molecular genetic blood group typing - bag health care · 6determine rhd zygosity of partners of...
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Application
Genotype multi-transfused recipients
Genotype patients after ABO-incompatible bone marrow transplantation
Determine RHD zygosity of partners of alloimmunized D-negative womenbefore pregnancies
Genotype Rh D negative donors with C or E in order to exclude the presence of the RHD gene and thus preventing anti-D alloimmunization of recipientscaused by hidden Rh D variants in RBC units
Identify genotype in case of weakly expressed Rh D (e.g. DEL) in donors
Confirm weak D genotype in recipients in order to avoid the donation of Rh D negative blood units
Quality control of serological methods
External Quality Assurance trials
Result according to Transfusion guideline
Rhesus D positiveCcD.ee Kell positive
microwell plateantiseradilution 1:5
agglutinationstrength (1) -1
tube testimmediate spin
questionableagglutinationwith monoclonalAnti-D
Final resultweak D type 1
Rhesus prophylaxisnot required
Transfusion of D positive red bloodcells is possible
Result according to Transfusion guideline
Rhesus D negative, Ccddee Kell negative
microwell plateantisera dilution 1:5
negative reaction
Final result: D category VI type II
Rhesus prophylaxis required
Transfusion of D negative red blood cells is required
Contents of our BAGene SSP kits
PCR plates or strips with prealiquoted, driedand colored reaction mixes containing allelespecific primers, internal control primers(specific for the HGH gene) and nucleotides.
10 x PCR buffer
PCR strip caps
Worksheets and Evaluation Diagrams
Instructions for Use
Evaluation of Results - RHD-RHCE genotyping
Test Procedure - Workflow
DNA Extraction 30 min
Preparation of Mastermix 30 min
PCR 1h 30 min
Gel Electrophoresis 30 min
Evaluation
Preparation of the PCR reaction mixes
BAGene DNA-SSP KitsPipetting Chart
10 x
PC
R-
buffe
r
DN
A-s
olut
ion
PCR reaction vessels:
Aqu
a de
st.
Taq-
Poly
mer
ase
Preparation of the mastermix depending on the number of reaction mixes
1 2 3 4
No. of mixes
DNA-solution (50-100 ng/µl)
Aqua dest. 10 x PCR-buffer Taq-Polymerase(5 U/µl)
total volume, app.
6 7 50 7 0,5 65 µl
7 9 70 9 0,7 90 µl
8 10 80 10 0,8 100 µl
12 14 112 14 1,1 140 µl
13 16 128 16 1,3 160 µl
16 19 152 19 1,5 190 µl
mix well (vortex)
add 10 µl to each well of the PCR-strips/-plates
10 µl
80 µl
10 µl0,8 µl
reaction mix
e.g. ABO-TYPE, KKD-TYPE orWeak D-TYPE
ABO-, KKD-, or Weak D-TYPE - 8 mixes
RH-TYPE - 13 mixes
D Zygosity-TYPE, MNS-TYPE preparation for 6 mixes
HPA-TYPE - 12 mixes
ABO-TYPE variant or Partial D-TYPE - 16 mixes
RHD-TYPE Asia - 7 mixes
Investigation StrategyABO blood group typing
Serological Determination
ABO unclear e.g. weak antigenexpression or weak and unexpected reactions in reverse testing respectively
unambiguous determinationof ABO including subgroups
confirmation bymolecular genetics
clarification by sequencing in case of ambiguous genotype
BAGeneABO-TYPE
BAGeneABO-TYPE variant
ABO genotype
O1
non O1
O2
nonO
2B non
BA
2non
A2
O1v
Ax/ B
3A
x / B3
Ael/ A
wA
wA
wA
3/ Bx
Bw
ABO Genotyping
BAGene ABO-TYPE variant genotype O1vAx
Investigation StrategyRhesus typing
Serological Determination
partial D
unambiguousdetermination
confirmation bymolecular genetics
weak D
ambiguous Rhesus phenotype
DEL UnclearC, c, E, e
BAGenePartialD-TYPE
BAGeneWeakD-TYPE
BAGene RH-TYPE
RHD / RHCE genotype
clarification by sequencing in case of ambiguous genotype
RHD, RHCE Genotyping
RH
D Intron 4 / Exon 7
RH
D Intron 7
RH
D(W
16X),RH
DΨ
non RH
DΨ
non RH
D(K
409K)
RH
D(K
409K)
RH
D(M
295I)
RH
D(IVS3+1G
>A)
Cde
s C
c E
e Cw
BAGene RH-TYPE genotype RHD(K409K) Ccee
Genotyping partial D
RH
D Exon 1
RH
D Exon 2 / C
RH
D Exon 3
RH
D Exon 4
RH
D Exon 5
RH
D Exon 6
RH
D Exon 7
RH
D Intron 7/ Exon 8
RH
D Exon 9
RH
D Exon 10
D cat. VII
DH
Mi
DN
B
D cat. II
DA
U
RH
D(K
409K)
BAGene Partial D-TYPE genotype D cat. VI type II
Genotyping weak D
weak D
type 1
weak D
type 2
weak D
type 3
weak D
type 4.0/4,1
weak D
type 4.2
weak D
type 5
weak D
type 11
weak D
type 15
BAGene Weak D-TYPE genotype weak D type 4.2
RHD Genotyping „Asia“
BAGene RHD-TYPE Asia genotype RHD positive / weak D type 17
RH
D Intron 4 / Exon 7
RH
D(K
409K)
non RH
D(K
409K)
weak D
type 15
non weak D
type 15
weak D
type 17
non weak D
type 17
RHD Zygosity
Dow
nstream
Rhesus B
ox
Hybrid
Rhesus B
ox
Dow
nstreamR
hesus Box
Hybrid
Rhesus B
ox
Dow
nstream
Rhesus B
ox
Hybrid
Rhesus B
ox
DD Dd dd
BAGene D Zygosity-TYPE
Genotyping KEL, JK, FY
KEL*1
KEL*2
JK*A
JK*B
FY*A
FY*B
FY*null01
FY*X
BAGene KKD-TYPE genotype KEL*1/KEL*2; JK*A/JK*B; FY*B/FY*null01
Genotyping HPA
BAGene HPA-TYPE genotype HPA-1a/a; 2a/b; 3a/a; 4a/a; 5a/b; 15 a/b
HPA
- 1a
HPA
- 1b
HPA
- 2a
HPA
- 2b
HPA
- 3a
HPA
- 3b
HPA
- 4a
HPA
- 4b
HPA
- 5a
HPA
- 5b
HPA
- 15a
HPA
- 15b
Genotyping MNS
BAGene MNS-TYPE genotype NSs
GYPA
- M
MN
S1
GYPA
- N
MN
S2
GYPB
- S M
NS3
GYPB
- s M
NS4
Molecular genetic blood group typing by the use of PCR-SSP techniqueM Prager, BAG Health Care, Lich, Germany
presented at the FDA Workshop on Molecular Methods in ImmunohematologyBethesda 25 – 26 Sept 2006published in Transfusion 2007;47:54S-59S.
ABO genotyping for diagnosis of unusual ABO blood groups:
A Comparative Study in German Blood Donor CentersM Prager, EA Scharberg, FF Wagner, J Burkhart, A Seltsam
Presentation at 40. Annual Meeting of the German Society for Transfusion Medicineand Immunohematology (DGTI) September 18-21, 2007
Recently published, presented soon