FLOW CYTOMETRY CHECKLISTwebapps.cap.org/.../flow_cytometry_sep07.pdf · FLOW CYTOMETRY OUTLINE ... records, including procedure manuals, quality control and proficiency testing records,

Revised: 09/27/2007

COMMISSION ON LABORATORY ACCREDITATION

Laboratory Accreditation Program

FLOW CYTOMETRY 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

FLOW CYTOMETRY

OUTLINE

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

GENERAL ISSUES ......................................................................................................................................................12 PROCEDURE MANUAL.............................................................................................................................................14 SPECIMEN COLLECTION AND HANDLING..........................................................................................................18 REAGENTS ..................................................................................................................................................................19 RECORDS AND REPORTS.........................................................................................................................................21 CONTROLS AND STANDARDS................................................................................................................................23 INSTRUMENTS AND EQUIPMENT..........................................................................................................................27

Flow Cytometers.......................................................................................................................................................27 Temperature-Dependent Equipment .........................................................................................................................31 Thermometers ...........................................................................................................................................................32 Centrifuges................................................................................................................................................................32

PROCEDURES AND TEST SYSTEMS ...........................................................................................................................33 IMMUNOPHENOTYPING ..........................................................................................................................................33

Blood Lymphocyte Subset Enumeration ..................................................................................................................34 CD34 Stem Cell Enumeration...................................................................................................................................37 Leukemia and Lymphoma ........................................................................................................................................39

DNA CONTENT AND CELL CYCLE ANALYSIS....................................................................................................43 PERSONNEL.....................................................................................................................................................................47 PHYSICAL FACILITIES ..................................................................................................................................................47 LABORATORY SAFETY.................................................................................................................................................51

FLOW CYTOMETRY (Web File) Page 2 of 51


SUMMARY OF CHANGES FLOW CYTOMETRY 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 FLO.05075 09/27/2007 FLO.18385 09/27/2007 FLO.23706 09/27/2007 FLO.13540 04/06/2006 FLO.16770 04/06/2006 FLO.23737 04/06/2006 FLO.24475 04/06/2006 REVISED Checklist Questions Question Effective Date FLO.10260 09/27/2007 FLO.23737 09/27/2007 FLO.23800 09/27/2007 FLO.21000 04/06/2006 DELETED Checklist Questions Question Effective Date FLO.10310 09/27/2007 FLO.30557 09/27/2007 FLO.20150 04/06/2006



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 Flow Cytometry 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 flow cytometry laboratory should be pathologists, clinical scientists or medical technologists who are actively involved with or have extensive recent experience in the practice of flow cytometry 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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PROFICIENCY TESTING

***************************************************************************** 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/).


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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. **NEW** 09/27/2007 FLO.05075 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]. FLO.10150 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


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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) 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]; 2) Brando B, Sommaruga E. Nationwide quality control trial on lymphocyte immunophenotyping and flow cytometer performance in Italy. Cytometry. 1993;14:294-306; 3) Homburger HA, et al. Assessment of interlaboratory variability of immunophenotyping: results of the College of American Pathologists flow cytometry survey. Ann NY Acad Sci. 1993;677:43-52; 4) Goguel AF, et al. Interlaboratory quality assessment of lymphocyte phenotyping. Etalonorme 1990-1992 surveys. Biol Cell. 1993;78:79-84; 5) 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; 6) 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; 7) Rosner E, et al. Assessment of the impact of a CD4+ T-cell testing laboratory improvement program. Arch Pathol Lab Med. 1998;122:512-519; 8) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; standard III. Northfield, IL: CAP, 1998; 9) Reilly JT, Barnett D. UK NEQAS for leukocyte immunophenotyping: the first 10 years. J Clin Pathol. 2001;54:508-511. FLO.10180 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. 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.


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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) Shahangian S, et al. A system to monitor a portion of the total testing process in medical clinics and laboratories. Feasibility of a split-specimen design. Arch Pathol Lab Med. 1998;122:503-511. FLO.10210 Phase II N/A YES NO Does the laboratory integrate all proficiency survey samples into the routine laboratory workload as much as possible, and are those samples analyzed by personnel who routinely test patient samples, using the same primary method systems as for patient samples? NOTE: Replicate analysis of proficiency samples is acceptable only if patient specimens are routinely analyzed in the same manner. If the laboratory uses multiple methods for an analyte, these 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. Records of these studies must be kept and can be an important part of the competency and continuing education documentation in the personnel files of the individuals. When external proficiency testing materials are not available, the semi-annual alternative performance assessment process should also be integrated within the routine workload. COMMENTARY: N/A REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 1992(Feb 28):7146 [42CFR493.801(b)]; 2) Shahangian S, et al. Toward optimal PT use. Med Lab Observ. 2000;32(4):32-43. **REVISED** 09/27/2007 FLO.10260 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):7173 [42CFR493.1407(e)(4)(iv)]; 2) 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; 3) Shahangian S, et al. Toward optimal PT use. Med Lab Observ. 2000;32(4):32-43; 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** 04/06/2006 FLO.13540 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** 04/06/2006 FLO.16770 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)]. **NEW** 09/27/2007 FLO.18385 Phase I N/A YES NO For laboratories that perform only interpretations of flow immunophenotyping data for leukemias and lymphomas, does the laboratory participate in a peer education program in interpretive flow cytometry of hematolymphoid neoplasia? NOTE: This checklist item applies to laboratories which do not perform staining and acquisition of flow cytometry data, but which receive [1] list mode files and/or [2] representative dot plots from an outside laboratory for interpretation. Programs dealing with analysis of flow data from hematolymphoid neoplasias and related benign conditions provide valuable educational opportunities for peer-performance comparisons. While not completely emulating the clinical setting involved in flow immunophenotyping, the peer data developed by these programs can provide a useful benchmark against which laboratory performance can be evaluated. COMMENTARY:



N/A REFERENCE: Clinical and Laboratory Standards Institute (CLSI). Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells—Second Edition. CLSI document H43-A2 (ISBN 1-56238-635-2). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA, 2007.

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QUALITY MANAGEMENT AND QUALITY CONTROL

***************************************************************************** ----------------------------------------------------------------- GENERAL ISSUES ----------------------------------------------------------------- FLO.20000 Phase II N/A YES NO Does the flow cytometry laboratory have a written quality management/quality control (QM/QC) program? NOTE: The QM/QC program in the flow cytometry 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 flow cytometry laboratory. COMMENTARY: N/A REFERENCES: 1) D'Hautcourt JL. Quality control procedures for flow cytometric applications in the hematology laboratory. Hematol Cell Ther. 1996;38:467-470; 2) Gratama JW, et al. Quality control of flow cytometric immunophenotyping of haematological malignancies. Clin Lab Haem. 1999;21:155-160.



FLO.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, consistent with good laboratory practice? COMMENTARY: N/A FLO.20020 Phase II N/A YES NO Is there evidence of ongoing evaluation of instrument maintenance and function, temperature, etc., for all procedures as required? COMMENTARY: N/A FLO.20050 Phase II N/A YES NO Is there documentation of corrective action taken when values for instrument function, temperature, etc., exceed defined tolerance limits? COMMENTARY: N/A FLO.20100 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 FLO.20200 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: 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** 04/06/2006 FLO.21000 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) 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]; 2) van Leeuwen AM. 6 Steps to building an efficiency tool. Advance/Lab. 1999:8(6):88-91; 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. FLO.21100 Phase II N/A YES NO Is there documentation of at least annual review of all policies and procedures in the flow cytometry laboratory section by the current laboratory director or designee? NOTE: The director must ensure that the collection of policies and 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. Signature or initials on each page of a procedure is not required. 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):7173 [42CFR493.1407(e)(13)]; 2) Borkowski A, et al. Intranet-based quality improvement documentation at the Veterans Affairs Maryland health care system. Mod. Pathol. 2001;14:1-5. FLO.21125 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)]. FLO.21150 Phase II N/A YES NO Does the laboratory have a system documenting that all personnel are knowledgeable about the contents of procedure manuals relevant to the scope of their testing activities? NOTE: The form of this system is at the discretion of the laboratory director. There is no specific requirement for annual procedure sign-off by testing personnel. COMMENTARY: N/A FLO.21210 Phase II N/A YES NO If there is a change in directorship, does the new director ensure (over a reasonable period of time) 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)]. FLO.21220 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)]. ---------------------------------------------------------------- SPECIMEN COLLECTION AND HANDLING ----------------------------------------------------------------- FLO.22000 Phase II N/A YES NO Are procedures adequate to verify sample identity and integrity (includes specimens of blood, body fluids and tissues)? COMMENTARY: N/A FLO.22050 Phase II N/A YES NO Are there documented criteria for the rejection of unacceptable specimens or the special handling of sub-optimal specimens? NOTE: This question does not imply that all suboptimal specimens are discarded or not analyzed, but rather that the laboratory should have a policy for dealing with such specimens. Laboratories should be alert to suboptimal specimen conditions such as hemolysis, lipemia or clotting, and these specimen conditions should be noted on the report. Specimens exhibiting extremes of these conditions where the accuracy of test results is significantly affected should be rejected. 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):7183 [42CFR493.1249(a) and (b)]; 2) National Institute for Allergy and Infectious Diseases/Division of AIDS guidelines for flow cytometric immunophenotyping, version 1.0, Jan 1993, sec 1.06.



FLO.22100 Phase II N/A YES NO Is the disposition of all unacceptable specimens documented in the patient report and/or quality management records? NOTE: This information is essential to proper patient test management and to the laboratory quality management program. COMMENTARY: N/A ----------------------------------------------------------------- REAGENTS ----------------------------------------------------------------- The laboratory has the responsibility for ensuring that all reagents, calibrators and controls, 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. FLO.23000 Phase II N/A YES NO Are reagents, calibrators, cellular controls, 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 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(c)]. FLO.23050 Phase II N/A YES NO Are all reagents used within their indicated expiration 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 [42CFR493.1252(d)]. FLO.23100 Phase II N/A YES NO Are reagents stored as recommended by the manufacturer? NOTE: Reagents must be stored as recommended by the manufacturer to prevent environmentally-induced alterations that could affect test performance. If ambient temperature is indicated, there must be documentation that the defined ambient temperature is maintained and corrective action is taken when tolerance limits are exceeded. COMMENTARY: N/A FLO.23150 Phase II N/A YES NO Are new reagent lots and/or shipments checked against old reagent lots or with suitable reference material before or concurrently with being placed in service? NOTE: The purpose is to determine that the new lot or shipment of test reagent gives a clinically comparable result to the old reagent. This may be accomplished by comparing the results of the old and new reagent tested in parallel on the same fresh control (patient or normal) or testing just the new



reagent on a standardized control with defined mean and reference range. This does not imply the need to compare the new reagent lot to a negative control. COMMENTARY: N/A FLO.23250 Phase II N/A YES NO Are the recommendations of the manufacturer for the proper use of reagents and controls in kit procedures followed? NOTE: If alternative procedures are used, the method accuracy must be evaluated and documented to justify the change. COMMENTARY: N/A REFERENCE: Caldwell CW. Analyte-specific reagents in the flow cytometry laboratory. Arch Pathol Lab Med. 1998;122:861-864. FLO.23300 Phase II N/A YES NO If there are multiple components of a reagent kit, does the laboratory only use within-kit lot components of reagents unless otherwise specified by the manufacturer? NOTE: If there are multiple components of a reagent kit, the laboratory must use components of reagent kits only with other kits that are in the same lot number unless otherwise specified by the manufacturer or accuracy/equivalency is verified 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. 2003(Jan 24):7164 [42CFR493.1252(d)]. ----------------------------------------------------------------- RECORDS AND REPORTS -----------------------------------------------------------------



FLO.23675 Phase II N/A YES NO If patient testing is performed using Class I analyte-specific reagents (ASR’s) obtained or purchased from an outside vendor, does the patient report include the disclaimer required by federal regulations? NOTE: ASR’s are antibodies, both polyclonal and monoclonal, specific receptor proteins, ligands, nucleic acid sequences, and similar reagents which, through specific binding or chemical reaction with substances in a specimen, are intended for use in a diagnostic application for identification and quantification of an individual chemical substance or ligand in biological specimens. An ASR is the active ingredient of an in-house-developed test system. This checklist question concerns Class I ASR’s. Class I ASR’s are not subject to preclearance by the U.S. Food and Drug Administration (FDA) or to special controls by FDA. Most ASR’s are Class I. Exceptions include those used by blood banks to screen for infectious diseases (Class II or III), or used to diagnose certain contagious diseases (e.g., HIV infection and tuberculosis) (class III). If the laboratory performs patient testing using Class I ASR’s, federal regulations require that the following disclaimer accompany the test result on the patient report: "This test was developed and its performance characteristics determined by (laboratory name). It has not been cleared or approved by the U.S. Food and Drug Administration." The CAP recommends additional language, such as "The FDA has determined that such clearance or approval is not necessary. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88) as qualified to perform high complexity clinical laboratory testing." The disclaimer is not required for tests using reagents that are sold in kit form with other materials or an instrument, nor reagents sold with instructions for use. The laboratory must establish the performance characteristics of tests using Class I ASR’s. COMMENTARY: N/A REFERENCES: 1) Department of Health and Human Services, Food and Drug Administration. Medical devices; classification/reclassification; restricted devices; analyte specific reagents. Final rule. Fed Register. 1997(Nov 21);62243 [21CFR809 and 864]; 2) Caldwell CW. Analyte-specific reagents in the flow cytometry laboratory. Arch Pathol Lab Med. 1998;122:861-864; 3) Graziano. Disclaimer now needed for analyte-specific reagents. CAP Today. 1998;12(11):5-11; 4) U.S. Department of Health and Human Services, Food and Drug Administration. Analyte Specific Reagents; Small Entity Compliance Guidance. http://www.fda.gov/cdrh/oivd/guidance/1205.html, February 26, 2003; 5) Shapiro JD and Prebula RJ. FDA’s Regulation of Analyte-Specific Reagents. Medical Devicelink, February 2003. http://www.devicelink.com/mddi/archive/03/02/018.html.


http://www.fda.gov./cdrh/oivd/guidance/1205.html

http://www.devicelink.com/mddi/archive/03/02/018.html


**NEW** 09/27/2007 FLO.23706 Phase II N/A YES NO Are gated dot plots and histograms retained for at least 10 years? COMMENTARY: N/A REFERENCE: CAP Policy PP, Retention of Laboratory Records and Materials. ----------------------------------------------------------------- CONTROLS AND STANDARDS ----------------------------------------------------------------- Controls are samples that act as surrogates for patient specimens. They are periodically processed like a patient sample to monitor the ongoing performance of the entire analytic process. Most quantitative tests are traditionally monitored with 2 levels of liquid control material (procedural control). This is done at a frequency within which the accuracy and precision of the measuring system is expected to be stable (based upon manufacturer's recommendations), but at least each day that patient testing is performed. The daily use of two levels of liquid control may NOT be required for certain test systems, where the daily use of instrument and/or electronic controls is demonstrably sufficient to validate that calibration status is maintained within acceptable limits. The daily use of 2 levels of instrument and/or electronic controls as the only QC system is acceptable only for unmodified test systems cleared by the FDA and classified under CLIA-88 as "waived" or "moderate complexity." The laboratory is expected to provide documentation of its validation of all instrument-reagent systems for which daily controls are limited to instrument and/or electronic controls, and the inspector will review these data to assess the adequacy of the QC system. This documentation must include the Federal complexity classification of the testing system AND data showing that calibration status is monitored. **NEW** 04/06/2006 **REVISED** 09/27/2007 FLO.23737 Phase II N/A YES NO Is the performance of reagents and staining procedures verified periodically by the use of positive controls?



NOTE: The source (type) of positive control(s) and their frequency of evaluation will vary by the particular flow cytometric application. The frequency should be: 1, each day of analysis for lymphocyte subset and CD34+ stem cell measurements, regardless of whether one- or two-platform methods are used; and 2, at least monthly for leukemia/lymphoma immunophenotyping. For single platform measurements of CD4+ lymphocyte and CD34+ stem cell concentrations, and for dual platform measurements of CD34+ stem cell concentrations, two levels of control are needed (see the next checklist question, below). For dual platform measurements of lymphocyte subsets (CD4+ lymphocytes), one level of positive control is sufficient. The source of control material should be 1, external positive controls (e.g. normal or commercial control(s)) for lymphocyte subset, CD34+ stem cell quantitations, and leukemia/lymphoma samples; or 2, internal positive controls only for leukemia/lymphoma samples. Such internal control cells are the variable numbers of residual normal cells in the patient's sample. Like the external controls, there must be written guidelines defining objective criteria for acceptable performance of the internal controls, and written documentation of the evaluation of the actual periodic performance. COMMENTARY: N/A **REVISED** 09/27/2007 FLO.23800 Phase II N/A YES NO For single platform QUANTITATIVE tests (e.g., CD4+, CD34+ cell concentrations), and dual platform quantitation of CD34+ stem cell concentrations, are at least 2 levels of positive cellular controls analyzed at least daily (or each time the flow cytometer is restarted) to verify the performance of reagents, preparation methods, staining procedures and the instrument? NOTE: One of the levels of these controls should be at (or near) clinical decision levels (e.g., low CD34). Examples would be a low CD4+ lymph count of 200 cell/uL in a HIV+ individual, or a 5 – 20 CD34+ stem cells/uL concentration in the peripheral blood of an individual being readied for peripheral stem cell pheresis. When commercial controls are not available, then the laboratory director must implement an equivalent procedure to meet the two-level requirement. This alternative must be deemed acceptable by the laboratory inspection team through LAP. 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. 1992(Feb 28):7146 [42CFR493.1256]. FLO.23925 Phase II N/A YES NO Has a statistically valid target mean and range been established or verified for each lot of control material? NOTE: For unassayed controls, the laboratory must establish a valid acceptable range by repetitive analysis in runs that include previously tested control material. For assayed controls, the laboratory must verify the recovery ranges supplied by the manufacturer. COMMENTARY: N/A FLO.24100 Phase II N/A YES NO Are there records to reflect the results of all control procedures? COMMENTARY: N/A FLO.24230 Phase II N/A YES NO Is there documented evidence of corrective action taken when quality control results exceed defined acceptability limits? NOTE: Patient 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 FLO.24250 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)]. FLO.24300 Phase II N/A YES NO Are the results of controls verified for acceptability before reporting results? NOTE: It is implicit in quality control that patient test results will not be reported when controls do not yield unacceptable results. 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)]. **NEW** 04/06/2006 FLO.24475 Phase II N/A YES NO Are quality control data reviewed and assessed at least monthly by the laboratory director or designee?



COMMENTARY: N/A FLO.24650 Phase II N/A YES NO If the laboratory has more than one method for performing tests for a given analyte, are they checked against each other at least twice a year for correlation of patient results? NOTE: This includes same or different instrument makes/models. This comparison must include all instruments. The use of fresh blood samples (rather than simply stabilized commercial controls with potential matrix effects) is important to directly address the issue of whether a patient sample yields the same results on all of the laboratory's instruments. Statistical agreement of commercial control materials across instruments does not guarantee comparability of patient specimen results. In cases when pre-analytical stability of patient specimens is a limiting factor, alternative protocols based on QC or reference materials may be necessary but the materials used should be validated to have the same response as fresh human samples for the instruments/methods involved. This checklist requirement applies only to instruments/methods accredited under a single CAP number. COMMENTARY: N/A REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Medicare, Medicaid and CLIA programs; CLIA fee collection; correction and final rule. Fed Register. 2003(Jan 24):5236 [42CFR493.1281(a)]. ---------------------------------------------------------------- INSTRUMENTS AND EQUIPMENT ----------------------------------------------------------------- ................................................................. Flow Cytometers ................................................................. 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. FLO.25000 Phase II N/A YES NO Is there a schedule or system AVAILABLE AT THE INSTRUMENT for the regular checking of the critical operating characteristics for all instruments in use? COMMENTARY: N/A FLO.25050 Phase II N/A YES NO Are instructions for instrument check systems documented (i.e., manufacturer's manual or written system prepared by the laboratory)? COMMENTARY: N/A FLO.25100 Phase II N/A YES NO Are function checks documented by the technical operator in a convenient manner to detect trends or malfunctions? NOTE: The daily and, more importantly, the periodic review of such data are best accomplished through graphical means. COMMENTARY: N/A FLO.25150 Phase II N/A YES NO Are there procedures for monitoring of optical alignment (where applicable) and instrument reproducibility at least daily (or after each time the flow cytometer is restarted), and is there documentation of this monitoring?



NOTE: Verifying reproducibility of instrument performance is an essential element of quality assurance within the laboratory. Instrument performance must be monitored under the same conditions used to run test samples. 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. FLO.25200 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 FLO.25250 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: The effective utilization of instruments by the technical staff depends upon the prompt availability of maintenance, repair, and service documentation (copies are acceptable). 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 FLO.25300 Phase II N/A YES NO Are records of maintenance kept and reviewed periodically by the person in charge of technical operations of the flow cytometry laboratory? COMMENTARY:



N/A FLO.30250 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. FLO.30260 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. FLO.30270 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 FLO.30280 Phase II N/A YES NO Are the results of instrument calibration and laser output checks (where appropriate) reviewed periodically by the person in charge of technical operations of the flow cytometry laboratory? COMMENTARY: N/A ................................................................. Temperature-Dependent Equipment ................................................................. FLO.30290 Phase II N/A YES NO Are temperatures checked and recorded appropriately for each of the following types of equipment?

1. Water baths 2. Incubators (where temperature control is necessary for a procedure) 3. 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 FLO.30320 Phase II N/A YES NO Have acceptable ranges been defined for all temperature-dependent equipment?



COMMENTARY: N/A ................................................................ Thermometers ................................................................. FLO.30360 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 FLO.30370 Phase II N/A YES NO Are all non-certified thermometers in use checked against an appropriate thermometric standard device before use? COMMENTARY: N/A ................................................................. Centrifuges ................................................................. FLO.30380 Phase II N/A YES NO Are all centrifuges clean and properly maintained (check lining for dried blood and other residue)?



COMMENTARY: N/A FLO.30390 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 FLO.30400 Phase II N/A YES NO Are the operating speeds of all centrifuges checked periodically as needed for the intended use and is this done in a safe manner? NOTE: For centrifuges having a safety mechanism preventing the opening of the lid while in operation, the checks of rpm should be performed only by an authorized service representative of the manufacturer. COMMENTARY: N/A

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PROCEDURES AND TEST SYSTEMS

***************************************************************************** NOTE: Reticulocyte quantification by flow cytometry is separately covered in the Hematology and Coagulation Checklist. ----------------------------------------------------------------- IMMUNOPHENOTYPING -----------------------------------------------------------------



................................................................. Blood Lymphocyte Subset Enumeration ................................................................. FLO.30430 Phase II N/A YES NO Is there a procedure in place to document specimen integrity? 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 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. FLO.30450 Phase II N/A YES NO Are specimens stored appropriately after initial processing? NOTE: As one 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.



FLO.30460 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. FLO.30470 Phase II N/A YES NO Are results of lymphocyte subset analysis corrected for gate purity as appropriate? NOTE: When >5% non-lymphocyte events are included in a gate, results must be corrected for the proportion of contaminating cells. One method uses low side scatter and bright CD45 fluorescence for identification of lymphocytes, where an assumption is made that the only cells meeting this criteria are lymphocytes, and therefore the lymphocyte purity of the gate is close to 100%. Other methods may also be appropriate, and must be documented. COMMENTARY: N/A REFERENCES: 1) National Institute for Allergy and Infectious Diseases/Division of AIDS. Revised 3 color supplement to flow cytometry guidelines, sec 5.02; 2) 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. FLO.30480 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. FLO.30500 Phase II N/A YES NO Are the results of analyses of patient's specimens reviewed by the laboratory director or designee, and are comments to clinicians reported when necessary to facilitate interpretation of the results? COMMENTARY: N/A FLO.30550 Phase II N/A YES NO Does the report include an established or verified reference interval for blood lymphocyte subsets appropriate for the age of the patient? NOTE: Age- and sex-specific reference intervals (normal values) must be determined by laboratory, if feasible. For example, a reference interval can be validated by testing samples from 20 healthy representative individuals; if no more than 2 results fall outside the proposed reference interval, that interval can be considered validated for the population studied (refer to CLSI guideline C28, referenced below). If this is not possible or practical, then the laboratory should carefully evaluate the use of published data for its own reference intervals, and retain documentation of this evaluation. COMMENTARY: N/A REFERENCES: 1) Knight JA. Laboratory issues regarding geriatric patients. Lab Med. 1997;28:458-461; 2) NCCLS. How to Define and Determine Reference Intervals in the Clinical Laboratory;



Approved Guideline—Second Edition. NCCLS document C28-A2 (ISBN 1-56238-406-6). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA 2000. ................................................................. CD34 Stem Cell Enumeration ................................................................. FLO.30564 Phase I N/A YES NO Is there a procedure in place to document CD34 cellular viability, where applicable? NOTE: Viability testing is not necessary on peripheral blood or apheresis specimens that are stained and analyzed within 4 hours of drawing (harvesting). Cord blood, bone marrow and samples more than four hours old should have total cellular viability evaluated as a minimum by dye exclusion. Analyses on these older samples are possible if the laboratory has verified the absence of clinically significant differences in CD34+ cells between the fresh and aged specimens. The viability dye 7-amino actinomycin-D (7-AAD) has been reported to provide excellent results in this analysis. COMMENTARY: N/A REFERENCES: 1) Owens M, Loken M. Peripheral blood stem cell quantitation, In Flow Cytometry Principles for Clinical Laboratory Practice. New York, NY: Wiley-Liss, 1995:111-127; 2) Keeney M., et al. Single platform flow cytometry absolute CD34+ cell counts based on the ISHAGE guidelines. Cytometry. 1998; 34:61-70; 3) Hubl W, et al. Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes. Cytometry.1998; 34:121-127; 4) Gratama J, et al. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. Cytometry. 1998;34:128-142. FLO.30571 Phase I N/A YES NO For apheresis samples stored more than 4 hours, is an aliquot of the product taken immediately before processing for freezing (autologous donors) or infusion (allogeneic donors)? NOTE: For apheresis samples stored for more than 4 hours before CD34 cell analysis, an aliquot of the product should be taken and tested for CD34+ cell numbers and viability immediately before infusion or processing for freezing. The viability assessment must be performed using a flow cytometric method with the viability dye included in the same tube with the CD34 and CD45 monoclonal antibodies for a precise determination of CD34+ cell viability. Estimates of total cellular viability (for example, trypan blue exclusion) may not be used as an alternative, as they may overestimate the viability of the much smaller (and more fragile) CD34 stem cell population. When for clinical reasons it is necessary to perform CD34 cell analysis on specimens stored for more than 4



hours, the report should include the number of hours elapsed prior to analysis of the specimen and a statement that laboratory viability testing is a reflection of the ability of CD34 cells to survive and proliferate in vivo. COMMENTARY: N/A REFERENCE: Keeney M, et al. Single platform flow cytometry absolute CD34+ cell counts based on the ISHAGE guidelines. Cytometry.1998;34:61-70. FLO.30578 Phase II N/A YES NO Are appropriately conjugated Class II or Class III anti-CD34 monoclonal antibodies used? NOTE: Class I reagents are not recommended. Class II reagents conjugated to FITC are not recommended. COMMENTARY: N/A FLO.30585 Phase II N/A YES NO Are a statistically valid number of CD34+ events collected to ensure clinically relevant precision and accuracy? NOTE: The maximum coefficient of variation for CD34+ cell counts should be 10%. To achieve this precision, a minimum of 100 CD34+ events should be counted, as recommended by the ISHAGE guidelines and European Working Group on Clinical Cell Analysis. If the CD34+ cell count in a sample is 0.13%, for example, then 75,000 events must be collected to reach a count of 100 CD34+ events. This level of precision is not required for extremely low counts, provided they are below clinical decision points. Precision is most important at clinical decision thresholds and laboratories should verify their precision at such decision points. COMMENTARY: N/A FLO.30592 Phase II N/A YES NO Are sequential (Boolean) gating techniques used to define the CD34+ stem cells?



COMMENTARY: N/A FLO.30599 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 results. It is essential that procedures are based on published work. COMMENTARY: N/A ................................................................. Leukemia and Lymphoma ................................................................. FLO.30610 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.



FLO.30640 Phase II N/A YES NO Does the laboratory use antibodies appropriate for the clinical situation? NOTE: The panel of monoclonal antibodies employed must be sufficiently comprehensive to address the clinical problem under consideration. Knowledge of the clinical situation and/or the morphologic appearance of the abnormal cells may help to guide antibody selection. Because antibodies vary in their degree of lineage specificity, and because many leukemias lack one or more antigens expected to be present on normal cells of a particular lineage, it is recommended that a certain degree of redundancy be built into a panel used for leukemia phenotyping. The Inspector should examine several reports and histograms of leukemias and/or lymphomas that have been recently analyzed by the laboratory. COMMENTARY: N/A REFERENCES: 1) 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; 2) Rimsza LM, et al. The presence of CD34+ cell clusters predicts impending relapse in children with acute lymphoblastic leukemia receiving maintenance chemotherapy. Am J Clin Pathol. 1998;110:313-320; 3) Siebert JD, et al. Flow cytometry utility in subtyping components of composite and sequential lymphomas. Am J Clin Pathol. 1998;110:536; 4) Kampalath B, et al. CD19 on T cells in follicular lymphocytic leukemia/small lymphocytic lymphoma, and T-cell-rich B-cell lymphoma: an enigma. Am J Clin Pathol. 1998;110:536; 5) Krasinskas AM, et al. The usefulness of CD64, other monocyte-associated antigens, and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation. Am J Clin Pathol. 1998;110:797-805. FLO.30670 Phase II N/A YES NO Are cell concentrations adjusted for optimal antibody staining? NOTE: Each laboratory must have a procedure to identify specimens with abnormal cell counts, and adjust concentrations for optimal antibody staining. 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. FLO.30720 Phase II N/A YES NO Are methods established to ensure that immunoglobulin staining is intrinsic and not extrinsic (cytophilic)? NOTE: The question is directed towards ensuring that the immunoglobulin light chain analysis includes only light chain synthesized by B cells (intrinsic light chain). Many cell types will bind serum immunoglobulin nonspecifically via Fc receptors (including B cells). To ensure that immunoglobulin staining detected by flow cytometry is intrinsic (on B cells) rather than cytophilic, a pan-B cell marker (e.g. CD19, CD20) may be included in the same tube as one or both anti-light chain reagents. The inclusion of both lambda and kappa light chain reagents in the same tube allows a clear delineation of non-specific binding, even on B cells. 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. FLO.30730 Phase II N/A YES NO Are there procedures established for distinguishing abnormal cells of interest from normal cells, based on their light scatter and fluorescence properties? NOTE: Generally, both neoplastic and non-neoplastic cells are acquired in any gate used for acquisition. Attempts must be made to distinguish them at the time of analysis. Appropriate procedures include use fluorescent antibodies, fluorescent dyes, light scatter measurements, or any combination thereof to select out the relevant cell subpopulation for further analysis. Morphologic evaluation is also a valuable parameter to improve analysis. COMMENTARY: N/A REFERENCES: 1) Muirhead KA, et al. Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory. Ann NY Acad Sci. 1986;468:113-127; 2)



American Society for Microbiology. Manual of clinical immunology, 4th ed. Washington, DC: ASM, 1992; 3) Sun T, et al. Gating strategy for immunophenotyping of leukemia and lymphoma. Am J Clin Pathol. 1997;108:152-157; 4) 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; 5) Macon WR, Salhany KE. T-cell subset analysis of peripheral T-cell lymphomas by paraffin section immunohistology and correlation of CD4/CD8 results with flow cytometry. Am J Clin Pathol. 1998;109:610-617; 6) Dunphy CH. Combining morphology and flow cytometric immunophenotyping to evaluate bone marrow specimens for B-cell malignant neoplasms. Am J Clin Pathol. 1998;109:625-630. FLO.30760 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. FLO.30790 Phase II N/A YES NO Does the final report include information about the immunophenotype of the abnormal cells, if identified, and comments necessary to facilitate the interpretation? NOTE: Clinical information and available pathologic material should be reviewed to select appropriate antibodies and avoid performing flow cytometry on normal tissues. Ideally, direct morphologic correlation should be done. Such review is appropriate for bone marrow samples, solid tissues, and blood in case of suspected lymphoproliferative disorders. In cases involving leukemia and lymphoma phenotyping, correlation should be made between the immunologic and pathologic results. The flow histograms, rather than just the percentage of positive cells, should be reviewed by the laboratory director in difficult cases. The peak channel and shapes of the curves may be helpful in identifying clonal populations.



COMMENTARY: N/A REFERENCES: 1) 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; 2) Nguyen AND, et al. A relational database for diagnosis of hematopoietic neoplasms using immunophenotyping by flow cytometry. Am J Clin Pathol. 2000;113:95-106. ----------------------------------------------------------------- DNA CONTENT AND CELL CYCLE ANALYSIS ----------------------------------------------------------------- FLO.31000 Phase II N/A YES NO Are there methods to ensure that specimens processed for DNA content and cell cycle analysis contain neoplastic cells of interest? NOTE: It is critical that specimens submitted for flow cytometric analysis are representative samples of the neoplastic disorder being characterized. In specimens in which no population of abnormal DNA content is detected, it is especially important to demonstrate that neoplastic cells are present in the sample run through the flow cytometer. This generally requires microscopic evaluation of the specimen by an anatomic or clinical pathologist. COMMENTARY: N/A FLO.31010 Phase II N/A YES NO Are there methods in place to account for cellular debris and aggregates? NOTE: Cellular debris can affect measurements of S-phase fraction, and aggregates can alter ploidy assessments; these need to be excluded from consideration. DNA analysis software programs generally provide options for debris subtraction and doublet discrimination. Each laboratory should incorporate such methods into their procedures. Confirmation with fluorescent microscopic examination of the stained nuclear suspension may provide additional documentation of cellular aggregates. COMMENTARY:



N/A FLO.31020 Phase II N/A YES NO Are criteria established for determining acceptable linearity for DNA content measurement using cells or particles of known relative fluorescence? COMMENTARY: N/A FLO.31050 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 FLO.31100 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.



FLO.31150 Phase I N/A YES NO Are there documented criteria that specify the type of neoplasms acceptable for DNA analysis? NOTE: The laboratory should show evidence that it restricts analysis to those neoplasms for which the literature supports significant independent prognostic significance for DNA ploidy and/or S-phase analysis. COMMENTARY: N/A REFERENCES: 1) DNA cytometry consensus conference. Cytometry. 1993;14:471-500; 2) Henson D, et al. College of American Pathologists Conference XXVI on clinical relevance of prognostic markers in solid tumors. Summary. Arch Pathol Lab Med. 1995;119:1109-1112. FLO.31200 Phase II N/A YES NO Are there documented criteria for acceptability of histograms for interpretation? COMMENTARY: N/A FLO.31250 Phase II N/A YES NO Are there documented criteria for specimen rejection for DNA content analysis? COMMENTARY: N/A FLO.31300 Phase II N/A YES NO Has the concentration of nucleic acid-specific dye been determined to be a saturating concentration? NOTE: Standard techniques use an excess concentration of fluorochrome since concentrations below saturation will make the cells appear hypoploid. COMMENTARY: N/A



REFERENCE: Shapiro HA. Practical flow cytometry. New York, NY: Alan R. Liss, 1985. FLO.31350 Phase II N/A YES NO Are control cells of known DNA content run with each specimen or batch of specimens to establish an acceptable CV for the G0/G1 peak and to determine the DNA index? NOTE: Repetitive analysis of the reference cells allows reference intervals to be established to determine an acceptable range of results. This can be used as a control for DNA staining and instrumental parameters used in the analysis. COMMENTARY: N/A REFERENCES: 1) Hiddemann W, et al. Convention on nomenclature for DNA cytometry. Cytometry. 1984;5:445-446; 2) 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. FLO.31400 Phase II N/A YES NO Are analytical criteria established for identification of an aneuploid cell population in the test specimen? NOTE: The ability to detect DNA aneuploidy by flow cytometric measurement depends upon the resolution of the DNA measurements, usually assessed by the coefficient of variation (CV) of the peaks. CVs should be reported for all clinical studies. The range of CVs is highly dependent on the tissue type and the way it is prepared. Histograms observed for clinical specimens often represent complex overlapping patterns because most tumor specimens contain a mixture of tumor cells, stromal cells, and inflammatory cells. Analysis of control cells is necessary to establish the CV for a normal diploid, G0/G1 peak. Periodic review of the CVs for control cells is necessary to ensure adequate functioning of the analytic procedure. An international workshop recommended that cells (or nuclei) should be termed as having an "abnormal DNA stemline" or "DNA aneuploidy" when at least two separate G0/G1 peaks are demonstrated. COMMENTARY: N/A



REFERENCES: 1) Hiddemann W, et al. Convention on nomenclature for DNA cytometry. Cytometry. 1984;5:445-446; 2) Coon JS, et al. Advances in flow cytometry for diagnostic pathology. Lab Invest. 1987;57:453-479. FLO.31450 Phase II N/A YES NO Are the results of analyses of patient's specimens reviewed by the laboratory director or designee, and are comments to clinicians reported when necessary to facilitate interpretation of the results? COMMENTARY: N/A REFERENCE: Coon JS, et al. Advances in flow cytometry for diagnostic pathology. Lab Invest. 1987;57:453-479.

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PERSONNEL

***************************************************************************** FLO.40000 Phase II N/A YES NO Does the person in charge of technical operations in flow cytometry have education equivalent to that of an MT(ASCP) and at least one year's experience in flow cytometry under a qualified director? COMMENTARY: N/A

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PHYSICAL FACILITIES

***************************************************************************** Sufficient space and utilities need to be provided for the overall workload of the flow cytometry section, and to meet all safety requirements



FLO.50000 Phase I N/A YES NO Is there adequate space for administrative functions? COMMENTARY: N/A FLO.50050 Phase I N/A YES NO Is there adequate space for clerical work? COMMENTARY: N/A FLO.50100 Phase I N/A YES NO Is there adequate space for technical work (bench space)? COMMENTARY: N/A FLO.50150 Phase I N/A YES NO Is there adequate space for instruments? COMMENTARY: N/A FLO.50200 Phase I N/A YES NO Is there adequate space for shelf storage? COMMENTARY: N/A



FLO.50250 Phase I N/A YES NO Is there adequate refrigerator/freezer storage space? COMMENTARY: N/A FLO.50300 Phase I N/A YES NO Is the available space efficiently utilized? COMMENTARY: N/A FLO.50350 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 FLO.50400 Phase I N/A YES NO Are floors and benches clean, free of clutter and well-maintained? COMMENTARY: N/A FLO.50450 Phase I N/A YES NO Are water taps, sinks, and drains adequate? COMMENTARY: N/A



FLO.50550 Phase I N/A YES NO Are electrical outlets adequate? COMMENTARY: N/A FLO.50600 Phase I N/A YES NO Is ventilation adequate? COMMENTARY: N/A FLO.50650 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 REFERENCE: College of American Pathologists. Medical laboratory planning and design. Northfield, IL: CAP, 1986. FLO.50700 Phase I N/A YES NO Is temperature/humidity control adequate? COMMENTARY: N/A FLO.50750 Phase I N/A YES NO Are telephones conveniently located, and are calls easily transferred?



COMMENTARY: N/A

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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 flow cytometry laboratory is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report. FLO.50850 Phase II N/A YES NO Do the procedure manuals contain information for safe handling of clinical samples and instruments with regard to infectious biohazard risks, cryogenic substances, carcinogenic dyes, and lasers? COMMENTARY: N/A
