histocompatibility checklist - college of american

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Histocompatibility Checklist

CAP Accreditation Program

College of American Pathologists 325 Waukegan Road Northfield, IL 60093-2750 www.cap.org

07.11.2011

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Histocompatibility Checklist 07.11.2011

Disclaimer and Copyright Notice If you are enrolled in the CAP's Laboratory Accreditation Program and are preparing for an inspection, you must use the Checklists that were mailed in your application or reapplication packet, not those posted on the Web site. The Checklists undergo regular revision and Checklists may be revised after you receive your packet. If a Checklist has been updated since receiving your packet, you will be inspected based upon the Checklists that were mailed. If you have any questions about the use of Checklists in the inspection process, please e-mail the CAP ([email protected]), or call (800) 323-4040, ext. 6065. The checklists used in connection with the inspection of laboratories by the Laboratory Accreditation Program of the College of American Pathologists have been created by the College and are copyrighted works of the College. The College has authorized copying and use of the checklists by College inspectors in conducting laboratory inspections for the CLA and by laboratories that are preparing for such inspections. Except as permitted by section 107 of the Copyright Act, 17 U.S.C. sec. 107, any other use of the checklists constitutes infringement of the College’s copyrights in the checklists. The College will take appropriate legal action to protect these copyrights. All Checklists are ©2011. College of American Pathologists. All rights reserved.

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Histocompatibility Checklist

TABLE OF CONTENTS

SUMMARY OF CHANGES .................................................................................................................................. 5 UNDERSTANDING THE 2010 CAP ACCREDITATION CHECKLIST COMPONENTS ..................................... 7 HOW TO INSPECT USING R.O.A.D INSPECTION TECHNIQUES................................................................... 8 INTRODUCTION ................................................................................................................................................. 9 PROFICIENCY TESTING .................................................................................................................................... 9 QUALITY MANAGEMENT AND QUALITY CONTROL ..................................................................................... 10

GENERAL ISSUES ........................................................................................................................................ 10 PROCEDURE MANUAL ................................................................................................................................ 12 SPECIMEN COLLECTION AND HANDLING ................................................................................................ 13 RESULTS REPORTING ................................................................................................................................ 16 RECORDS ..................................................................................................................................................... 16

Recipient and Donor Information Records ................................................................................................. 18 REAGENTS ................................................................................................................................................... 18 CONTROLS AND STANDARDS ................................................................................................................... 21 INSTRUMENTS AND EQUIPMENT .............................................................................................................. 23

Temperature-Dependent Equipment .......................................................................................................... 25 Thermometers ............................................................................................................................................ 27 Colorimeters, Spectrophotometers, and Fluoriometers ............................................................................. 28 Film Processing/Photographic Equipment ................................................................................................. 29 Fume Hoods and Biological Safety Cabinets ............................................................................................. 29 Electrophoresis Equipment ........................................................................................................................ 30 pH Meters ................................................................................................................................................... 31 Balances and Weights ................................................................................................................................ 31 Volumetric Glassware and Pipettes ........................................................................................................... 33

PROCEDURES AND TEST SYSTEMS ............................................................................................................ 35 LYMPHOCYTE ISOLATION .......................................................................................................................... 35 SEROLOGICAL PROCEDURES ................................................................................................................... 35

General ....................................................................................................................................................... 35 HLA Class I and II Antigen Typing ............................................................................................................. 36 Cytotoxicity Crossmatch ............................................................................................................................. 39

RED CELL TYPING ....................................................................................................................................... 40 FLOW CYTOMETRY ..................................................................................................................................... 43

Instrumentation and Phenotyping ............................................................................................................... 43 Flow Cytometry Crossmatch ...................................................................................................................... 46

MIXED LYMPHOCYTE CULTURE TESTING ............................................................................................... 48 HLA ANTIBODY SCREENING ...................................................................................................................... 49

Enzyme-Linked Immunosorbent Assays (ELISA) ...................................................................................... 51 MOLECULAR HLA ANTIGEN TYPING ......................................................................................................... 52 DONOR-RECIPIENT HISTOCOMPATIBILITY .............................................................................................. 59

Non-Renal Organ Transplants ................................................................................................................... 60 PERSONNEL ..................................................................................................................................................... 61

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SUMMARY OF CHECKLIST EDITION CHANGES Histocompatibility Checklist

07/11/2011 Edition

The following requirements have been added, revised, or deleted in this edition of the checklist, or in the two editions immediately previous to this one. If this checklist was created for a reapplication, on-site inspection or self-evaluation it has been customized based on the laboratory's activity menu. The listing below is comprehensive; therefore some of the requirements included may not appear in the customized checklist. Such requirements are not applicable to the testing performed by the laboratory. Note: For revised checklist requirements, a comparison of the previous and current text may be found on the CAP website. Click on Laboratory Accreditation, Checklists, and then click the column marked Changes for the particular checklist of interest. NEW Checklist Requirements None REVISED Checklist Requirements Requirement Effective Date HSC.10475 07/11/2011 HSC.20020 06/17/2010 HSC.20035 07/11/2011 HSC.20060 07/11/2011 HSC.21020 07/11/2011 HSC.21500 07/11/2011 HSC.21800 07/11/2011 HSC.22160 06/17/2010 HSC.22300 07/11/2011 HSC.22400 07/11/2011 HSC.22500 06/17/2010 HSC.23137 07/11/2011 HSC.23511 07/11/2011 HSC.24633 07/11/2011 HSC.26690 06/17/2010 HSC.31178 07/11/2011 DELETED Checklist Requirements Requirement Effective Date HSC.05000 07/10/2011 HSC.10000 07/10/2011 HSC.10200 07/10/2011 HSC.10400 07/10/2011 HSC.10550 07/10/2011 HSC.10650 07/10/2011 HSC.10750 07/10/2011 HSC.20005 06/16/2010 HSC.20100 07/10/2011

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HSC.20900 07/10/2011 HSC.20920 07/10/2011 HSC.20940 07/10/2011 HSC.20950 07/10/2011 HSC.20960 07/10/2011 HSC.21820 07/10/2011 HSC.22325 07/10/2011 HSC.22450 07/10/2011 HSC.22900 07/10/2011 HSC.22950 07/10/2011 HSC.24820 07/10/2011 HSC.31365 07/10/2011 HSC.50000 07/10/2011 HSC.50100 07/10/2011 HSC.50200 07/10/2011 HSC.50300 07/10/2011 HSC.50400 07/10/2011 HSC.50500 07/10/2011 HSC.50600 07/10/2011 HSC.50700 07/10/2011 HSC.50800 07/10/2011 HSC.50900 07/10/2011 HSC.51000 07/10/2011 HSC.51100 07/10/2011 HSC.51200 07/10/2011

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UNDERSTANDING THE CAP ACCREDITATION CHECKLIST COMPONENTS

To provide laboratories with a better means to engage in and meet their accreditation requirements, the CAP has enhanced the checklist content and updated its design. New components containing additional information for both the laboratory and inspectors include Subject Headers, Declarative Statements and Evidence of Compliance. See below for a definition of each new feature as an example of how they appear in the checklists.

Using Evidence of Compliance (EOC)

This component, which appears with several checklist requirements, is intended to:

1 Assist a laboratory in preparing for an inspection and managing ongoing compliance 2 Drive consistent understanding of requirements between the laboratory and the inspector 3 Provide specific examples of acceptable documentation (policies, procedures, records, reports,

charts, etc.) In addition to the Evidence of Compliance listed in the checklist, other types of documentation may be acceptable. Whenever a policy/procedure/process is referenced within a requirement, it is only repeated in the Evidence of Compliance if such statement adds clarity. All policies/procedures/processes covered in the CAP checklists must be documented. A separate policy is not needed for each item listed in EOC as it may be referenced in an overarching policy.

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HOW TO INSPECT USING R.O.A.D INSPECTION TECHNIQUES (Read, Observe, Ask, Discover)

CAP has streamlined the inspection approach used during onsite inspections and is now offering guidance to inspectors by providing assessment techniques to facilitate a more efficient, consistent, and effective inspection process. Specific inspector instructions are listed at the beginning of a grouping of related requirements. Rather than reviewing each individual requirement, CAP inspectors are encouraged to focus on the Inspector Instructions for a grouping of related requirements. Once an area of concern has been identified through "Read," "Observe," "Ask," "Discover," or a combination thereof, inspectors are encouraged to "drill down" to more specific requirements, when necessary and review more details outlined in the Evidence of Compliance statements. If a requirement is non-compliant, circle the requirement number to later list on the Inspector Summation Report. Inspectors may also make notes in the margins of the checklist document. Inspector Instructions and Icons used to evaluate a laboratory's performance now appear in several areas throughout the Inspector Checklists. Please note that all four R.O.A.D elements are not always applicable for each grouping, or sections of related requirements.

Inspector Instructions:

READ/review a sampling of laboratory documents. Information obtained from this review will be useful as you observe processes and engage in dialogue with the laboratory staff. (Example of the complimentary inspector instructions for Quality Management/Quality Control General Issues section appearing across checklists):

● Sampling of QM/QC policies and procedures

● Incident/error log and corrective action

OBSERVE laboratory practices by looking at what the laboratory personnel are actually doing and note if practice deviates from the documented policies/procedures. (Example)

● Observe the settings/QC range limits established in the laboratory LIS/HIS to ensure that the laboratory's stated ranges are accurately reflected

ASK open-ended, probing questions that start with phrases such as "tell me about..." or "what would you do if..." This approach can be a means to corroborate inspection findings that were examined by other techniques, such as Read & Observe. Ask follow-up questions for clarification. Include a variety of staff levels in your communication process. (Example)

● As a staff member, what is your involvement with quality management?

● How do you detect and correct laboratory errors?

DISCOVER is a technique that can be used to "drill down" or further evaluate areas of concern uncovered by the inspector. "Follow the specimen" and "teach me" are two examples of Discovery. Utilizing this technique will allow for the discovery of pre-analytic, analytic, and post-analytic processes while reviewing multiple requirements simultaneously. (Example)

● Select several occurrences in which QC is out of range and follow documentation to determine if the steps taken follow the laboratory policy for corrective action

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INTRODUCTION

Inspectors of a histocompatibility laboratory should be pathologists, clinical scientists or medical technologists who are actively involved with or have extensive experience in the practice of histocompatibility testing and are knowledgeable about current CAP Checklist and CLIA requirements. Inspectors preferably should have participated in a recent CAP Inspector Training activity. Inspectors should, to the greatest extent possible, be peers of the laboratory being inspected. An inspection of a laboratory section, or department will include the discipline-specific checklist(s), the Laboratory General Checklist, and the All Common Checklist. In response to the ongoing request to reduce the redundancy within the Accreditation Checklists, the CAP accreditation program is introducing the All Common Checklist (COM). The purpose of the All Common Checklist is to group together those requirements that were redundant in Laboratory General and the discipline-specific checklists. Therefore, the CAP centralized all requirements regarding: proficiency testing, procedure manuals, test method validations, and critical results into one checklist, the COM checklist. Note for non-US laboratories: Checklist requirements apply to non-US laboratories unless the checklist items contain a specific disclaimer of exclusion.

PROFICIENCY TESTING


● Are proficiency testing samples tested with the same cut-offs for clinical HLA antibody determination as clinical specimens?

● Are proficiency testing samples for HLA antigens tested to the same level of resolution as clinical specimens?

● Select a representative clinical report from each service area. Compare the extent of reporting for the relevant proficiency testing sample.

**REVISED** 07/11/2011 HSC.10475 PT Extent of Testing Phase II

Proficiency testing specimens are tested to the same extent as clinical specimens.

NOTE: Proficiency testing samples are to be tested in the same way as regular patient samples. They should not receive more extensive testing than would be performed under routine testing conditions. For example, high-resolution analysis and sequence-based typing may be used only if they are regularly used in similar clinical circumstances.

Evidence of Compliance:

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✓ Comparison of patient and proficiency testing work records demonstrating identification to the same extent

QUALITY MANAGEMENT AND QUALITY CONTROL

GENERAL ISSUES


● Sampling of QM/QC policies and procedures

● QM/QC program, including pre-analytic, analytic and post-analytic monitor records and corrective action when indicators do not meet threshold

● Incident/error log and corrective action

● Records of high school graduate high complexity test review by supervisor

● How do you evaluate data on the incident/error log? How do you determine appropriate corrective action?

● As a staff member, what is your involvement with quality management?

● How do you detect and correct laboratory errors?

● How do you control for inter-observer variation?

● How does your laboratory evaluate the test menu and level of resolution provided?

● Select several occurrences in which component QC is not acceptable and follow documentation to determine if the steps taken follow the laboratory policy for corrective action

● Follow an incident identified on the incident/error log and follow actions including notification and resolution

● Select several problems identified by the QM plan and follow tracking and corrective action. Determine if the methods used led to discovery and effective correction of the problem.

● Examine records (reagent, function checks, temperature records) for review by director or designee. Determine if exceptions have been investigated properly.

HSC.20000 Documented QM/QC Plan Phase II

The histocompatibility laboratory has a written quality management/quality control (QM/QC) program.

NOTE: The QM/QC program in the histocompatibility laboratory must be clearly defined and documented. The program must ensure quality throughout the pre-analytic, analytic, and post-analytic (reporting) phases of testing, including patient identification and preparation; specimen collection, identification, preservation, transportation, and processing; and accurate, timely result reporting. The program must be capable of detecting problems in the laboratory's systems, and identifying opportunities for system improvement. The laboratory must be able to develop plans of corrective/preventive action based on data from its QM system.

All QM requirements in the Laboratory General Checklist pertain to the histocompatibility laboratory. REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

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amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493.1227], 7176 [42CFR493.1445(e)(5)

HSC.20010 Specimen Collection Manual Phase II

There is a documented procedure describing methods for patient identification, patient preparation, specimen collection and labeling, specimen preservation, and conditions for transportation, and storage before testing, and this procedure is consistent with good laboratory practice.

**REVISED** 06/17/2010 HSC.20020 Ongoing Record Evaluation Phase II

There is evidence of monthly evaluation of records of instrument function and maintenance, temperature, etc. for all procedures as applicable, including refrigerators/freezers in which reagents or patient specimens are kept.

NOTE: For instruments requiring calibration, calibration must be verified at least every six months.

**REVISED** 07/11/2011 HSC.20035 Monthly QC Review Phase II

There is documentation of ongoing evaluation by the section director or designee of all of the following.

1. Control results of routine procedures 2. Reactivity of reagents 3. Instrument function checks 4. Temperature records

NOTE: Quality control records must be reviewed and assessed at least monthly by the laboratory director or designee.

The QC data for tests performed less frequently than once per month should be reviewed when the tests are performed.

HSC.20050 QC Corrective Action Phase II

There is documentation of corrective action taken when control results exceed defined acceptability limits.

NOTE: Patient/client test results obtained in an analytically unacceptable test run or since the last acceptable test run must be re-evaluated to determine if there is a significant clinical difference in patient/client results. Re-evaluation may or may not include re-testing patient samples, depending on the circumstances.

Even if patient samples are no longer available, test results can be re-evaluated to search for evidence of an out-of-control condition that might have affected patient results. For example, evaluation could include comparison of patient means for the run in question to historical patient means, and/or review of selected patient results against previous results to see if there are consistent biases (all results higher or lower currently than previously) for the test(s) in question). REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 1992(Feb 28):7176 [42CFR493.1445(e)]

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**REVISED** 07/11/2011 HSC.20060 Unusual Laboratory Results Phase II

There is a documented system in operation to detect and correct significant clerical and analytical errors, and unusual laboratory results, in a timely manner.

NOTE: One common method is review of results by a qualified person (technologist, supervisor, pathologist, laboratory director) before release from the laboratory, but there is no requirement for supervisory review of all reported data for single analyte tests that do not include interpretation. All tests that include an interpretation must be reviewed by the laboratory director or qualified designee before release from the laboratory. In computerized laboratories, there should be automatic "traps" for improbable results. The system for detecting clerical errors, significant analytical errors, and unusual laboratory results must provide for timely correction of errors, i.e. before results become available for clinical decision making. For confirmed errors detected after reporting, corrections must be promptly made and reported to the ordering physician or referring laboratory, as applicable.

Each procedure must include a listing of common situations that may cause analytically inaccurate results, together with a defined protocol for dealing with such analytic errors or interferences. This may require alternate testing methods; in some situations, it may not be possible to report results for some or all of the tests requested.

The intent of this requirement is NOT to require verification of all results outside the reference (normal) range.


✓ Record of review of results OR records of consistent implementation of the error detection system(s) defined in the procedure AND

✓ Records of timely corrective action of identified errors

HSC.20080 Supervisory Result Review Phase II

In the absence of on-site supervisors, the results of tests performed by personnel are reviewed by the laboratory director or general supervisor within 24 hours.

NOTE: This only applies to laboratories subject to US regulations. The CAP does NOT require supervisory review of all test results before or after reporting to patient records. Rather, this requirement is intended to address only that situation defined under CLIA for "high complexity testing" performed by trained high school graduates qualified under 42CFR493.1489(b)(5) when a qualified general supervisor is not present.


✓ Written policy defining the review process and personnel whose results require review AND

✓ Records of result review for specified personnel REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 1992(Feb 28):7182 [42CFR493.1463(a)(3) and 42CFR493.1463(c)], 7183 [42CFR493.1489(b)(1) and 42CFR493.1489(b)(5)]

PROCEDURE MANUAL


● Representative sample of procedures for completeness and director review. Current practice

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must match contents of procedures.

HSC.20200 Procedure Manual Phase II

The procedure manual contains specific instructions for test performance, preparation of reagents, and control methods for each of the following procedures.

1. Lymphocyte isolation 2. HLA-ABC serologic typing 3. HLA-DR, DQ serologic typing 4. DNA or RNA typing 5. Crossmatching-T cells (ABC loci) 6. Crossmatching-B cells (DR locus) 7. Flow cytometry crossmatching 8. Antibody screening and identification 9. ABO grouping 10. Mixed lymphocyte cultures (MLC) 11. Complement titration 12. Environmental control

SPECIMEN COLLECTION AND HANDLING


● Sampling of histocompatibility specimen collection/handling/tracking retrieval policies and procedures, including specimens for flow cytometry

● Sampling of specimen rejection records/log

● Evaluation records (specimen collection containers/anticoagulants) for preservation of sample integrity

● Donor arm preparation

● What are the specimen acceptability criteria for each specimen type?

● What is your course of action when you receive unacceptable/sub-optimal histocompatibility specimens?

● How does your laboratory ensure specimen identity throughout testing?

● How does your laboratory ensure appropriate storage of flow cytometry specimens?

● How does your laboratory ensure preservation of antibody integrity in recipient sera?

● Review records of unacceptable specimens and follow up. Determine if practice matches procedure.

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HSC.20970 Specimen Tracking Phase II

Procedures are adequate to maintain sample identity and integrity throughout all steps of the process.

NOTE: There must be a system in place to verify the identity of specimens, patients, donors, reagents; procedures utilized for testing; date and location of test performance; and the individuals performing the tests. REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 1992(Feb 28):7162 [42CFR493.1103] 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C2.120, C2.130. Richmond, VA:

UNOS, 2001

HSC.20976 Donor Arm Preparation Phase II

A documented procedure is followed for donor arm preparation that reduces the risk of bacterial contamination of the donor sample.

NOTE: The specific procedure used may vary but should include directions for the chemicals to be used, the time and manner that each is applied and the EXACT sequence and procedures so that bacterial contamination from removable surface microorganisms is minimized.

HSC.20982 Specimen Collection Procedures Evaluation Phase II

The laboratory evaluates its specimen collection procedures to ensure that the anticoagulant/preservation medium in use does not contribute to analytic interference in the assays to be performed, and that it preserves sample integrity as necessary.

NOTE: This may be done through some combination of direct testing by the laboratory, review of the clinical literature, and evaluation of information from manufacturers. It does not mandate exhaustive testing by each laboratory.


✓ Records documenting the evaluation of specimen collection procedures and anticoagulants in collection containers

HSC.20988 Specimen Integrity - Flow Cytometry Phase II

There is a procedure in place to document the integrity of specimens for flow cytometry.

NOTE: The yield of T lymphocytes from blood samples is affected by a number of factors. If specimens are not processed immediately after collection, the laboratory should verify that its anticoagulant, holding temperature and preparation method maintain specimen integrity. Selective loss of cell subpopulations and/or the presence of dead cells may lead to spurious results. Routine viability testing is not necessary on specimens of whole blood that are analyzed within 24 hours of drawing. Analyses on older samples are possible if the laboratory has verified the absence of statistical differences between the fresh and aged specimen phenotype fractions being evaluated.

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HSC.20994 Specimen Storage - Flow Cytometry Phase II

Flow cytometry specimens are stored appropriately after initial processing.

NOTE: For example, paraformaldehyde (0.5%) fixation of stained cells preserves cellular integrity and fluorescence for up to 5 days. Caution must be exercised in utilizing this procedure, as fluorescence may be diminished with some reagents and cytometers.


✓ Written procedure for specimen storage REFERENCES 1) American Society for Microbiology. Manual of clinical immunology, 4th ed. Washington, DC: ASM, 1992:940 2) Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved

Guideline—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007

HSC.21000 Unacceptable Specimens Phase II

There are documented criteria for unacceptable specimens.

NOTE: This requirement does not imply that all "unsuitable" specimens are discarded or not analyzed. If a sample is received that fails to meet acceptability criteria, there must be a mechanism to notify the requesting physician, and to note the condition of the sample on the report if the result is desired by the ordering physician. Some or all tests may not be analytically valid on such a specimen. The laboratory may wish to record that a dialogue was held with the physician, when such occurs.


✓ Records of rejected specimens REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7162 [42CFR493. 1291(c)(7)] 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C2.140. Richmond, VA: UNOS, 2001

**REVISED** 07/11/2011 HSC.21020 Disposition of Unacceptable Specimens Phase II

The disposition of all unacceptable specimens is documented in the patient report and/or quality management records.

NOTE: This information is essential to proper patient test management and to the laboratory quality management program.

HSC.21050 Recipient Sera Phase II

The most appropriate recipient sera are employed for final crossmatching.

NOTE: There must be a documented policy defining an appropriate crossmatch specimen to utilize in transplantation that takes into consideration the potential recipient's past pregnancies, past transplants, recent blood transfusions, and sensitization history. The specimens must have been properly handled and appropriately stored to preserve antibody integrity. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.230. Richmond, VA: UNOS, 2001

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HSC.21130 Specimen Retrieval Phase II

Procedures are adequate to ensure that patient specimens are easily retrievable.

REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493. 1278(f)]

RESULTS REPORTING


● Sampling of patient reports for completeness and review

● Sampling of reference laboratory accreditation records

● How are urgent results communicated?

● How do you store results for comparison with subsequent reports?

HSC.21250 Patient Report Requirements Phase II

Patient results are reported in a legible, easy to interpret format that clearly indicates the test method and delineates the clinical implications of the results.

NOTE: Interpretation of reports must document the identity of the Laboratory Director.

HSC.21275 Final Report Phase II

The final report includes an appropriate summary of the methods, the loci tested, the objective findings, all possible allele combinations, and an interpretation.

REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C5.000. Richmond, VA: UNOS, 2001

HSC.21281 Accreditation of Reference Laboratory Phase II

Outside reference laboratories are accredited by appropriate histocompatibility agencies (e.g. UNOS), and, for US laboratories, CLIA certified.


✓ Records verifying reference laboratory certification/accreditation in histocompatibility

HSC.21287 Report Review Phase II

All laboratory reports (including reports from outside referral laboratories) are reviewed by the section director (technical supervisor) or designee prior to release.

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RECORDS

The following types of records should be kept to the extent of services provided by the laboratory. If a service is not provided, mark the requirement "N/A."


● Record retention policy

● Sampling of stored specimen inventory records/log

● Sampling of transplant donor and recipient records

● Verification of UNOS patient data policy (interval of review documented)

● Sampling of UNOS patient histocompatibility data review and verification

● What process does your laboratory follow when resolving inter-laboratory HLA typing discrepancies?

● Review all records of a sampling of patient and donor histocompatibility results and reports to document completion of all steps in the process from specimen requisitions to final disposition. Determine if records provide an adequate audit trail of all activities.

HSC.21300 Record Retention Phase II

Records of results of all histocompatibility-testing reports are retained and easily accessible.

NOTE: The laboratory must retain all final and preliminary test results and records for an appropriate period. The following records must be retained for at least 2 years: specimen requisitions, patient and donor histocompatibility results and reports, identity of testing personnel, all blood bank typing/grouping and crossmatch studies, instrument printouts, accession records, all internal and external quality control records, proficiency testing records, and quality assurance records. These files should be retained in a manner that allows prompt retrieval of information. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C5.100, C5:110. Richmond, VA:

UNOS, 2001


A copy of each final report, all records of results, reagent lots, membranes, autoradiographs, gel photographs, and in situ hybridization slides, are retained in compliance with existing laws.


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HSC.21332 Stored Specimen Log Phase II

A log of all stored specimens is maintained to enable prompt retrieval for further testing.


✓ Electronic or paper inventory log of stored specimens

Recipient and Donor Information Records HSC.21350 UNOS Clinical Transplant Registry Phase II

The institution participates in and maintains records of patient and donor transplant information in the United Network for Organ Sharing (UNOS) Clinical Transplant Registry or its equivalent.

NOTE: The laboratory and/or transplant coordinator must maintain records on transplant recipients, including a history of prior transfusion, pregnancy, and prior transplants as well as PRA, date of transplant and outcome. In addition, there should be records of donor and recipient age, race, sex, ABO, and HLA types. The source of the donor kidney should be documented. This information can be maintained as part of an institutional registry. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C4.400. Richmond, VA: UNOS, 2001

HSC.21366 UNOS Record Review Phase II

There is documentation of periodic review and verification of UNOS patient histocompatibility data.

NOTE: Histocompatibility tests performed for organ transplantation (HLA typing, HLA antibody sensitization, unacceptable antigens during prior transplants or sensitization, and any pretransplant screening results) must be reviewed and verified when patients are placed on UNOS organ waiting lists. Changes or additions to the waiting lists must be verified. This patient documentation must be readily available for review, and maintained for at least 2 years. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C4.400. Richmond, VA: UNOS, 2001

HSC.21382 Discrepancy Resolution Phase II

There is a system to resolve HLA typing discrepancies between laboratories.

NOTE: There must be documentation of the steps taken to resolve discrepancies.


REAGENTS

The laboratory has the responsibility for ensuring that all reagents used, whether purchased or prepared by the laboratory, are appropriately reactive. The verification of reagent performance is required and must be documented. Any of several methods may be appropriate, such as direct analysis with reference materials, parallel testing of old vs. new reagents, and checking against routine controls. The intent of the requirements is for new reagents to be checked by an appropriate method and the results recorded before patient results are

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reported. Where individually packaged reagents/kits are used, there should be criteria established for monitoring reagent quality and stability, based on volume of usage and storage requirements. Processing of periodic "wet controls" to validate reagent quality and operator technique is a typical component of such a system.


● Sampling of test procedures for reagent storage and handling

● Sampling of new reagent/shipment validation records

● Inventory log

● Sampling of procedures for patient sample storage and handling

● Sampling of typing/screening tray records for completeness

● Validation studies for modified reagents

● Sampling of alarm monitoring records, if applicable

● Sampling of reagents (expiration date, labeling, storage)

● How do you validate and document new reagent lots?

● What are your laboratory's criteria for mixing components from one lot number of reagent kit with components from another lot number of kit?

● How do you ensure that all reagents are acceptable and in date?

● How does your laboratory manage and control reagent inventory?

HSC.21400 Reagent Labeling Phase II

Reagents and solutions are properly labeled, as applicable and appropriate, with the following elements.

1. Content and quantity, concentration or titer 2. Storage requirements 3. Date prepared or reconstituted by laboratory 4. Expiration date

NOTE: The above elements may be recorded in a log (paper or electronic), rather than on the containers themselves, providing that all containers are identified so as to be traceable to the appropriate data in the log. While useful for inventory management, labeling with "date received" is not routinely required. There is no requirement to routinely label individual containers with "date opened"; however, a new expiration date must be recorded if opening the container changes the expiration date, storage requirement, etc.


✓ Written policy defining elements required for reagent labeling REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7164 [42CFR493.1252(c)] 2) Clinical and Laboratory Standards Institute (CLSI). Laboratory Documents: Development and Control; Approved Guideline—Fifth

Edition. CLSI document GP2-A5 (ISBN 1-56238-600-X). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006

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**REVISED** 07/11/2011 HSC.21500 Reagent Expiration Date Phase II

For US laboratories, outdated reagents are handled in compliance with Food and Drug Administration requirements, and discarded/replaced as indicated.

NOTE: For laboratories not subject to US regulations, expired reagents may be used only under the following circumstances: 1. The reagents are unique, rare or difficult to obtain; or 2. Delivery of new shipments of reagents is delayed through causes not under control of the laboratory. The laboratory must document validation of the performance of expired reagents in accordance with written laboratory policy. Laboratories subject to US regulations must not use expired reagents. REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7164 [42CFR493.1252(d)]

HSC.21550 New Reagent Lot Verification Phase II

New reagent lots and/or shipments are checked against old reagent lots or with suitable reference material before or concurrently with being placed in service.

NOTE: There must be documentation that new reagent lots, batches and/or shipments (prepared commercially or locally) are checked against previous lots or known standards before or concurrent with being placed in service. Reagents include typing trays, primer, complement, and other materials used for patient testing.


✓ Written policy for the verification of new lots and shipments prior to use AND

✓ Records of verification of new reagents/shipments REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7166 [42CFR493.1256(e)]

HSC.21612 Reagent Tracking Phase II

There is a documented system for identification of the specific reagent lot numbers and shipments used for each assay.


amendments of 1988; final rule. Fed Register. 2004(Oct 1): 1038 [42CFR493.1256(a)] 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C3.500. Richmond, VA: UNOS, 2001 3) American Society for Histocompatibility and Immunogenetics. Standards for histocompatibility testing. Lenexa, KS: ASHI, 2005:

D.4.6.4.1

HSC.21675 Reagent Kit Components Phase II

Combinations of reagents from different lots are checked against old reagent lots or with suitable reference material before or concurrently with being placed in service.


✓ Written documentation defining allowable exceptions for mixing kit components from different lots

**REVISED** 07/11/2011

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HSC.21800 Specimen Storage Procedure Phase II

Procedures and conditions are established and documented for optimal reagent and patient sample storage.

NOTE 1: Written procedures should document storage conditions of frozen lymphocytes, frozen lymphocyte trays, and sera.

NOTE 2: Monitored alarms and back-up storage plans should be specified as applicable.


✓ Written procedure defining criteria for specimen/reagent storage with backup plan in case of equipment failure

REFERENCES 1) American Society for Histocompatibility and Immunogenetics. Standards for histocompatibility testing. Lenexa, KS: ASHI, 1995-2002:

C2.130, C2.310, C2.320

HSC.21810 Specimen Handling - Typing/Screening Trays Phase II

If typing trays and antibody screening trays are prepared locally, the records indicate source, bleeding date, donor, identification, and available volume for sera and a means of identifying, locating and collecting fresh donor cells.


amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493. 1278(a)(3)], 7171 [42CFR492. 1278(d)(3)]

HSC.21835 Modified Reagent Use Phase II

If reagents are used in a manner different than manufacturer's instructions, appropriate validation studies are performed and documented.


✓ Validation study data

CONTROLS AND STANDARDS


● Sampling of QC policies and procedures

● Sampling of lymphocyte preparation viability checks

● Sampling of QC records

● Control material (labeling)

● How do you determine when QC is unacceptable and corrective actions are needed?

● What is your course of action when QC for compatibility testing is not acceptable?

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● Select several occurrences in which QC is out of range and follow documentation to determine if the steps taken follow the laboratory policy for corrective action

HSC.21850 Daily Controls Phase II

Positive and negative controls are assayed daily, and there are positive controls for specific cell types (T cells, B cells, etc.), where available.

NOTE: Positive and negative controls must be run with each test procedure where appropriate. This must include daily positive controls for specific cell type (T cells, B cells, etc.), as well as appropriate antibody isotypes as needed for each assay. This must also include one positive control serum that is historically reactive to all Class I and/or Class II positive cells at the same dilutional titer as appropriate for the methodology utilized.


✓ Written QC procedure for each test describing the quality control materials used AND

✓ Records of control results REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7168 [42CFR493. 1256(d)(3)(iii)] 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.410, E2.420. Richmond, VA:

UNOS, 2001

HSC.21900 Control Labeling Phase II

Controls are properly labeled with content, lot number, date of preparation and expiration date.


✓ Written policy defining elements required for control labeling

HSC.21950 Viability Checks Phase II

Viability checks on lymphocyte preparations are performed and documented by recording negative control results or by performing a separate test each time they are used.

NOTE: Viability checks on the lymphocyte preparations must be performed and documented with each use. For cytotoxicity procedures, cell viability after initial incubation should be greater than 80% in the negative control well.


✓ Written procedure for evaluating viability of lymphocyte preparations REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.430. Richmond, VA: UNOS, 2001

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HSC.22070 Compatibility Testing Controls Phase II

The laboratory includes control material for each phase of compatibility testing.

NOTE: Results of patient testing must not be reported until control values are reviewed and found acceptable.


✓ Written QC procedure for compatibility testing that describes the quality control material used REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7171 [42CFR493. 1278(c) and (e)(3)]

HSC.22140 QC Handling Phase II

Control specimens are tested in the same manner and by the same personnel as patient samples.

NOTE: QC specimens must be analyzed by personnel who routinely perform patient testing - this does not imply that each operator must perform QC daily, so long as each instrument and/or test system has QC performed at required frequencies, and all analysts participate in QC on a regular basis. To the extent possible, all steps of the testing process must be controlled, recognizing that pre-analytic and post-analytic variables may differ from those encountered with patients.


✓ Records reflecting that QC is run by the same personnel performing patient testing REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7166 [42CFR493. 1256(d)(8)]

**REVISED** 06/17/2010 HSC.22160 QC Verification Phase II

The results of controls are verified for acceptability before reporting results.

NOTE: If the positive and negative controls do not give the expected outcome, the results are not reportable. The negative control serum is one that historically has been negative with all tested cells. The negative control may also originate from a non-sensitized male whose serum has been shown to be totally negative for cell death in cytotoxicity systems.


✓ Written policy/procedure stating that controls are reviewed and acceptable prior to reporting patient results AND

✓ Evidence of corrective action taken when QC results are not acceptable REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7166 [42CFR493. 1256(f)]

INSTRUMENTS AND EQUIPMENT

A variety of instruments and equipment are used to support the performance of analytical procedures. All instruments and equipment should be properly operated, maintained, serviced, and monitored to ensure that malfunctions of these instruments and equipment do not adversely affect the analytical results. The procedures

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and schedules for instrument maintenance must be as thorough and as frequent as specified by the manufacturer.


● Sampling of instrument(s) policies and procedures

● Sampling of instrument maintenance logs and repair records

● Instrument records (promptly retrievable)

HSC.22180 Routine Maintenance Schedule Phase II

There is a routine schedule or system for the regular checking of the critical operating characteristics of all instruments in use.


amendments of 1988; final rule. Fed Register. 1992(Feb 28):7164 [42CFR493.1256]

HSC.22200 Function Checks Phase II

Instructions for instrument function checks are available (i.e. manufacturer's manual or documented system prepared by the laboratory).



HSC.22250 Function Checks Phase II

Function checks are documented and conveniently located to detect trends or malfunctions.



**REVISED** 07/11/2011 HSC.22300 Instrument Tolerance Limits Phase II

Acceptable tolerance limits for instrument function are specified.


✓ Documentation of corrective action when tolerance limits are exceeded REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement


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HSC.22350 Instrument Troubleshooting Phase II

Instructions are provided for minor troubleshooting and repairs of instruments (such as manufacturer's service manual).

**REVISED** 07/11/2011 HSC.22400 Maintenance Records Phase II

Records are maintained for each instrument to document all repairs and service procedures and are readily available to the technical staff.

NOTE: Effective utilization of instruments by the technical staff depends upon the prompt availability of maintenance, repair, and service documentation. Laboratory personnel are responsible for the reliability and proper function of their instruments and must have access to this information. Off-site storage, such as with centralized medical maintenance or computer files, is not precluded if the inspector is satisfied that the records can be promptly retrieved. REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 1992(Feb 28):7146 [42CFR493.801(b)]

Temperature-Dependent Equipment


● Sampling of temperature logs (refrigerator, freezer, dialyzer bath, incubator, oven, water bath, heat block, thermocycler)

● Sampling of LN2 monitoring records

● What back-up options are available in the event of an electrical power outage?

● How is the storage unit alarm system monitored? How was the response time validated?

● How the laboratory ensure the individual wells of the thermocycler are maintaining accurate temperature?

● Review the calibration records of devices with built-in temperature sensors and controls. Determine if calibration accuracy is sufficient.

**REVISED** 06/17/2010 HSC.22500 Temperature Recording Phase II

Temperatures are checked and recorded daily for each of the following types of equipment.

1. Water baths and heating blocks 2. Incubators and ovens (where temperature control is necessary for a

procedure)

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3. Dialyzer baths 4. Refrigerators and freezers

NOTE: Temperature-dependent equipment containing reagents and patient specimens must be monitored daily, as equipment failures could affect accuracy of patient test results. Items such as water baths and heat blocks used for procedures need only be checked on days of patient testing.

The two acceptable ways of recording temperatures are: 1) recording the numerical temperature, or 2) placing a mark on a graph that corresponds to a numerical temperature (either manually, or using a graphical recording device). The identity of the individual recording the temperature(s) must be documented (recording the initials of the individual is adequate).

The use of automated (including remote) temperature monitoring systems is acceptable, providing that laboratory personnel have ongoing immediate access to the temperature data, so that appropriate corrective action can be taken if a temperature is out of the acceptable range. The functionality of the system must be documented daily. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.300. Richmond, VA: UNOS, 2001

HSC.22531 Alarm System Phase II

There is an audible alarm for each sample or reagent storage unit, and the alarm is monitored 24 hours per day (in laboratory or remotely).

NOTE: All storage units must have an audible alarm with continuous monitoring (in laboratory or remote). The laboratory should be able to demonstrate how this system works, and that there is a process to ensure a timely response to an alarm.

HSC.22562 Alarm System Checks Phase II

Alarm systems are checked at specified periodic intervals and results recorded.

NOTE: The alarm system must be checked at specified periodic intervals for both high and low settings to ensure proper function.

HSC.22593 Power Failure Back-up Phase II

The alarms continue to function if the power is interrupted.

NOTE: Alarm systems must have a source of power separate from the house current, in order to allow proper monitoring during power failures. This can be accomplished by a separate circuit, power failure alarm, or battery power.

HSC.22625 Cell Freezers Phase II

Cell freezers that use liquid nitrogen are monitored.

NOTE: The system must ensure that an adequate supply of liquid nitrogen is present to maintain optimal cell storage temperature.


✓ Written procedure defining method for monitoring liquid nitrogen (LN2) levels AND

✓ Records of monitoring of LN2 levels documented at defined frequency

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HSC.22750 Temperature Range Phase II

Acceptable ranges are defined for all temperature-dependent equipment.


✓ Temperature log or record with defined acceptable range

HSC.22775 Thermocycler Temperature Checks Phase II

Individual wells (or a representative sample thereof) of thermocyclers are checked for temperature accuracy before being placed in service and every 6 months thereafter.

NOTE: If it is not physically possible to check individual wells, a downstream measure of well-temperature accuracy (such as productivity of amplification) must be substituted to functionally meet this requirement. On thermocycler models where such is possible, individual wells must be checked for temperature accuracy before being placed in service and every 6 months thereafter.


✓ Written procedure for verification of thermocycler accuracy AND

✓ Records of thermocycler verification REFERENCES 1) Saunders GC, et al. Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA

analyses. Clin Chem. 2001;47:47-55

Thermometers


● Records of traceability to NIST calibration

● What is your laboratory's course of action prior to using non-certified thermometers?

● How do you know when to recertify a thermometer?

HSC.22800 NIST Thermometer Phase II

An appropriate thermometric standard device of known accuracy is available (guaranteed by manufacturer to meet NIST standards).

NOTE: Thermometers should be present on all temperature-controlled instruments and environments and checked daily. Thermometric standard devices should be recalibrated or recertified prior to the date of expiration of the guarantee of calibration.


✓ Thermometer certificate of accuracy

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HSC.22850 Non-Certified Thermometer Phase II

All non-certified thermometers in use are checked against an appropriate thermometric standard device before being placed in service.


✓ Written procedure defining criteria for verification of non-certified thermometers AND

✓ Records of verification prior to being placed in service

Colorimeters, Spectrophotometers, and Fluoriometers

The following requirements apply to stand-alone instruments; they are not applicable to instruments embedded in automated equipment for which the manufacturer's instructions should be followed.


● Spectrophotometer policy or procedure

● Sampling of manufacturer required system checks

● Filters (clean, not scratched or deteriorated)

**REVISED** 07/11/2011 HSC.23137 Absorbance/Linearity Phase II

Absorbance and/or linearity fluorescence is checked and documented at least annually or as often as specified by the manufacturer, with filters or standard solutions.


✓ Records of absorbance and linearity checks, as applicable

HSC.23324 Filter Photometers Phase II

Filters (filter photometers) are checked at least annually to ensure they are in good condition (e.g. clean, free of scratches).


✓ Records of filter checks documented at defined frequency

**REVISED** 07/11/2011 HSC.23511 Wavelength Calibration Phase II

Spectrophotometer (including ELISA plate readers) wavelength calibration, absorbance and linearity are checked at least annually (or as often as specified by the manufacturer), with appropriate solutions, filters or emission line source lamps, and the results documented.

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NOTE: Some spectrophotometer designs, e.g. diode array, have no moving parts that can alter wavelength accuracy and do not require routine verification. The manufacturer's instructions should be followed.


✓ Records of wavelength calibration checks, as applicable

HSC.23698 Stray Light Phase II

Stray light is checked at least annually with extinction filters or appropriate solutions, if required by the manufacturer.


✓ Records of stray light checks, as applicable

Film Processing/Photographic Equipment


● Sampling of film processing maintenance records

● Photographic equipment (clean, properly maintained) / camera mountings (secure, level)

HSC.23885 Film-Processing Phase II

Film-processing (developing) equipment is routinely serviced, repaired, and appropriately replenished with reagents, if maintained by the laboratory.

NOTE: If the laboratory uses another department's film processing equipment, the quality of the autoradiographs produced must be monitored and the appropriate personnel notified if corrective action is required.


✓ Records of maintenance and repairs documented at defined frequency

HSC.24072 Photographic Equipment Phase II

All photographic equipment in the laboratory is clean and properly maintained.

HSC.24259 Camera Mountings Phase I

Fixed camera mountings are secure and level.

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Fume Hoods and Biological Safety Cabinets


● BSC certification records

● Fume hood/chemical filtration unit (available)

HSC.24446 Fume Hood/Chemical Filtration Unit Phase II

A properly functioning fume hood (or chemical filtration unit) is available for any procedures using volatile chemicals.

**REVISED** 07/11/2011 HSC.24633 Biological Safety Cabinet Phase II

A biological safety cabinet (or hood) is available, when appropriate, and is certified at least annually to ensure that filters function properly and that airflow rates meet specifications.


✓ Maintenance schedule of BSC function checks AND

✓ Records of testing and certification REFERENCES 1) Classification of etiologic agents on the basis of hazard; US Department of Health, Education, and Welfare, PHS, Centers for Disease

Control, Office of Biosafety. Atlanta, GA. Reprinted September, 1976

Electrophoresis Equipment


● Sampling of voltage check records

● Electrophoresis equipment (clean, properly maintained)

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HSC.25007 Electrophoresis Equipment Phase II

All electrophoretic apparatus in the laboratory is clean and properly maintained (electrodes and buffer tank intact, power supply electrodes fit snugly, no build up of dried buffer).

HSC.25194 Voltage Reading Phase II

The displayed voltage reading is confirmed at least annually by a voltmeter or other suitable means for all power supplies in the laboratory.


✓ Records of annual voltage checks

pH Meters


● Sampling of pH meter policies or procedures

HSC.25381 pH Meters Phase II

There are documented procedures for operation, calibration, and function checks of pH meter(s).

HSC.25568 Buffers Phase II

High quality buffers (certified assay of content) are used for calibration of the pH meter(s).

Balances and Weights


● Sampling of analytic balance service records

● Sampling of analytic balance accuracy check records

● Analytic balance (mounting)

● Standard weights (maintained, appropriate class)

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HSC.25755 Balance Maintenance Phase II

Balances are cleaned, serviced, and checked at least annually by qualified service personnel.


✓ Records of balance maintenance

HSC.25942 Balance Mounting Phase II

Analytical balances are mounted such that vibrations do not interfere with readings.

HSC.26129 Standard Weights Phase II

Standard weights of the appropriate ANSI/ASTM Class are available for calibration.

NOTE: The verification of accuracy of the analytical balance must be performed on a regular schedule to ensure accurate creation of analytical calibrators and/or weighed-in controls from standard materials, as well as when gravimetrically checking the accuracy of pipettes.

There are three general types of balances in use. First, many contemporary balance designs use force transducers of various designs to provide mass readings. These balances typically have built-in certified calibration weights that are utilized automatically each time of use. The second type of balance employs a force transducer design that uses external weights for calibration each time the balance is used. Typically a single mass at the maximum weighing range, in conjunction with a zero point for the pan, is used for calibration of a force transducer balance design. The third type of balance, an older design, is a mechanical balance beam with internal moveable or external calibration weights. This design may have an electronic read-out.

In all cases, verification of accuracy over the weighing range with external calibrated masses is required on a periodic schedule appropriate to the use of the balance. Balances must be checked at least every 6 months. Accuracy must be verified when a new balance is installed and whenever a balance is moved.

External validation of accuracy requires the appropriate class of ASTM specification weights. ASTM Class 1 weights are appropriate for calibrating high precision analytical balances (0.01 to 0.1 mg limit of precision). ASTM Class 2 weights are appropriate for calibrating precision top-loading balances (0.001 to 0.01 g precision).w ASTM Class 3 weights are appropriate for calibrating moderate precision balances, (0.01 to 0.1 g precision).

Periodic external validation of accuracy is required to ensure that internal weights have not deteriorated from adsorption of surface film or corrosion; and to ensure that electronics remain correctly calibrated.


✓ Written procedure defining criteria for the use of standard weights for accuracy checks of analytical balances

REFERENCES 1) American Society for Testing and Materials. Standard specification for laboratory weights and precision mass standards, designation

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E 617-97 (Reapproved 2003). ASTM International, West Conshohocken, PA 19428-2959 (www.astm.org) 2) American Society for Testing and Materials. E 898-88 (Reapproved 2000). Standard method of testing top-loading, direct reading

laboratory scales and balances. ASTM International, West Conshohocken, PA 19428-2959 (www.astm.org)

HSC.26316 Weights Maintenance Phase II

Weights are well-maintained (clean, in a covered container, not corroded) and appropriate lifting or handling devices are available.

NOTE: Weights must be well-maintained (covered when not in use, not corroded) and only be handled by devices that will not allow residual contaminants to remain on the masses. Certified masses will only meet their specifications if maintained in pristine condition.

HSC.26503 Accuracy Checks Phase II

Results of periodic accuracy checks are recorded.

NOTE: Mass readings should be recorded in a log book. The deviations in log book readings should be no more than the precision required in the applications for which the balance is used. Acceptable ranges for readings should be specified.

Volumetric Glassware and Pipettes


● Pipette verification procedure

● Automatic pipette calibration procedure

● Sampling of pipette/dilutor checks

● What is your laboratory’s course of action prior to using non-certified volumetric pipettes?

● How does your laboratory ensure only dedicated pipettes are used for pre-amplification procedures?

**REVISED** 06/17/2010 HSC.26690 Glassware Accuracy Phase II

Volumetric glassware of certified accuracy (Class A, NIST Standard or equivalent) is in use, or if non-certified volumetric glassware is used, all items are checked for accuracy of calibration before initial use.

NOTE: The following table shows the American Society for Testing and Materials' calibration (accuracy) specifications for Class A volumetric pipettes:

Reconstitution of lyophilized calibrators, controls, proficiency testing materials, or other tasks requiring accurate volumetric measurement must be performed only with measuring devices of Class A accuracy, or those for which accuracy has been defined and deemed acceptable for the intended use.

If initial calibration is performed by the manufacturer or other outside facility, sufficient information must be provided to justify acceptance of the pipette's calibration based on the laboratory's documented specifications of acceptable bias and imprecision. The outside facility must also

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document the technique used to check calibration and ship the pipette in a manner that protects it from damage in transit.

Nominal Capacity (mL) Variation (± mL)

0.5-2 0.006

3-7 0.01

8-10 0.02

15-30 0.03

40-50 0.05

100 0.08


✓ Glassware marked Class A OR NIST certificate OR Validation study of accuracy for non-certified glassware

REFERENCES 1) Curtis RH. Performance verification of manual action pipets. Part I. Am Clin Lab. 1994;12(7):8-9 2) Curtis RH. Performance verification of manual action pipets. Part II. Am Clin Lab. 1994;12(9):16-17 3) Perrier S, et al. Micro-pipette calibration using a ratiometric photometer-reagent system as compared to the gravimetric method. Clin

Chem. 1995;41:S183 4) American Society for Testing and Materials. Standard specification for glass volumetric (transfer) pipets, designation E 969-95.

Philadelphia, PA: ASTM, 1995 5) Johnson B. Calibration to dye for: Artel's new pipette calibration system. Scientist. 1999;13(12):14 6) Connors M, Curtis R. Pipetting error: a real problem with a simple solution. Parts I and II. Am Lab News. 1999;31(13):20-22 7) Skeen GA, Ashwood ER. Using spectrophotometry to evaluate volumetric devices. Lab Med. 2000;31:478-479

HSC.26877 Pipette Accuracy - Non Class A Phase II

Non-class A pipettes that are used for quantitative dispensing of material are checked for accuracy and reproducibility at specified intervals, and results documented.

NOTE: Such checks are most simply done gravimetrically. This consists of transferring a number of measured samples of water from the pipette to a balance. Each weight is recorded, the weights are converted to volumes, then means (for accuracy), and SD/CV (for imprecision) are calculated. Alternative approaches include spectrophotometry or (less frequently) the use of radioactive isotopes, and commercial kits are available from a number of vendors. Computer software is useful where there are many pipettes, and provides convenient documentation. REFERENCES 1) Curtis RH. Performance verification of manual action pipets. Part I. Am Clin Lab. 1994;12(7):8-9 2) Curtis RH. Performance verification of manual action pipets. Part II. Am Clin Lab. 1994;12(9):16-17 3) Perrier S, et al. Micro-pipette calibration using a ratiometric photometer-reagent system as compared to the gravimetric method. Clin

Chem. 1995;41:S183 4) NCCLS. Laboratory Statistics—Standard Deviation; A Report. NCCLS document EP13-R (ISBN 1-56238-277-2). NCCLS, 940 West

Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1995 5) Bray W. Software for the gravimetric calibration testing of pipets. Am Clin Lab. Oct 1995 6) CLSI. Laboratory Instrument Implementation, Verification, and Maintenance; Approved Guideline. CLSI Document GP31-A. (ISBN 1-

56238-697-2). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898, USA, 2009 7) Johnson B. Calibration to dye for: Artel's new pipette calibration system. Scientist. 1999;13(12):14 8) Connors M, Curtis R. Pipetting error: a real problem with a simple solution. Parts I and II. Am Lab News. 1999;31(13):20-22 9) Skeen GA, Ashwood ER. Using spectrophotometry to evaluate volumetric devices. Lab Med. 2000;31:478-479

HSC.27064 Automatic Pipette Accuracy Phase II

Automatic pipettes are checked for accuracy of calibration and reproducibility before being placed in service and at regular intervals thereafter.

REFERENCES 1) Curtis RH. Performance verification of manual action pipets. Part I. Am Clin Lab. 1994;12(7):8-9 2) Curtis RH. Performance verification of manual action pipets. Part II. Am Clin Lab. 1994;12(9):16-17 3) Perrier S, et al. Micro-pipette calibration using a ratiometric photometer-reagent system as compared to the gravimetric method. Clin

Chem. 1995;41:S183 4) Bray W. Software for the gravimetric calibration testing of pipets. Am Clin Lab. Oct 1995

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5) CLSI. Laboratory Instrument Implementation, Verification, and Maintenance; Approved Guideline. CLSI Document GP31-A. (ISBN 1-56238-697-2). CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898, USA, 2009

6) Johnson B. Calibration to dye for: Artel's new pipette calibration system. Scientist. 1999;13(12):14 7) Connors M, Curtis R. Pipetting error: a real problem with a simple solution. Parts I and II. Am Lab News. 1999;31(13):20-22 8) Skeen GA, Ashwood ER. Using spectrophotometry to evaluate volumetric devices. Lab Med. 2000;31:478-479

HSC.27251 Dedicated Pipettes Phase II

Dedicated pipettors are used for pre-amplification procedures.

REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M2.120. Richmond, VA: UNOS, 2001

PROCEDURES AND TEST SYSTEMS


● If problems are identified during the review of the procedures and test systems, or when asking questions, further evaluate the laboratory’s responses, corrective actions and resolutions

● Select a representative assay and follow the entire process from specimen receipt to final result reporting

LYMPHOCYTE ISOLATION


● Lymphocyte isolation policy/procedures

HSC.27438 Lymphocyte Source Phase II

The source of the lymphocytes is documented.

NOTE: These may include blood, bone marrow, lymph nodes, spleen, or cultured cells. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C5.110. Richmond, VA: UNOS, 2001

SEROLOGICAL PROCEDURES

General


● Sampling of complement reagent validation records

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● How does your laboratory ensure cell death is appropriately measured?

HSC.27625 Scoring System Phase II

There is a scoring system in place for measuring cell death in cytotoxicity tests.

NOTE: There must be established limits for defining positive and negative results by approximate percentage of cell death. REFERENCES 1) REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.320. Richmond, VA:

UNOS, 2001

HSC.27812 QC - Complement Phase II

Each lot, batch and/or shipment of complement is checked for effectiveness before or during use for each specific target cell and each test method.

NOTE: Each lot, batch and/or shipment of complement must be evaluated to determine that it can mediate cytotoxicity when a specific antibody is present, and is not cytotoxic in the absence of a specific antibody. For each specific target cell (T cell, B cell, monocyte, etc.), complement cytotoxicity studies must be performed to determine optimal dilution for each type of cell tested by cytotoxicity. Two HLA antibodies should have variable antibody strengths when utilized in complement testing against 2 different known antigen-containing cells that are reactive to the antibodies. Alternatively, one antibody may be utilized at variable dilutions for complement testing.


✓ Written procedure for the validation of new complement reagents AND

✓ Records of validation of complement reagents REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7166 [42CFR493. 1256(e)] 2) United Network for Organ Sharing. Standard for Histocompatibility Testing. Richmond, VA: UNOS; E2.751; E2.752

HLA Class I and II Antigen Typing


● Sampling of HLA Class I & Class II Ag typing policies and procedures. Procedures should specify the level of resolution of HLA typing required for each tissue or organ transplanted.

● Sampling of HLA typing of solid organ and stem cell transplantation policies and procedures

● List of antigens defined by reagents used

● Sampling of typing reagent validation records

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● Sampling of typing tray QC records

● How does your laboratory select target cells to ensure the detection of WHO recognized antigens?

● What is your laboratory's course of action when an organ donor cannot be reliably HLA typed?

HSC.27999 Nomenclature Phase II

The HLA antigen assignments and their written designation conform to the most current World Health Organization Committee nomenclature on histocompatibility antigens.

NOTE: For example: Phenotype is HLA-A1,2; B51,B44; Cw3; DR1,4; DQ4,8;Dw1,Dw2. Genotype is HLA 1,B8,DR3,DQ6,Dw1/A2,B7,DR1,DQ2,Dw2.

Any newly discovered antigens not yet assigned by WHO Committee must be designated in such a manner as not to be confused with existing established terminology. All genotype and phenotype designations must also conform to WHO Committee recommendations. The laboratory must maintain a list of antigens defined by the reagents used. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing D1.100. Richmond, VA: UNOS, 2001

HSC.28092 Nomenclature - Leukocyte Phenotyping Phase II

There is documentation that the appropriate terminology is used for leukocyte phenotyping.

HSC.28186 Serologic Typing - Class I Phase II

Target cells are defined for serological determination of HLA Class I antigens, and selected to permit typing the antigens officially recognized by the WHO Committee for which reagents are readily available.

NOTE: Serological determination of HLA Class I antigens should be performed on T cells or mononuclear cell preparations. Local serological typing reagents must be supported by appropriate documentation of HLA specificity, using cells of known HLA types. The test must detect WHO recognized specificities. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.110, E2.310. Richmond, VA:

UNOS, 2001

HSC.28373 Serologic Typing - Class II Phase II

The methodology for serological Class II antigen typing defines the proportion of B-cells needed for optimal testing, and the specificities that are officially recognized by the WHO Committee and for which reagents are readily available.

NOTE: The method should produce at least 80% B-cell enriched. Documentation of B cell enrichment may not be necessary when procedural techniques already distinguish T- and B-lymphocytes, or when well-characterized antibodies are used that can only discriminate and

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identify Class II antigens. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.210, E2.310. Richmond, VA:

UNOS, 2001

HSC.28560 Class I Antigen Defined Phase II

Class I antigens are defined by at least 3 antisera, or by 2 antisera that are operationally monospecific.


✓ Written procedure for performing serologic determination of HLA Class I

HSC.28747 Class II Antigen Defined Phase II

Class II antigens are defined by at least 5 antisera, or by 3 antisera that are operationally monospecific.

NOTE: Class II antigen assignment by the use of operationally monoclonal antibodies to each DR and DQ antigen should be determined by (a) 2 antibodies directed to private epitope specificity, or (b) 1 antibody having private epitope specificity and 2 antibodies with public epitope specificity, or (c) 3 antibodies with partially non-overlapping antibodies directed at public epitope determinants.


✓ Written procedure for performing serologic determination of HLA Class II REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.630. Richmond, VA: UNOS, 2001

HSC.28934 Typing Trays Phase I

The typing trays used for disease association testing permit characterization of at least those antigens accepted by the World Health Organization (WHO) for which sera are readily available.


amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493. 1278(a) through (c)]

HSC.28996 Controls - Typing Trays Phase II

Each typing tray contains both a positive and a negative control.

NOTE: The positive control must be known to react with all cells expressing the class of antigens being tested at a titer comparable with the typing reagents. Each typing tray must also contain one negative control. Cell viability in the negative control must be sufficient to accurately interpret the results. The use of sufficiently discriminatory positive and negative controls also applies to assays in which cell viability is not required.

HSC.29058 Typing Reagent Validation Phase II

New typing reagents are validated using cell panels of known HLA Class I and Class II types.

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NOTE: Cell panels of known HLA Class I and II type must be used to validate new typing reagents. Panel cells should include at least one example of each HLA antigen the laboratory is able to define and be representative of the population served by the laboratory.


✓ Written procedure defining criteria for validation of Class I and II cell panels AND

✓ Validation study data for new typing reagents

HSC.29121 HLA Typing Post Blood Transfusion Phase II

There is a procedure describing what to do when an organ donor cannot be reliably HLA typed due to recent blood transfusions.

NOTE: If the potential donor was transfused during the week before HLA antigen testing, the results can only be interpretable if no extraneous antigens are detected. There must not be more than 2 antigens identified per locus. If there are requirements regarding accuracy of HLA antigen assignment in the cadaveric donor, the HLA typing should be done on cells from lymph nodes, spleen or by molecular DNA methods. Likewise, if HLA typing is unable to be accurately performed on a living donor or patient, it should be redone, using more sophisticated testing techniques to resolve initial inadequate typing results. Different techniques at different times may be necessary to deal with potential artifacts such as recent blood transfusions. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.510. Richmond, VA: UNOS, 2001

HSC.29150 HLA Typing Antigen/Allele Level Phase I

The laboratory performs HLA typing at the antigen or allele level appropriate for the individual transplant protocols supported (e.g. allele level typing for stem cell transplantation and antigen level typing for solid organ transplantation).


✓ Written procedures for HLA typing of solid organ and stem cell transplantation AND

✓ Patient reports and typing records

Cytotoxicity Crossmatch


● Sampling of cytotoxicity crossmatch policies and procedures including method sensitivity

● How does your laboratory ensure results of the final crossmatch are available prior to renal transplantation?

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HSC.29308 HLA Crossmatch Sensitivity Phase II

The technique used in the HLA crossmatch procedure is more sensitive than the basic NIH lymphocytotoxicity crossmatch.


✓ Documentation of method sensitivity REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.210. Richmond, VA: UNOS, 2001

HSC.29495 Crossmatch Phase II

The crossmatch procedure defines the patient sera and donor cells utilized for final crossmatch testing.

NOTE: Cellular targets for transplant crossmatches must include donor T-cells, and may include donor B-cells when appropriate. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.220. Richmond, VA: UNOS, 2001

HSC.29682 Specimen Handling Phase II

Patient samples for crossmatch testing are used undiluted, and kept frozen for a defined time post-transplantation.


✓ Written procedure for cytotoxicity crossmatch REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.310, H3.400. Richmond, VA:

UNOS, 2001

HSC.29869 Final Crossmatch Results Availability Phase II

There is a policy that defines when results of a final crossmatch are available before transplantation for renal transplant patients and for presensitized extrarenal transplant patients.

NOTE: Laboratories must be capable of performing prospective crossmatches and must have a written policy describing in what situations pre- or post- transplant crossmatching should be performed for all types of organ transplants. Results of the final crossmatch must be available before a kidney transplant is performed and before a non-renal transplant is performed in a patient known to be presensitized.


amendments of 1988; final rule. Fed Register. 1992(Feb 28):7170 [42CFR493.1265(a)(2)-(i)(3)] 2) United Network for Organ Sharing. Standard for histocompatibility testing. Richmond, VA: H3.100; I3.100

RED CELL TYPING


● Sampling of blood type/antibody screen policies and procedures

● Sampling of current typing sera/reagent package inserts, for consistency with written

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procedures

● Sampling of typing sera/reagent cell reactivity/anti-D QC records

● Sampling of patient records with forward and reverse grouping

● Record retention policy

● Sampling of historical record checks

● Sampling of typing sera/reagent cells (expiration date)

● If there has been an instance where the ABO and Rh typing results were not in agreement with the patient's historical record, further evaluate the laboratory's responses, corrective actions and resolutions

HSC.29877 Manufacturer Instructions Phase II

Typing sera and reagent cells are used as prescribed by their manufacturers.


✓ Written procedure consistent with manufacturer's instructions

HSC.29885 Manufacturer Instructions Phase II

Package inserts for the typing sera in use are available and typing sera are used according to the manufacturers' directions, or, if alternative procedures are used, they are evaluated to justify the changes.


✓ Written procedure consistent with manufacturer's instructions OR documentation of validation study if an alternative procedure is used

HSC.29893 Red Cell Typing Phase II

There is a test of each patient's blood sample with anti-A, anti-B, anti-D and A1 and B red cells.

NOTE: The ABO and the Rh type of the patient's red blood cells must be determined by an appropriate test procedure. Tests on each sample must include forward and reverse grouping. Discrepancies between cell and serum groups must be resolved before ABO group is assigned.


✓ Written procedure for ABO/Rh typing AND

✓ Logs or computer records with forward and reverse grouping

HSC.29901 A1 Subgroup Phase II

The specificity of the A1 subgroup of ABO testing is documented to distinguish A1 from

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other subgroups.

HSC.29909 QC - Typing Sera and Reagent Cells Phase II

Records document acceptable reactivity and specificity of typing sera and reagent cells on each day of use, including a check against known positive and negative cells or antisera, or manufacturer's directions for daily quality control are followed.

NOTE: Unless manufacturer instructions state otherwise, the following apply:

■ Each cell used for antibody detection must be checked each day of use for reactivity of at least one antigen using antisera of 1+ or greater avidity.

■ Typing reagents such as anti-D, anti-K, anti-Fy(a), etc., must be checked each day of use.

■ Anti-IgG reactivity of antiglobulin reagents may be checked during antibody screening and crossmatching.

■ Typing sera and reagent cells must be checked for reactivity and specificity on each day of use, including a check against known positive and negative cells or antisera.

This checklist requirement can be satisfied by testing one vial of each reagent lot each day of testing. REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement


HSC.29917 Expiration Dates Phase II

Typing sera and reagent cells are used within their indicated expiration date.

NOTE: Rare reagents may be used beyond their expiration date if appropriate positive and negative controls are run and react as expected. This exception is permitted by the FDA. This does NOT apply to reagents that are readily available. Refer to HSC.21500 for further information on the use of expired reagents by laboratories not subject to US regulations.

HSC.29925 Historical Record Check Phase II

ABO, Rh, and antibody screen test results are compared against the same tests performed previously to detect discrepancies.


✓ Written procedure defining process for the historical record check for ABO/Rh/antibody screen results AND

✓ Records of historical result comparisons


Immunohematology records and specimens are retained for an appropriate period.

NOTE: Records should be retained per the current CAP Guidelines, and in conformity with state and federal regulatory requirements. At the time of this Checklist edition, the Guidelines are as follows:

TYPE OF RECORD RETENTION PERIOD

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Patient records 10 years

Employee signatures, initials, and identification codes

10 years post employment

Quality Control Records 5 years

Specimens 7 days

Records, unexpected antibodies. Indefinitely

HSC.29941 Results Reporting Phase I

Criteria for the performance and interpretation of ABO antibody titers are defined and documented.

HSC.29949 Anti-D Controls Phase II

An appropriate control(s) is used for anti-D testing.

NOTE: High protein anti-D reagents require concurrent testing of an inert preparation of the manufacturer's diluent. Monoclonal anti-D reagents do not ordinarily require a separate reagent control. Spontaneous agglutination can be ruled out by observing negative reactions in any tube containing red cells and patient serum, or, alternatively, patient cells suspended in 5% bovine albumin.


✓ Written procedures defining controls used for anti-D testing AND

✓ Records of anti-D QC

FLOW CYTOMETRY

Instrumentation and Phenotyping


● Sampling of flow cytometry policies and procedures

● Sampling of QC policies and procedures (includes acceptable control type/frequency for each flow cytometric application)


● Sampling of optical alignment/laser output checks

● How does your laboratory monitor instrument reproducibility?

● How does your laboratory ensure each fluorochrome is appropriately calibrated?

● How does your laboratory determine appropriate color compensation settings?

● How does your laboratory ensure nucleic acid dye specificity?

HSC.29957 Daily QC Phase II

For quantitative tests (e.g. CD4+, CD34+ cell concentrations), at least 2 levels of positive cellular controls are analyzed daily to verify the performance of reagents, preparation

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methods, and staining procedures.

NOTE: One of the levels of these controls should be at (or near) clinical decision levels (e.g. low CD34).Control testing is not necessary on days when patient testing is not performed.


✓ Written procedure defining QC requirements for each test OR records of validation of an alternate/equivalent procedure when commercial controls are not available

✓ Records of QC results REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493.1252-1255]

HSC.29965 Optical Alignment Phase II

There are procedures for monitoring of optical alignment (where applicable) and instrument reproducibility on each day of use, and there is documentation of this monitoring.

NOTE: Instrument performance must be monitored under the same conditions used to run test samples. REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved


HSC.29973 Fluorochrome Standards Phase II

Appropriate standards for each fluorochrome, (e.g. fluorescent beads), are run each day that the instrument is used as part of the calibration process; and the results are recorded for quality control purposes.

NOTE: These steps are necessary to optimize the flow system and the optics of the instrument.


✓ Written procedure for calibration with fluorochrome standards REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI). Enumeration of Immunologically Defined Cell Populations by Flow Cytometry;

Approved Guideline—Second Edition. CLSI document H42-A2 (ISBN 1-56238-640-9). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007

HSC.29981 Color Compensation Settings Phase II

Procedures are established for determining appropriate color compensation settings.

NOTE: For two or more color analysis there must be a procedure to ensure that cells co-labeled with more than one fluorescent reagent can be accurately distinguished from cells labeled only with one reagent. Cells stained with mutually exclusive antibodies bearing the relevant fluorochromes are the proper reference material for establishing appropriate compensation settings. REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved


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HSC.29989 Laser Current Phase I

For laser instruments, there are procedures in place to ensure acceptable and constant laser current.

NOTE: For some instruments, current is a better gauge of laser performance than is power output, which may be relatively constant.

HSC.29997 Gating Techniques Phase II

Appropriate gating techniques are used to select the cell population for analysis.

NOTE: This may involve a combination of light scatter and/or fluorescence measurements. This is particularly important if the cell samples have a low lymphocyte count and/or a relatively high monocyte-granulocyte count. Lymphocyte gates may be validated using linear forward angle light scatter and 90-degree side scatter, or using CD45-FITC and CD14-PE monoclonal antibodies. REFERENCES 1) National Institute for Allergy and Infectious Diseases/Division of AIDS guidelines for flow cytometric immunophenotyping, ver 1.0, Jan

1993

HSC.30005 Markers/Cursors Phase II

There is a procedure to set markers (cursors) to distinguish fluorescence negative and fluorescence positive cell populations.

NOTE: Each laboratory must have a set of objective criteria to define the appropriate placement of markers (cursors) to delineate the population of interest. Isotypic controls may not be necessary in all cases, and cursor settings for the isotype control may not be appropriate for all markers. Cursor settings must be determined based on the fluorescence patterns from the negative and positive populations for CD3, CD4, and CD8. REFERENCES 1) National Institute of Allergy and Infectious Diseases/Division of AIDS flow cytometry guidelines, sec 3.09B and 5.03A 2) Sreenan JJ, et al. The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow

cytometry. Are they really necessary? Arch Pathol Lab Med. 1997;121:118-121

HSC.30013 Cellular Viability Phase II

There is a policy for determining when the percentage of viable cells in each test specimen should be measured.

NOTE: Selective loss of cell subpopulations and/or the presence of dead cells may lead to spurious results. Procedures must be in place to ensure that viable cells are analyzed. This does not mean that all specimens with low viability must be rejected. Finding an abnormal population in a specimen with poor viability may be valuable but the failure to find an abnormality should be interpreted with caution. If specimen viability is below the established laboratory minimum, test results are suspect and this should be noted in the test report. Routine viability testing may not be necessary on whole blood specimens that are analyzed within 24 hours of drawing. However, viability testing of other specimens such as disaggregated lymph node specimens is essential. REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved


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HSC.30021 Immunoglobulin Staining Phase II

Methods are established to ensure that immunoglobulin staining is intrinsic and not extrinsic (cytophilic).

NOTE: Many cell types will bind serum immunoglobulin nonspecifically via Fc receptors, and steps may have to be taken to ensure that immunoglobulin staining detected by flow cytometry is intrinsic rather than cytophilic. Histogram interpretation is valuable.


✓ Written procedure defining method to ensure intrinsic immunoglobulin staining REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved


HSC.30029 Cell Population Distinction Phase II

There is a procedure to distinguish fluorescence-negative and fluorescence-positive cell populations.

NOTE: This does not imply that a separate negative control sample must be run. It is possible to coordinate panels of monoclonal antibodies to compare the binding of monoclonal antibodies of the same subclass that typically have mutually exclusive patterns of reactivity of subsets of hematopoietic cells. In this way, test antibodies may also double as control reagents. REFERENCES 1) Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved


HSC.30037 Procedure Manual Phase II

The staining and analytical procedures described in the procedure manual are based upon established methodology (reference cited).

NOTE: Many different variables need to be controlled to ensure proper stoichiometry of dye binding to DNA. Therefore, it is essential that procedures adopted by a laboratory are based on published work.

HSC.30045 Specimen Treatment Phase II

Specimen treatment with nucleic acid dye includes treatment with RNAse if the dye is not specific for DNA.

NOTE: Certain dyes used to stain fixed cells, (e.g. ethidium and propidium iodide) bind to RNA. Prior treatment with RNAse eliminates artifactual broadening of the DNA content distributions that would result from fluorescence of complexes of the dye with RNA.


✓ Written procedure for specimen treatment with RNAse REFERENCES 1) Shapiro HA. Practical flow cytometry. New York, NY: Alan R. Liss, 1985

Flow Cytometry Crossmatch

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● Sampling of flow cytometry crossmatch policies and procedures

● Sampling of QC policies and procedures


● Sampling of positive cutoff validation records

● How has your laboratory established the cutoff for positive crossmatch results?

● Are cutoffs for crossmatches reviewed with the clinical transplant service?

● Have the cutoffs been correlated with signal strength or other measure of antibody concentration in the HLA antibody screen and detection methods used?

● How does your laboratory ensure separation of Class I & Class II antibodies?

HSC.30056 Crossmatch Phase II

The flow cytometry crossmatch identifies antibodies to T-cells.

NOTE: Two or multiple color techniques must be used to identify antibodies to T cells. If antibodies to B cells or other cells are reported, such target cells must also be identified properly. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.120. Richmond, VA: UNOS, 2001

HSC.30243 IgG Antibody Identification Phase II

IgG antibodies are identified by appropriately labeled heavy chain-specific F(ab')2 reagents.

REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.110. Richmond, VA: UNOS, 2001

HSC.30430 Sensitivity Phase II

There is documentation of the number of cells and volume of serum used for optimal sensitivity.

HSC.30617 Negative Control - Normal Human Serum Phase II

Normal human serum with demonstrated lack of reactivity against any potential target cell is used as a negative control.


✓ Written procedure describing controls used for flow cytometry crossmatch AND

✓ Records of control results REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.210. Richmond, VA: UNOS, 2001

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HSC.30804 Positive Control - Diluted Human Serum Phase II

The positive control is an appropriately diluted human serum containing suitable HLA antibodies of appropriate immunoglobulin class known to react with lymphocytes from all donors.


✓ Written procedure describing controls used for flow cytometry crossmatch AND

✓ Records of control results REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.220. Richmond, VA: UNOS, 2001

HSC.30991 Antibody Reagents Phase II

The antibody reagents (anti IgG, IgM, IgA, etc.) are used at a selected dilution for optimal sensitivity and class specificity.


✓ Written procedure describing controls used for flow cytometry crossmatch REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.230. Richmond, VA: UNOS, 2001

**REVISED** 07/11/2011 HSC.31178 Positive Crossmatch Results Cut-off Phase II

The cut-off for positive crossmatch results are determined by testing an appropriate number of sera from non-alloimmunized donors and established for all pertinent target cells (T-cells, B-cells, etc.).


✓ Records for the validation of the positive cut-off REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.240. Richmond, VA: UNOS, 2001

HSC.31552 HLA Class II Antibody Procedure Phase II

The procedure for HLA Class II antibodies readily separates Class I from Class II specificity.

REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.120. Richmond, VA: UNOS, 2001

MIXED LYMPHOCYTE CULTURE TESTING


● MLC testing procedure

● Sampling of instrument maintenance/QC records

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● Testing environment (aseptic conditions)

HSC.31739 Testing Environment Phase II

Testing is performed under aseptic conditions, with access to a laminar flow hood.

HSC.31926 Instrument Maintenance/QC Phase II

The instrument for counting tritiated thymidine is monitored and standardized according to manufacturer's recommendations.


✓ Written procedure for instrument monitoring and standardization AND

✓ Instrument maintenance records

HSC.32113 Incubator QC Phase II

On days of use, the incubator is monitored for temperature, CO2 concentration and humidity as defined by the procedure manual.


✓ Instrument QC records

HSC.32300 MLC QC Phase II

Autologous and at least 3 unrelated individuals or 2 unrelated individuals plus a pool of at least 3 or more unrelated individuals are used as sources for stimulator cells for each responder cell in the MLC.

NOTE: Proper controls must be used as sources of stimulator cells for each responder cell tested in the MLC. Cell viability must be appropriate and documented. Appropriate conditions (serum supplements, incubation time, etc) for MLC reactivity must be determined with at least 3 unrelated individuals or 2 unrelated individuals, plus a pool of at least 3 or more unrelated individuals as sources for stimulator cells for each responder cell.


✓ Written procedure for MLC testing REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing F1.400, F1.500. Richmond, VA:

UNOS, 2001

HLA ANTIBODY SCREENING


● Sampling of HLA antibody screening policies and procedures, including protocol for screening

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of each organ transplanted and the frequency of such screening

● Agreement for reflex testing using more sensitive screening method, if applicable

● Sampling of antibody identification QC records

● Sampling of initial and subsequent recipient sera screening records

● What is your laboratory's course of action for antibody identification/crossmatching for high risk patients?

● How does the laboratory determine cutoffs for identification of HLA antibody and assignment of unacceptable antigens for organ transplantation?

HSC.32487 Immunizing Event History Phase II

There is a system to document any potential immunizing event that could cause sensitization in a patient.

NOTE: There must be documentation of a policy that encourages a timely blood sample collection at 14 days after the potential immunizing event in a patient. This new sample should be available for use in antibody screening and in crossmatch studies. REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493.1252-1255()] 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H2.000. Richmond, VA: UNOS, 2001

HSC.32674 HLA Antibody Detection Phase II

The laboratory has the capability to detect HLA antibodies with sufficient sensitivity and to distinguish HLA antibodies from IgM autoantibodies or non-HLA antibodies.

NOTE: Methods to detect HLA antibodies must be more sensitive than the basic/NIH technique. Procedures to differentiate HLA antibody from autoantibodies must be present. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H2.000. Richmond, VA: UNOS, 2001

HSC.32861 Antigenic Diversity and Targets Phase II

There is sufficient antigenic diversity (individual antigens and/or crossreactive groups) for HLA Class I and II, and sufficient numbers of antigenic targets for optimal HLA antibody detection and specificity determination.

NOTE: There must be sufficient diversity for Class I and II HLA antigens and cross reactive groups, as well as sufficient numbers of well-characterized panel cells or HLA-purified protein targets for antibody detection and specificity determinations.


✓ Listing of antigenic targets for each panel cell REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing G1.310, G1:320. Richmond, VA:

UNOS, 2001

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HSC.33048 Antibody Detection and Specificity QC Phase II

For HLA antibody detection and specificity determinations, positive and negative controls are used, and sera tested undiluted and diluted when appropriate.


✓ Written procedure for QC for antibody detection and identification REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing G1.210. Richmond, VA: UNOS, 2001

HSC.33235 Target Source for Class I/II Antibody Determination Phase II

There is documentation that the appropriate target sources are used for separate HLA Class I and II antibody determination including appropriate methods to distinguish antibody mixtures.

NOTE: There must be documentation that the appropriate target sources are used for HLA Class I and II antibody determination. The targets for HLA Class I antibody determination should be blood, spleen, lymph nodes, and cell lines. In addition, well-characterized purified HLA protein targets may also be used. Class II antibodies are best detected utilizing B-lymphocytes, B-lymphoblastoid cell lines, CLL cells or specific Class II purified HLA protein. Mixtures must be defined by methods shown to distinguish Class I from Class II reactivity.


✓ Written procedure defining target sources used for HLA Class I/II antibody determination and to differentiate antibody mixtures

REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing G2.110, G2.120. Richmond, VA:

UNOS, 2001

HSC.33422 Recipient Sera Screening Phase II

All recipient sera are screened for HLA antibodies including, at least, an initial sample at the time of HLA typing, after sensitizing events and upon request.


✓ Written procedure defining criteria for screening recipient sera AND

✓ Records showing initial screening of recipient sera and all subsequent screening results REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493.1278(d)] 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H2.000. Richmond, VA: UNOS, 2001

HSC.33475 Antibody Identification/Crossmatching - High Risk Patients Phase I

The laboratory has a policy and/or procedures for antibody identification and crossmatching for patients at high risk of allograft rejection.

Enzyme-Linked Immunosorbent Assays (ELISA)


● Sampling of ELISA policies and procedures

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HSC.33609 ELISA Non-Specific Binding Phase II

There is an effective method for documenting and controlling non-specific binding of antibody, including the effectiveness of the washing step.

NOTE: There must be documentation of a methodology that evaluates and controls non-specific binding in the absence of HLA antigens in an ELISA assay. This must include documentation for the cut-off used to distinguish positive from negative results.


✓ Written procedure detailing methodology used to evaluate and control non-specific binding to include documentation of the cut-off used

HSC.33796 Cut-off Points Determination Phase II

There is documentation of how positive and negative cut-off points were determined.

MOLECULAR HLA ANTIGEN TYPING


● Sampling of molecular HLA antigen policies and procedures

● Specimen storage/handling policy


● Sampling of the following reports for completeness: HLA typing and engraftment, RFLP, stem cell engraftment

● Sampling of the following records: molecular weight marker, in-house probe labeling validation, nucleic acid measurement, electrophoretic gel interpretation, chimerism measurement

● Gel photographs (sufficient resolution, quality)

● Current databases of know sequences for all WHO-recognized alleles (available)

● Pre/post amplification areas (adequate physical separation)

● How does your laboratory ensure specimen identity through all phases of analysis?

● What is your laboratory's process for assessing the quality (intactness) of high molecular weight DNA or RNA?

● How does your laboratory avoid cross-contamination when performing amplification procedures?

● How does the laboratory ensure the level of resolution is adequate to support the clinical programs?

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HSC.33983 HLA Typing Level of Resolution Phase II

The level of resolution of HLA typing is adequate to support clinical programs.

NOTE: For proficiency testing specimens, the level of resolution must reflect the highest level of resolution that the laboratory performs for its patients. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E1.100. Richmond, VA: UNOS, 2001

HSC.34170 Specimen Identification Phase II

Sample identification is assured through all applicable phases of analysis, including all of the following.

1. Specimen receipt 2. Nucleic acid extraction 3. Nucleic acid quantification 4. Endonuclease digestion 5. Electrophoresis 6. Transfer 7. Hybridization 8. Detection 9. In situ hybridization 10. Enzymatic amplification 11. Photography 12. Storage

HSC.34357 Nucleic Acid Extraction/Purification Phase II

Nucleic acids are extracted and purified by methods reported in the literature, or there is validation of a method developed in-house.


✓ Records to support nucleic extraction/purification is performed by a validated method REFERENCES 1) Sambrook J, et al. Molecular cloning: A laboratory manual, second edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory

Press, 1989:E.3-E.4 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.110. Richmond, VA: UNOS, 2001

HSC.34544 Reverse Transcription Phase II

For RNA amplification methods, appropriate controls are used for reverse transcription.


HSC.34731 Specimen Handling/Storage Phase II

Handling and storage of nucleic acids are adequate to prevent degradation.


✓ Written procedure for specimen storage and handling

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REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.120, M1.130. Richmond, VA:

UNOS, 2001 2) Rainen L, et al. Stabilization of mRNA expression in whole blood samples. Clin Chem. 2002;48:1883-1890

HSC.34918 Nucleic Acid Quantity Phase II

The quantity of nucleic acid is measured, when appropriate.


✓ Written procedure defining conditions under which quantity of nucleic acid is measured AND

✓ Records of nucleic acid measurement REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.230. Richmond, VA: UNOS, 2001

HSC.35105 High Molecular Weight DNA/RNA Quality Phase II

The quality (intactness) of the high molecular weight DNA (or RNA) is assessed by gel electrophoresis or comparable method, when appropriate.


✓ Written procedure defining method for assessment of quality (intactness) REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.140. Richmond, VA: UNOS, 2001

HSC.35292 Analytical Gels Phase II

Standard amounts of nucleic acid are loaded on analytical gels, when possible.


HSC.35479 Audioradiographs/Gel Photographs Phase II

The test conditions for autoradiographs and gel photographs permit sufficient resolution and quality (low background, clear signal, absence of bubbles, etc.) to document the reported interpretation.


UNOS, 2001


Procedures are documented to prevent specimen loss, alteration, or contamination.

HSC.35853 Result Reporting - HLA Typing and Engraftment Phase II

For HLA typing and engraftment tests, sufficient information is documented on the nature of all restriction enzymes, probes, or primers used in an assay to permit interpretation and troubleshooting of test results.

NOTE: Items that must be defined are:

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1. The oligonucleotide sequence of probes and primers, the complementary sequence

recognized, and the target HLA locus and alleles 2. The HLA locus and allele designations recognized by the WHO for each combination

of positive results (hybridization for RFLP and SSOP and PCR product for SSP) 3. Ambiguous allele combinations 4. The HLA sequence database used 5. For RFLP, sites resistant to endonuclease digestion, and cross-hybridizing bands;

the labeling methods used, and standards for adequacy of hybridization or amplification

REFERENCES 1) McAlpine PJ, et al. The 1991 catalog of mapped genes and report of the nomenclature committee. Human gene mapping 11(1991).

Cytogenet Cell Genet. 1991, 58:5-102

HSC.35946 Result Reporting - RFLP Phase II

RFLP (restriction fragment length polymorphism) reports specify the probes, enzymes, fragment size and target DNA location.

HSC.36040 Enzymatic Amplification Procedures Phase II

Enzymatic amplification procedures (e.g. PCR) are designed to minimize carryover (false positive results) using appropriate physical containment and procedural controls.

NOTE: This item is primarily directed at ensuring adequate physical separation of pre- and post-amplification samples to avoid amplicon contamination. The extreme sensitivity of amplification systems requires that the laboratory take special precautions. For example, pre- and post-amplification samples should be manipulated in physically separate areas; gloves must be worn and frequently changed during processing; dedicated pipettes (positive displacement type or with aerosol barrier tips) must be used; manipulations must minimize aerosolization; following complete reagent addition to the reaction tubes, the patient samples should be added one at a time. The best way to avoid cross-contamination is to use the following order of preparation within an amplification run: actual samples, followed by positive controls, followed by negative controls. REFERENCES 1) Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989;339:237-238

HSC.36227 Mendelian Inheritance Phase II

Mendelian inheritance of the DNA system used is validated by family studies.


HSC.36414 Audioradiographs/Electrophoretic Gel Interpretation Phase II

Autoradiographs or electrophoretic gels are interpreted independently by at least 2 qualified readers using an objective method.


✓ Written procedure for interpretation of autoradiographs/electrophoresis gels AND

✓ Patient testing records or worksheets

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HSC.36601 End-Point Amplification QC Phase II

For end-point amplification assays such as sequence-specific priming, adequate internal controls are used, and criteria defined for a positive reaction.


HSC.36788 Daily Controls Phase II

Positive, negative and sensitivity controls are run for each assay, when available and appropriate.


✓ QC records documented at defined frequency REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.210. Richmond, VA: UNOS, 2001

HSC.36975 DNA Contamination Phase II

There is a procedure to detect and control for DNA contamination.

NOTE: Contamination must be monitored in different areas by swipe tests using the regular detection for testing. Results of monitoring and corrective action taken when contamination is detected must be documented. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M2.110, M2.120, M2.130, M2.510.

Richmond, VA: UNOS, 2001

HSC.37162 Molecular Weight Markers Phase II

Known molecular weight markers that span the range of expected bands are used for each electrophoretic run.


✓ Records of appropriate markers documented with each run REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.220. Richmond, VA: UNOS, 2001


For hybridization techniques and restriction fragment length polymorphism, the different components and steps are monitored and documented to be appropriate, including the amount and integrity of amplified product, the signal intensity produced by each probe (which is adequate to detect a single copy gene), performance of restriction enzymes, and size of digested products.


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HSC.37536 Pre-Analytic Testing Requirements Phase II

The conditions (temperature, salt concentration, probe concentration, etc.) for pre-hybridization, hybridization and autoradiography (or other read-out system) are optimized to consistently produce accurate results.


UNOS, 2001

HSC.37723 Probe Labeling Validation Phase II

The method of probe labeling is validated to detect the target sequence without a false positive signal for non-target sequences.


✓ Records of in-house validation study data REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.121. Richmond, VA: UNOS, 2001

HSC.37910 Re-Probing Phase II

If re-probing of the same membrane is performed, there is documentation that there is complete stripping of the previous probe before re-probing.


HSC.38097 Sequenced Based Typing Phase II

For sequence based typing, there is documentation that templates have sufficient specificity for a locus or allele; all steps are appropriately monitored; the quality of the electrophoretograms adequately support the sequence results; and the definition of a sequence follows a procedure for accurate assignment of HLA alleles.

NOTE: The documentation must include the HLA locus and allele specificity of the template, the source of the sequence data base used (annually updated), and procedures to resolve ambiguous combinations. Assignment of alleles for HLA loci must be done by comparing the sequences obtained by nucleotide assignment with the sequences of all alleles that are recognized by the WHO Current databases of known sequences for all WHO-recognized alleles must be immediately available.


HSC.38134 Stem Cell Engraftment Testing Phase II

For stem cell engraftment, samples from patient pre-transplant, donor, patient post-transplant, and an appropriate control are amplified and analyzed concurrently.


✓ Written procedure for stem cell engraftment testing

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HSC.38171 Internal Controls Phase II

Internal controls are used to determine appropriate genotypes or at least to distinguish patient from donor.

NOTE: Criteria for the acceptance or rejection of the amplification of a particular genetic locus or individual sample must be specified.


✓ Written procedure defining criteria for acceptance/rejection of amplification results

HSC.38208 Preferential Allele Amplification Phase II

Preferential allele amplification is considered in the interpretation of stem cell engraftment tests.

HSC.38245 Chimerism Phase II

There is documentation of the accuracy of quantitative methods used to measure chimerism.

NOTE: The accuracy of quantitative methods used to measure chimerism must be documented at least annually by controlled blood mixing or other suitable method. If results on cell subpopulations are reported there must be documentation of periodic testing of the purity of such cell subsets.

HSC.38284 Stem Cell Engraftment Procedure Phase II

For stem cell engraftment, the polymorphic nature of the DNA system used is detailed and documented in the literature.

NOTE: Available data must include: the type of polymorphism (i.e. single locus, multi-locus, simple diallelic, hypervariable); the chromosomal location if known; the restriction endonuclease and probe used to detect the polymorphism; the conditions of hybridization and sizes (or range of sizes) of variable and constant fragments produced; and population-specific allele frequency data, if available. REFERENCES 1) 1991 Report concerning recommendations of the DNA Commission of the International Society for Forensic Haemogenetics relating

to the use of DHA polymorphisms. Forensic Sci Intl. 1992;52:125-130 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing L2.222. Richmond, VA: UNOS, 2001

HSC.38471 Stem Cell Engraftment Phase II

For stem cell engraftment, independent segregation (e.g. location on separate chromosomes) is documented for all single locus probes tested in the DNA system used.

HSC.38658 Result Reporting Phase II

For stem cell engraftment or other individual identity purposes, the final report includes an appropriate summary of the methods, probes and endonucleases used, the loci or mutations tested, the objective findings and a clinical interpretation in a readily interpretable format.

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REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing L2.253. Richmond, VA: UNOS, 2001

DONOR-RECIPIENT HISTOCOMPATIBILITY


● Sampling of donor recipient histocompatibility policies and procedures

● Sampling of cell typing records

● Staffing/on call schedule

● What evidence, from family studies, does your laboratory use to support haplotype or other genetic reporting conclusions (four distinct haplotypes must be identified in a pedigree analysis for haplotype assignment)?

HSC.38845 HLA Identity Confirmation Phase II

HLA identity is confirmed in sibling donor-patient transplants.

NOTE: MLC testing may be used to confirm HLA identify among siblings. This can also be accomplished by molecular HLA determination for Class I and II.


✓ Written procedure defining confirmation method(s) used for HLA identity REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H5.200. Richmond, VA: UNOS, 2001

HSC.39032 Haplotype Reporting Phase II

If haplotypes are reported, there is sufficient documentation to support the haplotype assignments.

NOTE: When reporting haplotypes, homozygosity, blanks, recombination, or other genetic information, there must be sufficient evidence from family studies to support such conclusions. If probable haplotypes are reported, the report must indicate clearly that they are "probable". Reliable haplotype frequencies of the appropriate ethnic groups must be used. REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H5.100. Richmond, VA: UNOS, 2001

HSC.39219 Donor Typing Phase I

Pre-harvest donor materials are used whenever possible for donor HLA typing and recipient serum screening.

NOTE: Organ donors should be HLA-typed from any acceptable source of viable lymphocytes. Whenever possible, pre-harvest HLA testing and screening crossmatches should be done.


✓ Written procedure defining criteria for organ donor typing AND

✓ Record of typing and screening on pre-harvest donor materials, when possible

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HSC.39406 Recipient Typing Phase II

The HLA laboratory types all potential recipients, types cells from organ donors referred to the laboratory, and follows policies defining when antigen retyping and redefinition are required.


✓ Written procedures for typing potential recipients and organ donors with criteria for antigen retyping and redefinition AND

✓ Records of all cell typing REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2003(Jan 24):7170 [42CFR493.1278]

HSC.39499 Personnel Availability Phase II

Personnel are available at all times to perform testing as needed for organ transplantation.


✓ Staffing schedule OR on-call schedule if 24-hour staffing is not available

Non-Renal Organ Transplants


● Sampling of non-renal organ transplant policies and procedures

● Sampling of antibody screening records

● Sampling of crossmatch records

HSC.39593 Recipient Screening Phase II

Non-renal transplant recipients are screened for HLA antibodies as well as non-HLA antibodies and/or autoreactive antibodies.

NOTE: All potential transplant recipients of non-renal organs are screened for Class I antibodies and specificity determined if possible. It is also recommended to screen for Class II HLA antibodies, and to be able to differentiate HLA specificity from autoreactivity.


✓ Written procedure for antibody screening of non-renal transplant recipients AND

✓ Records of antibody screening on transplant recipients REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing I2.000. Richmond, VA: UNOS, 2001

HSC.39780 Prospective Crossmatch Phase II

Non-renal sensitized transplant recipients are prospectively crossmatched with their potential donor, whenever possible, before transplantation.

NOTE: The technique used for crossmatching in these patients must be one of enhanced

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sensitivity for antibody detection.


✓ Written procedure for crossmatch of non-renal sensitized transplant recipients AND

✓ Crossmatch records REFERENCES 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing I3.000. Richmond, VA: UNOS, 2001

HSC.39800 Emergency Non-renal Transplants Phase I

The laboratory documents, if known, the circumstances under which emergency transplant was performed for non-renal transplantation without HLA testing and/or final crossmatches.

NOTE: Records of the transplant must include any information provided to the laboratory by the patient's physician.

PERSONNEL


● Documentation of technical supervisor (section director)/testing personnel/clinical consultant education and experience

● Continuing education policy

● Sampling of continuing education records

HSC.40000 Technical Supervisor Qualifications Phase II

The technical supervisor (section director) of the histocompatibility section has the following qualifications.

1. Academic degree: MD, DO, or PhD in biological or clinical laboratory science, and

2. Laboratory training and experience: 4 years training and experience in histocompatibility, or 2 years training and experience in general immunology plus 2 years in histocompatibility.

NOTE: These qualifications fulfill the requirements of CLIA.


✓ Records of technical supervisor qualifications including degree or transcript, certification/registration, current license (if required) and work history in related field


amendments of 1988; final rule. Fed Register. 1992(Feb 28):7180 [42CFR493.1449(o)]

HSC.45000 Testing Personnel Qualifications Phase II

For laboratories subject to US federal regulations, all testing personnel meet CLIA requirements.

NOTE: There must be evidence in personnel records that all testing personnel have been

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evaluated against CLIA requirements, and that all individuals qualify.


✓ Records of qualifications including degree or transcript, certification/registration, current license (if required) and work history in related field


amendments of 1988; final rule. Fed Register. 1992(Feb 28):7175 [42CFR493.1423], 7183 [42CFR493.1489] 2) CLSI. Training and Competence Assessment; Approved Guideline—Third Edition. CLSI Document GP21-A3. (ISBN 1-56238-691-3).

CLSI, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA, 2009

HSC.46250 Qualification by Experience Phase II

General supervisor(s) and testing personnel (technologists) are qualified by experience in histocompatibility/transplantation.

NOTE: The general supervisor must have a minimum of 3 years experience in histocompatibility/transplantation. Technologists must have a minimum of 1 year experience in histocompatibility/transplantation. Testing personnel with less than 1 year experience must be supervised by qualified personnel.


✓ Documentation of work history in personnel file

HSC.47500 CE Program Phase II

There is a continuing clinical laboratory education program that addresses the areas of service offered by the laboratory.

NOTE: The laboratory must have a complete continuing clinical laboratory education program that meets the needs of the various types of laboratory personnel and addresses the areas of service offered by the laboratory. The laboratory must have documented evidence of sufficient continuing education credits for directors (50 hours annually), supervisors (27 hours annually), and other technical personnel (12 hours annually).

Evidence of Compliance:.

✓ Written policy for continuing education requirement for all personnel AND

✓ Records of continuing education

HSC.48750 Clinical Consultant Qualifications Phase II

The section director or other individual fulfills the responsibilities of clinical consultant as defined in CLIA.

NOTE: The clinical consultant must be an MD or DO with appropriate training and experience in the interpretation of histocompatibility / transplantation immunology test results, or a doctoral level clinical scientist certified by an approved specialty board.


✓ Records of clinical consultant qualifications including degree or transcript, certification/registration, current license (if required) and work history in related field AND

✓ Job description defining responsibilities of clinical consultant REFERENCES 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement

amendments of 1988; final rule. Fed Register. 2002(Oct 1):1043 [42CFR493.1417] and 1038 [42CFR493.1405(b)