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What Keeps us up at Night
The New Frontier in Blood Banking
HEA Molecular Testing
Sylvia M. Garza MT-P(AAB)
June 6, 2013
Objectives:
1. Define terms.
2. Discuss HEA molecular testing process.
3. Present case study demonstrating serological results that surprisingly differs from
molecular results.
4. Review conclusions.
2
Terminology Definitions 101
• Genotype- An individual’s actual genetic makeup.2(p621) Genotype of a person is the
complement of genes inherited by each person from his or her parents; the term is
frequently also used to refer to the set of alleles at a single gene locus.1(p334)
• Phenotype- The outward expression of genes (e.g., a blood type). On blood cells,
serologically demonstrable antigens constitute the phenotype, except those sugar
sites that are determined by transferases.2(p629)
• Polymorphism for blood groups- the occurrence in the same population, of two or
more alleles at one locus, each with appreciable (greater than 1%)
frequency.1(p335)
• Allele- One or two or more different genes that may occupy a specific locus on a
chromosome.2(613)
3
Terminology Definitions 101 cont.
• Locus- The site of a gene on a chromosome.2(p625)
• Chromosome- The structures within a nucleus that contain a linear thread of DNA,
which transmits genetic information. Genes are arranged along the strand of DNA
and constitute portions of the DNA.2(p616)
• DNA (Deoxyribonucleic Acid)- The chemical basis of heredity and carrier of genetic
information for all organisms, except RNA viruses. Structured as a double helix of
polymers of nucleotides, each containing bases (A, T, C, and G), sugar deoxyribose,
and three phosphates. They become monophosphates once they are built into DNA
molecules.2(p618)
4
BioArray Platform
• The HEA BeadChipTM (Bioarray Solutions, Immucor) analyzes 24 polymorphisms
associated with 38 Human Erythrocyte Antigens and phenotypic variants are
included in the BioArray Solutions HEA BeadChipTM kit.4(p4)
5
Principles of the Assay
• The BioArray Solutions HEA BeadChipTM Kit uses the proprietary Elongationmediated Multiplexed Analysis of Polymorphisms (eMAP®) technology to identify
the presence or absence of the selected alleles associated with a phenotype. After
multiplex PCR amplification and post-PCR, the single-stranded DNAs are incubated
on the BeadChipTM array. Elongation reaction extends and incorporates
fluorescently-labeled dNTP molecules only on those probes where the 3’ end
matches the annealed DNA. Elongation products of alleles A and B are
simultaneously detected by imaging the entire array.
6
Principles of the Assay cont.
4
Table 1: Genetic Markers for Red Blood Cell Antigens in the BeadChip Assay
• In this method, each probe is
covalently attached to a
distinguishable bead type. A
library of individual bead types
contain all of the probes of
interest. The library is
immobilized in the BeadChipTM
array, allowing for the detection
of the polymorphisms of interest.
Blood
Group
System
Analyte
Nucleotide
Polymorphism
ISBT
Phenotype
c/C
307 C>T
109 Ins
RH4, RH2
e/E
676 G>C
RH5, RH3
698 T>C
1910 C>T
961 T>C
125 G>A
KEL1, KEL2
KEL6, KEL7
KEL3, KEL4
FY1, FY2
RHCE*4,
RHCE*2
RHCE*5,
RHCE*3
RHCE*01.20.01,
RHCE01.20.02,
RHCE*01.20.04,
RHCE*01.20.05
KEL*01, KEL*02
KEL*06, KEL*07
KEL*03, KEL*04
FY*01, FY*02
-67 T>C*
FY-2
FY*02N.01
Fy [Fy(b+ )]
Jka/Jkb
265 C>T
838 G>A
FY2W
JK1, JK2
M/N
59 C>T
MNS1, MNS2
S/s
143 T>C
MNS3, MNS4
Silencing S
Ex5 230C>T
Silencing S
In5 g>t
Lua/Lub
Doa/Dob
Hy+/HyJo(a+)/Jo(a-)
230 A>G
793 A>G
323 G>T
350 C>T
LU1, LU2
DO1, DO2
DO4
DO5
FY*02M
JK*01, JK*02
GYPA*01,
GYPA*02
GYPB*03,
GYPB*04
GYPB*03N.01 or
GYPB*03N.02
GYPB*03N.03 or
GYPB*03N.04
LU*01, LU*02
DO*01, DO*02
DO*04
DO*05
LW a/LWb
308 A>G
LW5, LW7
LW*05, LW*07
2561 C>T
134 C>T
169 G>A
DI2, DI1
CO1, CO2
SC1, SC2
DI*02, DI*01
CO*01, CO*02
SC*01, SC*02
Rh
Vs
733 C>G,
1006 G>T
V
Kell
Duffy
K/k
Jsa/Jsb
Kpa/Kpb
Fya/Fyb
GATA
(Silencing FY)
x
Kidd
w
RH20
RH10
MNS
MNS-3, 5W
Lutheran
Dombrock
LandsteinerWiener
Diego
Colton
Scianna
ISBT Genotype
b
a
Di /Di
Coa/Cob
Sc1/Sc2
* The GATA mutation listed here has been previously reported at -33 and -46[8].
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Principles of the Assay cont.
• The BioArray Solution Array System (AIS400) is used to capture the fluorescent
signal from individual beads in an image of the entire array, determine the identity
of the bead by color and report the average signal intensity, coefficient of variance
of the intensities, and the number of beads measured for each type of probe.
8
Principles of the Assay cont.
• The HEA Analysis software in BioArray Solutions Information System (BASISTM)
imports the raw intensity output, assesses the validity of the internal controls, and
generates assay results.
9
Process
DNA
Extraction
• Whole blood
Multiplex
PCR
• Amplification with Phosphorylated Primers
Post PCR
Processing
• Cleanup and Digestion
Post PCR
Processing
• Annealing and Elongation
Assay Image
Acquisition and
BeadChipTM Analysis
6
Serology vs. Molecular Case Study
1. Patient Background
We followed an African American Woman (type A Neg) with Sickle Cell Anemia from
2006-2013 and reviewed her history. Between 2006-2013, 41 PRBC’s were transfused
with no reported transfusion reactions.
2.
Initial Serological Results
To obtain the patient’s phenotype, we elected to perform the hypotonic saline wash
technique due to the patient’s recent transfusions.
Patient’s Serological phenotype:
C+E-c+e+;M-N+S-s+;K- Fy(a-b-); Jk(a+b-)
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Serology vs. Molecular Case Study
2.
Initial Serological Results cont.
• Summation of Results 2006-2009:
-Anti-Fya, Anti-Jsa and Warm Autoantibody (autoanti-e)
• 2012 The transfusing facility reports that another facility identified:
Anti-E, Anti-K and Anti-C. So now the patient has Anti- Fya, -Jsa, -E, -K and Anti-C.
Anti-C, when the
patient’s serotypes
as C+ ????
8
9
Human Erythrocyte Antigen (HEA) Phenotyping by DNA Analysis Report
Report Date/Time:
May 15, 2013 06:49 AM
Lot #:
12-2
Chip Read Date/Time:
May 2, 2012 10:48 PM
Sample ID: Joseph, Judith
HEA82837_1
Chip ID:
North St. Petersburg, FL 33716
Blood Group
Antigen
Result
Rh
c
+
(0)*
+
0
0
+
0
+
0
+
+
0
0
0
0
+
0
+
0
+
0
+
+
0
0
+
+
+
+
0
+
0
++
C
e
E
Kell
K
k
Kpa
Kpb
Jsa
Jsb
Kidd
Jka
Jkb
Duffy
Fya
Fyb
MNS
M
N
S
s
Lutheran
Lua
Lub
Diego
Dia
Dib
Colton
Coa
Cob
Dombrock
Doa
Dob
Joa
Hy
Landsteiner-Wiener
LWa
LWb
Scianna
Sc1
Hemoglobin S
HbS
Sc2
Detail:
Florida Blood Services
10100 Martin Luther King Jr. St.
Notes
Possible VS+V-
Notes:
Status Code
CV - Coefficient of Variation of intensities exceeds recommended maximum
LS - Low Signal: signal intensity below recommended minimum
HB - High Background: exceeds recommended maximum
IC - Indeterminate call
Detail Code
Fyb(w) - Weak Fyb expression due to mutated FY265
Hy - or Jo(a) - Published reports indicate observation of a
"negative" phenotype; some RBC may react weakly
U(-) - Possible U(-) due to GYPB gene deletion
U(var) - Variant U detected involving S silencing mutation
Signature:
Date:
Name (Print):
Title:
Blood Group Phenotype DNA Analysis By: Elongation - mediated Multiplex Analysis of Polymorphisms
General Disclaimer
(0)* - Possible r' S
w - Weakly Expressed phenotype
14
Serology vs. Molecular Case Study
The (0)* may indicate a possible r ’ S
So what is r ’ S ? Why do we care?
So what is r ’ S ?
 Patient’s who are R2r ’ S or homozygous r ’ S will be hrB- and can produce anti-hrB.3(p65)
In our case, this is why we care.
 r ’ S phenotype also expresses partial C and these patients are at risk to form
anti-C.3(p65)
Could this explain our anti-C?
We need to look at the patient’s Genotype to see if the
patient is homozygous for r ’ S .
15
Human Erythrocyte Antigen (HEA) Genotyping by DNA Analysis Report
Report Date/Time:
May 15, 2013 06:50 AM
Lot #:
12-2
Chip Read Date/Time:
May 2, 2012 10:48 PM
Status:
Sample ID: Joseph, Judith
Chip ID: HEA82837_1
Florida Blood Services
10100 Martin Luther King Jr. St.
North St. Petersburg, FL 33716
Detail:
Ax(1)
Notes:
Polymorphism
RhCE-P103S
Result
Ax
AA
AA
BB
BB
BB
BB
BB
AA
BB
BB
BB
BB
BB
AA
BB
AA
AB
AA
AA
BB
AA
AA
AA
BB
RhCE-109Ins
RhCE-A226P
RhCE-L245V
RhCE-G336C
K1/K2
Kp
Js
JKA/JKB
FYA/FYB
GPA
GPBS
LUA/LUB
DIA/DIB
COA/COB
DO-793
DO-350
DO-323
LWA/LWB
SC1/SC2
GATA
GPB-Int5
GPB-230
FY-265
HbS173
Status Code
CV - Coefficient of Variation of intensities exceeds recommended maximum
LS - Low Signal: signal intensity below recommended minimum
HB - High Background: exceeds recommended maximum
IC - Indeterminate call
Approved By:
Signature:
Date:
Name (Print):
Title:
Notes
LOOK!!
L245V and G336C are
homozygous for B
Interpretation of HEA Genotyping
by DNA Analysis
Polymorphism
Result
Interpretation
RhCE L245V
BB
hr-B- also expresses partial C
RhCE G336C
BB
Very surprising.
FYA/FYB
BB
GATA
BB
Hmmm, OK.
[Red cells from patients that are FY*B but carry the GATA polymorphism are
phenotypically Fyb neg. Because carriers of this single nucleotide polymorphism do
express Fyb on cell types other than erythrocytes, they are not at risk for anti-Fyb
production.]3(p65)
18
Is Our Patient Remarkable?
Yes, this patient is remarkable. The 41 PRBC’s the patient was transfused didn’t cause
any transfusion reactions.
So we can deduce:
The patient didn’t make anti-hrB, even thought the patient is hrB-.
Or did make anti-hrB and the anti-hrB is clinically insignificant.
19
What Does This Mean Our Patient?
• Our patient is rare in phenotype, and genotype but having made anti- E, -C, -Fya,
and Jsa, locating antigen negative units should not be difficult.
• Patient’s Fyb is silenced
• This would eliminate the need for transfusing phenotypically Fy(a-b-) donor units. If
in the future we had to recommend phenotypically negative units, we can now
recommend genotypically negative units Fy(a-b+).
20
Conclusions
 Genotypes differ from phenotypes.
 Molecular testing helps supplement the limitations of serological techniques by
using DNA to analyze and predict red cell phenotypes by providing genotype
information.
 Genotyping is helpful for providing information for problematic serological cases
with complex or incomplete/questionable results.
 Though this relatively new tool in the field of blood banking has not replaced
serological testing, it continues to revolutionize and expand our knowledge of
immunohematology.
21
References
1. Roback JD, Grossman BJ, Harris T, Hillyer CD. Technical Manual. 17th ed.
Bethesda, Maryland: American Association of Blood Banks (AABB); 2011.
2. Harmening DM. Modern Blood Banking & Transfusion Practices. 6th ed.
Philadelphia, PA: F A Davis Company; 2012.
3. Ness PM, Moulds JM. BeadChip Molecular Immunohematology, Toward Routine
Donor and Patient Antigen Profiling by DNA Analysis. Springer Science+Business
Media; 2011.
4. HEA BeadChipTM Package Insert. Warren, NJ: BioArray Solutions; 2012.
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Thank You for Your Time
• Questions
23