Document
Olerup SSP
®
Product Insert
Page 1 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Olerup SSP®
Instructions for Use
For In Vitro Diagnostic Use
INTENDED USE
Olerup SSP® HLA Typing Kits are qualitative in vitro diagnostic kits for the DNA
typing of HLA Class I and HLA Class II alleles. The products are used by trained
professionals in medical settings for the purpose of determining HLA phenotype.
The source material tested is DNA.
SUMMARY AND EXPLANATION
Human leukocyte antigens (HLA) used to be determined by the lymphocytotoxicity
test. However, this test has been replaced by polymerase chain reaction (PCR)based DNA typing techniques due to its error rate and lack of allele level
resolution. In most PCR-based techniques, the PCR process is used only as an
amplification step of needed target DNA and a post-amplification step to
discriminate between the different alleles is required. In contrast, in the PCR-SSP
methodology (sequence-specific primer – SSP), the discrimination between the
different alleles takes place during the PCR process. This shortens and simplifies
the post-amplification step to a simple gel electrophoresis detection step. The SSP
test results are either positive or negative, which abolishes the need for
complicated interpretation of results. In addition, the typing resolution of the PCRSSP is higher than for other PCR-based typing techniques as each primer pair
defines two sequence motifs located in cis, i.e. on the same chromosome.
Furthermore, due to the synthetic nature of SSP reagents stability has been
improved and lot to lot variation reduced.
PRINCIPLE OF PROCEDURE
The PCR-SSP methodology is based on the principle that completely or almost
completely matched oligonucleotide primers without 3-‘end mismatches are more
efficiently used in the PCR reaction, than mismatched primers by thermo-stable
DNA polymerases without proof-reading properties. Primer pairs are designed to
be matched with single alleles or group(s) of alleles depending upon the degree of
typing resolution required. With strictly controlled PCR conditions, matched or
almost completely matched primer pairs allow amplification to occur, i.e. a positive
result, whereas mismatched primer pairs doesn’t allow amplification to occur, i.e. a
negative result.
After the PCR process, the amplified DNA fragments are size-separated e.g. by
agarose gel electrophoresis, visualized by staining with ethidium bromide and
exposure to ultraviolet light, documented by photography and interpreted.
Interpretation of PCR-SSP results is based on the presence or absence of specific
PCR product(s). The relative sizes of the specific PCR product(s) may be helpful
in the interpretation of the results. The PCR-SSP methodology for HLA was
originally described by O. Olerup in 1991 and 19921,2.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 2 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Since the PCR process may be adversely affected by various factors (e.g.
pipetting errors, too low DNA concentration, poor DNA quality, presence of PCR
inhibitors, thermal cycler accuracy) an internal positive control primer pair is
included in each PCR reaction2. The internal positive control primer pair matches
conserved regions of the human growth hormone gene, which is present in all
human DNA samples. In the presence of a specific PCR product of an HLA
allele(s), the product of the internal positive control band may be weak or absent.
The amplicons generated by the specific HLA primer pairs are shorter than the
amplicons of the internal positive control primer pair but larger than
unincorporated primers (see Expected values).
REAGENTS
A. Identification
®
The Olerup SSP typing kits contain dried, pre-optimized sequence-specific
primers for PCR amplification of HLA alleles and of the human growth
hormone gene, PCR Master Mix including Taq polymerase (“Master Mix”),
adhesive PCR seals and the Product Insert, which consists of the Instructions
for Use, Lot-specific Information and Worksheet.
The primer solutions are pre-aliquoted and dried in different 0.2 ml wells of
cut, thin-walled PCR trays. Each well of the tray contains a dried primer
solution consisting of a specific primer mix, i.e. allele- and group-specific HLA
primers as well as an internal positive control primer pair matching non-allelic
sequences and are ready for the addition of DNA sample, Master Mix, and
H2O.
Each low resolution (DQ low resolution from lot 06Y forward), and Combi
typing tray includes a negative control reaction well that detects the presence
®
of PCR products generated by more than 95% of the Olerup SSP HLA Class
I, DRB, DQB1, DPB1 and DQA1 amplicons as well as the amplicons
generated by internal positive control primer pairs.
As of lot series xxV, a Negative Control with the same composition as in the
low resolution kits is also included in all Olerup SSP high resolution kits.
The primers are designed for optimal PCR amplification when using the
Master Mix and the recommended DNA cycling program (see Programming
the Thermocycler).
See the provided lot-specific Specificity and Interpretation Tables or
Worksheet for the specific HLA alleles amplified by each primer mix.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 3 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
B. Warnings and Precautions
1. For In Vitro Diagnostic Use.
2. This product cannot be used as the sole basis for making a clinical
decision.
3. Biohazard Warning: All blood products should be treated as potentially
infectious. No known test method(s) can offer assurance that products
derived from human blood will not transmit infectious agents.
4. Biohazard Warning: The ethidium bromide used for staining DNA in the
agarose gel electrophoresis is a carcinogen. Handle with appropriate
personal protective equipment.
5. Caution: Wear UV-blocking eye protection, and do not view UV light
source directly when viewing or photographing gels.
6. Pipettes and other equipment used for post-PCR manipulations should
not be used for pre-PCR manipulations.
7. See Material Safety Data Sheet (http://www.olerup-ssp.com) for detailed
information.
C. Instructions for use
See Directions for Use.
D. Storage Instructions
Store kit components in the dark and at temperatures indicated on package
labels.
Use before the Expiration Date printed on package labels.
E: Purification or Treatment Required for Use
See Directions for Use.
F. Instability Indications
1. Do not use primer trays with cracks in the wells or damage to the upper
rim of the wells as this may cause evaporation during PCR
amplification. Do not use PCR cap strips with cracks as this may cause
evaporation during PCR amplification.
2. The pellets in the wells should be red in color. Yellow discoloration of
pellet may indicate degradation.
3. Master Mix should be red to purple in color. Yellow to orange
discoloration may indicate degradation.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 4 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
INSTRUMENT REQUIREMENTS
A. Instrument
A thermocycler with the following minimum specifications should be used:
• heated lid with a temperature of 104°C for oil-free operation
• sample block (aluminum, silver, or gold-plated silver) for use with
either a 96-well PCR plate or 0.2 ml thin-walled reaction tubes
• ramp rate of at least 0.7°C/sec
Note: Olerup SSP kits are validated using GeneAmp 9700 cycler set
to the 9600 mode. Higher ramp rates than the equivalent to the
described may have an effect on the typing results.
• temperature range of 4.0°C to 99.9°C
• temperature accuracy of ±0.25°C over the range of 35°C to 99.9°C
• sample block temperature uniformity of ≤0.75°C over the range of
55°C to 95°C
• temperature calibration traceable to a reference standard (i.e., NIST)
Program the thermocycler using the PCR Cycling Parameters in Section B
below.
For specific thermocycler information refer to the manufacturer’s user
manual. Thermocyclers should be calibrated according to ASHI (American
Society of Histocompatibility and Immunogenetics) or EFI (European
Federation of Immunogenetics) accreditation rules.
Program the thermocycler before starting the Directions for Use described
below.
B. PCR Cycling Parameters
1. 1 cycle
94oC
2. 10 cycles
94oC
65oC
3. 20 cycles
94oC
61oC
72oC
4. End - hold RT
4oC
2 min
10 sec.
60 sec.
10 sec.
50 sec.
30 sec.
denaturation
denaturation
annealing and extension
denaturation
annealing
extension
if less than 8 hours
if longer than 8 hours
Total reaction volume in each well, 10 l.
®
The same PCR Cycling Parameters are used for all the Olerup SSP kits.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 5 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
SPECIMEN COLLECTION AND PREPARATION
Extracted, highly pure DNA is needed for SSP typings. DNA samples to be
used for PCR-SSP HLA typing should be re-suspended in dH2O. The
A260/A280 ratio should be 1.6 – 2.0 by UV spectrophotometry for optimal band
visualization during electrophoresis.
We recommend automated DNA extraction with the QIAGEN EZ1 DSP DNA
Blood System. ACD blood should be used as starting material.
Alternatively, the DNA can be extracted by any preferred method yielding pure
DNA. When using alternative methods, the DNA concentration should be
adjusted to 30 ng/l. Do not use heparinised blood with these methods.
Recommended DNA concentration using:
EZ1-extracted DNA, 15 ng/l.
DNA extracted by other methods, 30 ng/l.
Concentrations exceeding 50 ng/l will increase the risk for nonspecific
amplifications and weak extra bands, especially for HLA Class I high resolution
SSP typings. If necessary, dilute the extracted DNA in dH2O.
DNA samples should not be re-suspended in solutions containing chelating
agents such as EDTA, above 0.5 mM in concentration.
DNA samples may be used immediately after extraction or stored at +4oC for up
to 2 weeks with no adverse effects on results. DNA samples can be stored at
-20oC or colder for 9 months. The purity and concentration of extracted DNA
samples that have been stored for a prolonged period should be tested for
acceptability prior to HLA typing.
DNA samples should be shipped at +4oC or colder to preserve their integrity
during transport.
PROCEDURE
A. Materials provided
®
1. Olerup SSP primer trays.
2. Master Mix (appropriate volume for the trays of the kit). The same
®
Master Mix is used for all Olerup SSP kits.
3. Adhesive PCR seals (appropriate number for the trays of the kit).
4. Product Insert.
B. Materials Required but not Provided
1. DNA isolation kit/equipment
2. UV spectrophotometer
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 6 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Pipetting devices. We recommend electronic single-channel dispenser
capable of dispensing 10 l aliquots for adding the DNA-Master MixdH20 mix to the tray wells.
Disposable pipette tips
Polypropylene tubes
Vortex mixer
Microcentrifuge
PCR tray rack
Thermocycler with heated lid for PCR with 96-well format, a temperature
gradient across the heating block ≤ 0.75°C, and tray/retainer for 0.2 ml
thin-walled reaction wells
Microwave oven or hot plate for heating agarose solutions
Electrophoresis grade agarose, e.g. FMC Seakem LE
0.5 x TBE buffer; 1 x TBE buffer is 89 mM Tris-borate, 2 mM disodium
EDTA, pH 8.0
Ethidium bromide dropper bottle, Product No. 103.301-10
Gel-loading pipetting device. We recommend 8 channel pipette for gelloading, 5-25 l adjustable volume
DNA size marker to cover range of 50 – 1 000 bp, e.g. 100 base pair
ladder, DNA Size Marker Product No. 103.202-100 or DNA Size Marker for short gel
runs 103.203-100
16. Electrophoresis apparatus/power supply
17. UV transilluminator
18. Photographic or image documentation system
C. Step-by-step Procedure
See Directions for Use.
DIRECTIONS FOR USE
A. Sample preparation
1. Purify genomic DNA from leukocyte sample by method of choice, see
Specimen Collection and Preparation above.
2. For specific information on sample preparation and storage, see
Specimen Collection and Preparation above.
3. Perform PCR amplification on purified DNA sample using an Olerup
®
SSP typing tray, or store DNA sample until ready to type.
B. Reagent/Equipment Preparation
®
1. Program a thermocycler to run the Olerup SSP PCR program, see
Instrument Requirements – PCR Cycling Parameters above.
2. Prepare electrophoresis gel, see section C – Gel Electrophoresis
Preparation below.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 7 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
C. Gel Electrophoresis Preparation
®
For Olerup SSP Gel System 96 (Product No. 103.101-01)
1. Set-up
 Level the casting chamber for 1 gel (Product No. 103.101-31) or the
casting chamber for 3 gels (Product No. 103.101-33) by using the leveling
bubble and the three height-adjustable legs.
 Place the gel tray(s) in the casting chamber.
2. 2% (w/v) Agarose Gel Preparation
Use a high quality electrophoresis grade agarose, capable of resolving
50 – 2 000 base pair fragments of DNA.
 To 5 ml of 10 x TBE (Tris Borate EDTA) buffer add 150 ml of
distilled water and 2 g of agarose in a 500 ml glass bottle.
 Dissolve the agarose by boiling in a microwave oven until a
homogenous solution of 100 ml is formed.
 Let the dissolved gel solution cool to 60oC, e.g. in a heating cabinet.
 Stain the gel prior to casting with ethidium bromide (10 mg/ml), 5 l
per 100 ml gel solution. For maximal ease of handling use our
ethidium bromide dropper bottles (Product No. 103.301-10). Note:
Ethidium bromide is a carcinogen. Handle with appropriate
personal protective equipment.
 Pour 100 ml of gel solution into the gel tray in the casting chamber.
Place 6 gel combs (Product No. 103.101-21) in the slots of the gel tray.
 Allow the gel to set for 15 minutes.
 Pour 750 ml of 0.5 x TBE buffer into the gel tank. Immerse the gel
tray in the gel box and carefully remove the 6 gel combs by lifting
them up.
Follow the manufacturer’s instructions for use when using alternative
electrophoresis systems. In order to be used with Olerup SSP® HLA Typing
Kits, these systems must be capable of resolving PCR products ranging from
50 to 1100 base pairs in size.
D.
Stepwise Procedure
1. Remove from the indicated storage temperature(s): the appropriate
number of DNA samples, the primer tray(s) and the volume of Master
Mix needed for the selected DNA sample(s)/primer tray(s). Thaw at
room temperature (20 to 25oC).
®
The same Master Mix is used for all for all Olerup SSP kits.
2. Mix DNA sample(s) briefly by vortexing.
3. Place the primer tray(s) in a PCR tray rack.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 8 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
4. Low and High resolution kits
 Vortex the Master Mix before taking aliquots.
 Using a manual single-channel pipette, add at room temperature
Master Mix and dH2O into a 0.5 ml or a 1.5 ml tube. (See table 1
below for appropriate amounts.)
 Cap the tube and vortex for 5 seconds. Pulse-spin the tube in a
microcentrifuge to bring all liquid down from sides of the tube.




Kits including Negative Control –
combi and low resolution kits (DQ low kits from lot 06Y forward), high
resolution kits from lot 01V forward:
Using a manual single-channel pipette, add 8 µl of the Master Mix –
dH2O mixture and 2 µl dH2O into the negative control well, i.e. the well
with the negative control primer pairs, of the primer tray.
Using a manual single-channel pipette, add at room temperature the
DNA sample to the remaining Master Mix – dH2O mixture. (See table 1
below for appropriate amounts.)
Cap the tube and vortex for 5 seconds. Pulse-spin the tube in a
microcentrifuge to bring all liquid down from sides of the tube.
Using an electronic single-channel dispenser aliquot 10 µl of the
sample reaction mixture into each well, except the negative control
well, of the primer tray.
Kits without Negative Control –
high resolution kits older than lot 01V, DQ low kits older than lot 06Y:
 Using a manual single-channel pipette, add at room temperature the
DNA sample to the Master Mix – dH2O mixture. (See table 1 below for
appropriate amounts.)
 Cap the tube and vortex for 5 seconds. Pulse-spin the tube in a
microcentrifuge to bring all liquid down from sides of the tube.
 Using an electronic single-channel dispenser aliquot 10 µl of the
sample reaction mixture into each well.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 9 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Table 1: Volumes of the components needed per test for different numbers of wells when
using Master Mix.
No. of
wells
per test
Volume of
Master Mix (µl)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
12
15
18
21
24
27
30
33
36
39
42
45
48
51
54
60
63
66
69
72
75
78
81
Volume of
DNA
sample (µl)
Volume
of dH2O
(µl)
No. of
wells
per
test
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
40
42
44
46
48
50
52
54
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
100
105
110
115
120
125
130
135
25
26
27
28
29
30
31
32
36
40
44
48
52
56
60
64
68
72
76
80
84
88
92
96
Volume of
Master Mix (µl)
87
90
93
96
99
102
105
108
126
138
150
162
174
186
198
210
228
240
252
264
276
288
300
312
Volume of
DNA
sample (µl)
58
60
62
64
66
68
70
72
84
92
100
108
116
124
132
140
152
160
168
176
184
192
200
208
Volume
of dH2O
(µl)
145
150
155
160
165
170
175
180
210
230
250
270
290
310
330
350
380
400
420
440
460
480
500
520
The recommended volumes listed above include volume to compensate for
pipette variations and for losses of liquid on the interior walls of the tubes.
5. Combi kits A-B-DR, A-B-C, A-B-DR-DQ and DQA-DQB-DR Enhanced
and the HLA-C high resolution for frequent alleles kit
 Vortex the Master Mix.
 Using a manual single-channel pipette, add at room temperature 520
µl dH2O into the provided 1.5 ml tube containing 312 µl Master Mix.
 Cap the tube and vortex for 5 seconds. Pulse-spin the tube in a
microcentrifuge to bring all liquid down from sides of the tube.
 Using a manual single-channel pipette, add 8 µl of the Master Mix –
dH2O mixture and 2 µl dH2O into the negative control well No. 96, i.e.
the well with the negative control primer pairs.
 Using a manual single-channel pipette, add at room temperature 206
µl DNA sample to the remaining Master Mix – dH2O mixture.
 Cap the tube and vortex for 5 seconds. Pulse-spin the tube in a
microcentrifuge to bring all liquid down from sides of the tube.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 10 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase

Using an electronic single-channel dispenser aliquot 10 µl of the
sample reaction mixture into each well, except the negative control
well No. 96, of the primer tray.
Important:
Be sure to apply the sample above the primers (dried at the bottom of each
well of the primer tray) to avoid cross-contamination between wells. Touch
the inside wall of the well with the pipette tip to allow the sample to slide
down to the bottom of the well. Check that all samples have dropped to the
bottom of each well. If not, tap the tray gently on the bench top so that all
samples settle at the bottom of the well before you begin PCR.
6. Cover the primer tray(s) with the provided adhesive PCR seals. Check
that all reaction wells are completely covered to prevent evaporative loss
®
during PCR amplification. The Olerup SSP Compression Pad (Product
No. 103.505-06) can be applied on top of the adhesive PCR seals to prevent
evaporation during thermal cycling.
7. Place the primer tray(s) in the thermocycler with a suitable tube-tray
adapter. Do not allow more than 5 minutes delay between PCR setup
and thermal cycling.
®
8. Enter your Olerup SSP program number. Specify a 10 µl reaction
volume.
9. Start the PCR program. The program takes approximately 1 hour and 20
minutes.
10. Remove the primer tray(s) from the thermocycler. Inspect the PCR tray
to make sure that there is approximately the same volume of fluid in
each PCR well. Electrophorese the samples, see the section E – Gel
Electrophoresis below. Interpret the typing results with the lot-specific
Interpretation and Specificity Tables or Worksheet, see Expected
Values below.
E. Gel Electrophoresis
1. After completing the PCR reaction orient the primer tray and gel box. The
order of the wells is from left to right and top to bottom.
2. Gently remove the strip lids without splashing the PCR products.
3. Load the PCR products in sequence to the 2 % agarose gel. (No addition
of gel loading buffer is necessary.) Use of an 8-channel pipette for gelloading is recommended.
4. Load a DNA size marker (100 base pair ladder, DNA Size Marker Product No.
103.202-100 or DNA Size Marker for short gel runs 103.203-100) in one well per
row.
5. Cover the gel box with the gel box lid
6. Electrophorese the gel in 0.5 x TBE buffer, without re-circulation of the
buffer, for 15-20 minutes at 8-10 V/cm.
7. Transfer the gel tray with the gel to a UV transilluminator.
8. Photograph the gel with or without the gel tray.
9. Mark the photograph according the rules of the laboratory.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 11 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
QUALITY CONTROL
ASHI HLA testing guidelines indicate that a negative (contamination) control well
must be included in each PCR setup. (Standards for Accredited Laboratories,
American Society for Histocompatibility and Immunogenetics, Final Revised
Standards Approved by the ASHI Board of Directors: August 31, 2007, Guidance
Final Version January 2008). A negative control well is included in all kits from
lot 01V and forward (the DQ low kit from lot 06Y and forward).
Refer to Gel Interpretation on page 14.
RESULTS
Lot-specific Cell Line Validation Sheets and Certificate of Analysis are provided
with each kit.
LIMITATIONS OF THE PROCEDURE
1. The PCR-SSP process requires highly controlled test conditions to ensure
adequate discriminatory amplification. The procedure described in
Instructions for Use must be strictly followed.
2. The extracted DNA sample is the template for the specific PCR
amplification process. The purified DNA should have an A260/A280 ratio
between 1.6 and 2.0 to obtain optimal band visualization by electrophoresis.
3. All instruments, e.g. thermocycler, pipetting devices, must be calibrated
according to the manufacturer’s recommendations.
4. Lot-specific information is given in the Product Insert: Lot-specific
Information and in the lot-specific Worksheet.
5. Based on testing performed, the following substances were evaluated with
three (3) extraction methods at the concentrations listed and found not to
impact test performance.
Extraction
Method
EZ1 DSP
DNA Blood
System
IFU-01
Rev. No.: 06
January 2015
Interfering
Substance
Bilirubin
Hemoglobin
Triglyceride
Protein
Interferent
concentration*
200mg/L
200g/L
30g/L
110g/L
QIAamp
DSP DNA
Blood Kit
Bilirubin
Hemoglobin
Triglyceride
Protein
200mg/L
200g/L
18.2g/L
77 - 96g/L
Gentra
PureGene
method
Bilirubin
Hemoglobin
Triglyceride
Protein
200mg/L
200g/L
18.2g/L
119 -146g/L
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 12 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
6. The PCR plates are compatible with the majority of thermocyclers on the
market. See the thermocycler compatibility table below. The table is
intended as a guide only.
Manufacturer
Biometra
Bio-Rad
Corbett Research
Eppendorf
Ericomp
ThermoHybaid
MJ Research™
MWG
ABI
Stratagene
Takara
Techne
G-Storm
IFU-01
Rev. No.: 06
January 2015
Compatibility Table
Thermocycler
Uno
Uno II
Tpersonal Cycler
T1 Thermocycler
T3 Thermocycler
TGradient
TRobot
Genecycler
iCycler™
MyCycler
Palm Cycler™
Mastercycler® Gradient
MasterCycler EP Gradient
M384
SingleBlock System
TwinBlock System
Deltacycler I
PCR Sprint
PCR Express, Px2, PxE
MultiBlock System and MBS®
Touchdown
Omnigene
Omn-E
PTC-200 DNA Engine™
PTC-225/220/221 DNA Tetrad™/ Dyad™
PTC-100™ with 96-well block
Primus 96
Primus 384
TheQ Lifecycler™
GeneAmp® 2400
Geneamp® 2700\2720
Geneamp® 9600
Geneamp® 9700
Geneamp® 9800 FAST Block
Robocycler
TP 240
TP 3000
TC-412/512
Touchgene Gradient
Flexigene
Genius
GS1/GS4/GSX
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
For In Vitro Diagnostic Use
0088
Olerup SSP
®
Product Insert
Page 13 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
EXPECTED VALUES
A. Data Analysis
Examine the gel photo carefully and determine the positive lanes.
1. A faster-migrating, shorter band will be seen in a gel lane if specific HLA
allele(s) was amplified. This indicates a positive test result.
a. Record the presence and absence of specific PCR products.
b. It is useful to monitor the relative lengths of the specific PCR
products as given in the lot-specific product inserts when
interpreting the gel results. Several lanes have two or more possible
lengths of specific PCR products. These wells contain multiple
primer pairs generating PCR products of different sizes depending
upon the HLA allele(s) of the sample DNA.
c. Match the pattern of gel lanes with specific PCR products with the
information in the lot-specific Interpretation and Specificity Tables to
obtain the HLA typing of the sample DNA.
2. An internal positive control band, slower-migrating and longer, should
be visible in all gel lanes, except in the negative control gel lane, as a
control of successful amplification. The internal positive control band
may be weak or absent in positive gel lanes.
a. Record the presence and relative lengths of the internal positive
control bands. The differently sized control bands will help in the
correct orientation of the typing as well as in kit identification.
b. Absence of internal positive control band with no specific PCR
product indicates failed PCR reaction.
i. If HLA alleles can be determined in the presence of failed PCR
reaction(s) and the failed PCR reaction(s) does not change the
allele assignment, then the test does not have to be repeated.
ii. If, however, if the failed PCR reaction(s) could change the HLA
allele assignment, then the typing must be repeated.
3. The presence of specific PCR product or internal positive control band
in negative control lane(s) indicates contamination with PCR product(s)
and voids all test results. Primer oligomers ranging from 40 to 60 base
pairs in size might be observed in the negative control lane(s). This
does not represent contamination.
IFU-01
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For In Vitro Diagnostic Use
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Olerup SSP
®
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Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
B. Gel Interpretation
Positive
Reaction
Well
Internal Positive
Control Band
Specific Band
Negative
Reaction
Failed PCR
Reaction
Primer Band
1. A DNA size marker (100 base pair ladder, DNA Size Marker Product No.
103.202-100 or DNA Size Marker for short gel runs 103.203-100) should be run
in one well per row of the gel or according to local laboratory
accreditation guidelines.
2. Bands longer than the internal positive control band might be obtained
and these should be disregarded in the interpretation of the typing
results.
3. Unused primers will form a diffuse band shorter band 50 base pairs.
4. Primer oligomer artefacts might be observed. These are longer than the
primer band but shorter than the specific bands.
SPECIFIC PERFORMANCE CHARACTERISTICS
Kit Lot Quality Control
Each primer solution is tested against a panel of 48 DNA samples from well
characterized cell lines of the IHWC, see the lot-specific Cell Line Validation
Sheet(s) in the Product Insert, Lot-specific Information.
Method Comparison Study 1
This was a multi-center study evaluating the agreement of the Olerup SSP®
DR Low Resolution HLA Typing Kit and the One Lambda Micro SSP TM HLA
DNA Typing Tray at three clinical laboratories in the United States.
The analyzed typing results of the Olerup SSP® DR Low Resolution HLA
Typing Kit and of the One Lambda Micro SSPTM HLA DNA Typing Tray
showed 98.4% (123 / 125; 95% CI: 94.3 – 99.8) agreement when two
ambiguous Olerup results are treated as discordant. Agreement was 100%
(123 / 123; 95% CI: 97.1 – 100) when the Olerup ambiguous results were
not included in the analysis, reflective of normal clinical practice.
Method Comparison Study 2
This study was designed to demonstrate agreement of HLA allele (A, B, C, DQ)
low resolution typing results obtained with the investigational Olerup SSP® HLA
Typing Kits and the reference One Lambda LABType SSO kits. ACD whole
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Olerup SSP HLA typing kits including Taq polymerase
blood samples were collected from 95 subjects distributed over 3 clinical sites in
the United States. DNA extraction was performed and the resulting purified
DNA was tested with the investigational Olerup SSP® and the reference One
Lambda LabType SSO HLA methods.
The overall agreement for Class I alleles was 99.6% (278 / 279; 95% CI: 98.0 100). For Class II alleles agreement was 100% (94 / 94; 95% CI: 96.2 – 100).
Table 1
Overall agreement of Olerup SSP® and
OneLambda SSO results for Class I and
Class II alleles.
Total
HLA
n/N
% agreement
Locus
(95% CI)
100
A
95 / 95
(96.2 – 100)
100
B
90 / 90
(96.0 – 100)
98.9
C
93 / 94
(94.2 – 100)
All Class I
99.6
278 / 279
Loci
(98.0 – 100)
Class II
100
94 / 94
Loci (DQ)
(96.2 – 100)
Kit Result Reproducibility Study.
This study compared Olerup SSP® HLA Typing results between three HLA
Testing Laboratories using a panel of 10 well characterized DNA samples
whose consensus results are included in the UCLA HLA DNA Bank for HLA
Class I (A, B and C), common Class II alleles (DRB1*, DRB3*/DRB4*/DRB5*,
and DQB1*) and less frequently investigated Class II alleles (DQA1*, DPA1*,
and DPB1*).
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January 2015
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Olerup SSP HLA typing kits including Taq polymerase
Table 2: Summary of Olerup SSP HLA Kit Reproducibility Study Results
HLA Allele Type
Typing Accuracy %
n/N
95% Conf. Interval (LL, UL)
Ambiguous result
Ambiguous result
treated as discordant
treated as
indeterminate and
excluded from analysis
Class I Low Resolution
(A and B combined)
98.3
(59/60)
91.1, 100
100
(59/59)
93.9, 100
Class I High Resolution
(A, B and C combined)
94.7
(142/150)
89.8, 97.7
98.6
(142/144)
95.1, 99.8
Class II Low Resolution
(DRB1* and
DRB3*/DRB4*/DRB5*)
100
(60/60)
94.0, 100
100
(60/60)
94.0, 100
Class II High Resolution
– Common alleles
(DRB1*,
DRB3*/DRB4*/DRB5*,
and DQB1*)
98.3
(118/120)
94.1, 99.8
100
(118/118)
96.9, 100
Class II High Resolution
Less frequently
investigated alleles
(DQA1*, DPA1*, and
DPB1*)
83.3
(75/90)
74.0, 90.4
86.2
(75/87)
77.2, 92.7
This study utilized a panel of ten (10) DNA samples with well characterized HLA
typing results.
The lower agreements observed for the less frequently investigated Class II
High Resolution alleles reflects the greater uncertainty in the “consensus
results” of the UCLA DNA samples considering the incomplete sequence
information available for the DQA1*, DPA1* and DPB1* alleles. For 9 out of the
11 discordant results observed during the reproducibility study (a DQA1*0505
call by Olerup SSP vs. DQA1*0501 “consensus typing”) all three testing sites
arrived at the same result indicating consistent Olerup DQA1* kit performance.
IFU-01
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January 2015
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Page 17 of 28
Instructions for Use
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Olerup SSP HLA typing kits including Taq polymerase
BIBLIOGRAPHY
1. Olerup O, Zetterquist H. HLA-DRB1*01 subtyping by allele-specific PCRamplification: A sensitive, specific and rapid technique. Tissue Antigens 1991:
37: 197-204.
2. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequencespecific primers (PCR-SSP) in 2 hours: An alternative to serological DR typing
in clinical practice including donor-recipient matching in cadaveric transplantations. Tissue Antigens 1992: 39: 225-235.
3. Current HLA alleles can be found at:
a. http://www.ebi.ac.uk/imgt/hla or
b. http://www.anthonynolan.org.uk/HIG
IFU-01
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Instructions for Use
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Olerup SSP HLA typing kits including Taq polymerase
TROUBLESHOOTING
Problem
No amplification (neither
amplification of the
internal control
fragments, nor specific
amplifications).
IFU-01
Rev. No.: 06
January 2015
Reason
Too low amount of DNA.
Action
Measure the DNA
concentration and see if
the amount added is
correct.
RNA contamination may
cause a
spectrophotometric
overestimation of DNA
concentration.
Repeat the DNA
extraction carefully with
freshly prepared
solutions.
We recommend
automated DNA
extraction with the
QIAGEN EZ1 DSP DNA
Blood System.
The DNA contains PCR
Measure the DNA
inhibitors, e.g. proteins,
quality. We recommend
ethanol (from precipitation an A260/A280 ratio of
steps), remaining matrixes 1.6 – 2.0 by UV
from solid-phase DNA
spectrophotometry.
purification products.
Follow the suppliers DNA
extraction protocol
exactly.
Re-extract the DNA.
We recommend
automated DNA
extraction with the
QIAGEN EZ1 DSP DNA
Blood System.
The DNA has been
Use non-heparinized
extracted from heparinized blood or use DNA
blood.
extraction protocols for
heparinized blood.
The DNA is dissolved in a Repeat the DNA
buffer containing EDTA.
extraction and dissolve
the DNA in dH20.
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Instructions for Use
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Olerup SSP HLA typing kits including Taq polymerase
Problem
Continuing:
No amplification (neither
amplification of the
internal control
fragments, nor specific
amplifications).
Reason
Accidental introduction of
bleach into test.
Kits are not stored at
adequate temperature.
Thermal cycler is not
working in a proper way.
Action
Review areas where
bleach might possibly be
introduced.
Store the kits at -20°C.
Calibrate the thermal
cycler and check the
PCR program.
A thermal cycler used for
routine PCR-SSP typing
should be calibrated
every 6-12 months.
Inadequate contact
Use correct tray/retainer
between thermal cycler
for 0.2 ml thin-walled
heating block and SSP
reaction wells, refer to
typing tray.
the thermal cycler
manual.
Random failure of
PCR seals/PCR tube caps Make sure the PCR
amplification (drop-outs). that are not tightly closed
seals/all caps are tightly
will lead to evaporation and closed. The Olerup SSP®
subsequent failure of
Compression Pad (Product
amplification.
No. 103.505-06) can be
applied on top of the
adhesive PCR seals to
prevent evaporation
during thermal cycling.
Gel-loading mistakes.
Check that the correct
number of wells has
been loaded and that
each well contains
approximately the same
volume of PCR mixture.
Use of non-calibrated
Calibrate all pipettes
pipettes.
routinely according to the
supplier’s
recommendations.
Pipetting errors.
Perform pipetting more
carefully.
Master Mix and sample
Mix briefly by vortexing
DNA have not been
before use. We
properly mixed before use. recommend to vortex
after each row.
Uneven volume of DNAPerform pipetting more
Master Mix mixture has
carefully.
been added to the wells.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
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Olerup SSP
®
Product Insert
Page 20 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Problem
Weak internal control
fragments.
Reason
Impure DNA.
Too low amount of DNA.
IFU-01
Rev. No.: 06
January 2015
Action
Measure the DNA
quality. The A260/A280
ratio should be 1.6 – 2.0
by UV
spectrophotometry.
RNA contamination may
cause a
spectrophotometric
overestimation of DNA
concentration.
Degraded DNA give rise
to smear in gel lanes.
Repeat the DNA
extraction carefully with
freshly prepared
solutions.
We recommend
automated DNA
extraction with the
QIAGEN EZ1 DSP DNA
Blood System.
Measure the DNA
concentration and adjust
to 30 ng/µl or to 15 ng/µl
for DNA extracted by the
QIAGEN EZ1 DSP DNA
Blood System.
RNA contamination may
cause a
spectrophotometric
overestimation of DNA
concentration.
Degraded DNA give rise
to smear in gel lanes.
Repeat the DNA
extraction carefully with
freshly prepared
solutions.
We recommend
automated DNA
extraction with QIAGEN
EZ1 DSP DNA Blood
System.
For In Vitro Diagnostic Use
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Page 21 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Problem
Continuing:
Weak internal control
fragments.
Reason
Too high annealing
temperature, the thermal
cycler is not calibrated.
The PCR Master Mix has
been stored at +4°C for
longer than 2 weeks.
Non-specific
amplification (ladders or
smears).
Use of excess DNA
sample.
Impure DNA.
IFU-01
Rev. No.: 06
January 2015
Action
Calibrate the thermal
cycler and check the
PCR program.
A thermal cycler used for
routine PCR-SSP typing
should be calibrated
every 6-12 months.
Properly store PCR
Master Mix.
Measure the DNA
concentration and adjust
30 ng/µl or to15 ng/µl for
DNA extracted by the
QIAGEN EZ1 DSP DNA
Blood System.
Some primer solutions
have a higher tendency
of giving rise to nonspecific amplification,
see footnotes in each lotspecific Specificity Table.
All fragments larger than
the internal control
fragment should be
disregarded when
interpreting the obtained
results.
Check the DNA quality.
Repeat the DNA
extraction.
We recommend
automated DNA
extraction with the
QIAGEN EZ1 DSP DNA
Blood System.
Some primer solutions
have a higher tendency
of giving rise to nonspecific amplification,
see footnotes in each lotspecific Specificity Table.
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Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Problem
Weaker and weaker
amplification signals
over time.
Reason
The ethidium bromide
agarose gel staining
solution is old.
One of the UV lamps is
broken.
Used too little DNA
sample.
Too high annealing
temperature, the thermal
cycler is not calibrated.
Strange amplification
patterns.
Incorrect lot-specific
Interpretation Table /
worksheet is used.
Incorrect order in gel
loading.
The amplification pattern
contains a false positive.
The amplification pattern
contains a false negative.
IFU-01
Rev. No.: 06
January 2015
Action
Prepare fresh ethidium
bromide solution to
achieve better staining of
the agarose gel and
better signal. The primer
clouds are easy to detect
if the staining of the
agarose gel is normal.
Check the UV light
equipment. The primer
clouds are easy to detect
if the UV light is normal.
Measure the DNA
concentration and adjust
to 30 ng/µl or to 15 ng/µl
for DNA extracted by the
QIAGEN EZ1 DSP DNA
Blood System.
Calibrate the thermal
cycler and check the
PCR program.
A thermal cycler used for
routine PCR-SSP typing
should be calibrated
every 6-12 months.
Check the lot number of
the product used and the
Interpretation Table /
worksheet used.
Check alignment of
mixes and gel lanes.
See below.
See below.
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Page 23 of 28
Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Problem
False positive
amplifications.
Reason
DNA contamination.
Impure DNA.
Use of excess DNA
sample.
Too low annealing
temperature.
Extensive delay between
PCR setup and start of
thermal cycling.
Delay between placing
typing trays in thermal
cycler and start of cycling.
IFU-01
Rev. No.: 06
January 2015
Action
Use gloves, pipette tips
containing barriers (filter
plugs) and separate
rooms for pre-PCR
handling and post-PCR
handling.
Assure accurate handling
of all samples, in all
steps.
Check for contamination
using Olerup SSP® Wipe
Test kit.
Measure DNA quality.
Follow the suppliers DNA
extraction protocol
exactly.
Try other DNA extraction
systems.
Re-extract the DNA.
We recommend automated DNA extraction
with the QIAGEN EZ1
DSP DNA Blood System.
Measure the DNA
concentration and adjust
to 30 ng/µl or to 15 ng/µl
for DNA extracted by the
QIAGEN EZ1 DSP DNA
Blood System.
Calibrate the thermal
cycler and check the
PCR program. A thermal
cycler used for routine
PCR-SSP typing should
be calibrated every 6 -12
months.
No more than a 5 minute
delay should be allowed
before thermal cycling.
Use pre-heated thermal
cycler.
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Product Insert
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Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Problem
Continuing:
False positive
amplifications.
Reason
Use of excess ethidium
bromide.
Incorrect interpretation of
an artefact as a specific
band.
The amplification pattern
contains a false positive.
False negative
amplifications.
Incorrect order in gel
loading.
The thermal cycler is not
properly calibrated.
Incorrect order in gel
loading.
IFU-01
Rev. No.: 06
January 2015
Action
Use recommended
amount of ethidium
bromide.
Check the lot-specific
Interpretation Table /
worksheet and Specificity
Table for correct band
size and foot notes.
Check if all specific
amplifications are correct
in size or if an artefact
(carry-over, primer dimer)
has been misinterpreted
as an amplification.
Check alignment of
mixes and gel lanes.
Calibrate the thermal
cycler and check the
PCR program. A thermal
cycler used for routine
PCR-SSP typing should
be calibrated every 6-12
months.
If not corrected by recalibration, re-type the
test with a reference
sample of the same
specificity. If confirmed
negative, contact
customer support.
Check alignment of
mixes and gel lanes.
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Instructions for Use
®
Olerup SSP HLA typing kits including Taq polymerase
Problem
Overall gel problems
(fuzzy gels and/or
smeared lanes).
IFU-01
Rev. No.: 06
January 2015
Reason
Degraded DNA sample.
Action
Appears as a smear in
the gel lanes. Isolate
DNA from a fresh
sample.
Heavy streaking in random Uneven suspensions of
wells.
DNA. Make sure sample
DNA is dissolved before
taking your aliquot.
Vortex diluted DNA
sample.
PCR product floated out of Carefully align pipette
well.
tips with gel wells and
dispense slowly.
The electrophoresis buffer Prepare new TBE buffer.
might be too warm.
Run at a lower voltage.
Incorrect percentage
Make sure the
agarose gel has been
recommended 2%
used.
agarose gel is used.
Agarose not completely
Shortly re-boil to melt the
dissolved.
agarose.
Incorrect TBE
Use the recommended
concentration.
0.5 x TBE concentration.
Gels too newly casted.
Gels are not ready for
use until 15 minutes after
casting.
Gels too old.
Do not cast gels too far
in advance.
The gel comb used has too Use thin combs (4 x 1
thick slots.
mm).
Gel tray not UV
Remove gel from gel tray
transparent.
before viewing.
Gel picture too bright.
Excess use of ethidium
bromide.
Check the camera
settings.
Gel picture too dark.
Use recommended
amount of ethidium
bromide.
Check the camera
settings.
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Olerup SSP HLA typing kits including Taq polymerase
Problem
General problems with
false negative
amplification or run-torun dependent problems
of such nature
IFU-01
Rev. No.: 06
January 2015
Reason
Action
Ramp rate setting too high. Olerup SSP kits are
validated using
GeneAmp 9700 cycler
set to the 9600 mode.
Higher ramp rates than
the equivalent to that
may have an effect on
the typing results.
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Olerup SSP HLA typing kits including Taq polymerase
TRADEMARKS USED IN THIS DOCUMENT/PRODUCT
®
Olerup SSP is a registered trademark of Olerup SSP AB.
QiagenTM is a trademark of QIAGEN.
WARRANTY
Olerup SSP AB warrants its products to the original purchaser against defects
in materials and workmanship under normal use and application. Olerup SSP
AB’s sole obligation under this warranty shall be to replace, at no charge, any
product that does not meet the performance standards stated on the product
specification sheet.
This warranty applies only to products that have been handled and stored in
accordance with Olerup SSP AB’s recommendations, and does not apply to
products that have been the subject of alternation, misuse, or abuse.
All claims under this warranty must be directed to Olerup SSP AB in writing and
must be accompanied by a copy of the purchaser’s invoice. This warranty is in
lieu of all other warranties, expressed or implied, including the warranties of
merchantability and fitness for a particular purpose. In no case shall Olerup
SSP AB be liable for incidental or consequential damages.
This product may not be reformulated, repacked or resold in any form without
the written consent of Olerup SSP AB, Franzengatan 5, SE-112 51 Stockholm,
Sweden.
Handle all samples as if capable of transmitting disease. All work should be
performed wearing gloves and appropriate protection.
GUARANTEE
®
Olerup SSP AB guarantees that the primers in the Olerup SSP typing trays have
the specificities given in the worksheet, lot-specific Specificity and Interpretation
Tables of the product insert.
When stored at –20oC, the dried primers are stable for 30 months from the date
of manufacture.
When stored at –20oC, the PCR Master Mix including Taq polymerase is stable
for 33 months from the date of manufacture.
Changes in revision R06 compared to R05:
1.
A Negative Control is included in all high resolution kits beginning lot xxV.
2.
A new combi kit, DQA-DQB- DR Enhanced, has been introduced.
3.
The combi kit DPB1-DQ-DR is no longer produced.
4.
For each new revision the changes are now specified.
Descriptions and protocols have been modified to reflect these changes.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
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Instructions for Use
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Olerup SSP HLA typing kits including Taq polymerase
ADDRESSES:
Manufacturer:
Olerup SSP AB, Franzengatan 5, SE-112 51 Stockholm, Sweden.
Tel: +46-8-717 88 27
Fax: +46-8-717 88 18
E-mail: [email protected]
Web page: http://www.olerup-ssp.com
Distributed by:
Olerup GmbH, Löwengasse 47 / 6, AT-1030 Vienna, Austria.
Tel: +43-1-710 15 00
Fax: +43-1-710 15 00 10
E-mail: [email protected]
Web page: http://www.olerup.com
Olerup Inc., 901 S. Bolmar St., Suite R, West Chester, PA 19382
Tel: 1-877-OLERUP1
Fax: 610-344-7989
E-mail: [email protected]
Web page: http://www.olerup.com
For information on Olerup SSP distributors worldwide, contact Olerup GmbH.
IFU-01
Rev. No.: 06
January 2015
For In Vitro Diagnostic Use
0088
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