforts are aimed at improving the algorithm for identification of AT
RISK patients and minimizing unnecessary RHIG administration to
NOT AT RISK patients.
Novaclone and/or enhancement techniques such as extended incubation
(or both) being considered.
An effort is being made to ensure that physicians & hospitals are aware of
RhD discrepant results detected in CBS prenatal laboratories, through the
use of coded comments & the inclusion of Novaclone results on patient
reports.
CBS is liaising with hospitals to minimize the number of discrepant RhD
typing results and the impact for patient care.
CBS Perinatal Committee
•Kathy O’Shea, BC & Y
•Gerri Growe, BC & Y
•Judith Hannon, AB
(Chair)
•Jean Ashdown, AB
•Debra Lane, MB
•Lee Grabner, MB
•Robert Fallis, MN
•Ted Alport, SK
•Greg Denomme, Toronto
•Janet Barnes, NRL
•Gwen Clarke, AB
•Gerri Barr, AB
•Vivian Stephens, BC & Y
•Judy Boland, MB
•Lynn Meilleur, MB
•Tony Dolnik, BC & Y
•Bernie Eurich, SK
•Satinder Walia, Toronto
•Heather Hume, Head Office
How to manage Variable D’s –
the hospital experience
“Variable” D results
• CBS perinatal results differ from hospital
results
• Previously positive; new reagent or
method, now negative
• Previously negative; new reagent or
method, now positive
• Doctors confused
• Lab credibility suffers a blow
Variable D results
• A particular combination of results does
not always predict what the “molecular” or
genetic D type really is
• Shall we call them all Rh negative?
Is all variability = Rh negative
in a recipient?
• Rh negative blood is a scarce resource
• Not all weak or partial D individuals are
at risk for allo immunization – in fact –
most are not!
• RhIg is a human blood product… should
we always err on the side of
administering RhIg?
Additional controversy
• Should 1+ be considered positive or negative?
– And the reaction strength is method specific
• Should front line staff be expected to record or
enter clear positive results as negative?
• Will the LIS allow blood group interpretation if
weak reactions are present and the
interpretation doesn’t match?
• What if a genotyping report is available?
Lab considerations
• What is the discrepancy due to
– Don’t forget mis- collection or misidentification
as a cause for discrepancy
• How do you report weak or variable
reactions
• How do you resolve discrepancies
between testing episodes or between labs
Clinical Considerations
• The risk of developing an anti D
• The risk of RhIg
• The risk of HDN
Mom and cord testing
• A pregnant woman is a potential recipient of fetal
red cells and may be immunized if she is weak
or partial D positive;
– Categorize as Rh negative if weak or discrepant
reactions
• A neonate is a potential blood donor (to mom)
and may immunize mom if weak or partial D
positive
– Categorize as Rh Positive for purposes of maternal
RhIg eligibility
– IAT (weak D testing) required for all Rh Negative
neonates/cord samples
Neonatal transfusion recipients
– Neonate/cord sample
may be Rh positive for
maternal RhIg
eligibility
– and Rh negative for
pre transfusion testing
– Cord samples should
not be treated as pre
transfusion samples
What’s a transfusion lab to do?!
• Know your reagent – is it limited
specificity and likely to pick up partial D…
or is it broad specificity and likely to make
all weak and partial D Rh Pos?
• Avoid weak D (IAT) testing as a routine
test in D negative individuals
• Consider using the same method/reagent
as nearby labs or sites that provide
reference testing
Continued….
• Determine the strength of reactivity that you
consider positive (is 1+ positive?)
• Standardize a method to be your “gold standard”
and use these results to guide your results – but
acknowledge the variability in your report
• Consider reference lab RHD genotyping to
resolve weakly reactive D and or discrepant
results
Donor Retesting and Cord samples
• Use broad specificity anti D reagents (a
monoclonal polyclonal IgM/IgG blend)
• Weak D testing must be done on Rh
negative or discrepant samples
• Cord weak D test results should be used
for determination of maternal RhIg
eligibility and not for pre transfusion
testing
Reporting
• Results discrepant from historical or
blood donor records should be explained
(admit defeat in your report)
• RHD genotyping (if done) should become
a part of the permanent record for that
individual –along with an explanatory
comment
• Cord and neonatal typing discrepancies
also require explanation
Example report:
• Rh(D) typing is variable with different
methods and reagents. This usually
indicates a weak or partial D antigen. RhIg
administration is at the discretion of the
patient and attending physician
Patient scenarios
• 37 year old G4P3; has never received
RhIg previously (always typed as Rh pos).
Now types with your new reagent/analyzer
as 1+ and negative. Has no detectable anti
D. Plans no further children with tubal
ligation to follow delivery…
recommendation?
Patient 2
• 24 year old G2P2 with discrepant D result
identified on postnatal sample for RhIg
eligibility (now is Rh positive; previously
typed as Rh negative) has previously
received RhIg… recommendation?
The End
Questions?