Molecular Analysis of Automated Phenotype Discrepancies among Blood Donors

Greg Denomme, PhDDirector of Immunohematology & Transfusion

ServicesBloodCenter of Wisconsin

Objectives

• Review how licensed serological reagents lead to blood group discrepancies

• Describe a molecular change resulting in the weak expression of A or B antigens

• Identify one impact of D antigen discrepancies among transfusion recipients or in pregnancy

• Understand how blood group discrepancies can be an indication of an underlying acquired disease

Blood Group Discrepancies

Definition:• A blood group discrepancy occurs when at least two

phenotyping results have different interpretations– When the forward and the reverse ABO typing do not agree– When two antisera results are different – one indicates the

antigen is expressed (positive) and the other not (negative)– When an historical phenotype does not match a current

phenotype

• A phenotype and genotype difference is deemed a discordance

What to do?Isolate the problem• Is the problem technical?

– Clerical check - mislabeled specimen– Laboratory failure - to add the reagent (repeat)– Interfering substance - repeat the test on same sample using

saline washed cells• Is the problem between two labs?

– Repeat the test – identify whether similar or different reagents• A true discrepancy is due to a problem with

– patient’s red cells– serum (often the case for ABO)– other

Kinds of Blood Group Discrepancies

• External Events– Recent transfusion– Bone marrow transplantation– Maternal-fetal hemorrhage

• Inherited– Variant blood group antigen– Microchimerism

• Acquired– Autoimmune disease (antigen blocking)– Leukemia (loss of antigenicity)

List of Technical Errors

• Clerical– Pre-lab: wrong patient, mislabeled specimen– In-lab: manual clerical error, computer entry error

• Methodological– Reagent not added– IFU not followed; wrong temperature or incorrect phase– Heavy red cell suspension; inexperienced operator

• Reagent or equipment problem (unlikely)– Using expired reagent– Contaminated reagent

Approach to Blood Group Discrepancies

• Patients – decreased antibody levels

• physiologic - newborns, elderly• immunodeficiency - congenital or acquired

– presence of auto- or allo-antibodies

• Donors– unusual blood group antigen?

• think genetic polymorphism

ABO Discrepancies – Serological Investigation

Discrepancy

Forward

Missing/Weak

A or B Subgroup

Disease (leukemia)

Extra

Acquired B

B(A) Phenotype

Rouleaux

Mixed Field

Group O Tx’n

BMT

Reverse

Missing/Weak

YoungElderly

Extra

Cold AutoAb

Cold AlloAb

Anti-A1

Rouleaux

Immuno-comp

Inherited Blood Group Discrepancies

Donor #4ABO Rh -B -A -A,B D1 D2 Control A1 B

B POS + - + + + - + -

PK7200 Data Log

Prediluted Olympus reagents used

Donor #4

Method -B -A -A,B A1 A2 B O

I.S. 4+ - 4+ 1+ - - -

RT 30’ 4+ 1+ 4+ 2+ - - -

Manual Bench Results

Immucor Gamma-clone reagents used

Unit returned to the blood center - Labelled B pos, tested AB pos at hospital

Donor ABO Discrepancy

261 467 526 703 796/803 1060802

ABO Subgroup Discrepancies

A1

B

O2

O1v

O1

∆G

A2

∆C

A1

Equal Crossover

Occurs in Meiosis during Metaphase II

Pair of chromosomes Non-sister chromatidexchange

Non-sister Chromatid Exchange

261 467 526 703 796/803 1060802

AX

B

O1v

O2

O1v

AX

Group A child from non-A parents

D Antigen Discrepancies

Population Study Rh+ Rh-

CaucasianKopec 1970

Wagner 1995Garratty 2004

82% 18%

Tunisia, Nigeria Ranque 1961Enosolease 2008 92-94% 6-8%

India Makroo 2013 94% 6%

Basques/Morocco Goti 1958Messerlin 1951 71% 29%

China Shao 2002 >99% 0.3%

Summarized from Weinstock ,C., Blood Transfusion 2014;12:3-6

Frequency of weak D expressionHopkins Scotland 1967 0.56%

Garretta France 1974 0.66%

Beck USA 1990 0.2%

Jenkins USA 2004 0.4%

Flegel Germany 2006 0.4%

Classification of D – potential for discrepancies

r’ haplotype: in trans with D (Ceppellini effect)

Weak D “types”: single amino acid changes Weak D Type 2

Partial and Category D: hybrid RHD alleles DVI, types I, II, III

Del: detection by adsorption/elution K409K, plus several others

RHD-deletion and non-functional RHD alleles

Ceppellini Effect

Ceppellini R., Dunn LC, Turri M. PNAS 1955;41:283-288

Prevalence of Weak D (Michigan Study)

Anti-D Score

PV GA BI Genotype MoAb8 8 5 DAR DV8 8 0 DAR DV8 10 5 Weak D Type 18 7 0 Weak D Type 18 10 5 Unknown8 10 6 Exon 4-5 CE hybrid8 10 5 Weak D Type 28 8 5 Weak D Type 28 7 0 Weak D Type 28 7 0 Weak D Type 2

Transfusion 2008;48:473-478

Not reported to make anti-D

Blood Donor D Antigen Discrepancy

• Hospital returns a unit to the blood center– Weak reaction noted with the weak D IAT

• Blood Center repeats the Rh typing– Ortho Bioclone 0– Immucor Gammaclone w+– RHD genotyping Weak D type 2 (RHD*01W.2)489 antigens/cell (using 59 anti-D clones on 1 sample)

• Why was the D antigen not detected?– Complete phenotype C+E+c+e+

• Weak D with a C allele in trans (Ceppillini effect)– cDE/Cde

Wagner FF. Blood;95-2699-708

D antigen expressing alleles

1 2 3 4 5 6 7 8 9 10 RHD1 2 3 4 5 6 7 8 9 10 DVI

1 2 3 4 5 6 7 8 9 10 RoHARCrawford

Allele Name Anti-D I.R.

YesWeak D Type 21 2 3 4 5 6 7 8 9 10 No

No

YesYes1 2 3 4 5 6 7 8 9 10

D Antigen Microchimerism – vw+ MF Agglutination

A single unit of blood can contain 4 - 20 mL of D+ blood

Wagner FF. BMC Genetics 2001;2:10-

Null Allele – Blood Group Discrepancies• Alloimmunized transfusion recipient (Polynesian)

– 54 yo with diabetes and chronic anemial– Multiple recent transfusions– increasing complex antibody investigation with time (responder)

• Multiple blood group antibodies– Anti-E, -c, -K, -Jka, -Cw

– Could not rule out -S, -Fyb, -Jkb

– Serum dilution study suggested an anti-Jkb is present– Given that an anti-Jka is present, an anti-Jk3 was considered– Children evaluated for possible donation

• Jk(a/b) expression - disparity noted– Eldest child Jk(a+b-)– Second child Jk(a+b+)

Rh-Hr Kell Duffy Kidd MNS P Ficin Saline IAT

D C E c e f Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb M N S S P1 Cell 16 512 1024

+ + 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 + + +s 1 3+ 1+ 0

+ + 0 0 + 0 0 0 0 + 0 + 0 + + 0 + 0 0 + + 0 +w 2 2+ w+ 0

+ 0 + + 0 0 0 0 0 + 0 + 0 + 0 + + + + + 0 + 0 3 3+ w+ 0

+ 0 0 + + + 0 + 0 + 0 + 0 + 0 0 + + + + + + +s 4 3+ 1+ 0

0 0 0 + + + 0 0 0 + 0 + 0 + 0 + + + + + 0 + 0 5 4+ 1+ w+

0 0 + + + + 0 0 0 + 0 + 0 + + + + + + 0 + 0 +w 6 3+ w+ 0

0 0 0 + + + 0 0 + + 0 + 0 + 0 + + 0 + + 0 + +w 7 3+ w+ 0

0 0 0 + + + 0 0 0 + 0 + 0 + + + + 0 0 + 0 + +s 8 2+ w+ 0

0 0 0 + + + 0 0 0 + 0 + 0 + + 0 0 + + + + + +w 9 3+ 2+ 2+

0 0 0 + + + 0 0 0 + 0 + 0 + + + 0 + + 0 + 0 0 10 4+ 2+ 2+

+ + 0 0 + 0 0 0 + + 0 + 0 + 0 + 0 + + + + + +s 11 3+ 2+ 1+

Intron SNP - JKnull Phenotype

Protein Truncated protein – Jknull

DNA: JKA/JKBRBCs: Jk(a+b-) Can make anti-Jk(b)!

Protein Jka

Exon 5 Exon 6 Exon 7 Exon 8mRNA JKA

Allele 1 Exon 8Exon 7Exon 6 Exon 9Exon 5GT….GA

Exon 5 Intron 5mRNA Exon 6

Allele 2

JKB

Exon 8Exon 7Exon 6 Exon 9Exon 5

JK intron 5

GT…AA

JK∆6 allele – alteration of intron 5 splice site

Wildtype JK∆6

Intron 5 Exon 6 Intron 5 Exon 6

M 1 2 3 4 5 6

Acquired Blood Group Discrepancies – Loss of Ag

• Leukemia and pre-leukemia

• ABO discrepancies in healthy blood donors should be investigated (serological or molecular)– Identification of an ABO subgroup allele rules out a blood

disorder

Bianco T. Blood 2001;97:3633-3639

Acquired Blood Group Discrepancies - BlockingPassive Antigen Blocking• D antigen typing in hemolytic disease

– maternal serum anti-D; titer 64– Neonate Hgb 97 g/L; DAT 2+– RhD typing performed – negative (Why?)

• Direct agglutinating anti-D is IgM.– All D antigen sites are blocked by maternal anti-D

IgMMaternal anti-D

blocking

Bosco AM. Transfusion 2009;49:750-756 Summarizes acquired Ag blocking

Blood Group Discrepancies• Technical problems are common and easy to address• Obtaining medical history and speaking with the patient

can resolve some external events– Maternal-fetal bleed requires additional intervention to consider

additional doses of RhIG• Inherited variant alleles

– ABO subgroups can be solved with adsorption/elution studies• Molecular typing may be necessary

– RHD variant cause problems with patients and donors– Other variants are ‘uncovered’ during Ab investigations– Chimerism is very rarely observed

• Acquired disorders– Associated with known diseases– Suspected when observed in healthy blood donors

Thank you

E-mail: greg.denomme@bcw.edu