AllSet+ Gold SSP - БиоХимМак fileThe Invitrogen™ Corporation HLA AllSet+™ Gold SSP is a PCR‐based HLA typing evaluation method designed to provide low to high resolution

USER GUIDE

AllSet+™ Gold SSP Instructions for Use

For Both Low and High Resolution Kits

Publication Number MAN0002563 Revision 07

For In Vitro Diagnostic Use

2 AllSet+™ Gold SSP

Disclaimers LIFE TECHNOLOGIES CORPORTATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

Limited Use Label Licenses: No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008. Professional Use Only HLA typing using the AllSet+™ Gold SSP Kits must be performed in the presence of an HLA Lab Director, Technical Supervisor and/or general Supervisor following accepted laboratory accreditation standards (ASHI). These products are for professional use only. Trademarks The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

QIAamp is a registered trademark of Qiagen GMBH Corp. All other trademarks are the sole property of their respective owners. Copyright ©2012, Life Technologies Corporation. All rights reserved.

Information in this document is subject to change without notice.

AllSet+™ Gold SSP 3

Contents

Product Information ....................................................................... 4 Intended Use ................................................................................................ 4 Product description ....................................................................................... 4

Kit usage ................................................................................................. 4 AllSet+™ Gold Kit contents and storage ........................................................ 5 Materials and equipment required but not included ...................................... 6 General purpose supplies required but not included ..................................... 7

Methods .......................................................................................... 8 Before starting .............................................................................................. 8

Follow these sample guidelines ............................................................... 8 Sample setup .......................................................................................... 9 Pre-Amplification set up (perform in pre-PCR area) ................................ 9

Amplification ............................................................................................... 10 Load the AllSet+™ Gold SSP Tray ......................................................... 10 PCR amplification set up (perform in the post-PCR area) ..................... 11

Gel electrophoresis ..................................................................................... 12 General directions ................................................................................. 12 Procedure .............................................................................................. 12

Interpretation ............................................................................................... 14 Troubleshooting .......................................................................................... 16

Appendix A: Limitations and Cautions ...................................... 20

Precautions ................................................................................................. 20 Performance Characteristics ....................................................................... 20

Appendix B: Safety ...................................................................... 21 Chemical safety .......................................................................................... 22 Biological hazard safety .............................................................................. 23 Documentation and Support ....................................................................... 24

Obtain SDSs .......................................................................................... 24 Obtain support ....................................................................................... 24 Limited Product Warranty ...................................................................... 24

IVD Symbols ............................................................................................... 25 References ................................................................................................. 26

Product Information Product description


Product Information

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix in this document.

Intended Use The Invitrogen™ Corporation HLA AllSet+™ Gold SSP is a PCR‐based HLA typing evaluation method designed to provide low to high resolution of the HLA Class I (A, B, C) and Class II (DRB, DQB) Loci using genomic DNA. HLA AllSet+™ Gold SSP is designed to use typing evaluation Worksheets or UniMatch® Plus software.

Product description

Kit usage

The AllSet+™ Gold Kit is a PCR‐based method designed to provide low to high resolution of the various HLA Class I and II types. Formulations of allele or group specific primer sets are used to amplify genomic DNA using a 96‐well thermal tray. Setup includes:

1. Mixing a reaction buffer with a human genomic DNA sample and Taq DNA Polymerase.

2. Dispensing the mixture to the AllSet+™ Gold Tray.

3. Sealing.

4. Thermal cycling.

After cycling is complete, the PCR products are loaded onto a 2% agarose gel for electrophoresis. After electrophoresis, the ethidium bromide stained gel is photographed and interpreted using a worksheet for the specific amplification patterns. The test can be completed in 2.5 hours post DNA isolation. (Times vary depending on make and model of the thermal cycler used.) The AllSet+™ Gold method is based on sequence specific primer (SSP) amplification methods previously published. 1‐3 The primer sets amplify the alleles described by the international nomenclature committee of WHO.1 For details, see the Worksheet, Primer Mix Specificity Table and Ambiguity List provided with each kit. The method has been extensively tested with reference DNA from the International Workshops, UCLA Reference DNA Panel samples, and other well‐characterized, serotyped samples.

Product Information AllSet+™ Gold Kit contents and storage


AllSet+™ Gold Kit contents and storage Item Description Quantity Storage

96-well test plates Each well in the PCR tube contains a dried SSP primer solution consisting of allele- and/or group-specific primers as well as a control primer pair matching non-allelic sequences. The control primer pair amplifies a fragment of a conserved gene present in all samples.

10 plates 4°C to -20°C in a non-frost free

freezer

Aliquots of optimized PCR buffer

PCR buffer containing dNTPs and Gel Loading Buffer. 10 each

4°C to -20°C in a non-frost free

freezer PCR sealing sheets Self-adhesive plastic sheets for sealing

test plates. 11 sheets —

CD ROM • Instructions for Use • Translated Instructions For Use • Gel Documentation Form • Worksheets • Primer Mix Specificity Table • Certificate of Analysis / Tray

Configuration

1 each —

Product Information Materials and equipment required but not included


Materials and equipment required but not included

Item Source

Taq DNA Polymerase, 5 units/µL

• The following enzymes are validated for use with the AllSet+™ Gold products: • Life Technologies Recombinant Taq DNA Polymerase • Roche Molecular Systems Taq DNA Polymerase • Perkin Elmer Ampli-Taq DNA Polymerase • Fisher Taq DNA Polymerase • Advanced Biotechnologies Ltd. Taq DNA Polymerase.

• Other DNA polymerase enzymes must be validated by the user.

96-well thermal cycler with heated lid

96-well x 0.2-mL thermal cycler with heated lid: • Temperature range ≥ 4°C–99.9°C • Thermal Accuracy of +/− 0.75°C • Ramping speed of ≥ 1°C per second

Note: This kit has been validated with thermal cyclers meeting the above specifications. Using different equipment will require user validation of thermal cycling parameters.

Recommended Electrophoresis System

Item Life Technologies Cat. no.

E-Gel® 96 2% Agarose 8-Pak A10570 E-Gel® 48 2% Agarose 8-Pak A10571 Electro-Fast® Electrophoresis System: 96 sample lanes plus separate marker loading lanes

920001

Owl Centipede™ Extra Wide Minigel System, Model #: D3-14 with 4 Microtiter combs, 25 teeth, 1.5-mm thick

800001D

Recommended Gel Documentation System

UV transilluminator

Product Information General purpose supplies required but not included


General purpose supplies required but not included IMPORTANT! The customer is responsible for validating all general purpose supplies before use, and for compliance with regulatory requirements that pertain to their procedures and uses of the instrument.

All of the items in this table can be purchased from any major laboratory supplier.

Item Description

Sterile, molecular grade water —

Pipettes and tips

• 1–10 µL • 10–200 µL • 100–1000 µL

Electronic dispensing pipettes • 100–250 µL capacity • Capable of dispensing 8 µL aliquots

DNA Molecular Weight markers to cover range of 50—2000 bp

Life Technologies PCR Markers, Cat. no. 74601250 (recommended)

DNA Grade Agarose Life Technologies, Cat. no. 75000500 (recommended)

TBE electrophoresis buffer 0.5X concentration

Ethidium bromide Caution: Ethidium bromide is a mutagen. Handle with appropriate personal protective equipment.

10 mg/mL

Electrophoresis Power supply —

Methods Before starting


Methods

Before starting

Follow these sample guidelines

• DNA sample in TE buffer or sterile water from human nucleated cells purified by any preferred method. The final concentration of the DNA prior to PCR‐SSP should be approximately 50 ng/μL.

• DNA isolated from blood samples should be collected in EDTA or ACD anticoagulated tubes.

Note: Do not use heparinized samples. Heparin may inhibit DNA amplification.

• Good quality DNA is critical to achieve an optimal result with the AllSet+™ Gold SSP System. Good quality DNA means that:

• The OD260/280 is between 1.7 and 1.9 on diluted sample measured by UV spectrophotometry

• When checked on agarose gel electrophoresis, the major portion of DNA runs slower than the 9.4 kb band of Hind III digested Lambda DNA marker

• Extracted DNA should be diluted in distilled water.

Note: Isolate DNA by any qualified protocol that produces high‐purity and high‐quality DNA. The Life Technologies DNA Isolation Kit (Cat. no. 761001D) and QIAamp® System are validated for this use.

Methods Before starting


Sample setup

Note: This procedure should be performed in 2 areas of the laboratory (Pre‐PCR Amplification and Post‐PCR Amplification) as indicated in the following instructions.

Pre-Amplification set up (perform in pre-PCR area)

PCR mix • Dependent on the number of PCR reactions per test

• Use the following table for required volumes

10 µL well/test PCR Buffer (µL) Water (µL) DNA (50 ng/µL) Taq (µL) 96 460 608 125 7 48 230 304 62.5 3.5 32 153 203 42 2.3 24 115 152 31.3 1.8 16 80 105.8 21.7 1.2 8 66 87 18 1

Tray Layout The correct orientation of the tray is identified by the blue contamination well in the last well of the test.

8 16 24 32 40 48 56 64 72 80 88 96

7 15 23 31 39 47 55 63 71 79 87 95

6 14 22 30 38 46 54 62 70 78 86 94

5 13 21 29 37 45 53 61 69 77 85 93

4 12 20 28 36 44 52 60 68 76 84 92

3 11 19 27 35 43 51 59 67 75 83 91

2 10 18 26 34 42 50 58 66 74 82 90

1 9 17 25 33 41 49 57 65 73 81 89

AllSet+™ Gold SSP Tray

Methods Amplification


Amplification

Load the AllSet+™ Gold SSP Tray

1. Open the pouch containing the trays. Remove a tray from the bottom of the stack. Return remaining trays to the pouch and reseal.

2. Remove the Taq DNA Polymerase from the freezer and keep chilled during setup (e.g., on ice).

3. Add water and Taq polymerase (5U/μL) to the pre‐aliquotted PCR Buffer. Mix well.

4. Add 10 μL of this mixture (from step 3) to the contamination control tube containing blue dye.

5. Add Sample DNA (50 ng/μL) to the remaining buffer mixture. Mix well.

6. Dispense 10 μL into each of the remaining wells. Be careful to dispense the drops onto the side walls of the wells, near each well’s top, allowing the dispensed drop to slide down. Do not allow the pipette tip to come in contact with the well contents.

7. Ensure all liquid is at the bottom of the tray. Cover the tray with a seal or caps and close tightly.

8. The samples are now ready for PCR amplification. Store remaining kit reagents at 4°C to ‐20°C.

Methods Amplification


PCR amplification set up (perform in the post-PCR area)

1. Place the sample in the thermal cycler.

2. Program the thermal cycler using the following parameters:

Step Temperature (°C) Time (seconds) Action A denaturation step 96 60 Denature

5 cycles 96 70 72

25 50 45

Denature Anneal Extend

21 cycles 96 65 72

25 50 45


4 cycles 96 55 72

25 60

120


Hold 4 User-specified time —

Note: Obtain rapid ramp times (~1°C per second) and precise temperature control for optimal results. For specific thermal cycler information, refer to the manufacturer’s handbook.

3. Start the program.

4. When the program is complete, remove the samples from the thermal cycler.

Note: After thermal cycling, remove the tray and proceed to gel electrophoresis. If you are not performing electrophoresis immediately, store the tray at 4º C for up to 1 week.

Methods Gel Electrophoresis


Gel electrophoresis

General directions

Use the following procedure to detect PCR amplification using agarose gel electrophoresis.

Procedure

Prepare a 2% agarose gel 1. Add 2 g of agarose (Life Technologies Cat. no. 75000500) to 100 mL 0.5x TBE

buffer to prepare a 2% (w/v) agarose gel.

2. Dissolve the agarose by boiling in a microwave oven until thoroughly dissolved.

3. Cool to 60°C.

4. Add ethidium bromide to a final concentration of 0.5 μg/mL gel.

5. Cast a 3–4 mm thick gel with 3‐mm wide wells.

6. Allow the gel to set for at least 30 minutes.

Run the gel 1. Transfer the agarose gel to a submarine gel electrophoresis unit.

Note: Cover the gel with 0.5X TBE buffer to a depth of approximately 1–2 mm above the gel surface.

2. Carefully remove the comb.

3. Load 5 μL of PCR marker to the appropriate lane(s) of the gel. (See the Gel Documentation Form.)

4. Load the entire PCR product in sequence directly and carefully onto the gel.

5. Run gels at 10 volts per centimeter gel length. For the Owl Centipede™ unit, electrophoretically separate the DNA at 150 volts for 18–23 minutes or until the orange dye front in the PCR Marker approaches the next row of wells.

Note: The purple sample dye will be at approximately 300 bp after running the gel for 18–23 minutes.

6. Turn off the power, disconnect electrodes and remove the gel.

Note: To aid in troubleshooting and technical support it is helpful for Life Technologies Corporation to obtain an original gel photo. For this purpose, the user may want to take additional photos.

Examine the gel

Examine the gel photo carefully under UV illumination and document by photography. Determine the positive lanes to interpret the results.

Note: The same 0.5x TBE buffer can be used several times, but performance may be enhanced by using fresh buffer.

Methods Gel electrophoresis


WARNING! Ethidium Bromide is a powerful mutagen. Wear nitrile gloves and eye protection when handling gels or solutions containing Ethidium Bromide.

Methods Interpretation


Interpretation • Affix the gel photo to the Gel Documentation Form.

On the Gel Documentation Form, “M” refers to the marker lane. • Examine the gel photo carefully and determine the positive lanes. See the

following table for guidelines regarding examining the gel photo.

Observation Explanation A control band in each lane Each lane of the gel, containing a loaded sample, should show a control

band except the lane which contains the contamination control well. See the Gel Documentation Form for details on the internal control sizes.

Inefficient amplification of the control

The control band may or may not amplify efficiently when there is specific product present due to substrate competition. The control primers are present in lower concentration in order to favor the allele specific reaction.

Weak bands above the internal control (except the 200-base pair internal control) appearing in all lanes

These are not of particular concern and are an indication of robust amplification.

Absent control bands with no specific amplification

Failed reactions. • 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.

• If, however, there is an apparent homozygous result, or the missed reaction could change an allele assignment, the typing must be repeated.

Weak bands of incorrect product size are present

Disregard the weak bands if the overall strength and clarity of the amplification is good.

Diffuse band below 50 base pairs

Unused primers will form this band.

Fuzzy band below 80 base pairs

Primer-dimer usually appears above the primer band, but below the area where specific product is found.

Several lanes have 2 or more possible sizes of PCR products

These wells have multiplexed primer pairs which give rise to different, amplicons depending upon the allele present. Refer to the Primer Mix Specificity Table (provided with each kit) for further information for allele assignment.

Resolution detail See the worksheet provided with the kit for details on alleles.

Methods Interpretation


Observation Explanation

False negative reactions Can be caused by inefficient amplification, poor quality of DNA, uneven placement of the plate in the block, temperature variations across the wells of the thermal cycler itself, or inadequate thermal cycler calibration. • False negative reactions rarely occur when the control band is present. • The false negatives may be due to a new or yet uncharacterized allele.

The contamination control well contains primer pairs that amplify DNA produced by either specific PCR amplifications or genomic DNA

• If the buffer/Taq mixture was added as directed, any band in this lane is evidence of contamination and the results of the test are invalid.

• A primer dimer band of <80 base pairs may be present. This does not invalidate the test. Primer dimers are known to occur occasionally.

1. Confirm the approximate product size using the Gel Documentation Form or Worksheet.

2. Mark the positive lanes on the worksheet.

3. Using a highlighting pen, highlight each positive lane (column) with a vertical line running through the chart columns from top to bottom.

4. Align a ruler across the first row of the worksheet and scan from left to right. Examine each row from top to bottom, making the first allele assignment only where all the black boxes for that allele choice fall within the highlighted lanes.

Note: We recommend using UniMatch™ software for interpretation.

5. Assign the second allele choice, if needed, by continuing to scan the remaining alleles as above. Be sure to complete an entire review of the worksheet to determine all possible allele assignments. You must account for all positive lanes at least once. However, lanes can be used to assign more than one allele.

6. Be certain that all the lanes required for a particular allele are positive before making a specific allele assignment. Refer to Limitations and Cautions for reaction patterns that do not give a typing result.

7. When using the AllSet+™ Gold Kit for high resolution assignments, the assignments should not be made for allele groups exhibiting positive reactions other than the groups for which the test was designed, i.e., B*15 assignments should not be made from an AllSet+™ Gold B*35 High Resolution Kit.

8. Serological equivalent data is assigned based on the rel_dna_ser.txt file available from http://hla.alleles.org/wmda. Assignment of a given allele is based on the Unambiguous Serology and Expert Assigned designations present in this file. In the event an allele has both an Unambiguous Serology assignment and an Expert Assigned assignment, the worksheets and UniMatch™ will list the Unambiguous Serology assignment first followed by the Expert Assigned assignment in parenthesis.

As an example, the A*66:02 allele would be listed as having the following serological equivalent: A66(34).

It is important to note that this should not be mistaken as a serological ambiguity.

Methods Troubleshooting


Troubleshooting

Observation Possible Causes Recommended Action Intense smear of high molecular weight DNA present on gel photos of amplified products

Excess sample DNA, favoring nonspecific PCR products

Use the recommended amount of sample DNA.

Overall poor or absent amplification indicated by weak control bands, or absent or negative allele specific bands

Insufficient amount of DNA used in the amplification reaction

Check the volume and concentration of DNA. Adjust the DNA concentration to 50 ng/μL.

The DNA contains PCR inhibitors e.g. proteins, ethanol (from precipitation steps)

Measure the DNA purity. Try an alternative DNA extraction method.

The DNA has been extracted from heparinized blood

Use non-heparinized blood.

Impure DNA Measure the DNA quality. If necessary, repeat the DNA extraction with freshly prepared solutions.

Annealing temperature too high Check that the PCR parameters have been programmed correctly. Professionally calibrate thermal cyclers at least once every 12 months.

Poor quality of the Taq Polymerase

Use a validated DNA polymerase.



Observation Possible Causes Recommended Action Random amplification failure

PCR tubes that are not tightly closed will lead to evaporation and subsequent failure of amplification

• Make sure PCR sealing sheets or caps are affixed properly – use a capping tool.

• Check the heated lid of the Thermal cycler. 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.

Using non-calibrated pipettes Calibrate all pipettes routinely according to the supplier’s recommendations.

Sample DNA, Taq polymerase or PCR pre-mixtures have not been mixed properly before use

Mix briefly by vortexing before use.

The DNA is not mixed adequately with PCR buffer

Mix thoroughly before adding the mixture to the tray.

The DNA is not evenly re-suspended in diluent

Pipet the DNA up and down several times to aid mixing; alternatively, heat DNA on a heat block 10 minutes at 70ºC to dissolve.

Uneven volume of buffer/Taq/DNA solution added

Exercise caution when dispensing samples. Make sure all of the reaction mixture is contained under the paraffin oil.

False positive amplifications

Contamination • Use gloves, pipette tips containing filter plugs, and separate areas for pre and post-PCR.

• Employ careful and accurate sample handling in all steps.

• Check for amplification in the Contamination Control mix.

Impure DNA Measure the DNA quality. If necessary, repeat the DNA extraction with freshly prepared solutions.

Annealing temperature too low Check that the PCR parameters have been programmed correctly. Professionally calibrate the thermal cycler at least once every 12 months.

Excess DNA or Taq Polymerase Measure the DNA with UV spectrophotometry. Extensive delay between PCR setup

and start of thermal cycling No more than a 5-minute delay should be allowed before thermal cycling.

Incorrect order in gel loading Check alignment of mixes and gel lanes. Interpretation of primer dimer as

specific bands Check correct band size.



Observation Possible Causes Recommended Action False negative amplifications Annealing temperature too high Check that the PCR parameters have

been programmed correctly. Professionally calibrate thermal cycler at least once every 12 months.

Insufficient amount of DNA used in amplification

Check the volume and concentration of DNA.

Impure DNA Measure the DNA quality. Repeat the DNA extraction with freshly prepared solutions.

The amount of DNA Taq Polymerase used is too low

Check the volume carefully.

The quality of the Taq Polymerase is poor

Use a validated DNA polymerase.

Incorrect order in gel loading Check the alignment of mixes and gel lanes.

Overestimating DNA concentration measured by spectrophotometry

RNA contamination Run a small aliquot of sample DNA (about 200 ng) in a 0.7% agarose gel and compare it with a DNA marker of known concentration.

Overall fuzzy bands, smeared lanes

The gel is too thin due to excess evaporation while heating

Compensate for lost volume by adding water.

Agarose not completely dissolved Boil for an additional 30 seconds after melting.

Overheating gel, too high voltage Use lower voltage. TBE concentration too high The concentration should be 0.5X TBE. Heavy streaking in random wells

can be caused by uneven suspensions of DNA

Using an 8 channel pipette, mix the PCR product up and down 2 times before loading.

Rapid release of amplified product during gel loading can cause product to float out of well

Use slow, steady pipetting when loading gel.

Gel picture too dark Forgot to add ethidium bromide, or added the wrong amount

Use 2 µL ethidium bromide (10 mg/mL) for each 100 mL agarose solution.

Gel tray not UV transparent Remove the gel from the tray before viewing.

Incorrect camera setting Increase exposure time or aperture setting.

Gel picture too bright Excess amount of ethidium bromide

Use 2 µL ethidium bromide (10 mg/mL) for each 100 mL agarose solution.

Incorrect camera setting Decrease the exposure time or aperture setting.



Observation Possible Causes Recommended Action Occasional faint lanes The product floated out of well Pipette tips need to be properly aligned

with gel wells. Non-specific amplification (ladders or smears)

Impure DNA Measure the DNA quality. Repeat the DNA extraction with freshly prepared solutions.

Increasingly weak amplification signals over time

The ethidium bromide agarose gel staining solution is old

Prepare fresh ethidium bromide solution to achieve better staining of the gel.

Broken UV lamp Check the UV light equipment. Incorrect Kit Storage Follow storage instructions on product

packaging. Strange amplification patterns Incorrect interpretation table is

used Check that the lot number of the product used is the same as the lot number on the interpretation documents.

The amplification pattern contains a ‘false positive’

Check that all amplifications are the correct size. An artifact (carry-over, primer-dimer) may have been mis-interpreted as a specific amplification.

New allele, not covered by the interpretation table

Repeat the typing, if reproducible, contact technical support for information on amplification patterns of recently described alleles.

Amplicon fails to load properly The running buffer and gel buffer are not the same strength

• Use the same buffer for gel and running buffer; 0.5 x TBE is recommended.

• Pipet properly. The running buffer is at the wrong

concentration 0.5 x TBE is recommended.

Air bubbles in the pipette Pipet carefully.

Appendix A: Limitations and Cautions Precautions


Appendix A: Limitations and Cautions

Precautions • Before implementing the AllSet+™ Gold SSP method in your laboratory,

perform quality assurance and quality control for amplification based methods using known molecularly typed samples. Such samples can be obtained from the International Workshop Reference Cell Panel and the UCLA DNA Reference panel. Be sure to consult the Primer Mix Specificity Table and Ambiguity List for details on coverage.

• All primer mixes used in this kit have been tested with well‐characterized, molecularly typed DNA samples when available. Due to the lack of available reference material for all published alleles, some primer mixes may not be tested with positive control DNA. Refer to the Certificate of Analysis for detailed information. Kits are confirmed to cover 95% of HLA allele frequencies in the world population.

• HLA typing using AllSet+™ Gold SSP must be performed in the presence of a qualified Director, Technical Supervisor, and/or general Supervisor following accepted laboratory accreditation standards. We must emphasize that these products are for professional use only.

• All typing results should be confirmed using another typing method.

• In some cases, the SSP kit can give greater than 2‐digit resolution results. Low‐resolution kits are designed to be used for low‐resolution results; therefore, any result greater than 2‐digit resolution must be confirmed with a high‐resolution method before reporting.

Performance Characteristics • A comparison of AllSet+™ Gold SSP and Life Technologies Corporation SSP

UniTray® SSP typing methodologies was performed to assess the accuracy of the AllSet+™ Gold SSP typing kits. In the comparison, seventeen random samples were collected and analyzed for their HLA allelic typing using various Class I and II AllSet+™ Gold SSP and SSP UniTray® typing kits. The results show a 100% positive concordance (17/17) between the two methods.

• A Certificate of Analysis, which describes the performance characteristics of each manufactured batch, is included with each batch of kit. If additional information is requested, contact Technical Support at 1‐888‐821‐4443 (USA), 44 (0)141 814 6305 (Europe).

Appendix B: Safety General Safety Warning


Appendix B: Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.

• Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device.

• Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the “Documentation and Support” section in this document.

• All testing should be performed in accordance with local, regional and national acceptable laboratory accreditation standards and/or regulations.

Appendix B: Safety Chemical Safety


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions:

• Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document.

• Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing).

• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood).

• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturerʹs cleanup procedures as recommended in the SDS.

• Handle chemical wastes in a fume hood. • Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)

• After emptying a waste container, seal it with the cap provided. • Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory. • Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations. • IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

Appendix B: Safety Biological hazard safety


Biological hazard safety

WARNING! – BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following:

In the U.S.: • U.S. Department of Health and Human Services guidelines published

in Biosafety in Microbiological and Biomedical Laboratories found at: www.cdc.gov/biosafety

• Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR§1910.1030), found at: www.access.gpo.gov/nara/cfr/waisidx_01/ 29cfr1910a_01.html

• Your company’s/institution’s Biosafety Program protocols for working with/handling potentially infectious materials.

• Additional information about biohazard guidelines is available at: www.cdc.gov

In the EU: Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization (WHO) Laboratory Biosafety Manual, third edition, found at: www.who.int/csr/resources/publications/biosafety/ WHO_CDS_CSR_LYO_2004_11/en/


Documentation and Support

Obtain SDSs

Safety Data Sheets (SDSs) are available from www.lifetechnologies.com/support

For the SDSs of chemicals not distributed by Life Technologies, contact the chemical manufacturer.

Obtain support

For the latest services and support information for all locations, go to:

www.lifetechnologies.com/support

At the website, you can:

• Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support ([email protected])

• Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents

• Obtain information about customer training

• Download software updates and patches

Limited Product Warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.


IVD Symbols The symbols used on all labels and packaging conform to the harmonized standard EN980.

Symbol Description

In Vitro Diagnostic Medical Device

Manufacturer

Number of Tests

Consult Instructions for Use

Temperature Limitation (range)

Lower Temperature Limitation

Upper Temperature Limitation

Use By

Catalog Number

Batch Code

Read SDS Read Safety Data Sheet

Keep Away from Sunlight

Warning: Product may contain biohazardous material

Serial Number

Date of Manufacture

Authorized Representative in the European Community

Warning: Attention, see instructions for use

Caution, Risk of Electric Shock


References 1. Current HLA alleles can be found at www.anthonynolan.org.uk/HIG. 2. Olerup, O. & Zetterquist, H. (1992). HLA-DR typing by PCR amplification with

sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation [see comments]. Tissue Antigens 39(5), 225-35.

3. Bunce, M., O'Neill, C. M., Barnardo, M. C. N. M., Krausa, P., Browning, M. J., Morris, P. J. & Welsh, K. I. (1995b). Phototyping: Comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilising sequence-specific primers (PCR-SSP). Tissue Antigens 46(5), 355-367.

4. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350-4.

5. Wu, D. Y., Ugozzoli, L., Pal, B. K. & Wallace, R. B. (1989). Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A 86(8), 2757-60.

6. Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N., Smith, J. C. & Markham, A. F. (1989). Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17(7), 2503-16.

7. Richmond, Y. and McKinney, W. (eds.) 1993. Biosafety in microbiological and biomedical laboratories. HHS Publication Number (CDC) 93-8395.

8. National committee for Clinical Laboratory Standards. Protection of Laboratory Workers from Infectious Disease Transmitted by Blood, Body Fluids and Tissue. Tentative Guideline. NCCLS Document M29-T Villanova, PA:NCCLS, 1989.

9. National Committee for Clinical Laboratory Standards. Protection of Laboratory Workers from Infectious Disease Transmitted by Blood, Body Fluids and Tissue. Tentative Guideline. NCCLS Document M29-T Villanova, PA:NCCLS, 1989.

10. Satsangi J, Jewell DP, Welsh K, Bunce M, Bell JI.Effect of heparin on polymerase chain reaction. Lancet. 1994 Jun 11;343(8911):1509-10.

12 February 2013

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AllSet+ Gold SSP - БиоХимМак fileThe Invitrogen™ Corporation HLA AllSet+™ Gold SSP is a PCR‐based HLA typing evaluation method designed to provide low to high resolution