molecular genetic blood group typing - bag health care · 6determine rhd zygosity of partners of...

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