commission on laboratory …webapps.cap.org/apps/docs/laboratory_accreditation/...do if the sd or cv...

Revised: 09/27/2007

COMMISSION ON LABORATORY ACCREDITATION

Laboratory Accreditation Program

HISTOCOMPATIBILITY CHECKLIST

Disclaimer and Copyright Notice The College of American Pathologists (CAP) Checklists are posted on the CAP's Web site for information only. If you are enrolled in the CAP's Laboratory Accreditation Program and are preparing for an inspection, you must use the Checklists that were mailed in your application or reapplication packet, not those posted on the Web site. The Checklists undergo regular revision and Checklists may be revised after you receive your packet. If a Checklist has been updated since receiving your packet, you will be inspected based upon the Checklists that were mailed. If you have any questions about the use of Checklists in the inspection process, please e-mail the CAP ([email protected]), or call (800) 323-4040, ext. 6065. All Checklists are ©2007. College of American Pathologists. All rights reserved.

College of American Pathologists Revised: 09/27/2007

HISTOCOMPATIBILITY (Web File) of 87

HISTOCOMPATIBILITY

OUTLINE

SUMMARY OF CHANGES ...............................................................................................................................................3 INSPECTION TECHNIQUES – KEY POINTS..................................................................................................................4 INTRODUCTION................................................................................................................................................................6 LABORATORY SAFETY...................................................................................................................................................6 PROFICIENCY TESTING ..................................................................................................................................................6 QUALITY MANAGEMENT AND QUALITY CONTROL.............................................................................................12

GENERAL ISSUES ......................................................................................................................................................12 PROCEDURE MANUAL.............................................................................................................................................15 SPECIMEN COLLECTION AND HANDLING..........................................................................................................19 RESULTS REPORTING...............................................................................................................................................23 RECORDS.....................................................................................................................................................................24

Recipient and Donor Information Records ...............................................................................................................25 REAGENTS ..................................................................................................................................................................26 CONTROLS AND STANDARDS................................................................................................................................30 INSTRUMENTS AND EQUIPMENT..........................................................................................................................32

Temperature-Dependent Equipment .........................................................................................................................34 Thermometers ...........................................................................................................................................................37 Centrifuges................................................................................................................................................................37 Spectrophotometers ..................................................................................................................................................38 Film Processing/Photographic Equipment................................................................................................................39 Fume Hoods and Biological Safety Cabinets............................................................................................................40 Electrophoresis Equipment .......................................................................................................................................41 pH Meters .................................................................................................................................................................41 Balances and Weights ...............................................................................................................................................42 Volumetric Glassware and Pipettes ..........................................................................................................................44

PROCEDURES AND TEST SYSTEMS ...........................................................................................................................46 LYMPHOCYTE ISOLATION......................................................................................................................................46 SEROLOGICAL PROCEDURES.................................................................................................................................47

General......................................................................................................................................................................47 HLA Class I and II Antigen Typing..........................................................................................................................48 Cytotoxicity Crossmatch...........................................................................................................................................52

RED CELL TYPING.....................................................................................................................................................53 FLOW CYTOMETRY ..................................................................................................................................................56

Instrumentation and Phenotyping .............................................................................................................................57 Flow Cytometry Crossmatch ....................................................................................................................................62

MIXED LYMPHOCYTE CULTURE TESTING .........................................................................................................64 HLA ANTIBODY SCREENING..................................................................................................................................65

Enzyme-Linked Immunosorbent Assays (ELISA) ...................................................................................................68 MOLECULAR HLA ANTIGEN TYPING...................................................................................................................68 DONOR-RECIPIENT HISTOCOMPATIBILITY........................................................................................................79

Non-Renal Organ Transplants ..................................................................................................................................80 PERSONNEL.....................................................................................................................................................................81 PHYSICAL FACILITIES ..................................................................................................................................................83



SUMMARY OF CHANGES HISTOCOMPATIBILITY Checklist

9/27/2007 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.05000 09/27/2007 HSC.10475 09/27/2007 HSC.21612 09/27/2007 HSC.10650 10/31/2006 HSC.10750 10/31/2006 REVISED Checklist Questions Question Effective Date HSC.10550 09/27/2007 HSC.29909 09/27/2007 HSC.20100 10/31/2006 DELETED Checklist Questions Question Effective Date HSC.10600 09/27/2007 HSC.20070 10/31/2006



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.

CONTINUING EDUCATION INFORMATION Beginning January 2008, you may earn continuing education credits (CME/CE) by completing an online Inspection Preparation activity that includes review of this checklist. Prior to reviewing the checklist, log on to the CAP Web site at <www.cap.org <http://www.cap.org>>, click the Education Programs tab, then select Laboratory Accreditation Program (LAP) Education Activities, and Inspection Preparation for complete instructions and enrollment information. __________________________________________________________________________________ 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

**************************************************************** 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 Documents Document 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 Questions Observing and asking questions accomplish the following:

1. Verifies that the actual practice matches the written policy or procedure 2. Ensures that the laboratory processes are appropriate for the testing performed 3. 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

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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 could not identify an obvious 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 results 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

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

***************************************************************************** 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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Definitions: Proficiency testing (PT) is defined as determination of laboratory testing performance by means of interlaboratory comparisons, in which a PT program periodically sends multiple specimens to members of a group of laboratories for analysis and/or identification; the program then compares each laboratory’s results with those of other laboratories in the group and/or with an assigned value…(adapted from Clinical Laboratory Standards Institute Harmonized Terminology Database; available at http://www.nccls.org/). Alternative assessment is defined as determination of laboratory testing performance by means other than PT--for example, split-sample testing, testing by a different method, etc. The laboratory must participate in a CAP or CAP-approved program of interlaboratory comparison testing, when available, appropriate to the scope of the laboratory. **NEW** 09/27/2007 HSC.05000 Phase I N/A YES NO Does the laboratory’s current CAP Activity Menu accurately reflect the testing performed? NOTE: An accurate Activity Menu is required to properly assess a laboratory’s compliance with proficiency testing requirements. The accuracy of the Activity Menu can be assessed by inquiry of responsible individuals, and by examination of the laboratory’s test requisition(s), computer order screens, procedure manuals, or patient reports. All tests performed by the laboratory should be listed on the Activity Menu, and visa versa. If tests are identified that are not included on the laboratory’s test menu, the inspector should contact the CAP (800-323-4040) for instructions. Please note that unusual or esoteric tests performed in the laboratory section may not be specifically listed on the laboratory's activity menu but may be identified on the activity menu as a miscellaneous code. Further information may be found with the laboratory's instrumentation list. Some activities are also included on the Master Activity Menu using more generic groupings or panels instead of listing the individual tests. The Master Activity Menu represents only those analytes that are directly measured. Calculations are not included. 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. 2004(Oct 1): 985 [42CFR493.51].

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HSC.10000 Phase II N/A YES NO Does the laboratory participate in the appropriate required CAP Surveys or another proficiency testing (PT) program accepted by CAP for the patient testing performed? NOTE: The list of analytes for which CAP requires proficiency testing is available on the CAP website [http://www.cap.org/] or by phoning 800-323-4040 (or 847-832-7000), option 1. A 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 another proficiency testing provider accepted by CAP. Laboratories will not be penalized if they are unable to participate in an oversubscribed program. If unable to participate, however, the laboratory must implement an alternative assessment procedure for the affected analytes. For regulated analytes, if the CAP and CAP-accepted 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: Integrating Five Key Quality System Components; Approved Guideline—Second Edition. NCCLS document GP22-A2 (ISBN 1-56238-552-6). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004; 4) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; standard III. Northfield, IL: CAP, 1998. HSC.10200 Phase II N/A YES NO 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 practice. Participation in ungraded/educational proficiency testing programs also satisfies this checklist question. Semiannual alternative assessment must be performed on tests for which PT is not available.

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The list of analytes for which CAP requires proficiency testing is available on the CAP website [http://www.cap.org/] or by phoning 800-323-4040 (or 847-832-7000), option 1. 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. HSC.10400 Phase II N/A YES NO 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. With respect to morphologic examinations (identification of cell types and microorganisms; review of electrophoretic patterns, etc.), group review and consensus identifications are permitted only for unknown samples that would ordinarily be reviewed by more than one person in an actual patient sample. If the laboratory uses multiple methods for an analyte, proficiency samples should be analyzed by the primary method. The educational purposes of proficiency testing are best served by a rotation that allows all technologists to be involved in the proficiency testing program. Proficiency testing records must be retained 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, if practical. 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.

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**NEW** 09/27/2007 HSC.10475 Phase I N/A YES NO Are proficiency testing specimens tested to the same extent as clinical specimens? NOTE: Protocols for additional testing that are used for routine patient samples, including reflex testing to clarify results, should be followed for proficiency testing specimens. However, proficiency samples should not receive more extensive testing than routine patient samples. COMMENTARY: N/A **REVISED** 09/27/2007 HSC.10550 Phase II N/A YES NO Is there ongoing evaluation of PT and alternative assessment results, with prompt corrective action taken for unacceptable results? NOTE: Compliance with this item can be examined by selecting a sample of PT evaluation results and alternative assessment records. Special attention should be devoted to unacceptable results. Compliance requires that all of the following are true:

1. There is documented evidence of ongoing review of all PT reports and alternative assessment results by the laboratory director or the director’s designee. Reviews should be completed within one month of the date reports and results become available to the laboratory.

2. All “unacceptable” PT results and alternative assessment test result have been investigated.

3. Corrective action has been initiated for all unacceptable PT and alternative assessment results. Corrective action is appropriate to the nature and magnitude of the problem; it might consist of staff education, instrument recalibration, change in procedures, institution of new clerical checks, discontinuation of patient testing for the analyte or discipline in question, or other appropriate measures.

4. Primary records related to PT and alternative assessment testing are retained for two years (unless a longer retention period is required elsewhere in this checklist for specific analytes or disciplines). These include all instrument tapes, work cards, computer printouts, evaluation reports, evidence of review, and documentation of follow-up/corrective action.

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) Clinical and Laboratory Standards Institute (CLSI). Using Proficiency Testing to Improve the Clinical Laboratory; Approved Guideline—Second Edition. CLSI document GP27-A2 (ISBN 1-56238-632-8). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007; 4) Zaki Z, et al. Self-improvement by participant interpretation of proficiency testing data from events with 2 to 5 samples. Clin Chem. 2000;46:A70. **NEW** 10/31/2006 HSC.10650 Phase II N/A YES NO Is there a policy that prohibits interlaboratory communication about proficiency testing samples until after the deadline for submission of data to the proficiency testing provider? 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)(3)]; 2) Bierig JR. Comparing PT results can put a lab’s CLIA license on the line. Northfield, IL: College of American Pathologists CAP Today. 2002;16(2):84-87. **NEW** 10/31/2006 HSC.10750 Phase II N/A YES NO Is there a policy that prohibits referral of proficiency testing specimens to another laboratory? NOTE: Under CLIA-88 regulations, there is a strict prohibition against referring proficiency testing specimens to another laboratory. In other words, the laboratory may not refer a proficiency testing specimen to a laboratory with a different CLIA number (even if the second laboratory is in the same health care system). COMMENTARY: N/A



REFERENCE: Department of Health and Human Services, Centers for Medicare & Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 1992(Feb 28): [42CFR493.801(b)(4)].

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QUALITY MANAGEMENT AND QUALITY CONTROL

***************************************************************************** ----------------------------------------------------------------- GENERAL ISSUES ----------------------------------------------------------------- HSC.20000 Phase II N/A YES NO Does the histocompatibility laboratory have a written quality management/quality control (QM/QC) program? NOTE: The QM/QC program in the histocompatibility laboratory must be clearly defined and documented. The program must ensure quality throughout the 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. All QM questions in the Laboratory General Checklist pertain to the histocompatibility laboratory. 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). HSC.20005 Phase II N/A YES NO 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?



NOTE: Each person must be tested at least once annually for each test he or she performs, using previously tested samples or split samples. COMMENTARY: N/A REFERENCE: United Network for Organ Sharing Standard for Histocompatibility Testing C4.140. HSC.20010 Phase II N/A YES NO 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: N/A HSC.20020 Phase II N/A YES NO 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 HSC.20035 Phase II N/A YES NO Is there documentation of ongoing evaluation by the section director or designee of all of the following?

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

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



COMMENTARY: N/A HSC.20050 Phase II N/A YES NO Is there documentation of corrective action taken when control results exceed defined tolerance limits? NOTE: Patient/client test results obtained in an analytically unacceptable test run or since the last acceptable test run must be re-evaluated to determine if there is a significant clinical difference in patient/client results. Re-evaluation may or may not include re-testing patient samples, depending on the circumstances. Even if patient samples are no longer available, test results can be re-evaluated to search for evidence of an out-of-control condition that might have affected patient results. For example, evaluation could include comparison of patient means for the run in question to historical patient means, and/or review of selected patient results against previous results to see if there are consistent biases (all results higher or lower currently than previously) for the test(s) in question). 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):7176 [42CFR493.1445(e)]. HSC.20060 Phase II N/A YES NO Is there a documented system in operation to detect and correct significant clerical and analytical errors, and unusual laboratory results, in a timely manner? NOTE: The laboratory must have a documented system in operation to detect and correct significant clerical and analytical errors, and unusual laboratory results. One common method is review of results by a qualified person (technologist, supervisor, pathologist) before release from the laboratory, but there is no requirement for supervisory review of all reported data. The selective use of delta checks also may be useful in detecting clerical errors in consecutive samples from the same patient/client. In computerized laboratories, there should be automatic "traps" for improbable results. The system for detecting clerical errors, significant analytical errors, and unusual laboratory results must provide for timely correction of errors, i.e., before results become available for clinical decision making. For suspected errors detected by the end user after reporting, corrections must be promptly made if such errors are confirmed by the laboratory.



Each procedure must include a listing of common situations that may cause analytically inaccurate results, together with a defined protocol for dealing with such analytic errors or interferences. This may require alternate testing methods; in some situations, it may not be possible to report results for some or all of the tests requested. The intent of this requirement is NOT to require verification of all results outside the reference (normal) range. COMMENTARY: N/A HSC.20080 Phase II N/A YES NO 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: 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):7182 [42CFR493.1463(a)(3) and 42CFR493.1463(c)], 7183 [42CFR493.1489(b)(1) and 42CFR493.1489(b)(5)]. ----------------------------------------------------------------- PROCEDURE MANUAL ----------------------------------------------------------------- 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. **REVISED** 10/31/2006 HSC.20100 Phase II N/A YES NO Is a complete procedure manual available at the workbench or in the work area?

NOTE 1: The use of inserts provided by manufacturers is not acceptable in place of a procedure manual. However, such inserts may be used as part of a procedure description, if the insert accurately and precisely describes the procedure as performed in the laboratory. Any variation from this printed or 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. However, procedures must be available to laboratory personnel when the electronic versions are inaccessible (e.g., during laboratory information system or network downtime); thus, the laboratory must maintain either paper copies or electronic copies on CD or other media that can be accessed via designated computers. All procedures, in either electronic or paper form, must be readily available for review by the inspector at the time of the CAP inspection. Electronic versions of procedures 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 annual review). Documentation of review of electronic procedures may be accomplished by including statements such as “reviewed by [name of reviewer] on [date of review]” in the electronic record. Alternatively, paper review sheets may be used to document review of electronic procedures. Documentation of review by a secure electronic signature is NOT required.

COMMENTARY:



N/A 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) Clinical and Laboratory Standards Institute (CLSI). Laboratory Documents: Development and Control; Approved Guideline—Fifth Edition. CLSI document GP2-A5 (ISBN 1-56238-600-X). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006. HSC.20200 Phase II N/A YES NO Does the procedure manual contain specific instructions for test performance, preparation of reagents, and control methods for each of the following procedures?

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

NOTE: The Inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report. COMMENTARY: N/A HSC.20900 Phase II N/A YES NO 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. HSC.20920 Phase II N/A YES NO Does the director (or a designee who meets CAP director qualifications) 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: 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):7164 [42CFR493.1251(d)]. HSC.20940 Phase II N/A YES NO 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:



N/A HSC.20950 Phase II N/A YES NO 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: 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):7164 [42CFR493.1251(d)]. HSC.20960 Phase II N/A YES NO 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: 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):7164 [42CFR493. 1105(a)(2);493.1251(e)]. ----------------------------------------------------------------- SPECIMEN COLLECTION AND HANDLING ----------------------------------------------------------------- HSC.20970 Phase II N/A YES NO Are procedures adequate to maintain sample identity and integrity throughout all steps of the process? NOTE: There must be a system in place to verify the identity of specimens, patients, donors, reagents; procedures utilized for testing; date and location of test performance; and the individuals performing the tests.



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):7162 [42CFR493.1103]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C2.120, C2.130. Richmond, VA: UNOS, 2001. HSC.20976 Phase II N/A YES NO 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 HSC.20982 Phase II N/A YES NO 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 HSC.20988 Phase II N/A YES NO 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 HSC.20994 Phase II N/A YES NO 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) Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007. HSC.21000 Phase II N/A YES NO 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: 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):7162 [42CFR493. 1291(c)(7)]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C2.140. Richmond, VA: UNOS, 2001. HSC.21020 Phase II N/A YES NO Is the disposition of any unacceptable specimens documented in the patient report and/or quality management records? COMMENTARY: N/A HSC.21050 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.230. Richmond, VA: UNOS, 2001. HSC.21130 Phase II N/A YES NO Are procedures adequate to ensure that patient specimens are easily retrievable? 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. 1278(f)].



----------------------------------------------------------------- RESULTS REPORTING ----------------------------------------------------------------- HSC.21250 Phase II N/A YES NO 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: N/A HSC.21275 Phase II N/A YES NO 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. HSC.21281 Phase II N/A YES NO 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



HSC.21287 Phase II N/A YES NO 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 ----------------------------------------------------------------- RECORDS ----------------------------------------------------------------- The following types of records should be kept to the extent of services provided by the laboratory. If a service is not provided, mark the question "N/A." HSC.21300 Phase II N/A YES NO 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. HSC.21316 Phase II N/A YES NO 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: N/A



REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C5.110. Richmond, VA: UNOS, 2001. HSC.21332 Phase II N/A YES NO Is a log of all stored specimens maintained to enable prompt retrieval for further testing? COMMENTARY: N/A ................................................................. Recipient and Donor Information Records ................................................................. HSC.21350 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C4.400. Richmond, VA: UNOS, 2001. HSC.21366 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C4.400. Richmond, VA: UNOS, 2001. HSC.21382 Phase II N/A YES NO Is there a system to resolve HLA typing discrepancies between laboratories? NOTE: There must be documentation of the steps taken to resolve discrepancies. COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C4.250. Richmond, VA: UNOS, 2001. ----------------------------------------------------------------- REAGENTS ----------------------------------------------------------------- The laboratory has the responsibility for ensuring that all reagents used, whether purchased or prepared by the laboratory, are appropriately reactive. The verification of reagent performance is required and must be documented. Any of several methods may be appropriate, such as direct analysis with reference materials, parallel testing of old vs. new reagents, and checking against routine controls. The intent of the 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. HSC.21400 Phase II N/A YES NO Are reagents and solutions properly labeled, as applicable and appropriate, with the following elements?

1. Content and quantity, concentration or titer



2. Storage requirements 3. Date prepared or reconstituted by laboratory 4. Expiration date

NOTE: The above elements may be recorded in a log (paper or electronic), rather than on the containers themselves, providing that all containers are identified so as to be traceable to the appropriate data in the log. While useful for inventory management, labeling with "date received" is not routinely required. There is no requirement to routinely label individual containers with "date opened"; however, a new expiration date must be recorded if opening the container changes the expiration date, storage requirement, etc. 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) Clinical and Laboratory Standards Institute (CLSI). Laboratory Documents: Development and Control; Approved Guideline—Fifth Edition. CLSI document GP2-A5 (ISBN 1-56238-600-X). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006. HSC.21500 Phase II N/A YES NO For U.S. laboratories, are outdated reagents handled in compliance with Food and Drug Administration requirements, and discarded/replaced as indicated? 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):7164 [42CFR493.1252(d)]. HSC.21550 Phase II N/A YES NO 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** 09/27/2007 HSC.21612 Phase II N/A YES NO Is there a documented system for identification of the specific reagent lot numbers and shipments used for each assay? 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. 2004(Oct 1): 1038 [42CFR493.1256(a)]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C3.500. Richmond, VA: UNOS, 2001; 3) American Society for Histocompatibility and Immunogenetics. Standards for histocompatibility testing. Lenexa, KS: ASHI, 2005: D.4.6.4.1. HSC.21675 Phase II N/A YES NO 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 HSC.21800 Phase II N/A YES NO Are procedures and conditions established and documented for reagent and patient sample storage, including procedures for frozen lymphocytes, frozen lymphocyte trays, and sera?



NOTE: Alarms are required to ensure that the materials have been properly stored and maintained. COMMENTARY: N/A REFERENCE: American Society for Histocompatibility and Immunogenetics. Standards for histocompatibility testing. Lenexa, KS: ASHI, 1995-2002: C2.130, C2.310, C2.320. HSC.21810 Phase II N/A YES NO 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: 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. 1278(a)(3)], 7171 [42CFR492. 1278(d)(3)]. HSC.21820 Phase II N/A YES NO 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: N/A HSC.21835 Phase II N/A YES NO If reagents are used in a manner different than manufacturer’s instructions, have appropriate validation studies been performed and documented? COMMENTARY: N/A



----------------------------------------------------------------- CONTROLS AND STANDARDS ----------------------------------------------------------------- HSC.21850 Phase II N/A YES NO 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: 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):7168 [42CFR493. 1256(d)(3)(iii)]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.410, E2.420. Richmond, VA: UNOS, 2001. HSC.21900 Phase II N/A YES NO Are controls properly labeled with content, lot number, date of preparation and expiration date? COMMENTARY: N/A HSC.21950 Phase II N/A YES NO 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:



N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.430. Richmond, VA: UNOS, 2001. HSC.22070 Phase II N/A YES NO Does the laboratory include control material for each phase of compatibility testing? NOTE: Results of patient testing must not be reported until control values are reviewed and found acceptable. 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):7171 [42CFR493. 1278(c) and (e)(3)]. HSC.22140 Phase II N/A YES NO Are control specimens tested in the same manner and by the same personnel as patient samples? NOTE: 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. 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(d)(8)]. HSC.22160 Phase II N/A YES NO 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: 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(f)]. ----------------------------------------------------------------- INSTRUMENTS AND EQUIPMENT ----------------------------------------------------------------- A variety of instruments and equipment are used to support the performance of analytical procedures. All instruments and equipment should be properly operated, maintained, serviced, and monitored to ensure that malfunctions of these instruments and equipment do not adversely affect the analytical results. The 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. HSC.22180 Phase II N/A YES NO Is there a routine schedule or system for the regular checking of the critical operating characteristics of all instruments in use? 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):7164 [42CFR493.1256]. HSC.22200 Phase II N/A YES NO Are instructions for instrument function checks available (i.e., manufacturer's manual or documented system prepared by the laboratory)?



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 [42CFR493.1255]. HSC.22250 Phase II N/A YES NO Are function checks documented and conveniently located to detect trends or malfunctions? 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):7164 [42CFR493.1256]. HSC.22300 Phase II N/A YES NO Are tolerance limits for acceptable function documented for specific instruments wherever appropriate? 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):7164 [42CFR493.1254]. HSC.22325 Phase II N/A YES NO Is there a written procedure for implementing corrective action when tolerance limits of instrument function checks are exceeded? COMMENTARY: N/A



HSC.22350 Phase II N/A YES NO Are instructions provided for minor troubleshooting and repairs of instruments (such as manufacturer's service manual)? COMMENTARY: N/A HSC.22400 Phase II N/A YES NO Are records maintained for each instrument to document all repairs and service procedures? COMMENTARY: N/A HSC.22450 Phase II N/A YES NO 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 .................................................................



HSC.22500 Phase II N/A YES NO Are temperatures checked and recorded appropriately for each of the following types of equipment?

1. Water baths and heating blocks 2. Incubators and ovens (where temperature control is necessary for a procedure) 3. Dialyzer baths 4. Refrigerators and freezers

NOTE: Temperature-dependent equipment containing reagents and patient specimens must be monitored daily, as equipment failures could affect accuracy of patient test results. Items such as water baths and heat blocks used for procedures need only be checked on days of patient testing. The Inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report. COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.300. Richmond, VA: UNOS, 2001. HSC.22531 Phase II N/A YES NO 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 HSC.22562 Phase II N/A YES NO 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 HSC.22593 Phase II N/A YES NO 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 failure alarm, or battery power. COMMENTARY: N/A HSC.22625 Phase II N/A YES NO 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: N/A HSC.22750 Phase II N/A YES NO Have acceptable ranges been defined for all temperature-dependent equipment? COMMENTARY: N/A HSC.22775 Phase II N/A YES NO 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 ................................................................. HSC.22800 Phase II N/A YES NO Is an appropriate thermometric standard device of known accuracy available (NIST certified or guaranteed by manufacturer to meet NIST standards)? NOTE: Thermometers should be present on all temperature-controlled instruments and environments and checked daily. Thermometric standard devices should be recalibrated or recertified prior to the date of expiration of the guarantee of calibration. COMMENTARY: N/A HSC.22850 Phase II N/A YES NO Are all non-certified thermometers in use checked against an appropriate thermometric standard device before being placed in service? COMMENTARY: N/A ................................................................. Centrifuges .................................................................



HSC.22900 Phase I N/A YES NO Are all centrifuges in the histocompatibility laboratory clean and properly maintained? COMMENTARY: N/A HSC.22950 Phase II N/A YES NO Is there a documented protocol and schedule for maintenance of all centrifuges (cleaning, changing brushes, etc.)? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.600. Richmond, VA: UNOS, 2001. ................................................................. Spectrophotometers ................................................................. HSC.23137 Phase II N/A YES NO Are absorbance and photometric linearity checked periodically with filters or standards solutions, if required by the instrument manufacturer? COMMENTARY: N/A HSC.23324 Phase II N/A YES NO Are filters (filter photometers) checked periodically to ensure they are in good condition (e.g., clean, free of scratches)? COMMENTARY: N/A



HSC.23511 Phase II N/A YES NO 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: N/A HSC.23698 Phase II N/A YES NO Is stray light checked periodically with extinction filters or appropriate solutions, if required by the manufacturer? COMMENTARY: N/A ................................................................ Film Processing/Photographic Equipment ................................................................. HSC.23885 Phase II N/A YES NO 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: N/A HSC.24072 Phase II N/A YES NO Is all photographic equipment in the laboratory clean and properly maintained? COMMENTARY:



N/A HSC.24259 Phase I N/A YES NO Are fixed camera mountings secure and level? COMMENTARY: N/A ................................................................. Fume Hoods and Biological Safety Cabinets ................................................................. HSC.24446 Phase II N/A YES NO Is a properly functioning fume hood (or chemical filtration unit) available for any procedures using volatile chemicals? COMMENTARY: N/A HSC.24633 Phase II N/A YES NO Is a biological safety cabinet (or hood) available, when appropriate? COMMENTARY: N/A 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. HSC.24820 Phase II N/A YES NO 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: N/A ................................................................. Electrophoresis Equipment ................................................................. HSC.25007 Phase II N/A YES NO 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: N/A HSC.25194 Phase II N/A YES NO Is the displayed voltage reading periodically confirmed by a voltmeter or other suitable means for all power supplies in the laboratory? COMMENTARY: N/A ................................................................. pH Meters ................................................................. HSC.25381 Phase II N/A YES NO Are there documented procedures for operation, calibration, and function checks of pH meter(s)? COMMENTARY: N/A



HSC.25568 Phase II N/A YES NO Are high quality buffers (certified assay of content) used for calibration of the pH meter(s)? COMMENTARY: N/A ................................................................. Balances and Weights ................................................................. HSC.25755 Phase II N/A YES NO Are balances cleaned, serviced, and checked periodically by qualified service personnel? COMMENTARY: N/A HSC.25942 Phase II N/A YES NO Are analytical balances mounted such that vibrations do not interfere with readings? COMMENTARY: N/A HSC.26129 Phase II N/A YES NO 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). HSC.26316 Phase II N/A YES NO 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

http://www.astm.org/



HSC.26503 Phase II N/A YES NO Are results of periodic accuracy checks recorded? NOTE: Mass readings should be recorded in a log book. The deviations in log book readings should be no more than the precision required in the applications for which the balance is used. Acceptable ranges for readings should be specified. COMMENTARY: N/A ................................................................ Volumetric Glassware and Pipettes ................................................................. HSC.26690 Phase II N/A YES NO 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

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



REFERENCES: 1) Curtis RH. Performance verification of manual action pipets. Part I. Am Clin Lab. 1994;12(7):8-9; 2) Curtis RH. Performance verification of manual action pipets. Part II. Am Clin Lab. 1994;12(9):16-17; 3) Perrier S, et al. Micro-pipette calibration using a ratiometric photometer-reagent system as compared to the gravimetric method. Clin Chem. 1995;41:S183; 4) American Society for Testing and Materials. Standard specification for glass volumetric (transfer) pipets, designation E 969-95. Philadelphia, PA: ASTM, 1995; 5) Johnson B. Calibration to dye for: Artel's new pipette calibration system. Scientist. 1999;13(12):14; 6) Connors M, Curtis R. Pipetting error: a real problem with a simple solution. Parts I and II. Am Lab News. 1999;31(13):20-22; 7) Skeen GA, Ashwood ER. Using spectrophotometry to evaluate volumetric devices. Lab Med. 2000;31:478-479. HSC.26877 Phase II N/A YES NO 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: N/A REFERENCES: 1) Curtis RH. Performance verification of manual action pipets. Part I. Am Clin Lab. 1994;12(7):8-9; 2) Curtis RH. Performance verification of manual action pipets. Part II. Am Clin Lab. 1994;12(9):16-17; 3) Perrier S, et al. Micro-pipette calibration using a ratiometric photometer-reagent system as compared to the gravimetric method. Clin Chem. 1995;41:S183; 4) NCCLS. Laboratory Statistics—Standard Deviation; A Report. NCCLS document EP13-R (ISBN 1-56238-277-2). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, 1995; 5) Bray W. Software for the gravimetric calibration testing of pipets. Am Clin Lab. Oct 1995 (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.



HSC.27064 Phase II N/A YES NO Are automatic pipettes checked for accuracy of calibration and reproducibility before being placed in service and at regular intervals thereafter? COMMENTARY: N/A 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. HSC.27251 Phase II N/A YES NO Are dedicated pipettors used for pre-amplification procedures? COMMENTARY: N/A 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

***************************************************************************** ----------------------------------------------------------------- LYMPHOCYTE ISOLATION -----------------------------------------------------------------



HSC.27438 Phase II N/A YES NO Is the source of the lymphocytes documented? NOTE: These may include blood, bone marrow, lymph nodes, spleen, or cultured cells. COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C5.110. Richmond, VA: UNOS, 2001. ----------------------------------------------------------------- SEROLOGICAL PROCEDURES ----------------------------------------------------------------- ................................................................. General ................................................................. HSC.27625 Phase II N/A YES NO 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: N/A REFERENCE: REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.320. Richmond, VA: UNOS, 2001. HSC.27812 Phase II N/A YES NO 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 ................................................................. HSC.27999 Phase II N/A YES NO 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. Any newly discovered antigens not yet assigned by W.H.O. Committee must be designated in such a manner as not to be confused with existing established terminology. All genotype and phenotype designations must also conform to W.H.O. Committee recommendations. The laboratory must maintain a list of antigens defined by the reagents used. COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing D1.100. Richmond, VA: UNOS, 2001.



HSC.28092 Phase II N/A YES NO Is there documentation that the appropriate terminology is used for leukocyte phenotyping? COMMENTARY: N/A HSC.28186 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.110, E2.310. Richmond, VA: UNOS, 2001. HSC.28373 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.210, E2.310. Richmond, VA: UNOS, 2001.



HSC.28560 Phase II N/A YES NO Are Class I antigens defined by at least 3 antisera, or by 2 antisera that are operationally monospecific? COMMENTARY: N/A HSC.28747 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.630. Richmond, VA: UNOS, 2001. HSC.28934 Phase I N/A YES NO 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: 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. 1278(a) through (c)].



HSC.28996 Phase II N/A YES NO 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 HSC.29058 Phase II N/A YES NO 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 HSC.29121 Phase II N/A YES NO 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: N/A



REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E2.510. Richmond, VA: UNOS, 2001. ................................................................. Cytotoxicity Crossmatch ................................................................. HSC.29308 Phase II N/A YES NO Is the technique used in the HLA crossmatch procedure more sensitive than the basic NIH lymphocytotoxicity crossmatch? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.210. Richmond, VA: UNOS, 2001. HSC.29495 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.220. Richmond, VA: UNOS, 2001. HSC.29682 Phase II N/A YES NO Are patient samples for crossmatch testing used undiluted, and kept frozen for a defined time post-transplantation? COMMENTARY: N/A



REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H3.310, H3.400. Richmond, VA: UNOS, 2001. HSC.29869 Phase II N/A YES NO 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 ----------------------------------------------------------------- HSC.29877 Phase II N/A YES NO Are typing sera and reagent cells used as prescribed by their manufacturers? COMMENTARY: N/A HSC.29885 Phase II N/A YES NO 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 HSC.29893 Phase II N/A YES NO 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 HSC.29901 Phase II N/A YES NO Is the specificity of the A1 subgroup of ABO testing documented to distinguish A1 from other subgroups? COMMENTARY: N/A **REVISED** 09/27/2007 HSC.29909 Phase II N/A YES NO 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, or are manufacturer’s directions for daily quality control followed? NOTE: Unless manufacturer instructions state otherwise, the following apply:

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

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

• Anti-IgG reactivity of antiglobulin reagents may be checked during antibody screening and

crossmatching.



• Typing sera and reagent cells must be checked for reactivity and specificity on each day of use,

including a check against known positive and negative cells or antisera. This checklist requirement can be satisfied by testing one vial of each reagent lot each day of testing. 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):7171 [42CFR493.1256]. HSC.29917 Phase II N/A YES NO 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 HSC.29925 Phase II N/A YES NO Are ABO, Rh, and antibody screen test results compared against the same tests performed previously to detect discrepancies? COMMENTARY: N/A HSC.29933 Phase II N/A YES NO 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 PERIOD

Patient records 10 years Employee signatures, initials, and identification codes

10 years post employment

Quality Control Records 5 years Specimens 7 days Records, unexpected antibodies. Indefinitely

COMMENTARY: N/A HSC.29941 Phase I N/A YES NO Have criteria for the performance and interpretation of ABO antibody titers been defined and documented? COMMENTARY: N/A HSC.29949 Phase II N/A YES NO 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 ----------------------------------------------------------------- FLOW CYTOMETRY -----------------------------------------------------------------



……………………………………………………….. Instrumentation and Phenotyping ……………………………………………………….. HSC.29957 Phase II N/A YES NO 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). Control testing is not necessary on days when patient testing is not performed. 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 [42CFR493.1252-1255]. HSC.29965 Phase II N/A YES NO 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? NOTE: Instrument performance must be monitored under the same conditions used to run test samples. COMMENTARY: N/A REFERENCE: Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007.



HSC.29973 Phase II N/A YES NO 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? NOTE: These steps are necessary to optimize the flow system and the optics of the instrument. COMMENTARY: N/A REFERENCE: Clinical and Laboratory Standards Institute (CLSI). Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline—Second Edition. CLSI document H42-A2 (ISBN 1-56238-640-9). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007. HSC.29981 Phase II N/A YES NO Are procedures established for determining appropriate color compensation settings? NOTE: For two or more color analysis there must be a procedure to ensure that cells co-labeled with more than one fluorescent reagent can be accurately distinguished from cells labeled only with one reagent. Cells stained with mutually exclusive antibodies bearing the relevant fluorochromes are the proper reference material for establishing appropriate compensation settings. COMMENTARY: N/A REFERENCE: Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007. HSC.29989 Phase I N/A YES NO For laser instruments, are there procedures in place to ensure acceptable and constant laser current? NOTE: For some instruments, current is a better gauge of laser performance than is power output, which may be relatively constant. COMMENTARY:



N/A HSC.29997 Phase II N/A YES NO 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. Lymphocyte gates may be validated using linear forward angle light scatter and 90-degree side scatter, or using CD45-FITC and CD14-PE monoclonal antibodies. COMMENTARY: N/A REFERENCE: National Institute for Allergy and Infectious Diseases/Division of AIDS guidelines for flow cytometric immunophenotyping, ver 1.0, Jan 1993. HSC.30005 Phase II N/A YES NO Is there a procedure to set markers (cursors) to distinguish fluorescence negative and fluorescence positive cell populations? NOTE: Each laboratory must have a set of objective criteria to define the appropriate placement of markers (cursors) to delineate the population of interest. Isotypic controls may not be necessary in all cases, and cursor settings for the isotype control may not be appropriate for all markers. Cursor settings must be determined based on the fluorescence patterns from the negative and positive populations for CD3, CD4, and CD8. COMMENTARY: N/A REFERENCES: 1) National Institute of Allergy and Infectious Diseases/Division of AIDS flow cytometry guidelines, sec 3.09B and 5.03A; 2) Sreenan JJ, et al. The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow cytometry. Are they really necessary? Arch Pathol Lab Med. 1997;121:118-121. HSC.30013 Phase II N/A YES NO Is there a policy for determining when the percentage of viable cells in each test specimen should be measured?



NOTE: Selective loss of cell subpopulations and/or the presence of dead cells may lead to spurious results. Procedures must be in place to ensure that viable cells are analyzed. This does not mean that all specimens with low viability must be rejected. Finding an abnormal population in a specimen with poor viability may be valuable but the failure to find an abnormality should be interpreted with caution. If specimen viability is below the established laboratory minimum, test results are suspect and this should be noted in the test report. Routine viability testing may not be necessary on whole blood specimens that are analyzed within 24 hours of drawing. However, viability testing of other specimens such as disaggregated lymph node specimens is essential. COMMENTARY: N/A REFERENCE: Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007. HSC.30021 Phase II N/A YES NO Are methods established to ensure that immunoglobulin staining is intrinsic and not extrinsic (cytophilic)? NOTE: Many cell types will bind serum immunoglobulin nonspecifically via Fc receptors, and steps may have to be taken to ensure that immunoglobulin staining detected by flow cytometry is intrinsic rather than cytophilic. Histogram interpretation is valuable. COMMENTARY: N/A REFERENCE: Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007. HSC.30029 Phase II N/A YES NO 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: N/A REFERENCE: Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007. HSC.30037 Phase II N/A YES NO Are the staining and analytical procedures described in the procedure manual based upon established methodology (reference cited)? NOTE: Many different variables need to be controlled to ensure proper stoichiometry of dye binding to DNA. Therefore, it is essential that procedures adopted by a laboratory are based on published work. COMMENTARY: N/A HSC.30045 Phase II N/A YES NO Does specimen treatment with nucleic acid dye include treatment with RNAse if the dye is not specific for DNA? NOTE: Certain dyes used to stain fixed cells, (e.g., ethidium and propidium iodide) bind to RNA. Prior treatment with RNAse eliminates artifactual broadening of the DNA content distributions that would result from fluorescence of complexes of the dye with RNA. COMMENTARY: N/A REFERENCE: Shapiro HA. Practical flow cytometry. New York, NY: Alan R. Liss, 1985.



……………………………………………………….. Flow Cytometry Crossmatch ……………………………………………………….. HSC.30056 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.120. Richmond, VA: UNOS, 2001. HSC.30243 Phase II N/A YES NO Are IgG antibodies identified by appropriately labeled heavy chain-specific F(ab’)2 reagents? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.110. Richmond, VA: UNOS, 2001. HSC.30430 Phase II N/A YES NO Is there documentation of the number of cells and volume of serum used for optimal sensitivity? COMMENTARY: N/A HSC.30617 Phase II N/A YES NO Is normal human serum with demonstrated lack of reactivity against any potential target cell used as a negative control?



COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.210. Richmond, VA: UNOS, 2001. HSC.30804 Phase II N/A YES NO 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 REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.220. Richmond, VA: UNOS, 2001. HSC.30991 Phase II N/A YES NO Are the antibody reagents (anti IgG, IgM, IgA, etc.) used at a selected dilution for optimal sensitivity and class specificity? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.230. Richmond, VA: UNOS, 2001. HSC.31178 Phase II N/A YES NO Has the cut-off for positive crossmatch results been established for all pertinent target cells (T-cells, B-cells, etc.)? COMMENTARY: N/A



REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.240. Richmond, VA: UNOS, 2001. HSC.31365 Phase II N/A YES NO Has the cut-off for positive crossmatches been determined by testing an appropriate number of sera from non-alloimmunized donors? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.240. Richmond, VA: UNOS, 2001. HSC.31552 Phase II N/A YES NO Does the procedure for HLA class II antibodies readily separate Class I from Class II specificity? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing N2.120. Richmond, VA: UNOS, 2001. ----------------------------------------------------------------- MIXED LYMPHOCYTE CULTURE TESTING ----------------------------------------------------------------- HSC.31739 Phase II N/A YES NO Is testing performed under aseptic conditions, with access to a laminar flow hood? COMMENTARY: N/A



HSC.31926 Phase II N/A YES NO Is the instrument for counting tritiated thymidine monitored and standardized according to manufacturer’s recommendations? COMMENTARY: N/A HSC.32113 Phase II N/A YES NO On days of use, is the incubator monitored for temperature, CO2 concentration, and humidity as defined by the procedure manual? COMMENTARY: N/A HSC.32300 Phase II N/A YES NO 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. ----------------------------------------------------------------- HLA ANTIBODY SCREENING -----------------------------------------------------------------



HSC.32487 Phase II N/A YES NO 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. HSC.32674 Phase II N/A YES NO 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 REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H2.000. Richmond, VA: UNOS, 2001. HSC.32861 Phase II N/A YES NO 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. HSC.33048 Phase II N/A YES NO For HLA antibody detection and specificity determinations, are positive and negative controls used, and sera tested undiluted and diluted when appropriate? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing G1.210. Richmond, VA: UNOS, 2001. HSC.33235 Phase II N/A YES NO 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. HSC.33422 Phase II N/A YES NO 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:



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 [42CFR493.1278(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) ................................................................. HSC.33609 Phase II N/A YES NO 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 HSC.33796 Phase II N/A YES NO Is there documentation of how positive and negative cutoff points were determined? COMMENTARY: N/A ----------------------------------------------------------------- MOLECULAR HLA ANTIGEN TYPING -----------------------------------------------------------------



HSC.33983 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing E1.100. Richmond, VA: UNOS, 2001. HSC.34170 Phase II N/A YES NO Is sample identification assured through all applicable phases of analysis, including all of the following?

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

NOTE: The inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report. COMMENTARY: N/A HSC.34357 Phase II N/A YES NO Are nucleic acids extracted and purified by methods reported in the literature, or is there validation of a method developed in-house?



COMMENTARY: N/A REFERENCES: 1) Sambrook J, et al. Molecular cloning: A laboratory manual, second edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989:E.3-E.4; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.110. Richmond, VA: UNOS, 2001. HSC.34544 Phase II N/A YES NO For RNA amplification methods, are appropriate controls used for reverse transcription? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M2.430. Richmond, VA: UNOS, 2001. HSC.34731 Phase II N/A YES NO Is handling and storage of nucleic acids adequate to prevent degradation? COMMENTARY: N/A REFERENCES: 1) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.120, M1.130. Richmond, VA: UNOS, 2001; 2) Rainen L, et al. Stabilization of mRNA expression in whole blood samples. Clin Chem. 2002;48:1883-1890. HSC.34918 Phase II N/A YES NO Is the quantity of nucleic acid measured, when appropriate? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.230. Richmond, VA: UNOS, 2001.



HSC.35105 Phase II N/A YES NO Is the quality (intactness) of the high molecular weight DNA (or RNA) assessed by gel electrophoresis or comparable method, when appropriate? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.140. Richmond, VA: UNOS, 2001. HSC.35292 Phase II N/A YES NO Are standard amounts of nucleic acid loaded on analytical gels, when possible? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.230. Richmond, VA: UNOS, 2001. HSC.35479 Phase II N/A YES NO 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.



HSC.35666 Phase II N/A YES NO Are procedures documented to prevent specimen loss, alteration, or contamination? COMMENTARY: N/A HSC.35853 Phase II N/A YES NO 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 combinations 4. The HLA sequence database used 5. For RFLP, sites resistant to endonuclease digestion, and cross-hybridizing bands; the

labeling methods used, and standards for adequacy of hybridization or amplification 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. HSC.35946 Phase II N/A YES NO Do RFLP (restriction fragment length polymorphism) reports specify the probes, enzymes, fragment size and target DNA location? COMMENTARY: N/A



HSC.36040 Phase II N/A YES NO 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: N/A REFERENCE: Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989;339:237-238. HSC.36227 Phase II N/A YES NO Has Mendelian inheritance of the DNA system used been validated by family studies? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.510. Richmond, VA: UNOS, 2001. HSC.36414 Phase II N/A YES NO Are autoradiographs or electrophoretic gels interpreted independently by at least 2 qualified readers using an objective method? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.320. Richmond, VA: UNOS, 2001.



HSC.36601 Phase II N/A YES NO For end-point amplification assays such as sequence-specific priming, are adequate internal controls used, and criteria defined for a positive reaction? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M2.630. Richmond, VA: UNOS, 2001. HSC.36788 Phase II N/A YES NO Are positive, negative and sensitivity controls run for each assay, when available and appropriate? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.210. Richmond, VA: UNOS, 2001. HSC.36975 Phase II N/A YES NO 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: N/A 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.



HSC.37162 Phase II N/A YES NO Are known molecular weight markers that span the range of expected bands used for each electrophoretic run? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M1.220. Richmond, VA: UNOS, 2001. HSC.37349 Phase II N/A YES NO 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 REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.322. Richmond, VA: UNOS, 2001. HSC.37536 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.113, M3.131. Richmond, VA: UNOS, 2001.



HSC.37723 Phase II N/A YES NO Has the method of probe labeling been validated to detect the target sequence without a false positive signal for non-target sequences? COMMENTARY: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.121. Richmond, VA: UNOS, 2001. HSC.37910 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.140. Richmond, VA: UNOS, 2001. HSC.38097 Phase II N/A YES NO 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 REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing M3.251. Richmond, VA: UNOS, 2001.



HSC.38134 Phase II N/A YES NO 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 HSC.38171 Phase II N/A YES NO 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 HSC.38208 Phase II N/A YES NO Is preferential allele amplification considered in the interpretation of stem cell engraftment tests? COMMENTARY: N/A HSC.38245 Phase II N/A YES NO 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



HSC.38284 Phase II N/A YES NO For stem cell engraftment, is the polymorphic nature of the DNA system used detailed and documented in the literature? NOTE: Available data must include: the type of polymorphism (i.e., single locus, multi-locus, simple diallelic, hypervariable); the chromosomal location if known; the restriction endonuclease and probe used to detect the polymorphism; the conditions of hybridization and sizes (or range of sizes) of variable and constant fragments produced; and population-specific allele frequency data, if available. COMMENTARY: N/A REFERENCES: 1) 1991 Report concerning recommendations of the DNA Commission of the International Society for Forensic Haemogenetics relating to the use of DHA polymorphisms. Forensic Sci Intl. 1992;52:125-130; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing L2.222. Richmond, VA: UNOS, 2001. HSC.38471 Phase II N/A YES NO 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: N/A HSC.38658 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing L2.253. Richmond, VA: UNOS, 2001.



----------------------------------------------------------------- DONOR-RECIPIENT HISTOCOMPATIBILITY ----------------------------------------------------------------- HSC.38845 Phase II N/A YES NO 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: N/A REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H5.200. Richmond, VA: UNOS, 2001. HSC.39032 Phase II N/A YES NO 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 REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing H5.100. Richmond, VA: UNOS, 2001. HSC.39219 Phase I N/A YES NO 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: N/A HSC.39406 Phase II N/A YES NO 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: 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 [42CFR493.1278]. HSC.39499 Phase II N/A YES NO Are personnel available at all times to perform testing as needed for organ transplantation? COMMENTARY: N/A ................................................................. Non-Renal Organ Transplants ................................................................. HSC.39593 Phase II N/A YES NO Are non-renal transplant recipients screened for HLA antibodies as well as non-HLA antibodies and/or autoreactive antibodies? NOTE: 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. COMMENTARY: N/A



REFERENCE: United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing I2.000. Richmond, VA: UNOS, 2001. HSC.39780 Phase II N/A YES NO 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.

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PERSONNEL

***************************************************************************** HSC.40000 Phase II N/A YES NO 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, and 2. Laboratory training and experience: 4 years training and experience in

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

NOTE: These qualifications fulfill the requirements of CLIA 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)].



HSC.45000 Phase II N/A YES NO 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 and Competence Assessment; Approved Guideline—Second Edition. NCCLS document GP21-A2 (ISBN 1-56238-531-3). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004. HSC.46250 Phase II N/A YES NO 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. Testing personnel with less than 1 year experience must be supervised by qualified personnel. COMMENTARY: N/A HSC.47500 Phase II N/A YES NO Is there a continuing clinical laboratory education program that addresses the areas of service offered by the laboratory? NOTE: The laboratory must have a complete continuing clinical laboratory education program that meets the needs of the various types of laboratory personnel and addresses the areas of service offered by the laboratory. The laboratory must have documented evidence of sufficient continuing education credits for directors (50 hours annually), supervisors (27 hours annually), and other technical personnel (12 hours annually).



COMMENTARY: N/A HSC.48750 Phase II N/A YES NO 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. HSC.50000 Phase II N/A YES NO 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. HSC.50100 Phase I N/A YES NO Is there adequate space for technical work (bench space)? 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. HSC.50200 Phase I N/A YES NO Is there adequate space for instruments? 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. HSC.50300 Phase I N/A YES NO Is there adequate refrigerator and freezer storage space? 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.



HSC.50400 Phase I N/A YES NO Is the space available efficiently utilized? 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. HSC.50500 Phase II N/A YES NO Is sufficient space available so that there is no compromise of the quality of work, (including quality control activities) or safety of personnel? 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):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. HSC.50600 Phase I N/A YES NO Are floors, benches, and sinks clean, free of clutter, and well maintained? COMMENTARY: N/A HSC.50700 Phase I N/A YES NO Are water taps, sinks, and drains adequate? 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):7163 [42CFR493.1101(a)]. HSC.50800 Phase I N/A YES NO Are electrical outlets adequate? 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):7163 [42CFR493.1101(a)]. HSC.50900 Phase I N/A YES NO 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: 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):7163 [42CFR493.1101(a)]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.200. Richmond, VA: UNOS, 2001. HSC.51000 Phase I N/A YES NO Is ventilation adequate? 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):7163 [42CFR493.1101(a)]; 2) United Network for Organ Sharing. Bylaws Appendix B. Standard for histocompatibility testing C1.200. Richmond, VA: UNOS, 2001. HSC.51100 Phase I N/A YES NO Is ambient (room) temperature adequately controlled? 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):7163 [42CFR493.1101(a)]. HSC.51200 Phase I N/A YES NO Are telephones conveniently located, and are calls easily transferred? 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):7163 [42CFR493.1101(a)].