Serologic Weak D Phenotype to RHD Genotyping: How will I ... Program Handouts...Testing Platform...
Transcript of Serologic Weak D Phenotype to RHD Genotyping: How will I ... Program Handouts...Testing Platform...
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Serologic Weak D Phenotype
to RHD Genotyping:
How will I know?
Part 1
Sue Johnson, MSTM, MT(ASCP)SBB
Director, Clinical Education
BloodCenter of Wisconsin
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Commentary
It’s time to phase-in RHD genotyping
for patients with a serologic
weak D phenotype
Sandler SG, Flegel WA, Westhoff CM, Denomme GA,
Delaney M, Keller MA, Johnson ST, Louis Katz,
Queenan JT, Vassallo RR, Simon CD
Transfusion 2015 Mar;55(3):680-9
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How will I know?
Objectives
• Identify causes for variability of expression of
RhD antigen
• Describe specificity, sensitivity & intended use
of current commercial RhD typing reagents
used in test tube & automated methods.
• List challenges of typing for RhD by
serologic methods.
• Define management & transfusion options for
patients with weak or variable RhD typing.
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CONTRIBUTORS
OF VARIABILITY
VARIABLES
RHD Gene Weak D C in Trans
to RHD
Partial D Del
D epitopes on
RhCE Protein
ceCF ceHAR
Anti-D Reagents Polyspecific
Slide and
Modified Tube
Human IgG
Monoclonal
IgG
Monoclonal
IgM
Monoclonal
IgM
Human IgG
Monoclonal
Blends
Testing Platform Test Tubes
IS & IAT
Column
Agglutination
Solid Phase Liquid
Microtiter
Individual being
Rh Typed
Transfusion
Recipient
Obstetrical
Patient
Cord Blood Donor Blood
Variables Impacting Rh Typing
Transfusion Technology Report Vol. #013 Immucor, Inc.
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2 3 4 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 10
Locus 1 Locus 2
RH Genes – Rh Positive
RHD
RHCE
Chromosome 1
Locus 1 - presence of RHD
codes for the presence of D or
no D.
Locus 2 - presence of RHCE
codes for Ce, CE, cE, ce.
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RhD Protein
COOH
417 NH2
53 107 167 230
321
358
391
Vestibule
282
•Crosses RBC membrane 12 times
•No sugars attached
http://www.jic.ac.uk/corporate/about/publications/advances/images_10/protein.jpg
231 347
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11 72
32 53 94 107 158 167 230 231 282 290 347 358
75 131 135 186 201 263 266 321 324 391
417
RhD differs from RhCE by 34 to 37 amino acids =
E= C=
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RhD Negative
• Deletion of RHD
• Inactivating mutations of RHD
• RHD in African Americans
• Hybrid RHD-CE-D in African backgrounds
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1 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 10
Locus 1 Locus 2
Exons
RH Genes in Rh Negative Caucasians
No D antigens ce antigens
RHCE
Locus 1 deletion of RHD therefore, no D antigen.
Chromosome 1
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2 3 4 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 10
Locus 1 Locus 2
Exons Exons
RH Genes in Rh Negative - African Background
No D antigen C/c and E/e antigens
RHD
RHCE
Locus 1 – 37 bp insertion & several mutations in
RHD results in no product
Chromosome 1
RHD Allele frequency of ~.030 African Americans*
*Reid ME, et al Blood Cells, Molecules & Diseases 2013
Chou S, et al. Blood 2013
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2 3 4 9 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 10
Locus 1 Locus 2
Rh (D) Negative – African Background
RHD
RHCE
Chromosome 1
C/c and E/e antigens No D antigen
Locus 1 – RHCE inserted in RHD results in no
D antigen and weak C.
Allele frequency of hybrid RHD-CE-D is ~ .029-.09*
*Reid ME, et al Blood Cells, Molecules & Diseases 2013
Chou S, et al. Blood 2013
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What About
Weak Expression of D?
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Traditional Weak D
Reactivity with Anti-D
• Agglutinated with some anti-D on
direct agglutination (IS)
• Negative on direct agglutination (IS)
• D antigen detected by IAT only
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Commentary
It’s time to phase-in RHD genotyping
for patients with a serologic
weak D phenotype
Sandler SG, Flegel WA, Westhoff CM, Denomme GA,
Delaney M, Keller MA, Johnson ST, Louis Katz,
Queenan JT, Vassallo RR, Simon CD
Transfusion 2015 Mar;55(3):680-9
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Serologic Weak D Phenotype
Definition
• Anti-D reagent agglutinates RBCs
weakly (≤ 2+) or not at all by test
tube method at IS, but agglutinating
moderately or strongly with
antihuman globulin
Identified by weak reactivity or
by discordant typing results
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Frequency of Serologic Weak D
• RhD-negative individuals
• Estimated to be 2.9% among
a mixed population
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Weakened Expression of D
2 Categories
• Not at risk of making anti-D
• At risk of making anti-D
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WEAK D
Not at Risk of Making Anti-D
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Weak Expression of D Not at Risk of Making Anti-D
• C in trans with RHD
(Ceppellini effect)
• r’ haplotype (R1r’ – DCe/Ce)
• Weak D “Types”: amino acid change(s),
usually a single change
• Types 1, 2, 3
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Weak D Types
Most Not At Risk of Making Anti-D
• Changes in regions of RHD predicted to be
in the RBC membrane or inside RBC
• Less Rh protein in RBC membrane
• Can type as Rh-positive or Rh-negative by
direct agglutination with monoclonal (IgM)
anti-D reagents
IS D IAT Ct. IAT
Anti-D 0 3 0
IS
Anti-D w+ - 2+ or
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11 72
32 53 94 107 158 167 230 231 282 290 347 358
75 131 135 186 201 263 266 321 324 391
417
Type 2
Gly(385)Ala
Type 1 Val(270)Gly
Account for 80-90% of Weak D
Not at risk of making Anti-D
Type 3 Ser(3)Cys
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Normal RhD Positive
Normal Rh Protein
Normal RhD antigen
~ Antigens/RBC*
DCe/ce 9900-14,600
DcE/ce 12,000-19,700
DCe/DCe 14,500–22,800
*Daniels G. Human Blood Groups
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Weak D Type 1- 81
Fewer Copies of Rh Protein
Normal RhD antigen
~ Antigens/RBC*
Type 1 759
Type 2 491
Type 3 1948
*Blood 2000;95:2699-2708
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WEAK EXPRESSION OF D
At Risk of Making Anti-D
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Weak Expression of D
At Risk of Making Anti-D
• Partial Ds: hybrid RHD alleles
• DVI
• Others…
• Del: detected by adsorption/elution
• D epitopes on RHCE gene
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Partial D
• Lack exofacial epitopes or have
altered exofacial epitopes
• Hybrid proteins
• Missense mutations affecting
exofacial protein
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Partial D
Missing RhD Epitopes
Normal RhD antigen
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Partial D
Altered RhD Epitopes
Normal RhD antigen
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Partial D – European Ancestry
• DNB, DVI and DVII most common in
European ancestry
• DVI
• 2 reports of fatal hydrops fetalis • Transfusion.1983 Mar-Apr;23(2):91-4
• Obstet Gynecol. 2003 Nov;102(5 Pt 2):1143-5 2003
• Anti-D reagents designed to be neg
at IS/pos at IAT
IS D IAT Ct. IAT
Anti-D 0 3 0
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Partial D – African American
• DIIIa & DIVa most common
• Type as RhD positive at IS
IS
Anti-D 3+
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Deletion of exon 9 in Asians occurs in 10-30%
Del
•Type as D-negative (IS & IAT), only adsorb & elute anti-D
•Severely reduced protein
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D Epitope on RHCE Genes
• ceHAR - formally known as R0Har or DHAR
• ceCF (ceRT, ceSL) - Crawford phenotype
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1 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 10
Locus 1 Locus 2
Exons
No D antigens ce antigens
RHCE
ceHAR results from one RHD exon inserted into the RHCE gene.
D Epitope on RHCE Gene - ceHAR
IS
Anti-D 3+
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ceHAR Phenotype: Reactivity with Reagent Anti-D
Anti-D RBCs
Reagent IgM IgG ceHAR
Gamma-Clone GAMA401 F8D8 Pos*
Immucor-4 MS201 MS26 Pos*
Immucor-5 TH28 MS26 Pos*
Ortho Bioclone MAD2 Human
polyclonal
Neg
Ortho (ID-MTS) MS201 Pos
Biotest (Bio-Rad) BS232 BS221
H41 11B7
Pos
Quotient - Alpha LDM1 Pos
Quotient - Delta LDM1 ESD1M Pos
*Positive reactions often weaker at IAT
AABB Tech Manual, 18th ed. 2014
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1 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 10
Locus 1 Locus 2
Exons
No D antigens ce antigens
RHCE
ceCF results from 3 nucleotide changes,
48G>C, 697C>G, 733C>G in RHce
gene.
D Epitope on RHce Gene - ceCF
GammaClone
IS
Anti-D 3+
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Anti-D Reagents: Reactions with Crawford Phenotype RBCs
Anti-D RBCs
Reagent IgM IgG Crawford
IS/IAT
GammaClone GAMA401 F8D8 Pos/Neg
Immucor-4 MS201 MS26 Neg/Neg
Immucor-5 TH28 MS26 Neg/Neg
Ortho Bioclone MAD2 Human
polyclonal
Neg/Neg
Ortho (ID-MTS) MS201 Neg/Neg
Biotest (Bio-Rad) BS232 BS221
H41 11B7
Neg/Neg
Quotient - Alpha LDM1 Neg/Neg
Quotient - Delta LDM1 ESD1M Neg/Neg
AABB Tech Manual, 18th ed. 2014
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Anti-D
Reagents & Methods
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Monoclonal Reagent Types
• Blend of monoclonal & polyclonal antibodies
• Blend of two or more monoclonal antibodies,
each secreted by a different cell line
• IgG or IgM, or combination of IgG + IgM
• Why?
• D antigen has >30 different epitopes
• Variant D antigens
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FDA Approved Reagent Anti-D - Tubes
Anti-D
Reagent IgM IgG
Gamma-Clone GAMA401 F8D8
Immucor-4 MS201 MS26
Immucor-5 TH28 MS26
Ortho Bioclone Tube MAD2 Human polyclonal
Bio-Rad Seraclone - Blend BS232 BS221
H41 11B7
Bio-Rad Seraclone - 226 BS226
Quotient (Alba) – Alpha LDM1
Quotient (Alba) – Beta LDM3
Quotient (Alba) – Delta* LDM1
ESD1-M
Quotient (Alba) – Blend LDM3 EDS1
*Not for patient testing, detects DVI at IS
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FDA Approved Reagent Anti-D - Other Methods
Anti-D
Anti-D Method IgM IgG
Immucor – Series 4 Galileo Echo®/Neo® MS201 MS26
Immucor– Series 5 Galileo Echo®/Neo® TH28 MS26
Ortho Gel/Provue® MS201
PK1 PK7200®/PK7300® P3X61
PK2 PK7200®/PK7300® HM10
Blend PK7200®/PK7300® P3X61
P3X21223B10
P3X290
P3X35
Bio-Rad 226 Tango® BS226
Bio-Rad 232 Tango® BS232
Solidscreen II Blend
(weak D testing)
Tango® H411B7
BS221
Grifols DG Gel/Erytra® P3x61
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Specificity of Anti-D CFR 660.26
• Panel of antigen positive RBCs must be
tested and include at least 3 donors of
each phenotype:
• DCe/ce, Dce/ce
• dCe/ce, dce/cE
• Group A, B and O dce/dce
• Most include testing with DVI
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Sensitivity of Anti-D
• Reagent Anti-D compared to Reference
Blood Grouping Sera (FDA) for potency
• Monoclonal Anti-D WILL NOT detect all
weak D and partial D
• Weak D Test (IAT/AHG) enhances
reactivity with most examples of weak
D and partial D
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Human IgG Anti-D
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Monoclonal IgM/IgG ANTI-D
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Monoclonal IgM/IgG ANTI-D #1
Direct Agglutination - IS
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Monoclonal IgM/IgG ANTI-D #1
Weak D Test - IAT
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Monoclonal IgM/IgG ANTI-D #2
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Method Variability
• Test Tube Methods
• Reagents used
• Technologist-dependent
• Weak D IAT/AHG or not…
• Other Methods
• Liquid microtiter
• Column Agglutination
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Direct Agglutination in Test Tubes
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Reading Direct Agglutination
ABO vs. RhD Reagents
Anti-A / Anti-B Anti-D
Antibody Class IgM IgM or
IgM & IgG
Antigen Structure
Carbohydrate
Integral Membrane
Protein
Antigen Sites/RBC ~2 – 10 x 105
~ 9,900 – 33,000
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Reading Direct Agglutination
Shake, Rock, Roll, Swirl, Tilt, or some combination… Grade as soon as
RBC button is
resuspended
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Microplate ABO/Rh Typing
Fluid Phase – Hemagglutination by Settling
www.beckmancoulter.eu/diagnostics/3108_PK7300.htm
PK7300
Settling Method
Neg Pos
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Microplate ABO/Rh Typing Fluid Phase – Hemagglutination
Galileo Echo® & NEO ®
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Microtiter Plate ABO/Rh Typing
Hemagglutination Method
Monoclonal Control
Anti-A
Anti-B
Anti-D Series 4
Anti-D Series 5
A1 Cells
B Cells
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Microplate ABO/Rh Typing Hemagglutination by Aggitation
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Column Agglutination Hemagglutination Methods
http://www.grifols.com
www.ortho-wire.com/en/blood-group-serology/learning-library/AgAb/page5.cfm
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Questionable RhD Typing Results
Using Automation
Microplate
Anti-D ? Or NTD
Discordant with historical typing
Gel
Anti-D ? or <2+
or
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RhD Typing Results
Test Tube Method
Tube IS IAT
Anti-D 0-2+ 2-4+
Consider RHD genotyping
Tube IS
Anti-D 3-4+
or
Serologic Weak D Phenotype
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Reasons to Resolve Weak Expression
Pregnancy
• Avoid giving RhIG to women who do not
need it (Rh status is confirmed for
historical discrepancies)
• Resolve early in pregnancy to eliminate
false-positive rosette tests
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Rosette Test Results
Negative Control Positive Control
Weak D+ Mom Courtesy of MR Combs
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Reasons to Resolve Weak Expression
Transfusion
• Conserve Rh-negative blood for D-
negative recipients (high risk of
making anti-D)
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Sandler SG, et al. Commentary – Transfusion March 2015)
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Objectives
• Identify causes for variability of expression of
RhD antigen
• Describe specificity, sensitivity & intended use
of current commercial RhD typing reagents
used in test tube & automated methods.
• List challenges of typing for RhD by
serologic methods.
• Define management & transfusion options for
patients with weak or variable RhD typing.
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References • Wagner FF, Gassner C, Mu¨ller TH, et al. Molecular basis of weak D
phenotypes. Blood 1999; 93:385–393.
• Wagner FF, Frohmajer A, Ladewig B, Eicher NI, Lonicer CB, Mu¨ller TH,
Siegel MH, Flegel WA. Blood 2000;95:2699-2708.
• Denomme GA, Wagner FF, Fernandes BJ, et al. Partial D, weak D types,
and novel RHD alleles among 33 864 multiethnic patients: implications for
anti-D alloimmunization and prevention. Transfusion 2005; 45:1554–1560.
• Flegel WA, Denomme GA, Yazer MH. On the complexity of D antigen
typing: a handy decision tree in the age of molecular blood group
diagnostics. J Obstet Gynaecol Can. 2007;29:746-52.
• Flegel WA. How I manage donors and patients with a weak D phenotype.
Curr Opin Hematol 2006;13:476–483.
• Denomme GA, Dake LR, Vilensky D, Ramyar L, Judd WJ. Rh
discrepancies caused by variable reactivity of partial and weak D types with
different serologic techniques.Transfusion 2008;48:473-476.
• Roback JD, Grossman BJ, Harris T, Hillyer CD. Technical Manual, 18th ed.
2014.
• Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller
MA, Johnson ST, Louis Katz, Queenan JT, Vassallo RR, Simon CD,
Transfusion. 2015 Mar;55(3):680-9.
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References • Flegel WA. Molecular genetics and clinical applications for RH.
Transfusion and Apheresis Science 2011;44:81-91.
• Sandler SG, Li W, Langeberg AL, Landy HJ. New Laboratory
Procedures and Rh Blood Type Changes in a Pregnant Woman. Obstet
Gynecol 2012;119:426–8.
• Wang D, Lane C, Quillen K. Prevalence of RhD variants, confirmed by
molecular genotyping, in a multiethnic prenatal population. Am J Clin
Pathol 2010;134:438-442
• Credidio DC, Pellegrino J, Castilho L, Serologic and molecular
characterization of D variants in Brazilians: impact for typing and
transfusion strategy. Immunohematology 2011;27:6-11.
• Abelrazik AM, Elshafie SM, Ahmed GME, Abdelaziz HM. Combining
serology and molecular typing of weak D role in improving D typing
strategy in Egypt. Transfusion. 2013 Nov;53(11 Suppl 2):2940-4
• Chou ST, Jackson T, Vege S, Smith-Whitley K, Friedman DF, Westhoff
CM. High prevalence of red blood cell alloimmunization in sickle cell
disease despite transfusion from Rh-matched minority donors. Blood.
2013 Aug 8;122(6):1062-71.
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References
• Reid ME, Hipsky CH, Hue-Roye, K, Hoppe C. Genomic analysis of RH
alleles to improve transfusion therapy in patients with sickle cell
disease. Blood Cells Mol Dis. 2014 Apr;52(4):195-202
• Garratty G, Glynn SA, McEntire for the Retrovirology Epidemiology
Donor Study. ABO and Rh(D) phenotype frequencies of different
racial/ethnic groups in the United States. Transfusion 2004;44:703-6.
• Haspel RH, Westhoff CM. How do I manage Rh typing in obstetrical
patients. Transfusion 2015;55;470–474.
• Cannon M, Pierce R, Taber EB, Schucker J. Fatal hydrops fetalis
caused by anti-D in a mother with partial D. Obstet Gynecol. 2003
Nov;102(5 Pt 2):1143-5.
• Lacey PA, Caskey CR, Werner DJ, Moulds JJ. Fatal hemolytic disease
of a newborn due to anti-D in an Rh-positive Du variant mother.
Transfusion. 1983 Mar-Apr;23(2):91-4.