HISTOCOMPATIBILITY€¦ · Web viewNominal 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 COMMENTARY: Volumetric glassware must be

Revised: 10/06/2005

HISTOCOMPATIBILITY

This College of American Pathologists (CAP) Laboratory Accreditation Program (LAP) Checklist is provided as a Microsoft® Word 2000 electronic file for convenience and for educational purposes. It represents the fully-approved version for use in the LAP as of the date given in the header.

Newer approved versions of this Checklist may be found via the Internet at the CAP Web site (http://www.cap.org/apps/docs/laboratory_accreditation/checklists/checklistftp.html) for both viewing and download to your computer.

If you are currently enrolled in the CAP LAP and are preparing for an inspection, please note:

The Checklists undergo frequent revision, and the contents may have changed after you receive your inspection packet. If a Checklist has been updated since receiving your packet, you will be inspected based upon the Checklists that were mailed to you in your application or reapplication packet.

For questions about the use of Checklists in the inspection process, please e-mail the CAP at [email protected], or call (800) 323-4040, ext. 6065. Suggestions for content improvement should be sent by e-mail to LAP at [email protected].

All checklists are © 2005 College of American Pathologists. All rights reserved.

http://www.cap.org/apps/docs/laboratory_accreditation/checklists/checklistftp.html

College of American Pathologists Revised: 10/06/2005

OUTLINE

SUMMARY OF CHANGESINSPECTION TECHNIQUES – KEY POINTSINTRODUCTIONLABORATORY SAFETYPROFICIENCY TESTINGQUALITY CONTROL AND QUALITY MANAGEMENT

SUPERVISIONPROCEDURE MANUALSPECIMEN COLLECTION AND HANDLINGRESULTS REPORTINGRECORDS

Recipient and Donor Information RecordsREAGENTSCONTROLS AND STANDARDSINSTRUMENTS AND EQUIPMENT

Temperature-Dependent EquipmentThermometersCentrifugesSpectrophotometersFilm Processing/Photographic EquipmentFume Hoods and Biological Safety CabinetsElectrophoresis EquipmentpH MetersBalances and WeightsVolumetric Glassware and Pipettes

PROCEDURES AND TEST SYSTEMSLYMPHOCYTE ISOLATIONSEROLOGICAL PROCEDURES

GeneralHLA Class I and II Antigen TypingCytotoxicity Crossmatch

RED CELL TYPINGFLOW CYTOMETRY

Instrumentation and PhenotypingFlow Cytometry Crossmatch

MIXED LYMPHOCYTE CULTURE TESTINGHLA ANTIBODY SCREENING

Enzyme-Linked Immunosorbent Assays (ELISA)MOLECULAR HLA ANTIGEN TYPINGDONOR-RECIPIENT HISTOCOMPATIBILITY

Non-Renal Organ TransplantsPERSONNELPHYSICAL FACILITIES

HISTOCOMPATIBILITY Page 2 of 91


SUMMARY OF CHANGESHISTOCOMPATIBILITY Checklist

10/6/2005 Edition

The following questions 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 questions included may not appear in the customized checklist. Such questions are not applicable to the testing performed by the laboratory.

Note: For revised checklist questions, 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 Questions

Question Effective Date HSC.20035 12/29/2004HSC.20976 12/29/2004HSC.20982 12/29/2004HSC.20988 12/29/2004HSC.20994 12/29/2004HSC.21281 12/29/2004HSC.21287 12/29/2004HSC.21293 12/29/2004HSC.21675 12/29/2004HSC.22325 12/29/2004HSC.22531 12/29/2004HSC.22562 12/29/2004HSC.22593 12/29/2004HSC.28092 12/29/2004HSC.28996 12/29/2004HSC.29058 12/29/2004HSC.29877 12/29/2004HSC.29885 12/29/2004HSC.29893 12/29/2004HSC.29901 12/29/2004HSC.29909 12/29/2004HSC.29917 12/29/2004HSC.29925 12/29/2004HSC.29933 12/29/2004HSC.29941 12/29/2004HSC.29949 12/29/2004HSC.29957 12/29/2004



HSC.29965 12/29/2004HSC.29973 12/29/2004HSC.29981 12/29/2004HSC.29989 12/29/2004HSC.29997 12/29/2004HSC.30005 12/29/2004HSC.30013 12/29/2004HSC.30021 12/29/2004HSC.30029 12/29/2004HSC.30037 12/29/2004HSC.30045 12/29/2004HSC.35946 12/29/2004HSC.38134 12/29/2004HSC.38171 12/29/2004HSC.38208 12/29/2004HSC.38245 12/29/2004HSC.39499 12/29/2004HSC.46250 12/29/2004HSC.47500 12/29/2004HSC.48750 12/29/2004

REVISED Checklist Questions

Question Effective Date HSC.10000 12/29/2004HSC.10200 12/29/2004HSC.22775 12/29/2004HSC.26129 12/29/2004HSC.26503 12/29/2004HSC.39780 12/29/2004HSC.50000 12/29/2004

DELETED Checklist Questions

Question Effective Date HSC.21200 12/29/2004HSC.60000 12/29/2004



The checklists used in connection with the inspection of laboratories by the Commission on Laboratory Accreditation (“CLA”) 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.

IMPORTANT: The contents of the Laboratory General Checklist are applicable to the Histocompatibility section of the laboratory.

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INSPECTION TECHNIQUES – KEY POINTS

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I. READ – OBSERVE – ASK – the three methods of eliciting information during the inspection process. These three methods may be used throughout the day in no particular order. Plan the inspection in a way that allows adequate time for all three components.

READ = Review of Records and DocumentsDocument review verifies that procedures and manuals are complete, current, available to staff, accurate and reviewed, and describe good laboratory practice. Make notes of any questions you may have, or processes you would like to observe as you read the documentation.

OBSERVE – ASK = Direct Observation and Asking QuestionsObserving and asking questions accomplish the following:

1. Verifies that the actual practice matches the written policy or procedure2. Ensures that the laboratory processes are appropriate for the testing performed3. Ensures that outcomes for any problem areas, such as PT failures and issues/problems

identified through the quality management process, have been adequately investigated and resolved

4. Ensures that previously cited deficiencies have been corrected

Use the following techniques: Observe laboratory practices – look at what the laboratory is actually doing. Compare the

written policy/procedure to what you actually observe in the laboratory to ensure the written policy/procedure accurately reflects laboratory practice. Note if practice deviates from the documented policies/procedures.

Ask open ended, probing questions – these are starting points that will allow you to obtain large amounts of information, and help you clarify your understanding of the documentation you’ve seen



and observations you’ve made. This eliminates the need to ask every single checklist question, as the dialogue between you and the laboratory may address multiple checklist questions.

Ask open-ended questions that start with phrases such as “show me how…” or “tell me about …” or “what would you do if…”. By asking questions that are open-ended, or by posing a hypothetical problem, you will avoid “cookbook” answers. For example, ask “Could you show me the specimen transport policy and show me how you ensure optimum specimen quality?” This will help you to determine how well the technical staff is trained, whether or not they are adhering to the lab’s procedures and policies, and give you a feel for the general level of performance of the laboratory.

Ask follow-up questions for clarification. Generally, it is best not to ask the checklist questions verbatim. For example, instead of asking the checklist question “Is there documentation of corrective action when control results exceed defined tolerance limits?” ask, “What would you do if the SD or CV doubles one month?” A follow-up probing question could be, “What would you do if you were unable to find a cause for the change in SD or CV?”

II. Evaluate Selected Specimens and Tests in Detail

For the Laboratory General Checklist: Follow a specimen through the laboratory. By following a specimen from collection to test result, you can cover multiple checklist questions in the Laboratory General checklist: questions on the specimen collection manual; phlebotomy; verbal orders; identification of patients and specimens; accessioning; and result reporting, including appropriate reference ranges, retention of test records, maintaining confidentiality of patient data, and proper handling of critical values and revisions to reports.

For the individual laboratory sections: Consult the laboratory’s activity menu and focus on tests that potentially have the greatest impact on patient care. Examples of such tests include HIV antibodies, hepatitis B surface antigen, urine drugs of abuse, quantitative beta-hCG, cultures of blood or CSF, acid-fast cultures, prothrombin time and INR reporting, and compatibility testing and unexpected antibody detection. Other potentially high-impact tests may be identified by looking at very high or low volume tests in the particular laboratory, or problems identified by reviewing the Variant Proficiency Testing Performance Report.

To evaluate preanalytic and postanalytic issues: Choose a representative specimen and “follow" the specimen through the laboratory or section of the laboratory, reviewing appropriate records in the preanalytic and postanalytic categories.

To evaluate analytic processes: Choose 2 or 3 analytes and perform a comprehensive review of records, including procedure manuals, quality control and proficiency testing records, instrument maintenance records and method performance validations for the last 2 years, selecting timeframes at the beginning, mid-point, and end of this timeframe. Compare instrument print-outs to patient reports and proficiency testing results to ensure accurate data entry. If problems are identified, choose additional tests or months to review.



III. Verify that proficiency testing problem have been resolved: From the inspector’s packet, review the Variant PT Performance Report that identifies, by analyte, all of the PT scores below 100%. Correlate any PT problems to QC or maintenance records from the same time period. Be thorough when reviewing these representative records, selecting data from the beginning, middle and end of the period since the last on-site inspection.

IV. Review correction of previous deficiencies: Review the list of deficiencies from the previous on-site inspection provided in the inspector’s packet. Ensure that they have been appropriately addressed.

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INTRODUCTION

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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-88 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.

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LABORATORY SAFETY

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The inspector should review relevant questions from the Safety section of the Laboratory General checklist, to assure that the histocompatibility laboratory is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report.

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PROFICIENCY TESTING

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The laboratory must participate in a CAP or CAP-approved program of interlaboratory comparison testing, when available, appropriate to the scope of the laboratory.



**REVISED** 12/29/2004

HSC.10000 Phase II N/A YES NO

Is the laboratory enrolled in the appropriate required CAP Surveys or CAP-approved alternative proficiency testing program appropriate for the testing performed?

NOTE: The list of analytes for which CAP requires proficiency testing is available on the CAP website [http://www.cap.org/apps/docs/laboratory_accreditation/ptgraded.html] or by phoning 800-323-4040 (or 847-832-7000), option 1. The laboratory’s participation in proficiency testing must include all analytes on this list for which it performs patient testing. Participation in proficiency testing may be through CAP Surveys or a CAP-approved proficiency testing provider. Laboratories will not be penalized if they are unable to enroll in an oversubscribed program. If unable to enroll, however, the laboratory must implement an alternative assessment procedure for the affected analytes. For regulated analytes, if the CAP and CAP-approved alternative PT programs are oversubscribed, CMS requires the laboratory to attempt to enroll in another CMS-approved PT program.

COMMENTARY:

N/A

REFERENCES: 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C4.100. Richmond, VA: UNOS, 2001; 2) Westgard JO, et al. Laboratory precision performance. State of the art versus operating specifications that assure the analytical quality required by clinical laboratory improvement amendments proficiency testing. Arch Pathol Lab Med. 1996;120:621-625; 3) NCCLS. Continuous quality improvement: essential management approaches and their use in proficiency testing; proposed guideline GP22-P. Wayne, PA: NCCLS, 1997; 4) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; standard III. Northfield, IL: CAP, 1998.

**REVISED** 12/29/2004


For tests for which CAP does not require PT, does the laboratory at least semiannually 1) participate in external PT, or 2) exercise an alternative performance assessment system for determining the reliability of analytic testing?

NOTE: Appropriate alternative performance assessment procedures may include: split sample analysis with reference or other laboratories, split samples with an established in-house method, assayed material, regional pools, clinical validation by chart review, or other suitable and documented means. It is the responsibility of the laboratory director to define such alternative performance assessment procedures, as applicable, in accordance with good clinical and scientific laboratory


http://www.cap.org/apps/docs/laboratory_accreditation/ptgraded.html


practice. Participation in ungraded/educational proficiency testing programs also satisfies this checklist question.

COMMENTARY:

N/A

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):7184 [42CFR493.1236(c)(1); 2) NCCLS. Assessment of Laboratory Tests When Proficiency Testing is Not Available; Approved Guideline. NCCLS document GP29-A [ISBN 1-56238-479-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002.


Does the laboratory integrate all proficiency testing samples within the routine laboratory workload, and are those samples analyzed by personnel who routinely test patient samples, using the same primary method systems as for patient samples?

NOTE: Replicate analysis of proficiency samples is acceptable only if patient specimens are routinely analyzed in the same manner. If the laboratory uses multiple methods for an analyte, proficiency samples should be analyzed by the primary method. There must not be any interlaboratory communication on proficiency testing data before results reporting. The educational purposes of proficiency testing are best served by a rotation that allows all technologists to be involved in the proficiency testing program. Records of these studies must be kept and can be an important part of the competency and continuing education documentation in the personnel files of the individuals. When external proficiency testing materials are not available, the semi-annual alternative performance assessment process should also be integrated within the routine workload.

COMMENTARY:

N/A

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)]; 2) Shahangian S, et al. Toward optimal PT use. Med Lab Observ. 2000;32(4):32-43.


Does each technologist performing tests participate in and record results of blind testing of a previously tested sample at least monthly to document reproducibility of results, and does this testing program cover all clinical tests that the technologist performs?



COMMENTARY:

All technologists reporting results must test a previously tested sample at least monthly, or utilize a split sample, to document reproducibility. Each person must be tested at least once annually for each test he or she performs.

REFERENCE: United Network for Organ Sharing Standard for Histocompatibility Testing C4.140.


Is there evidence of evaluation and, if indicated, corrective action in response to "unacceptable" results on the proficiency testing reports and results of the alternative performance assessment system?

NOTE: The evaluation must document the specific reason(s) for the "unacceptable" result(s) and actions taken to reduce the likelihood of recurrence. This must be done within one month after the laboratory receives its proficiency testing evaluation. In addition, each ungraded challenge, each educational challenge, and each episode of nonparticipation must be reviewed and corrective action instituted as appropriate.

COMMENTARY:

N/A

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 [42CFR493.1236]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C4.120. Richmond, VA: UNOS, 200; 3) NCCLS. Using proficiency testing (PT) to improve the clinical laboratory; approved guideline GP27-A. Wayne, PA: NCCLS, 1998.


Is there documented evidence of ongoing evaluation by the laboratory director or designee of proficiency testing and alternative performance assessment results?

COMMENTARY:

There must be documented evidence of ongoing evaluation by the laboratory director or designee of proficiency testing and alternative performance assessment 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. 1992(Feb 28):7176 [42CFR493.1445(e)(4)]; 2) American Society for Histocompatibility and Immunogenetics.



Standards for histocompatibility testing. Lenexa, KS: ASHI, 1995-2002: C4.400; 3) NCCLS. Using proficiency testing (PT) to improve the clinical laboratory; approved guideline GP27-A. Wayne, PA: NCCLS, 1998.

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QUALITY CONTROL AND QUALITY MANAGEMENT

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SUPERVISION

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Before patient results are reported, QC data must be judged acceptable. The laboratory director or designee must review QC data at least monthly. Beyond these specific requirements, a laboratory may (optionally) perform more frequent review at intervals that it determines appropriate. Because of the many variables across laboratories, the CAP makes no specific recommendations on the frequency of any additional review of QC data.

All quality management (QM) questions in the Laboratory General Checklist pertain to the histocompatibility laboratory.


Does the histocompatibility laboratory have a written QC/QM program?

NOTE: The QM/QC program in the histocompatibility laboratory must be clearly defined and documented. The program must ensure quality throughout the preanalytic, 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.

Appropriate laboratory personnel must judge QC data acceptable before patient results are reported. The laboratory director or designee must review QC data at least monthly. Beyond these specific requirements, a laboratory may (optionally) perform more frequent review at intervals that it determines appropriate. Because of the many variables across laboratories, the CAP makes no specific recommendations on the frequency of any additional review of QC data.

COMMENTARY:

N/A



REFERENCE: 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 [42CFR493.1227], 7176 [42CFR493.1445(e)(5).


Is there 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 is this procedure consistent with good laboratory practice?

COMMENTARY:

The laboratory must have a completely documented procedures describing methods for patient identification, patient preparation, specimen collection and labeling, specimen preservation, conditions for transportation, and storage before testing. Such protocols must be consistent with good laboratory practice.


Is there evidence of ongoing evaluation of instrument function and maintenance, temperature, etc.?

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

COMMENTARY:

N/A

**NEW** 12/29/2004


Is there documentation of ongoing evaluation by the section director or designee of all of the following?

1. Control results of routine procedures2. Reactivity of reagents3. Instrument function checks4. Temperature records

COMMENTARY:



N/A


Is there documentation of corrective action taken when controls, etc., exceed defined tolerance limits?

NOTE: Where practical, patient test results obtained in an analytically unacceptable test run or since the last acceptable test run must be evaluated to determine if the accuracy of those results could be affected in a way that would be clinically significant and alter patient care.

COMMENTARY:

There must be adequate documentation of corrective actions taken when controls, etc., exceed defined tolerance limits. Where practical, patient test results obtained in an analytically unacceptable test run or since the last acceptable test run must be evaluated to determine if the accuracy of those results could be affected in a way that would be clinically significant and alter patient care.

REFERENCE: 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)].


Is there a documented system in operation to detect and correct significant clerical errors, significant analytical errors, and unusual laboratory results that could affect patient management?

NOTE: The laboratory must have a documented system in operation to detect and correct significant clerical errors, significant analytical errors, and unusual laboratory results that could affect patient management. The selective use of delta checks also may be useful in detecting clerical errors in consecutive samples from the same patient. In computerized laboratories, there should be automatic "traps" for improbable results.

COMMENTARY:

N/A


Does the system provide for the timely correction of errors?



COMMENTARY:

The system for detecting clerical errors, significant analytical errors, and unusual laboratory results must provide for timely correction of errors. For suspected errors detected by the end user after reporting, corrections must be promptly made if such errors are confirmed by the laboratory.


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

NOTE: This only applies to laboratories subject to CLIA-88. The CAP does NOT require supervisory review of all test results before or after reporting to patient records. Rather, this question is intended to address only that situation defined under CLIA-88 for "high complexity testing" performed by trained high school graduates qualified under 42CFR493.1489(b)(5) when a qualified general supervisor is not present.

COMMENTARY:

In the absence of on-site supervisors, the results of tests performed by personnel must be reviewed by the laboratory director or general supervisor within 24 hours. The CAP does not require supervisory review of all test results before or after reporting to patient records. Rather, this question is intended to address only that situation defined under CLIA-88 for "high complexity testing" performed by trained high school graduates qualified under 42CFR493.1489(b)(5) when a qualified general supervisor is not present. It is only applicable to laboratories subject to CLIA-88.

REFERENCE: 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)].

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PROCEDURE MANUAL

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The procedure manual should be used by personnel at the workbench and should include: test principle, clinical significance, specimen type, required reagents, test calibration, quality control, procedural steps, calculations, reference intervals, and interpretation of results. The manual should address relevant pre-analytic and post-analytic considerations, as well as the analytic activities of the laboratory. The specific style and format of procedure manuals are at the discretion of the laboratory director.



The inspection team should review the procedure manual in detail to understand the laboratory's standard operating procedures, ensure that all significant information and instructions are included, and that actual practice matches the contents of the procedure manuals.


Is a complete procedure manual available at the workbench or in the work area?

NOTE 1: The use of inserts provided by a manufacturer is not acceptable in place of a procedure manual. However, such inserts may be used as a part of a procedure description, if the insert accurately and precisely describes the procedure as performed in the laboratory. Any variation from this printed/electronic procedure must be detailed in the procedure manual. In all cases, appropriate reviews must occur.

NOTE 2: A manufacturer's procedure manual for an instrument/ reagent system may be acceptable as a component of the overall departmental procedures. Any modification to or deviation from the procedure manual must be clearly documented.

NOTE 3: Card files or similar systems that summarize key information are acceptable for use as quick reference at the workbench provided that:

a. A complete manual is available for reference.b. The card file or similar system corresponds to the complete manual and is subject to

document control.

NOTE 4: Electronic (computerized) manuals are fully acceptable. There is no requirement for paper copies to be available for the routine operation of the laboratory, so long as the electronic versions are readily available to all personnel. Such electronic versions must be subjected to proper document control (i.e., only authorized persons may make changes, changes are dated/signed (manual or electronic), and there is documentation of periodic review). Current paper copies of electronically stored procedures should be available at the time of the CAP inspection, or rapidly generated at the request of the Inspector.

COMMENTARY:

A documented procedure manual must be developed for the histocompatibility section of the laboratory and be available at the workbench. Its elements should include: test principle, clinical significance, specimen type(s), required reagents, calibration, quality control, procedural steps, calculations, reference intervals, and interpretation, as applicable.

REFERENCES: 1) Van Leeuwen AM. 6 Steps to building an efficiency tool. Advance/Laboratory. 1999:8(6):88-91; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C3.100. Richmond, VA: UNOS, 2001; 3) Borkowski A, et al. Intranet-based quality improvement documentation at the Veterans Affairs Maryland health care system. Mod.



Pathol. 2001;14:1-5; 4) NCCLS. Clinical laboratory technical procedure manuals - fourth edition; approved guideline GP2-A4. Wayne, PA: NCCLS, 2002.


Does the procedure manual contain specific instructions for test performance, preparation of reagents, and control methods for each of the following procedures?

1. Lymphocyte isolation2. HLA-ABC serologic typing3. HLA-DR, DQ serologic typing4. DNA or RNA typing5. Crossmatching-T cells (ABC loci)6. Crossmatching-B cells (DR locus)7. Flow cytometry crossmatching8. Antibody screening and identification9. ABO grouping10. Mixed lymphocyte cultures (MLC)11. Complement titration12. Environmental control

NOTE: The Inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report.

COMMENTARY:

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

1. Lymphocyte isolation2. HLA-ABC serologic typing3. HLA-DR, DQ serologic typing4. DNA or RNA typing5. Crossmatching-T cells (ABC loci)6. Crossmatching-B cells (DR locus)7. Flow cytometry crossmatching8. Antibody screening and identification9. ABO grouping10. Mixed lymphocyte cultures (MLC)11. Complement titration12. Environmental control

Specific details of non-compliance are identified in Part B (deficiency summary) of the Inspector's Summation Report.




Is there documentation of at least annual review of all policies and procedures in the histocompatibility laboratory by the current section director?

NOTE: There must be documentation of at least annual review of all policies and procedures in the histocompatibility laboratory by the current section director. The section director is responsible for ensuring that the collection of technical protocols is complete, current, and has been thoroughly reviewed by a knowledgeable person. Technical approaches must be scientifically valid and clinically relevant. To minimize the burden on the laboratory and reviewer(s), it is suggested that a schedule be developed whereby roughly 1/12 of all procedures are reviewed monthly. Paper/electronic signature review must be at the level of each procedure, or as multiple signatures on a listing of named procedures. A single signature on a title page or index of all procedures is not sufficient documentation that each procedure has been carefully reviewed. Changes to procedures must be initialed and dated by the section director.

COMMENTARY:

N/A

REFERENCES: 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C3.200. Richmond, VA: UNOS, 2001; 2) Borkowski A, et al. Intranet-based quality improvement documentation at the Veterans Affairs Maryland health care system. Mod. Pathol. 2001;14:1-5.


Does the director or designee review and approve all new policies and procedures, as well as substantial changes to existing documents, before implementation?

NOTE: Current practice must match the policy and procedure documents.

COMMENTARY:

The director or designee must review and approve all new policies and procedures, as well as substantial changes to existing documents before implementation. Current practice must match these documents.

REFERENCE: 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 [42CFR493.1251(d)].




Does the laboratory have a system documenting that all personnel are knowledgeable about the contents of procedure manuals (including changes) relevant to the scope of their testing activities?

NOTE: This does not specifically require annual procedure sign-off by testing personnel. The form of this system is at the discretion of the laboratory director.

COMMENTARY:

The laboratory must have a system documenting that all personnel are knowledgeable about the contents of procedure manuals (including changes) relevant to the scope of their testing activities. This does not specifically require annual procedure sign-off by testing personnel. The form of this system is at the discretion of the laboratory director.


If there is a change in directorship, does the new director ensure (over a reasonable period) that laboratory procedures are well-documented and undergo at least annual review?

COMMENTARY:

If there is a change in directorship of the laboratory, the new director must ensure (over a reasonable period) that all histocompatibility laboratory procedures are well-documented and undergo at least annual review.



When a procedure is discontinued, is a paper or electronic copy maintained for at least 2 years, recording initial date of use, and retirement date?

COMMENTARY:

A paper or electronic copy of a discontinued procedure must be maintained for at least 2 years, recording initial date of use, and retirement date.

REFERENCE: 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. 1105(a)(2);493.1251(e)].



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SPECIMEN COLLECTION AND HANDLING

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Are procedures adequate to maintain sample identity and integrity throughout all steps of the process?

COMMENTARY:

There must be a system in place to verify the identity of specimens, patients, donors, reagents, and 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.

**NEW** 12/29/2004


Is a documented procedure 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. IF at all practical, the inspector should observe donor arm preparation.

COMMENTARY:

N/A



**NEW** 12/29/2004


Has the laboratory evaluated 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.

COMMENTARY:

N/A

**NEW** 12/29/2004


Is there 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.

COMMENTARY:

N/A

**NEW** 12/29/2004


Are flow cytometry specimens 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.



COMMENTARY:

N/A

REFERENCES: 1) American Society for Microbiology. Manual of clinical immunology, 4th ed. Washington, DC: ASM, 1992:940; 2) NCCLS. Clinical applications of flow cytometry: immunophenotyping of leukemic cells; approved guideline H43-A. Wayne, PA: NCCLS, 1998.


Are there documented criteria for unacceptable specimens?

NOTE: This question 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.

COMMENTARY:

Documented criteria must be available for unacceptable specimens. This 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 still 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.

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.


Is the disposition of any unacceptable specimens documented in the patient report and/or quality management records?

COMMENTARY:

A record of any rejected specimens must be maintained in the patient report and/or quality management records. This information is essential to proper patient test management and to the laboratory quality management program.




Are the most appropriate recipient sera 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.

COMMENTARY:

The most appropriate recipient sera must be employed for final crossmatching. 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.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.230. Richmond, VA: UNOS, 2001.


Are procedures adequate to ensure that patient specimens are easily retrievable?

COMMENTARY:

Procedures must be adequate to ensure that patient specimens can be easily retrieved.

REFERENCE: 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)].

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RESULTS REPORTING

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Are patient results 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.



COMMENTARY:

Patient results must be reported in a legible, easy to interpret format that clearly indicates the test method and delineates the clinical implications of the results, as well as documenting the identity of the laboratory director.


Does the final report include an appropriate summary of the methods, the loci tested, the objective findings, all possible allele combinations, and an interpretation?

COMMENTARY:

N/A

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

**NEW** 12/29/2004


Are outside reference laboratories accredited by appropriate histocompatibility agencies (UNOS, NMDP), and, for US laboratories, CLIA 88 certified?

NOTE: Reference laboratories must have a valid accreditation in histocompatibility.

COMMENTARY:

N/A

**NEW** 12/29/2004


Are all laboratory reports (including reports from outside referral laboratories) reviewed by the section director (technical supervisor) or designee prior to release?

COMMENTARY:

N/A



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RECORDS

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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 question "N/A."


Are records of results of all histocompatibility-testing reports 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.

COMMENTARY:

N/A

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C5.100, C5:110. Richmond, VA: UNOS, 2001.


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

COMMENTARY:

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



Is a log of all stored specimens maintained to enable prompt retrieval for further testing?



COMMENTARY:

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

.................................................................

Recipient and Donor Information Records

.................................................................


Does the institution participate in and maintain 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.

COMMENTARY:

The laboratory must participate in and maintain records of patient and donor transplant information in the United Network for Organ Sharing (UNOS) Clinical Transplant Registry or its equivalent. Complete recipient and donor information must be maintained, including history of prior transfusion, pregnancy, 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.



Is there 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.



COMMENTARY:

There must be documentation of periodic review and verification of UNOS patient histocompatibility. 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.



Is there a system to resolve HLA typing discrepancies between laboratories?

COMMENTARY:

There must be a system to document any tissue-typing discrepancies between laboratories, and what steps were taken to resolve the discrepancy.


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REAGENTS

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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 questions is for new reagents to be checked by an appropriate method and the results recorded before patient results are 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.


Are reagents and solutions properly labeled, as applicable and appropriate, with the following elements?



1. Content and quantity, concentration or titer2. Storage requirements3. Date prepared or reconstituted by laboratory4. 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. The inspector will describe specific issues of non-compliance in the Inspector's Summation Report.

COMMENTARY:

N/A

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) NCCLS. Clinical laboratory technical procedure manuals - fourth edition; approved guideline GP2-A4. Wayne, PA: NCCLS, 2002.


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

COMMENTARY:

For U.S. laboratories, outdated reagents must be discarded and replaced routinely and handled in compliance with Food and Drug Administration requirements, and discarded/replaced as indicated.



Are new reagent lots, batches and/or shipments 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.

COMMENTARY:

N/A

REFERENCE: 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)].

**NEW** 12/29/2004


Are combinations of reagents from different lots checked against old reagent lots or with suitable reference material before or concurrently with being placed in service?

COMMENTARY:

N/A


Are procedures and conditions established and documented for reagent and patient sample storage, including procedures for frozen lymphocytes, frozen lymphocyte trays, and sera?

COMMENTARY:

The storage procedures for preparation of all reagents and storage conditions for reagents and reagent cells must be defined, followed, and documented to ensure proper reactivity and retrievability. Alarms are required to ensure that the materials have been properly stored and maintained.

REFERENCE: American Society for Histocompatibility and Immunogenetics. Standards for histocompatibility testing. Lenexa, KS: ASHI, 1995-2002: C2.130, C2.310, C2.320.


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

COMMENTARY:



Complete records and documentation must be available for all locally prepared reagents, including antisera and reagent cells.

REFERENCE: 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(a)(3)], 7171 [42CFR492. 1278(d)(3)].


Is there a system in place to ensure optimal storage and maintenance of all reagents and stored patient sera, including, where appropriate, monitored alarm systems and alternate storage plans?

COMMENTARY:

Sera stored for further study and all reagents must be optimally maintained. This must include monitored alarms to indicate malfunction of freezers or refrigerators, and a plan to provide for alternative storage.


If reagents are used in a manner different than manufacturer’s instructions, have appropriate validation studies been performed and documented?

COMMENTARY:

Appropriate validation studies must be performed and documented when reagent use does not follow manufacturer’s instructions.

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CONTROLS AND STANDARDS

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Are positive and negative controls assayed daily, and are there 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.

COMMENTARY:

Positive and negative controls must be run with each test procedure, where available. 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.

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.


Are controls properly labeled with content, lot number, date of preparation and expiration date?

COMMENTARY:

Controls must be properly identified with content, lot number, date of preparation and expiration date.


Are viability checks on lymphocyte preparations 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.

COMMENTARY:

Viability checks of the lymphocyte preparation 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.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.430. Richmond, VA: UNOS, 2001.




Does the laboratory include control material for each phase of compatibility testing?

COMMENTARY:

The laboratory must run control material through each phase of compatibility testing. Results of patient testing must not be reported until control values are reviewed and found acceptable.

REFERENCE: 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)].


Are control specimens tested in the same manner and by the same personnel as patient samples?

COMMENTARY:

It is implicit in quality control that control specimens are tested in the same manner as patient specimens. Moreover, 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.

REFERENCE: 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)].


Are the results of controls 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.

COMMENTARY:

Controls must be reviewed before reporting patient results. 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.

REFERENCE: 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)].

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INSTRUMENTS AND EQUIPMENT

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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 inspection team should review the procedures for instrument/equipment operations, maintenance, and monitoring records to ensure that these devices are properly used. The procedures and schedules for instrument maintenance must be as thorough and as frequent as specified by the manufacturer.


Is there a routine schedule or system for the regular checking of the critical operating characteristics of all instruments in use?

COMMENTARY:

A documented maintenance and function verification schedule must be established for all instruments in use.

REFERENCE: 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):7164 [42CFR493.1256].


Are instructions for instrument check systems available (i.e., manufacturer's manual or documented system prepared by the laboratory)?

COMMENTARY:

Specific instructions for checking instrument function must be available. A manufacturer's manual or laboratory prepared instructions is acceptable.





Are function checks conveniently documented to detect trends or malfunctions?

COMMENTARY:

Function checks must be documented in a manner to allow convenient detection of trends or malfunctions.



Are tolerance limits for acceptable function documented for specific instruments wherever appropriate?

COMMENTARY:

Tolerance limits for instrument function checks must be defined and documented (where appropriate).


**NEW** 12/29/2004


Is there a written procedure for implementing corrective action when tolerance limits of instrument function checks are exceeded?

COMMENTARY:

N/A




Are instructions provided for minor troubleshooting and repairs of instruments (such as manufacturer's service manual)?

COMMENTARY:

Instructions for minor troubleshooting and repairs must be available.


Are records maintained for each instrument to document all repairs and service procedures?

COMMENTARY:

Documentation of instrument service and repairs must be maintained.


Are instrument maintenance, service and repair records (or copies) promptly available to, and usable by, the technical staff operating the equipment?

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.

COMMENTARY:

N/A

REFERENCE: 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

.................................................................




Are temperatures checked and recorded appropriately for each of the following types of equipment?

1. Water baths and heating blocks2. Incubators and ovens (where temperature control is necessary for a procedure)3. Dialyzer baths4. 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 Inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report.

COMMENTARY:

Temperatures must be checked and recorded appropriately for each of the following types of equipment.

1. Water baths and heating blocks2. Incubators and ovens (where temperature control is necessary for a procedure)3. Dialyzer baths4. Refrigerators and freezers

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.


**NEW** 12/29/2004


Is there an audible alarm for each sample or reagent storage unit, and is the alarm 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.

COMMENTARY:



N/A

**NEW** 12/29/2004


Are alarm systems 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.

COMMENTARY:

N/A

**NEW** 12/29/2004


Will 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 fail-ure alarm, or battery power.

COMMENTARY:

N/A


Are cell freezers that use liquid nitrogen monitored?

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

COMMENTARY:

Liquid nitrogen-based cell freezers must be monitored to ensure that an adequate supply of nitrogen is present to maintain optimal cell storage temperature.




Have acceptable ranges been defined for all temperature-dependent equipment?

COMMENTARY:

An acceptable temperature range must be defined for all temperature-dependent equipment.

**REVISED** 12/29/2004


Are individual wells (or a representative sample thereof) of thermocyclers 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.

COMMENTARY:

N/A

REFERENCE: 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

.................................................................


Is an appropriate thermometric standard device of known accuracy available (NIST certified or guaranteed by manufacturer to meet NIST standards)?

COMMENTARY:

A thermometric standard device of known accuracy (NIST certified or guaranteed to meet NIST standards) must be available for use by the laboratory.




Are all non-certified thermometers in use checked against an appropriate thermometric standard device before being placed in service?

COMMENTARY:

All thermometers must be checked for accuracy against a thermometric device of known accuracy.

.................................................................

Centrifuges

.................................................................

HSC.22900 Phase I N/A YES NO

Are all centrifuges in the histocompatibility laboratory clean and properly maintained?

COMMENTARY:

Improvement is needed in the care and maintenance of the centrifuges in use.


Is there a documented protocol and schedule for maintenance of all centrifuges (cleaning, changing brushes, etc.)?

COMMENTARY:

A documented maintenance protocol and schedule for centrifuges must be defined and implemented.


.................................................................

Spectrophotometers

.................................................................




Are absorbance and photometric linearity checked periodically with filters or standards solutions, if required by the instrument manufacturer?

COMMENTARY:

Absorbance and photometric linearity must be checked periodically with filters or standards solutions, if so specified by the instrument manufacturer.


Are filters (filter photometers) checked periodically to ensure they are in good condition (e.g., clean, free of scratches)?

COMMENTARY:

Filters (filter photometers) must be checked periodically to be in good condition (e.g., clean, free of scratches).


Is spectrophotometer (including ELISA plate readers) wavelength calibration, absorbance and linearity checked at least every 6 months (or as required by the manufacturer) with appropriate solutions, filters, or emission line source lamps?

COMMENTARY:

Spectrophotometer wavelength calibration must be checked and/or documented regularly with appropriate solutions, filters or emission line source lamps, if required by the manufacturer.


Is stray light checked periodically with extinction filters or appropriate solutions, if required by the manufacturer?

COMMENTARY:

Stray light must be checked periodically with extinction filters or appropriate solutions, if required by the manufacturer.



................................................................

Film Processing/Photographic Equipment

.................................................................


Is film-processing (developing) equipment 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.

COMMENTARY:

There must be documentation of routine service and repair of equipment, and/or appropriately replenishing of reagents. 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.


Is all photographic equipment in the laboratory clean and properly maintained?

COMMENTARY:

There must be improvement in cleanliness and maintenance of the photographic equipment in the laboratory.


Are fixed camera mountings secure and level?

COMMENTARY:

Fixed camera mountings must be secure and level.



.................................................................

Fume Hoods and Biological Safety Cabinets

.................................................................


Is a properly functioning fume hood (or chemical filtration unit) available for any procedures using volatile chemicals?

COMMENTARY:

A properly functioning fume hood (or chemical filtration unit) must be available for procedures that use volatile chemicals.


Is a biological safety cabinet (or hood) available, when appropriate?

COMMENTARY:

A biological safety cabinet (or hood) must be available.

REFERENCE: 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.


Is the biological safety cabinet (or hood) certified at least annually to ensure that filters are functioning properly and that airflow rates meet specifications?

COMMENTARY:

There must be documentation of annual certifications that the biological safety cabinet (or hood) filters are functioning properly and that airflow rates meet specifications.

.................................................................

Electrophoresis Equipment

.................................................................




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

COMMENTARY:

Electrophoretic apparatus in the laboratory must be kept clean and properly maintained (electrodes and buffer tank not intact, tight power supply electrodes, no build up-of dried buffer, etc.).


Is the displayed voltage reading periodically confirmed by a voltmeter or other suitable means for all power supplies in the laboratory?

COMMENTARY:

The displayed voltage reading of all laboratory power supplies must be confirmed periodically using a voltmeter or other appropriate means.

.................................................................

pH Meters

.................................................................


Are there documented procedures for operation, calibration, and function checks of pH meter(s)?

COMMENTARY:

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


Are high quality buffers (certified assay of content) used for calibration of the pH meter(s)?

COMMENTARY:

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



.................................................................

Balances and Weights

.................................................................


Are balances cleaned, serviced, and checked periodically by qualified service personnel?

COMMENTARY:

Balances must be cleaned, serviced, and checked periodically by qualified service personnel.


Are analytical balances mounted such that vibrations do not interfere with readings?

COMMENTARY:

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

**REVISED** 12/29/2004


Are standard weights of the appropriate ANSI/ASTM Class 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.

COMMENTARY:

N/A

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


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

COMMENTARY:

N/A

**REVISED** 12/29/2004


Are results of periodic accuracy checks recorded?


http://www.astm.org/


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.

COMMENTARY:

N/A

................................................................

Volumetric Glassware and Pipettes

.................................................................


Is volumetric glassware of certified accuracy (Class A, NIST Standard or equivalent) in use, or if non-certified volumetric glassware is used, are all items 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:

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

COMMENTARY:

Volumetric glassware must be of certified accuracy (Class A, NIST standard or equivalent), and checked for accuracy of calibration before initial use. Reconstitution of lyophilized calibrators, controls, or proficiency testing materials, or any 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.

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.


Are non-class A pipettes that are used for quantitative dispensing of material 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.

COMMENTARY:

Non-class A pipettes used for quantitative dispensing of material must be checked for accuracy and reproducibility at specified periodic intervals. Results of such checks must be documented. 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, and 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. Wayne, PA: NCCLS, 1995; 5) Bray W. Software for the gravimetric calibration testing of pipets. Am Clin Lab. Oct 1995 (available on the internet at http://www.labtronics.com/pt_art.htm); 6) Kroll MH, et al (eds). Laboratory instrument evaluation, verification & maintenance manual, 5th edition. Northfield, IL: College of American Pathologists, 1999:126-127; 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.




Are automatic pipettes checked for accuracy of calibration and reproducibility before being placed in service and at regular intervals thereafter?

COMMENTARY:

Automatic pipettes must be 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 (available on the internet at http://www.labtronics.com/pt_art.htm); 5) Kroll MH, et al (eds). Laboratory instrument evaluation, verification & maintenance manual, 5th edition. Northfield, IL: College of American Pathologists, 1999:126-127; 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.


Are dedicated pipettors used for pre-amplification procedures?

COMMENTARY:

Dedicated pipettors must be used for pre-amplification procedures.

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

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PROCEDURES AND TEST SYSTEMS

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LYMPHOCYTE ISOLATION

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Is the source of the lymphocytes documented?

NOTE: These may include blood, bone marrow, lymph nodes, spleen, or cultured cells.

COMMENTARY:

The source of the lymphocytes must be documented.


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SEROLOGICAL PROCEDURES

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.................................................................

General

.................................................................


Is there 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.

COMMENTARY:

For cytotoxicity testing procedures, there must be established limits for defining positive and negative results by approximate percentage of cell death.

REFERENCE: REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.320. Richmond, VA: UNOS, 2001.


Is each lot, batch and/or shipment of complement 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.

COMMENTARY:

N/A

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

.................................................................


Do 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.

COMMENTARY:

The HLA antigen assignment in written reports must conform to the most current World Health Organization (W.H.O.) nomenclature. Any newly discovered antigens not yet assigned by W.H.O. Committee must be designated alternatively in such a manner as not to be confused with existing established terminology. All genotype and phenotype designations must also conform to W.H.O. Committee recommendations. The laboratory must maintain a list of antigens defined by the reagents used.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing D1.100. Richmond, VA: UNOS, 2001.



**NEW** 12/29/2004


Is there documentation that the appropriate terminology is used for leukocyte phenotyping?

COMMENTARY:

N/A


Are target cells defined for serological determination of HLA Class I antigens, and selected to permit typing the antigens officially recognized by the W.H.O. 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 W.H.O. recognized specificities.

COMMENTARY:

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 laboratory must be able to type for the W.H.O recognized specificities for which reagents are readily available.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.110, E2.310. Richmond, VA: UNOS, 2001.


Does the methodology for serological Class II antigen typing define the proportion of B-cells needed for optimal testing, and the specificities that are officially recognized by the W.H.O. 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 identify class II antigens.

COMMENTARY:



Determination of HLA class II antigen typing must be performed on B-cell preparations where the percentage of B-lymphocytes is documented by a method that is at least 80% B-cell enriched. Documentation of this enrichment may not be necessary when procedural techniques already distinguish T- and B-lymphocytes, or when well-characterized antibodies are utilized that can only discriminate and identify class II antigens. The laboratory must be able to type for the specificities that are officially recognized by the W.H.O. Committee and for which reagents are readily available

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.210, E2.310. Richmond, VA: UNOS, 2001.


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

COMMENTARY:

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


Are Class II antigens 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.

COMMENTARY:

Class II antigen assignment by the use of operationally monoclonal antibodies to each DR and DQ antigen must 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.





Do 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?

COMMENTARY:

Trays for disease association studies must define, at a minimum, those major specificities for which sera are readily available.

REFERENCE: 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(a) through (c)].

**NEW** 12/29/2004


Does each typing tray contain 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.

COMMENTARY:

N/A

**NEW** 12/29/2004


Are new typing reagents validated using cell panels of known HLA class I and class II types?

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.

COMMENTARY:

N/A




Is there 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 questions 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.

COMMENTARY:

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


.................................................................

Cytotoxicity Crossmatch

.................................................................


Is the technique used in the HLA crossmatch procedure more sensitive than the basic NIH lymphocytotoxicity crossmatch?

COMMENTARY:

Techniques for HLA crossmatching in transplantation must use a method that is more sensitive than the basic NIH lymphocytotoxicity procedure.





Does the crossmatch procedure define 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.

COMMENTARY:

A policy must define what patient’s sera are used in the final crossmatch. Cellular targets for transplant crossmatches must include donor T-cells, and may include donor B-cells when appropriate.



Are patient samples for crossmatch testing used undiluted, and kept frozen for a defined time post-transplantation?

COMMENTARY:

Patient serum samples for crossmatching in transplant patients must be used undiluted. Additional testing with serum dilutions is also appropriate. Sera must be kept frozen and available for retrieval as needed for a defined period post-transplantation.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.310, H3.400. Richmond, VA: UNOS, 2001.


Is there a policy that defines when results of a final crossmatch must be 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.

COMMENTARY:

N/A

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):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

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**NEW** 12/29/2004


Are typing sera and reagent cells used as prescribed by their manufacturers?

COMMENTARY:

N/A

**NEW** 12/29/2004


Are package inserts for the typing sera in use available and are typing sera used according to the manufacturers' directions, or, if alternative procedures are used, have they been evaluated to justify the changes?

COMMENTARY:

N/A



**NEW** 12/29/2004


Is there 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.

COMMENTARY:

N/A

**NEW** 12/29/2004


Is the specificity of the A1 subgroup of ABO testing documented to distinguish A1 from other subgroups?

COMMENTARY:

N/A

**NEW** 12/29/2004


Do 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?

NOTE 1: Records of specific reactivity checks for A cells, B cells, anti-A, and anti-B may be omitted when forward and reverse grouping results consistently agree.

NOTE 2: Each cell used for antibody detection must be checked each day of use for reactivity of at least one antigen using antisera of 1+ to 3+ avidity.

NOTE 3: Typing reagents such as anti-D, anti-K, anti-Fy(a), etc., must be checked each day of use.

NOTE 4: Anti-IgG reactivity of antiglobulin reagents may be checked during antibody screening and crossmatching.



This Checklist item can be satisfied by testing one vial of each reagent lot each day of testing.

COMMENTARY:

N/A


**NEW** 12/29/2004


Are typing sera and reagent cells 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.

COMMENTARY:

N/A

**NEW** 12/29/2004


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

COMMENTARY:

N/A

**NEW** 12/29/2004


Are immunohematology records and specimens 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 PERIODPatient records 10 yearsEmployee signatures, initials, and identification codes

10 years post employment

Quality Control Records 5 yearsSpecimens 7 daysRecords, unexpected antibodies. Indefinitely

COMMENTARY:

N/A

**NEW** 12/29/2004


Have criteria for the performance and interpretation of ABO antibody titers been defined and documented?

COMMENTARY:

N/A

**NEW** 12/29/2004


Are appropriate control(s) 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.

COMMENTARY:

N/A



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FLOW CYTOMETRY

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………………………………………………………..

Instrumentation and Phenotyping

………………………………………………………..

**NEW** 12/29/2004


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

NOTE: One of the levels of these controls should be at (or near) clinical decision levels (e.g., low CD34).

COMMENTARY:

For quantitative lymphocyte subset measurements, positive cellular controls at a minimum of 2 levels must be analyzed daily to verify performance of reagents, preparation methods, and staining procedures. One of the levels of these controls should be at (or near) clinical decision levels (e.g., low CD34).

REFERENCE: 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].

**NEW** 12/29/2004


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

COMMENTARY:



Verifying reproducibility of instrument performance is an essential element of quality assurance within the laboratory. Instrument performance must be monitored under the same conditions used to run test samples.

REFERENCE: NCCLS. Clinical applications of flow cytometry: immunophenotyping of leukemic cells; approved guideline H43-A. Wayne, PA: NCCLS, 1998.

**NEW** 12/29/2004


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

COMMENTARY:

Appropriate standards for each fluorochrome must be run each day the instrument is used and the results recorded for quality control purposes. These steps are necessary to optimize the flow system and the optics of the instrument.

REFERENCE: NCCLS. Clinical applications of flow cytometry: quality assurance and immunophenotyping of lymphocytes; approved guideline H42-A. Wayne, PA: NCCLS, 1998.

**NEW** 12/29/2004


Are procedures established for determining appropriate color compensation settings?

COMMENTARY:

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.




**NEW** 12/29/2004


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

COMMENTARY:

The laboratory should have procedures in place to ensure that the laser current is acceptable and constant. For some instruments, current is a better gauge of laser performance than is power output, which may be relatively constant.

**NEW** 12/29/2004


Are appropriate gating techniques 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.

COMMENTARY:

Gating techniques must be used to select the cell population for analysis. Examples include lymphocyte gates set using linear forward angle light scatter and 90-degree side scatter, or may be validated using CD45-FITC and CD14-PE monoclonal antibodies.

REFERENCE: National Institute for Allergy and Infectious Diseases/Division of AIDS guidelines for flow cytometric immunophenotyping, ver 1.0, Jan 1993.

**NEW** 12/29/2004


Is there a procedure to set markers (cursors) to distinguish fluorescence negative and fluorescence positive cell populations?

COMMENTARY:

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.

**NEW** 12/29/2004


Is there a policy for determining when the percentage of viable cells in each test specimen should be measured?

NOTE: 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.

COMMENTARY:

The quality of the result and the utility of the information are directly related to the viability of the cell population examined. Procedures must be in place to ensure that viable cells are analyzed. Selective loss of cell subpopulations and/or the presence of dead cells may lead to spurious results. 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. If specimen viability is below the established laboratory minimum, test results are suspect and this should be noted in the test report.

REFERENCE: Immunophenotyping of leukemic cells; approved guideline H43-A. Wayne, PA: NCCLS, 1998.

**NEW** 12/29/2004


Are methods established to ensure that immunoglobulin staining is intrinsic and not extrinsic (cytophilic)?

COMMENTARY:



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.

REFERENCE: Clinical applications of flow cytometry: immunophenotyping of leukemic cells; approved guideline H43-A. Wayne, PA: NCCLS, 1998.

**NEW** 12/29/2004


Is there 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.

COMMENTARY:

There must be a procedure to distinguish fluorescence-negative and fluorescence-positive cell populations. Test antibodies may double as control reagents. However, there must be procedures in place to compare displays of test data to similar displays of designated controls. 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.


**NEW** 12/29/2004


Are the staining and analytical procedures described in the procedure manual based upon established methodology (reference cited)?

COMMENTARY:

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.



**NEW** 12/29/2004


Does specimen treatment with nucleic acid dye include treatment with RNAse if the dye is not specific for DNA?

COMMENTARY:

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.

REFERENCE: Shapiro HA. Practical flow cytometry. New York, NY: Alan R. Liss, 1985.

………………………………………………………..

Flow Cytometry Crossmatch

………………………………………………………..


Does the flow cytometry crossmatch identify 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.

COMMENTARY:

For flow cytometry crossmatches, 2 or multiple color techniques must be used to identify antibodies to T-cells.

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


Are IgG antibodies identified by appropriately labeled heavy chain-specific F(ab’)2 reagents?

COMMENTARY:

For flow cytometry crossmatches, IgG antibodies must be identified by appropriately labeled heavy chain-specific F(ab’)2 reagents.





Is there documentation of the number of cells and volume of serum used for optimal sensitivity?

COMMENTARY:

For flow cytometry crossmatches, the number of cells and volume of serum used must be documented for optimal sensitivity.


Is normal human serum with demonstrated lack of reactivity against any potential target cell used as a negative control?

COMMENTARY:

For flow cytometry crossmatches, normal human serum with demonstrated lack of reactivity against potential target cells used must be used as a negative control.



Is the positive control an appropriately diluted human serum containing suitable HLA antibodies of appropriate immunoglobulin class known to react with lymphocytes from all donors?

COMMENTARY:

N/A





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

COMMENTARY:

For flow cytometric crossmatch methods, the antibody reagents (anti IgG, IgM, IgA, etc.) must be used at a selected dilution for optimal sensitivity and class specificity.



Has the cut-off for positive crossmatch results been established for all pertinent target cells (T-cells, B-cells, etc.)?

COMMENTARY:

The cut-off for positive flow cytometric crossmatch results must be established for all pertinent target cells (T-cells, B-cells, etc.).



Has the cut-off for positive crossmatches been determined by testing an appropriate number of sera from non-alloimmunized donors?

COMMENTARY:

The cut-off for positive flow cytometric crossmatch results must be determined by testing an appropriate number of sera from non-alloimmunized donors.



Does the procedure for HLA class II antibodies readily separate Class I from Class II specificity?



COMMENTARY:

The flow cytometric crossmatch testing procedure for HLA class II antibodies must readily separate Class I from Class II specificity.


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MIXED LYMPHOCYTE CULTURE TESTING

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Is testing performed under aseptic conditions, with access to a laminar flow hood?

COMMENTARY:

Mixed lymphocyte culture testing must be performed under aseptic conditions, with access to a laminar flow hood.


Is the instrument for counting tritiated thymidine monitored and standardized according to manufacturer’s recommendations?

COMMENTARY:

The instrument for counting tritiated thymidine in MLC testing must be monitored and standardized according to manufacturer’s recommendations.


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

COMMENTARY:

The incubator for MLC testing must be monitored on days of patient testing for temperature, CO2 concentration, and humidity as defined by the procedure manual.




Are autologous and at least 3 unrelated individuals or 2 unrelated individuals plus a pool of at least 3 or more unrelated individuals 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.

COMMENTARY:

N/A

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing F1.400, F1.500. Richmond, VA: UNOS, 2001.

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HLA ANTIBODY SCREENING

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Is there 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.

COMMENTARY:

N/A

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.




Does the laboratory have 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.

COMMENTARY:

N/A



Is there 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 crossreactive groups, as well as sufficient numbers of well-characterized panel cells or HLA-purified protein targets for antibody detection and specificity determinations.

COMMENTARY:

N/A

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing G1.310, G1:320. Richmond, VA: UNOS, 2001.


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

COMMENTARY:

For HLA antibody detection and specificity determination, positive and negative controls must be used and patient sera must be tested undiluted. When needed for further antibody characterization, dilutions may be appropriate.



REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing G1.210. Richmond, VA: UNOS, 2001.


Is there 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.

COMMENTARY:

N/A

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing G2.110, G2.120. Richmond, VA: UNOS, 2001.


Are all recipient sera screened for HLA antibodies including, at least, an initial sample at the time of HLA typing, after sensitizing events and upon request?

COMMENTARY:

There must be a system in place determining that all recipient sera are periodically screened for HLA antibodies. This must include at least an initial sample at the time of HLA typing, after sensitizing events, and upon request.

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. .................................................................

Enzyme-Linked Immunosorbent Assays (ELISA)

.................................................................




Is there 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.

COMMENTARY:

N/A


Is there documentation of how positive and negative cutoff points were determined?

COMMENTARY:

There must be documentation of how positive and negative cutoff points were determined in ELISA antibody detection methods.

-----------------------------------------------------------------

MOLECULAR HLA ANTIGEN TYPING

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Is the level of resolution of HLA typing 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.

COMMENTARY:

The HLA typing resolution level must be adequate to support clinical programs. For proficiency testing specimens, the level of resolution must reflect the highest level of resolution that the laboratory performs for its patients.





Is sample identification assured through all applicable phases of analysis, including all of the following?

1. Specimen receipt2. Nucleic acid extraction3. Nucleic acid quantification4. Endonuclease digestion5. Electrophoresis6. Transfer7. Hybridization8. Detection9. In situ hybridization10. Enzymatic amplification11. Photography12. Storage

NOTE: The inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report.

COMMENTARY:

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

1. Specimen receipt2. Nucleic acid extraction3. Nucleic acid quantification4. Endonuclease digestion5. Electrophoresis6. Transfer7. Hybridization8. Detection9. In situ hybridization10. Enzymatic amplification11. Photography12. Storage


Are nucleic acids extracted and purified by methods reported in the literature, or is there validation of a method developed in-house?



COMMENTARY:

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

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.


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

COMMENTARY:

Appropriate controls must be employed for reverse transcription methods that use RNA.



Is handling and storage of nucleic acids adequate to prevent degradation?

COMMENTARY:

Nucleic acid handling and storage must be adequate to prevent degradation.

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.


Is the quantity of nucleic acid measured, when appropriate?

COMMENTARY:

The quantity of nucleic acid must be measured in a procedure where it is appropriate to do so.





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

COMMENTARY:

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



Are standard amounts of nucleic acid loaded on analytical gels, when possible?

COMMENTARY:

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



Do 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?

NOTE: Autoradiographs and gel photographs must be of sufficient resolution and quality (low background, clear signal, absence of bubbles, etc.) to permit the reported interpretation.

COMMENTARY:

N/A

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.240, M1.250. Richmond, VA: UNOS, 2001.




Are procedures documented to prevent specimen loss, alteration, or contamination?

COMMENTARY:

Procedures must be documented to prevent specimen loss, alteration, or contamination.


For HLA typing and engraftment tests, is sufficient information 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:

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 combinations4. The HLA sequence database used5. For RFLP, sites resistant to endonuclease digestion, and cross-hybridizing bands; the

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

COMMENTARY:

N/A

REFERENCE: 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.

**NEW** 12/29/2004


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

COMMENTARY:

N/A




Are enzymatic amplification procedures (e.g., PCR) designed to minimize carry over (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.

COMMENTARY:

Enzymatic amplification procedures (e.g., PCR) must be designed to minimize carry over (false positive results) using appropriate physical containment and procedural controls. 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.

REFERENCE: Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989;339:237-238.


Has Mendelian inheritance of the DNA system used been validated by family studies?

COMMENTARY:

The DNA system used must be validated by family studies to demonstrate Mendelian inheritance.





Are autoradiographs or electrophoretic gels interpreted independently by at least 2 qualified readers using an objective method?

COMMENTARY:

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



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

COMMENTARY:

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



Are positive, negative and sensitivity controls run for each assay, when available and appropriate?

COMMENTARY:

Positive, negative and sensitivity controls must be run for each assay, when available and appropriate?



Is there 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.

COMMENTARY:

DNA 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.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M2.110, M2.120, M2.130, M2.510. Richmond, VA: UNOS, 2001.


Are known molecular weight markers that span the range of expected bands used for each electrophoretic run?

COMMENTARY:

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



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

COMMENTARY:

N/A





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

COMMENTARY:

The conditions (temperature, salt concentration, probe concentration, etc.) for pre-hybridization, hybridization and autoradiography must have been optimized to consistently produce accurate results.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.113, M3.131. Richmond, VA: UNOS, 2001.


Has the method of probe labeling been validated to detect the target sequence without a false positive signal for non-target sequences?

COMMENTARY:

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



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

COMMENTARY:

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





For sequence based typing, is there 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 W.H.O. Current databases of known sequences for all W.H.O.-recognized alleles must be immediately available.

COMMENTARY:

N/A


**NEW** 12/29/2004


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

COMMENTARY:

N/A

**NEW** 12/29/2004


Are internal controls 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.

COMMENTARY:



N/A

**NEW** 12/29/2004


Is preferential allele amplification considered in the interpretation of stem cell engraftment tests?

COMMENTARY:

N/A

**NEW** 12/29/2004


Is there documentation of the accuracy of quantitative methods used to measure chimerism?

NOTE: The accuracy of quantitative methods used to measure chimerism must be documented periodically 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.

COMMENTARY:

N/A


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

NOTE: Data that should be available 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.

COMMENTARY:

The polymorphic nature of the DNA system must be detailed and documented in the literature. Data that must be available 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 the 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.


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

COMMENTARY:

Independent segregation (e.g., location on separate chromosomes) of all single-locus probes tested in the DNA system used must be documented.


For stem cell engraftment or other individual identity purposes, does the final report include 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?

COMMENTARY:

Final reports must include 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. There must be documentation that supports the interpretation of the results.

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

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DONOR-RECIPIENT HISTOCOMPATIBILITY

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Is HLA identity 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.

COMMENTARY:

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.



If haplotypes are reported, is there 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.

COMMENTARY:

N/A



Are pre-harvest donor materials 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.

COMMENTARY:

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.




Does the HLA laboratory type all potential recipients, type cells from organ donors referred to the laboratory, and follow policies defining when antigen retyping and redefinition are required?

COMMENTARY:

HLA-A, B (including Bw4 and Bw6) and DR typing must be done on all recipients and donors . There must be policies to define when antigen redefinition and retyping are necessary.

REFERENCE: 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].

**NEW** 12/29/2004


Are personnel available at all times to perform testing as needed for organ transplantation?

COMMENTARY:

N/A

.................................................................

Non-Renal Organ Transplants

.................................................................


Are non-renal transplant recipients screened for HLA antibodies as well as non-HLA antibodies and/or autoreactive antibodies?

COMMENTARY:

All potential transplant recipients of non-renal organs must be 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.

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing I2.000. Richmond, VA: UNOS, 2001.



**REVISED** 12/29/2004


Are non-renal sensitized transplant recipients prospectively crossmatched with their potential donor, whenever possible, before transplantation?

NOTE: The technique used for crossmatching in these patients must be one of enhanced sensitivity for antibody detection.

COMMENTARY:

N/A

REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing I3.000. Richmond, VA: UNOS, 2001.

*****************************************************************************

PERSONNEL

*****************************************************************************


Does the technical supervisor (section director) of the histocompatibility section have the following qualifications?

1. Academic degree: MD, DO, or PhD in biological or clinical laboratory science, and2. 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 88.

COMMENTARY:

N/A

REFERENCE: 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):7180 [42CFR493.1449(o)].




For laboratories subject to US federal regulations, do all testing personnel meet CLIA-88 requirements?

NOTE: There must be evidence in personnel records that all testing personnel have been evaluated against CLIA-88 requirements, and that all individuals qualify.

COMMENTARY:

N/A

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):7175 [42CFR493.1423], 7183 [42CFR493.1489]; 2) NCCLS. Training verification for laboratory personnel; approved guideline GP21-A. Wayne, PA: NCCLS, 1996.

**NEW** 12/29/2004


Are general supervisor(s) and testing personnel (technologists) 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.

COMMENTARY:

N/A

**NEW** 12/29/2004


Is there an in-service continuing clinical laboratory education program that addresses the areas of service offered by the laboratory?

NOTE: The laboratory must have a complete in-service 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).



COMMENTARY:

N/A

**NEW** 12/29/2004


Does the section director or other individual fulfill the responsibilities of clinical consultant as defined in CLIA 88?

NOTE: The clinical consultant must be an M.D. or D.O. 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.

COMMENTARY:

N/A

REFERENCE: 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).

*****************************************************************************

PHYSICAL FACILITIES

*****************************************************************************

Sufficient space and utilities need to be provided for the overall workload of the histocompatibility section, and to meet all safety requirements.

**REVISED** 12/29/2004


Is there adequate space for administrative and clerical functions?

NOTE: Additional space must be provided for administrative and clerical functions.

COMMENTARY:

N/A



REFERENCES: 1) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; Standard II. Northfield, IL: CAP, 1998; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.100. Richmond, VA: UNOS, 2001.


Is there adequate space for technical work (bench space)?

COMMENTARY:

Additional space should be provided for technical work (bench space).



Is there adequate space for instruments?

COMMENTARY:

Additional space should be provided for instruments.



Is there adequate refrigerator and freezer storage space?

COMMENTARY:

Additional space should be provided for refrigerator storage.

REFERENCES: 1) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; Standard II. Northfield, IL: CAP, 1998; 2) United Network for



Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.100. Richmond, VA: UNOS, 2001.


Is the space available efficiently utilized?

COMMENTARY:

Existing space should be more efficiently utilized.



Is the space available so there is no compromise of the quality of work, safety of personnel, or limitation of quality control activities?

COMMENTARY:

Existing space limitations were so severe as to interfere with the quality of work, the safety of personnel, and/or the ability of personnel to carry out adequate quality control procedures with appropriate documentation.

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):7163 [42CFR493.1101(a)]; 2) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; Standard II. Northfield, IL: CAP, 1998; 3) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.100. Richmond, VA: UNOS, 2001.


Are floors, benches, and sinks clean, free of clutter, and well maintained?

COMMENTARY:

Floors, benches, and sinks appeared cluttered or otherwise deteriorated. Additional cleaning and maintenance procedures need to be implemented.




Are water taps, sinks, and drains adequate?

COMMENTARY:

Water taps, sinks and drains should be improved to support the types of procedures and workload of the laboratory.

REFERENCE: 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):7163 [42CFR493.1101(a)].


Are electrical outlets adequate?

COMMENTARY:

Electrical outlets should be improved to support the types of procedures and workload of the laboratory.

REFERENCE: 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):7163 [42CFR493.1101(a)].


Is lighting adequate?

NOTE: Direct sunlight should be avoided because of its extreme variability and the need for low light levels necessary to observe various computer consoles, etc. Lighting control should be sectionalized so general levels of illumination can be controlled in areas of the room if desired.

COMMENTARY:

Lighting should be improved.

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):7163 [42CFR493.1101(a)]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.200. Richmond, VA: UNOS, 2001.




Is ventilation adequate?

COMMENTARY:

Ventilation should be improved to support the types of procedures and workload of the laboratory.

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):7163 [42CFR493.1101(a)]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.200. Richmond, VA: UNOS, 2001.


Is ambient (room) temperature adequately controlled?

COMMENTARY:

Ambient (room) temperature should be adequately controlled to support the types of procedures and workload of the laboratory.



Are telephones conveniently located, and are calls easily transferred?

COMMENTARY:

Telephones - number and/or location should be improved to support the types of procedures and workload of the laboratory.

