User Manual Protrans HLA SSP Kits

User Manual Protrans HLA SSP Kits
i
User Manual
Protrans HLA SSP Kits
Molecular genetic DNA Typing, SSP technique
PROTRANS
Cyclerplate System
REF
200 070
200 080
200 030
200 020
200 010
200 011
200 050
200 090
200 040
201 201
200 048
200 049
IIVDI
l
H
i
PROTRANS
Domino System
Article
HLA- A*
HLA- B*
HLA- DRB1*
HLA- A*,-B*,-DRB1*
HLA- A*,- B*,- C*
V
C
0197
HLA- A*,- B*
HLA- DRB1*,- DQB1*
HLA- C*
Negative Control HLA
V
HLA- DQB1* high
C
HLA- DQB1* low
REF
201 071
201 072
201 070
201 077
201 073
201 074
201 075
201 076
201 078
201 079
201 080
Article
HLA- DRB1* 01
HLA- DRB1* 03
HLA- DRB1* 04
HLA- DRB1* 07/*09
HLA- DRB1* 08/*12
HLA- DRB1* 11
HLA- DRB1* 13
V
C
0197
HLA- DRB1* 14
HLA- DRB1*15
HLA- DRB1*16
HLA- DRB1* 15/*16
HLA- DQA1* high
For In Vitro Diagnostic use
Buffer D, R and Y
- 20°C
Cyclerplates and Domino Strips
2°- 8°C
See label on the Testkit
User Manual
Warning and Precautions
Protrans medizinische diagnostische Produkte GmbH
M
D- 68766 Hockenheim, Ketschau 2
Tel.: 0049-(0)6205–2929910 Fax: 0049-(0)6205-2929920
www.protrans.info - [email protected]
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
1 of 16
Content
1.
1.1
1.2
1.3
2.
2.1
2.2
2.3
3.
3.1
4.
4.1
4.2
5.
5.1
5.2
6.
6.1
6.2
6.3
7.
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
7.10
8.
8.1
8.2
8.3
8.4
8.5
9.
10.
11.
12.
Content
Quality and Certification
License ARMS™ technology
Symbols
Introduction
Intended Use
Summary
Principle of the Tests
Reagents
Contents of the Protrans SSP Kits
Warning and Precautions
Storage and shelf life
Instrument requirements
Programming the thermocycler
Specimen collection and preparation
DNA Isolation
Non-acceptable specimens
Materials
Materials provided
Additional materials, reagents and equipment required but not supplied
PCR – Polymerase Chain Reaction
Precautionary measures
Pre-PCR area
Post-PCR area
Instruction for PROTRANS PCR-SSP DNA Typing Kits
Preparation of the PCR
Preparation of the Master Mix for the PROTRANS PCR-SSP System
Dispensing the Master Mix in the PROTRANS Cyclerplates or Domino-Strips
Capping the PROTRANS Cyclerplates or Domino-Strips
Table: PROTRANS Cyclerplate System and PROTRANS Buffers
Table: Volume of PROTRANS Buffers and Taq Polymerase for Mastermix
Table: Number and Positions of Primer Mixes of PROTRANS Cyclerplate System
Table: PROTRANS Domino System and PROTRANS Buffers
Table: Volume of PROTRANS Buffers and Taq Polymerase for Mastermix
Table: Number and Positions of Primer Mixes of PROTRANS Domino System
Post PCR
Gel electrophoresis
Performing Gel electrophoresis
Result Interpretation and result documentation
Result Interpretation
Gel Interpretation
Limitations of the Procedure
Quality Control
Trouble Shooting
Literature
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
page
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Quality and Certification
We declare under our sole responsibility that the mentioned products are in compliance with the essential
requirements of the Directive IVDD 98/79 EG and EN ISO 13485:2003 and EN ISO 9001:2000 on design,
production and sales of HLA tissue typing products and are marked with the C symbol.
License ARMS™ technology
With the Protrans SSP Typing Kits the ARMS™ technology is used. The Products are sold under license of BTG
International Limited UK and DxS Ltd. UK. ARMS™ is the subject of worldwide patent property and is a trademark
of the AstraZeneca group of companies.
Symbols
Country
REF
NC
DE
GB
Artikelnummer
Article number
ES
IT
FR
Artikulo número
control negativo
Codice prodotto
controllo negativo
Numéro de l´article contrôle negatif
Negativkontrolle
negative controll
I
IVD
H
in-vitro Diagnostik
For in vitro diagnostic use
Lagern bei
Store at
Sólo para el diagnóstico in vitro
Solo per la diagnostica in vitro
Pour le diagnostic invitro
Almacenar
Conservare a
Conserver à
Haltbar bis
Expiration
Date
Caducidad
Scadenza
Péremption
1. Introduction
1.1 Intended Use
The Protrans HLA Sequence Specific Primer (SSP) Kits are intended for the determination of HLA-class I or class II alleles based on the PCR-SSP method.
1.2 Summary and Explanation
The HLA-system is a complex, co-dominantly inherited system of antigens which plays an important role in the
immune system by enabling it to distinguish „self“ from „non self“. In organ transplantation, HLA compatibility
between donor and recipient is one of the major determinants of transplant outcome. For this reason, the
determination of the individual combinations of HLA antigens is used as a basis for the selection of donors and
recipients.
New dimensions have been opened up to modern diagnostics by the development of DNA-based test methods.
HLA antigens partly differ from each other only by single amino acids within the polypeptide chain. Recognition of
these largely identical structures by serological means is almost impossible; for this reason, the resolution
capacity of the method is limited. As the DNA sequences of most important HLA alleles are now known,
variations in sequences can be identified at the DNA level with the help of synthetic oligo nucleotides. Utilizing
amplifications of genomic DNA (PCR, Polymerase Chain Reaction) together with specific primer pairs (SSP,
Sequence Specific Primer) it is possible to identify a large number of HLA alleles by molecular test methods.
1.3 Principle of the Test
Protrans HLA SSP Kits are intend to determine HLA-class I or –class II alleles based on a PCR method using
sequence specific primer (PCR-SSP). The principle of the SSP method is to generate an amplificate only when
the sequence of a primer is perfectly complementary to the target sequence of a DNA sample. On the other
hand, non-complementary primer do not bind to the DNA and for this reason no amplification takes place.
Using agarose gel electrophoresis, the amplified DNA can be determined. Successful amplification will generate
a DNA fragment of defined length which appears as a band in the gel. If no amplification takes place, no specific
band is seen.
The composition of the primer mixes in each well of the Cyclerplates and Domino Strips permits positive
identification of different specific alleles of each HLA locus.
The Protrans SSP kits are in Vitro Diagnostic Test systems for the diagnosis of the immune system of organ
donors and recipients and of patients receiving blood component substitution therapy.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
3 of 16
2. Reagents
2.1 Contents of the Protrans SSP Kits
1
Component 1
Cyclerplates or Domino Strips
2
Component 2
HLA class 1 Buffer D and Buffer Y
HLA class 2 Buffer R and Buffer Y
HLA class 1 HLA class 2 in combination Buffer D and Buffer Y
H
2-8°C
Oligonucleotides (dried)
H
Master Mix for Amplification
-20°C
PROTRANS Cyclerplates and PROTRANS Domino Strips
contain pre-pipetted, dried, specific Primer Mixes (oligonucleotides).
The positions and specificities of the Primer Mixes are LOT – specific and described in the Kit Inserts
Reaction Protocol, Primer MixTable, Amplification Table and Typing Table.
Number of Primer Mixes of each Testkit in the PROTRANS Cyclerplate System refer to: Table 7.7
Number of Primer Mixes of each Testkit in the PROTRANS Domino System refer to: Table 7.10
The PROTRANS Cyclerplates are fit into each other and packed in zip-lock pouches. Each pouch contains
10 or 20 Cyclerplates. The pouch and the upper Cyclerplate are marked with a label.
Name of the product, specificities of the pre-pipetted Primer Mixes, LOT number, storage temperature and
expiration date are written on the label.
Name of the product and LOT number are printed on each PROTRANS Cyclerplate.
Each well of the PROTRANS Cyclerplate is marked with a digit-letter combination from A1 to H12. Digits are
visible on the top, letters on the left edge of the Cyclerplate.
Primer Mix 1 of each Testkit is located at position H1.
For better orientation the lower edge of the Cyclerplate has been marked with a black line.
96 wells
8
7
6
5
4
3
2
1
16
15
14
13
12
11
10
9
24
23
22
21
20
19
18
17
32
31
30
29
28
27
26
25
40
39
38
37
36
35
34
33
48
47
46
45
44
43
42
41
56
55
54
53
52
51
50
49
64
63
62
61
60
59
58
57
72
71
70
69
68
67
66
65
80
79
78
77
76
75
74
73
88
87
86
85
84
83
82
81
96
95
94
93
92
91
90
89
The PROTRANS Domino Strips are fit into each other and packed in zip-lock pouches. Each pouch contains
12, 24 or 30 Domino Strips. The pouch is marked with a label.
On the label Name of the product, specificities of the pre-pipetted Primer Mixes, LOT number, storage
temperature and expiration date are written.
PROTRANS Domino System
Position Primer Mix 1 Domino Strip
8 wells
8
7
6
5
4
3
2
1
User Manual
16 wells
8
7
6
5
4
3
2
1
16
15
14
13
12
11
10
9
24 wells
8
7
6
5
4
3
2
1
16
15
14
13
12
11
10
9
PROTRANS HLA SSP Cyclerplate and Domino System
32 wells
24
23
22
21
20
19
18
17
8
7
6
5
4
3
2
1
Version 2.4
16
15
14
13
12
11
10
9
Rev.: 2009 10 20
24
23
22
21
20
19
18
17
page
32
31
30
29
28
27
26
25
4 of 16
2.2 Warning and Precautions:
IIVDI Reagents only for In Vitro Diagnostic use
The PROTRANS Testkits must be performed by well-trained and authorised laboratory technicians.
All reagents should be handled in accordance to good laboratory practice using appropriate
precautions.
In addition, handle all patient samples as potentially infectious. Do not pipette by mouth.
All used PCR-Cyclerplates and Domino Strips should be treated as potentially infectious and should
be destroyed according to the valid national guidelines.
Do not use reagents which are expired. See expiration date printed on the label.
Pre-PCR and Post-PCR rooms must be strictly separated.
Use separate pipettes in the Pre-PCR area and in the Post-PCR area
Ethidium bromide used for staining of DNA is a potential carcinogen. Always wear protective gloves
when handling Ethidium bromide and stained gels. Waste management according to national
guidelines.
Wear UV-blocking eye protection and avoid direct UV light when viewing or photographing gels.
See Material Safety Data Sheet (MSDS) for detailed information. Available from Protrans.
2.3 Storage and shelf life
Kit Components
Cyclerplates 96-well PCR plates
Domino-Strips
32-, 24-, 16-, or 8-well PCR-Strips
Ingredients
Primer (DNA Oligo-Nucleotides)
Cresol Red
I
H
2-8°C
See label
Ammonium Sulfate, Tris-Buffer, MgCl2, Glycerol,
-20°C See label
Cresol Red, dNTPs (Deoxyribonucleotides)
-20°C See label
Watery solution
Buffer
Y
Once the test kits have been opened, the remaining unused Cyclerplates or Domino Strips must be kept
closed in their original package. Reseal the ziplock to prevent moisture accumulation during storage.
Buffer
User Manual
D
R
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
5 of 16
3. Instrument requirements
3.1 Programming the Thermocycler
Thermal Cycling Program
For optimal results it is important to obtain rapid ramp times (1° - 2,5°C/s) and precise temperature control. The
following thermal cycler profile is optimized and validated with the thermocyclers given in 5.2.3. for use with the
PROTRANS SSP Cyclerplate System and Domino System.
The final volume of the amplification mix is
Initial denaturation
Denaturation
Annealing and Extension
Denaturation
Annealing
Extension
Hold
10 µl.
94°C
94°C
65°C
94°C
61°C
72°C
4°C
2 min
15 sec
60 sec
15 sec
50 sec
30 sec
15 min
Hold
10 cycles
20 cycles
Hold
See 5.2.3: list of validated Thermocyclers with 96-well block and heated lid.
4. Specimen collection and preparation
4.1 DNA Isolation
Genomic DNA may be obtained from all nucleated cells. Starting materials are EDTA- or citrate blood, buffy coat
or cell suspensions. A vast range of various protocols exist for the isolation of DNA. For PCR-SSP testing only
those methods which provide DNA of adequate quality and quantity should be considered.
The Protrans DNA Extraction Kit PROTRANS DNA Box 500/5000 provides DNA of high stability and quality.
All DNA extraction methods must be validated before routine use.
The concentration of the DNA should be adjusted to 50 – 100 ng/µl.
The ratio (purity) A260/A280 should be 1.6 – 1.8.
Concentration and purity of the DNA is of decisive importance for optimal test results.
DNA sample may be used immediately after isolation or stored at –20°C or below for extended period of time
(over one year) with no adverse effects on the results.
For storage and stability information of isolated DNA please refer to the technical information provided by the
DNA extraction test kit manufacturer.
Use either EDTA- or Citrate-blood samples for DNA extraction.
Do not use heparinized blood samples. Heparin inhibits the PCR.
4.2. Non-acceptable specimens
Contamination of the DNA by PCR inhibitors, such as haemoglobin, heparin, ethanol, ficoll-separated specimens
(buffy coat, cell suspensions), etc. can result in serious interference with the PCR reaction.
For this reason, heparin blood is not acceptable as a starting material for DNA isolation.
If the patient is on heparin therapy, use an alternative source of DNA.
Avoid the use of lipemic or haemolysed specimens.
The use of specimens collected without anticoagulant or frozen/thawed multiple times is not recommended since
these conditions may not provide sufficient quantity or quality of DNA for testing.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
6 of 16
5. Materials
5.1 Materials Provided
See 2.1 contents of the PROTRANS SSP kits.
5.2 Additional materials, reagents and equipment required but not supplied
All reagents and equipment other than recommended requires validation by the user.
5.2.1
UV – spectrophotometer for photometric DNA measurement
e.g. Lambda Scan 200, MWG www.THE.MWG.com
PC photometer program: KC 4, Bio-Tek Instruments, Inc. www.biotek.com
Use of other UV-spectral photometers must be validated by the user.
5.2.2
DNA Taq Polymerase
The following enzymes are validated for use with the PROTRANS SSP Kits:
•
AmpliTaq, Perkin Elmer, 5U/µl, Cat.No. N8010060
•
MBI Fermentas DNA Taq Polymerase, 5U/µl, Cat.No. EP0402
Use of other DNA Taq Polymerase enzymes must be validated by the user.
5.2.3
Thermocycler
The following Thermocyclers with 96-well block and heated lid are validated for use
with the PROTRANS SSP Kits:
•
PE 2700, Applied Biosystems, www.appliedbiosystems.com
(production PE 9600, Applied Biosystems, stopped)
•
PTC-100, PTC-200 MJ Research, Inc., www.mjr.com
Thermocyclers other than the recommended have to be user-validated.
Thermocyclers which have no adjustable pressure plate require an adaptor mat in order
to guarantee optimal heat transfer from the heat cover to the PCR tubes.
5.2.4
Pipettes
•
•
•
5.2.5
Disposables
•
Suitable Filtertips specific for each pipettes
•
•
5.2.6
Adjustable pipettes for volumes:
o 1-10µl
o 10-100µl
o 100-1000µl
Eppendorf Multipette Type 4720
8-channel pipette, e.g. 5-50µl adjustable pipetting volume
(Finnpipette, ThermoLabsystems Cat.No. 4510020)
1,5 ml polypropylene reaction tubes (e.g. Eppendorf Safe-Lock, Cat.No. 0030 120.86)
0.5 ml Combitips for Eppendorf Multipette Type 4720
Gel - Electrophoresis
Agarose (for molecular biology)
TAE–Buffer (1x) – electrophoresis buffer
•
TAE = Tris-buffer / conc. Acetic Acid (CH3COOH) / 0,5 M NA2-EDTA ph 8.0
Distilled water (dH2O)
Ethidium bromide solution (10mg/ml); Caution: Ethidium bromide is mutagenic (see 2.2.)
Magnetic stirrer with hotplate or microwave oven
DNA molecular weight marker (50 – 1000 bp ladder)
Power Supply
PROTRANS Electrophoresis System (PROTRANS Electrophoresis Unit or Gel-Check
REF 210 000, 210 001)
Gel documentation System
•
Polaroid camera with UV Filter and Polaroid film type 667
•
Gel documentation system
•
Transilluminator (312nm) UV-light
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
7 of 16
6. PCR – Polymerase Chain Reaction
6.1 Precautionary measures
PCR is an extremely sensitive method which can efficiently amplify even the smallest amounts of DNA.
It follows from this that even traces of contaminating DNA in a sample can be amplified in the PCR reaction and
falsifies the test results. One particular source of contamination is amplified DNA coming into contact with
samples which are still to be amplified. To avoid contamination with amplified material, it is recommended that
the work areas be strictly separated as follows:
6.2 Pre – PCR area:
All work carried out before PCR (DNA isolation and storage, preparations for the PCR, production and storage of
reagents and solutions for DNA extraction and PCR).
When working in the pre-PCR area, pipettes with aerosol protection should be used (filtered tips).
It is recommended to use a negative control with every amplification to detect contaminations.
6.3 Post – PCR area:
Thermocycler, gel electrophoresis, evaluation, storage of amplified DNA. Equipment and consumables from the
post-PCR area must not be taken into the pre-PCR area.
7 Instruction for PROTRANS SSP DNA Typing Kits
7.1 Preparation of the PCR
7.1.1
7.1.2
7.1.3
7.1.4
7.1.5
Out of the refrigerator collect the desired PCR-Cyclerplates or PCR-Domino-Strips.
Out of the freezer (-20°C) collect the DNA Taq Polymerase and
the corresponding Protrans Buffers. See Tables 7.5 and 7.8.
Place the PCR-Cyclerplates or the PCR-Domino-Strips and the DNA Taq Polymerase into the cooled
(-20°C) PROTRANS „PCR Workstation“ or into a 96-well PCR-rack.
With the Protrans Cutter (scissors) it is possible to cut the PCR-Cyclerplates
into single tests.
Thaw the Protrans Buffers D or R and Buffer Y.
7.2 Preparation of Master Mix for the Protrans PCR-SSP System
7.2.1
For each DNA sample to be tested pipette the appropriate volume of Buffer D or R and Buffer Y
and DNA Taq Polymerase into a 1,5ml reaction tube. See Tables 7.6 and Table 7.9.
7.2.2
Vortex Mastermix well
7.2.3
7.2.4
7.2.5
When using a negative control pipette 10 µl of this Mastermix without the DNA
into the appropriate well. See Tables 7.6, 7.7 and 7.9, 7.10.
Add to each Mastermix the appropriate volume of DNA recommended for the test
See Tables 7.6 and 7.9.
Vortex Mastermix well
7.3 Dispensing the Master Mix in the Protrans PCR-Cyclerplates or PCR-Domino-Strips
7.3.1
Dispense 10 µl of the Mastermix into each well of the PCR-Cyclerplate or PCR-Domino-Strip but not
into the negative control.
When pipetting at the upper edge of each well the drop will run down to the bottom. The bottom of the
wells must not be touched with the tip.
7.4 Capping the Protrans PCR-Cyclerplates or PCR-Domino-Strips
7.4.1
7.4.2
7.4.3
User Manual
Ensure that the Mastermix is at the bottom of all wells by either centrifugation or gentle tapping the
Cyclerplate or Domino-Strip onto the work bench.
Visual control ensures that the Mastermix is at the bottom of each well.
Seal Cylerplates or Domino-Strips carefully using the caps provided or with re-usable PROTRANS
24-/96-well Coverplates. Ensure caps completely seal the wells to prevent evaporation.
Place the Cyclerplate or Domino-Strips into Thermocycler and start amplification.
Final volume in each well is 10 µl.
If unable to start amplification immediately after PCR set-up, the PCR-Cyclerplates or -Domino-Strips
must be stored at 2°-8° C. Start amplification within 2 hours.
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
8 of 16
PROTRANS Cyclerplate System
7.5
REF
Article
Buffer
Buffer
200 070
HLA- A*
D
Y
200 080
HLA- B*
D
Y
200 090
HLA- C*
D
Y
200 030
HLA- DRB1*
R
Y
200 040
HLA- DQB1* low
R
Y
200 020
HLA- A*,- B*,- DRB1*
D
Y
200 010
HLA- A*,- B*,- C*
D
Y
200 011
HLA- A*,- B*
D
Y
201 201
Negative Control HLA
D or R
Y
200 050
HLA- DRB1*- DQB1*
R
Y
200 048
HLA- DQB1* high
R
Y
200 049
HLA- DQA1* high
R
Y
7.6
Volumes of PROTRANS Buffers and Taq Polymerase for the Master Mix
200 070
HLA- A*
D
70
Y
140
1,6 µl -
DNA
50-100
ng/µl
50 µl
200 080
HLA- B*
D
140
Y
280
3,3 µl -
100 µl
200 090
HLA- C*
D
70
Y
140
1,6 µl A3, 6, 9, 12
50 µl
200 030
HLA- DRB1*
R
70
Y
140
1,6 µl A3, 6, 9, 12
50 µl
200 040
HLA- DQB1* low
R
46
Y
94
1,1 µl C2, 4, 6, 8, 10, 12
34 µl
200 020
HLA- A*,- B*,- DRB1*
D
280
Y
560
6,5 µl A12
200 µl
200 010
HLA- A*,- B*,- C*
D
280
Y
560
6,5 µl A12
200 µl
200 011
HLA- A*,- B*
D
210
Y
420
5,0 µl H10
150 µl
201 201
Negative Control HLA
200 050
HLA- DRB1*- DQB1*
R
115
Y
230
2,8 µl A 3,9 C 5,11
200 048
HLA- DQB1* high
R
140
Y
280
3,3 µl B 6,12
200 049
HLA- DQA1* high
R
70
Y
140
1,6 µl A 3,6,9,12
REF
7.7
Protrans SSP
Cyclerplate System
Buffer/µl
Taq
Buffer/µl
D or R
Y
NC
in Position
Volume as indicated for each REF
85 µl
100 µl
50 µl
Number and Positions of Primer Mixes of PROTRANS Cyclerplate System
200 070
Protrans Cyclerplate
System
HLA- A*
200 080
HLA- B*
2x 48
H1, 7
200 090
HLA- C*
4x 24
H1, 4, 7, 10
200 030
HLA- DRB1*
4x 24
H1, 4, 7, 10
A 3, 6, 9, 12
200 040
HLA- DQB1* low
6x 14
H1, 3, 5, 7, 9, 11
C 2, 4, 6, 8, 10, 12
200 020
HLA- A*,- B*,- DRB1*
96
A*: H1; B*: H4; DRB1*: H10
A12
200 010
HLA- A*,- B*,- C*
96
A*: H1; B*: H4; Cw*: H10
A12
200 011
HLA- A*,- B*
73
A*: H1; B*: H4
H10
200 050
HLA- DRB1*- DQB1*
2x 38
DRB1* H1, H7; DQB1* H4, H10
A3, C5, A9, C11
200 048
HLA- DQB1* high
2x 47
H1, H7
B6, B12
200 049
HLA- DQA1* high
4x 24
H1, H4, H7, H10
A3, A6, A9, A12
REF
User Manual
Number of
Primer Mixes
4x 24
Position of
1. Primer Mix
H1, 4, 7, 10
PROTRANS HLA SSP Cyclerplate and Domino System
Position NC
-
Version 2.4
A 3, 6, 9, 12
Rev.: 2009 10 20
page
9 of 16
PROTRANS Domino System
7.8
REF
Article
Buffer
Buffer
201 071
HLA- DRB1* 01
R
Y
201 072
HLA- DRB1* 03
R
Y
201 070
HLA- DRB1* 04
R
Y
201 077
HLA- DRB1* 07/*09
R
Y
201 073
HLA- DRB1* 08/*12
R
Y
201 074
HLA- DRB1* 11
R
Y
201 075
HLA- DRB1* 13
R
Y
201 076
HLA- DRB1* 14
R
Y
201 078
HLA-DRB1*15
R
Y
201 079
HLA-DRB1*16
R
Y
201 080
HLA- DRB1* 15/*16
R
Y
7.9
Volumes of PROTRANS Buffers, Taq Polymerase and DNA for the Master Mix
Protrans SSP Domino
System
Buffer/µl
Buffer/µl
201 071
HLA- DRB1* 01
R
46
Y
94
1,1 µl
13
DNA
50-100
ng/µl
34 µl
201 072
HLA- DRB1* 03
R
70
Y
140
1,6 µl
20
50 µl
201 070
HLA- DRB1* 04
R
95
Y
190
2,2 µl
32
70 µl
201 077
HLA- DRB1* 07/*09
R
40
Y
80
1,0 µl
12
30 µl
201 073
HLA- DRB1* 08/*12
R
70
Y
140
1,6 µl
24
50 µl
201 074
HLA- DRB1* 11
R
95
Y
190
2,2 µl
30
70 µl
201 075
HLA- DRB1* 13
R
70
Y
140
1,6 µl
24
50 µl
201 076
HLA- DRB1* 14
R
75
Y
145
1,7 µl
27
55 µl
201 078
HLA-DRB1*15
R
70
Y
140
1,6 µl
24
50 µl
201 079
HLA-DRB1*16
R
40
Y
80
1,0 µl
-
30 µl
201 080
HLA- DRB1* 15/*16
R
46
Y
94
1,1 µl
16
34 µl
REF
7.10
Taq
Polymerase
NC
in Position
Number and Positions of Primer Mixes of PROTRANS Domino System
201 071
Protrans
Domino System
HLA- DRB1* 01
201 072
HLA- DRB1* 03
20
1
20
201 070
HLA- DRB1* 04
32
1
32
201 077
HLA- DRB1* 07/*09
12
1
12
201 073
HLA- DRB1* 08/*12
24
1
24
201 074
HLA- DRB1* 11
30
1
30
201 075
HLA- DRB1* 13
24
1
24
201 076
HLA- DRB1* 14
27
1
27
201 078
HLA-DRB1*15
24
1
24
201 079
HLA-DRB1*16
12
1
-
201 080
HLA- DRB1* 15/*16
16
1
16
REF
User Manual
Number
Primer Mixe
13
Position
1.Primer Mix
1
Position
NC
13
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
10 of 16
8. Post PCR
After thermal cycling, remove the PCR-Cyclerplate or -Domino-Strips and proceed to gel electrophoresis. If
not performing electrophoresis immediately, store the Cyclerplate at 4°C for up to two days; or at –20°C for
longer.
8.1 Gel electrophoresis
The PCR products are identified using agarose gel electrophoresis followed by the detection of the DNA
bands in UV light.
8.2 Performing gel electrophoresis
A 2% solution of agarose is prepared by boiling 4g of agarose in 200ml of 1x TAE using a magnetic stirring
hotplate or microwave oven until the solution becomes clear. Allow the solution to cool to below 60°C before
adding 10µl of ethidium bromide solution.
Ethidium bromide – solution (10mg/ml)
Dissolve 100mg of ethidium bromide in 10ml of distilled water. Store at 2-8°C protected from light.
Caution: Ethidium bromide is mutagenic and toxic. Always wear protective gloves when working
with ethidium bomide (also in diluted form). In case of contact with the skin, wash off immediately
with copious amounts of water.
Place the PROTRANS UV Gel Tray on a horizontal surface. Fill the agarose solution into the PROTRANS UV
Gel Tray avoiding air bubbles, and place the PROTRANS Gel Combs (4 combs with x 4x25 slots, each
containing 10µl) into the gel. The distance of the combs’ teeth correspond to that of a standard 8-channel
pipette which allows rapid load of the samples onto the gel.
After polymerization (about 30-60 minutes at room temperature) remove the combs and place the gel into the
gel chamber that has been filled with 1x TAE buffer. The level of electrophoresis buffer should be 2-3 mm
above the gel surface and completely cover the gel.
Carefully remove the caps or Protrans Coverplates from the Cyclerplates or Domino strips and pipette all
PCR products with a 8 channel pipette into the slots of the gel. Make sure that the order in which you load the
samples is standardized. The PROTRANS PCR Primer Mixes are containing Loading Buffer (glycerol and
cresol-red) don´t use an additional loading buffer.
Caution:
Sudden movement of the Cyclerplate or Domino Strip can disperse amplified product,
contaminating the laboratory and may require repetition of the test.
To measure the size of the PCR Products a molecular weight marker standard (50–1000 bp ladder) can be
included in each row.
Run the PROTRANS Electrophoresis for about
20 min at 150 V
the PROTRANS Gel Check for about
5 min at 200 V
8.3 Result Interpretation and result documentation
Place the gel on a UV Transilluminator or on the PROTRANS UV Transilluminator
(suitable face protection against UV radiation should be worn). For interpretation and documentation of the
results place the PROTRANS Number-Plate on the Gel and take a Polaroid picture
8.4 Result Interpretation
Each of the PROTRANS primer mixes contains a non-allelic amplification control primer pair which amplifies
either a part (89 bp) of the β-globin gene (HLA class I) or a part (440 bp) of the C-reactive-protein gene (HLA
class II). The concentration of these primer pairs is lower than the allele specific primer pairs and their
purpose is to provide an internal control of successful PCR amplification. This amplification generally always
occurs, i.e. both in presence or in absence of an allele-or group-specific PCR fragment. The control band can
therefore generally be seen in all PCR reactions. In the presence of an allele specific PCR product the control
band can appear weaker or is completely missing. This is not a limitation of the method, as the specific band
provides a check on the success of the PCR amplification.
The composition of the primer mixes permits positive identification of the HLA characteristics. The
interpretation is based on whether a specific band is present or not. The size of the amplified DNA fragment
does not need to be taken into consideration when evaluating the test, nevertheless it might be helpful for the
test interpretation. For evaluation the pattern of the specific bands is transferred to the LOT specific Protrans
Reaction Protocol supplied and the typing result read off with the aid of the reaction pattern. For the
interpretation the Primer Mix tables and the Amplification tables are very helpful. Additionally the Score
Program (www.ihwg.org) can be used for detailed result interpretation.
If the testkit contains a negative control, any band in this amplification is evidence of contamination. The
results of the test would be invalid and the test must be repeated.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
11 of 16
8.5 Gel Interpretation
HLA class I
PROTRANS
Positive reaction
HLA class II
Gel pocket
Gel pocket
Specific band
>100-900bp
Control band
440bp
Control band
89 bp
Specific band
<260bp
Primer cloud
Primer cloud
1.
If weak bands of incorrect size are present, disregard them if the overall strength and clarity of the
amplification is good.
2.
Unused primer will form a diffuse band below 50 base pair.
3.
Occasionally the formation of primer-dimers (< 80 bp) can be observed.
The primer-dimers do not invalidate the test. A wrong interpretation of the result can be avoided by
checking the approximate product size against the correct product size of the PROTRANS Primer Mix
Table (see package insert).
4.
Some of the primer mixes of HLA-A (mixes 5, 16, 19 and 24) and of HLA-B (mixes 5, 9, 43 and 47) will
give short PCR products (< 200 bp).
These PCR products might be difficult to distinguish from the 89 bp control PCR product.
In general, these specific products will give a much stronger signal than that of the controls and will not
have migrated as far into the gel as the control bands. If you are not sure whether the strong signal is
due to a specific or a generic PCR product, you might let the gel run for an additional 15 minutes at a
lower voltage. This way the specific band will be separated from the control band and you will be able to
clearly see a double signal at this position: a very strong, specific, and a weaker, shorter, control band.
5.
Some lanes have two or more different sizes of PCR products. These wells have multiplexed primer
pairs which give rise to different amplicons depending upon the allele present. Refer to the locus specific
tables in the LOT specific kit inserts
6.
False negative reactions can be caused by
•
inefficient amplification
•
poor quality of DNA
•
uneven placement of the PCR plate in the block of the thermocycler
•
temperature variations across the wells of the thermal cycler itself
•
inadequate cycler calibration
7.
It is possible, in rare instances, that the false negatives are due to a new or yet uncharacterized allele.
In such cases it is recommended to repeat the test using another technique (SBT)
8.
The test must be repeated if
•
absent control bands with no specific amplification are indicating failed reactions
•
there is an apparent homozygous result, or the missed reaction could change an allele assignment
•
the reaction pattern gives no clear result
•
the reaction pattern shows the prescence of 3 alleles
•
the negative control is not negative
9.
If alleles can be determined in the presence of a failed PCR reaction, and that failed reaction does not
change the allele assignment, the test does not need to be repeated.
10. For detailled informations refer to the HLA locus- and LOT specific kit inserts.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
12 of 16
9. Limitation of the Procedure
1.
Intensity of positive bands will vary due to the quality and amount of the PCR product.
Quality of the PCR product will directly affect the intensity of the specific and the control bands
visualized through the UV Transilluminator.
In case of missing internal control bands and no accounted alleles the test should be repeated.
2.
DNA samples may be used immediately after isolation or stored at –20°C or below for extended
periods of time (over 1 year) with no adverse effects on test results.
3.
Performance of the test can only be guaranteed if enclosed instructions are strictly adhered to.
4.
The SSP HLA Typing tests should only be used for initial HLA typing.
Other clinical and diagnostics findings should be used in addition,
when determining suitability for transplant.
5. Use of the PROTRANS SSP HLA typing kits cannot resolve all combinations.
10. Quality Control
Each manufactured LOT is checked against a panel of DNA samples representative of specific detection by the
primer. See certificates of Analysis for each LOT.
All PROTRANS SSP kits
are produced on the basis of the quality standards
EN ISO 9001:2008,
EN ISO 13485:2003 + AC:2007
and attachment IV of the IVDD 9879/EG.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
13 of 16
11. Troubleshooting
Problem
Possible Cause
Solution
No PCR products.
Multiple thawing and freezing of the
Mastermix (Buffer D and R).
Aliquot the Mastermix and freeze aliquots:
For the PROTRANS Cyclerplate System make
aliquots of 280µl and freeze them.
For the PROTRANS Domino System make
aliquots of 23, 46, 69 and 92µl and freeze them.
Dry PCR tubes
after amplification.
Wells not sealed properly.
Repeat test and seal wells properly.
No Ethidium bromide in the gel.
Re-stain gel
Control of Thermocycler by
Protrans Cycler Check, REF 200 100
Repeat test with validated Thermocycler
(see 5.2.3).
The described program applies to the recommended thermal cyclers. Other thermal cycler as
recommended have to be user-validated.
Repeat test with correct amplification program
Repeat test with validated DNA Taq Polymerase
(see 5.2.2).
Repeat test with correct DNA concentration.
Repeat test with a new, re-extracted
DNA sample.
Repeat test with a new, re-extracted
DNA sample out of EDTA-/Citrate-blood.
Repeat test with a known reference DNA to
verify activity of Taq Polymerase.
Control of thermocycler by
Protrans Cycler Check, REF 200 100
Repeat test with validated Thermocycler
(see 5.2.3).
The described program applies to the recommended thermal cyclers. Other thermal cycler as
recommended have to be user-validated.
The provided rack from PE (sample holder)
must not be used in combination with the
96-well PCR Cyclerplates.
Repeat test with re-extracted DNA, from
appropriate sample (EDTA or citrate blood).
Use approx. 100 ng/µl DNA.
After washing the DNA pellet with wash buffer
including ethanol, allow sample to dry thoroughly
Repeat test with correct DNA concentration.
Repeat test with a new, re-extracted
DNA sample.
Repeat test with a known reference DNA to
verify activity of Taq Polymerase.
Control of Thermocycler by
Protrans Cycler Check, REF 200 100.
Repeat test with validated Thermocycler
(see 5.2.3).
The described program applies to the recommended thermal cyclers. Other thermal cycler as
recommended have to be user-validated.
Repeat test with correct amplification program.
Inefficient Thermocycler, e.g. block defect.
No visible bands in gel.
Incorrect Thermocycler program used.
Wrong Taq Polymerase used.
DNA concentration used was too high/low.
Degraded DNA (smears in gel lanes).
PCR inhibitors in the genomic DNA or
impure DNA (see 4.2).
Lack of DNA Taq Polymerase activity.
Inefficient Thermocycler, e.g. block defect.
Weak specific bands.
Contact of the PCR plate to the block of
the thermocycler is not correct.
No control bands
in the gel.
PCR inhibitors (heparin, ficoll, ethanol)
in the genomic DNA.
Impure DNA (see 4.2).
DNA concentration used was too high/low.
Degraded DNA (smears in gel lanes).
Lack of DNA Taq Polymerase activity.
Inefficient Thermocycler, e.g. block defect.
Non-specific bands.
Incorrect thermocycler program used.
DNA concentration used was too high.
Wrong Taq Polymerase used.
No visible control bands Inefficient thermocycler, e.g. block defect.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Repeat test with correct DNA concentration.
Repeat test with validated DNA Taq Polymerase
(see 5.2.2).
Control of thermocycler by
Protrans Cycler Check REF 200 100.
Repeat test with validated Thermocycler
(see 5.2.3).
The described program applies to the recommended thermal cyclers. Other thermal cycler as
recommended have to be user-validated.
Version 2.4
Rev.: 2009 10 20
page
14 of 16
Problem
Possible Cause
Contact of the PCR plate to the block of
No visible control bands. the thermocycler is not correct.
Incorrect Thermocycler program used.
Wrong Taq Polymerase used.
In case of a homozygous typing result.
No visible control bands
(continued).
DNA not evenly re-suspended in original
diluent or in Mastermix.
Inefficient Thermocycler,
e.g. block defect.
Specific bands
occasionally missing.
Contact of the PCR plate to the block of
the Thermocycler is not correct.
DNA not evenly re-suspended in original
diluent or in Mastermix.
Wrong loading of the agarose gel.
Inefficient Thermocycler,
e.g. block defect.
Excess of DNA.
False positive bands.
Excess of DNA Taq Polymerase.
Extensive delay between PCR set-up and
start of thermal cycling.
Mis-interpretation of primer-dimer as
specific amplification.
DNA contaminated with other DNA or
PCR product.
Gel is too thin due to excess evaporation
while heating.
Agarose not completely dissolved.
Overall fuzzy bands,
smeared lanes.
Occasional faint lanes.
Overheating gel, too high voltage .
Heavy streaking in random wells can be
caused by uneven suspensions of DNA .
Rapid release of amplified product during
gel loading can cause product to float out
of well.
Product floated out of gel slot.
Wrong amount of Ethidium bromide used.
Gel picture too dark.
Gel tray not UV-transparent.
Incorrect camera setting.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Solution
The provided rack from PE (sample holder)
must not be used in combination with the
96-well PCR Cyclerplates.
Repeat test with correct amplification program.
Repeat test with validated DNA Taq Polymerase
(see 5.2.2).
Repeat test.
Vortex DNA sample and Mastermix thoroughly
before PCR set-up and repeat test .
If two alleles can be positively identified, no
action need to be taken.
In all other cases it is recommended to repeat
the test.
Control of Thermocycler by
Protrans Cycler Check REF 200 100.
Repeat test with validated Thermocycler
(see 5.2.3).
The described program applies to the recommended thermal cyclers. Other thermal cycler as
recommended have to be user-validated.
The provided rack from PE (sample holder)
must not be used in combination with the
96-well PCR Cyclerplates.
Vortex DNA sample and Mastermix thoroughly
before PCR set-up and repeat test .
If two alleles can be positively identified, no
action need to be taken.
In all other cases it is recommended to repeat
the test.
Control of gel and gel lanes concerning the
loaded primer mixes.
Control of Thermocycler by
Protrans Cycler Check REF 200 100.
Repeat test with validated Thermocycler
(see 5.2.3).
The described program applies to the recommended thermal cyclers. Other thermal cycler as
recommended have to be user-validated.
Repeat test with adequate amount of DNA
Concentration ~ 100ng/µl.
Repeat test with adequate amount of Taq.
Validate each LOT of DNA Taq Polymerase
before using it routinely.
Start amplification directly after PCR set-up or
store pipetted Cyclerplate at 2°-8°C until start of
amplification (max. for 2 hours).
Check correct band size.
Separate strictly pre-PCR area from post-PCR
area. Perform wipe test.
Compensate for lost volume by adding water.
Boil for an additional 30 seconds after melting.
Lower voltage.
Using an 8 channel pipettor, mix the PCR
product up and down twice before loading.
Use slow, steady pipetting when loading gel.
Pipette tip needs to be properly aligned with the
slots of the agarose gel.
Use 5µl Ethidium bromide (10mg/ml) for each
100ml gel solution.
Remove gel from tray before viewing.
Use PROTRANS Electrophoresis Equipment
(REF 210 000).
Increase exposure time or aperture setting.
Version 2.4
Rev.: 2009 10 20
page
15 of 16
Problem
Gel picture too bright.
Possible Cause
Excessive amount of Ethidium bromide
used.
Incorrect camera setting
Solution
Use 5µl ethidium bromide (10mg/ml) for each
100ml gel solution.
Increase exposure time or aperture setting.
11. Literatur / References / Références Bibliographiques/ Bibliografia
1.
U. Forssmann, J. Mytilineos, S. Scherer, and G. Opelz: A method for HLA-DQA Typing by the PCR-SSP
Technique. Transplant Int 1994, 7: 515-518.
2.
J. Mytilineos, M. Lempert, D. Middleton, F. Williams, C. Cullen, S. Scherer, and G: Opelz: HLA Class I DNA
Typing of 215 “HLA-A, -B, -DR zero mismatched Kidney transplants”. Tissue Antigens 1997, 50: 355-358.
3.
J. Mytilineos, U. Christ, M. Lempert, and G: Opelz: Comparison of typing results by serology and PCR with
sequence specific primer for HLA-Cw in 650 individuals. Tissue Antigens 1997, 50: 395-400.
4.
J. Mytilineos, M. Lempert, S. Scherer, V. Schwarz, and G: Opelz: Comparison of serological and DNA PCR-SSP
typing results for HLA-A and HLA-B in 421 black individuals. Human Immunology 1998, 59: 512-517.
5.
J.G. Bodmer, S.G.E. Marsh, E.D. Albert et al.: Nomenclature for factors of the HLA system, 1996. Human
Immunology V 53: 98-129, March 1997.
6.
Arnett, K.L. and Parham, P.: HLA Class I nucleotide sequences, 1995. Tissue Antigens V 46: 217-257, 1995.
7.
Olerup, O. and Zetterquist, H.: HLA-DR typing by PCR amplification with sequence specific primer (PCR-SSP) in
2 hours. An alternative to serological DR typing in clinical practice including donor-recipient matching in
cadaveric transplantations. Tissue Antigens V 39: 225-235, 1992.
8.
Specific enzymatic amplification of DNA in vitro: The Polymerase Chain Reaction, Mullis et all, Cold Spring
Harbour Symposia on quantitive biology, Vol. 51, No.1, p. 263-273; 1986.
9.
Bunce, O'Neill, Barnado, Krausa, Browning, Morris, Welsh (1995), Phototyping: comprehensive DNA Typing for
HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 mixes utilizing sequence-specific primer
(PCR-SSP), Tissue Antigens 46: 355-367.
10. Blasczyk, Hahn, Wehling, Huhn, Salama (1995), Complete subtyping of the HLA-A locus by sequence specific
amplification followed by direct sequencing or single-strand conformation polymorphism analysis, Tissue
Antigens 46: 86-95.
User Manual
PROTRANS HLA SSP Cyclerplate and Domino System
Version 2.4
Rev.: 2009 10 20
page
16 of 16
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