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Master Flow Cytometry Checklist - College of American Pathologists

1. 30 of 36 Flow Cytometry Checklist 07 11 2011 e How does your laboratory distinguish neoplastic from non neoplastic cells e How does your laboratory distinguish between intrinsic and extrinsic immunoglobulin staining NEW 06 17 2010 FLO 30605 Immunophenotyping Data Phase Il If flow leukemia lymphoma immunophenotyping data from an outside facility i e a technical flow laboratory are interpreted the laboratory ensures the following 1 The technical flow laboratory s panel of monoclonal antibodies are sufficiently comprehensive to address the clinical problem under consideration 2 The technical flow laboratory uses appropriate gating techniques 3 The final report includes information about the immunophenotype of normal and abnormal cells and includes comments necessary to facilitate the interpretation 4 Gated dot plots and histograms are retained for 10 years List mode files that include analysis gates are acceptable FLO 30610 Cellular Viability Phase Il There is a policy for determining when the percentage of viable cells in each test specimen should be measured NOTE Selective loss of cell subpopulations and or the presence of dead cells may lead to spurious results Procedures must be in place to ensure that viable cells are analyzed This does not mean that all specimens with low viability must be rejected Finding an abnormal population in a specimen with poor viability may be valuable but the failure to
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 etal 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 Apheresis Specimen Handling Phase For apheresis samples stored more than 4 hours an aliquot of the product is 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 sh
3. 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 FLO 31100 Specimen Treatment Phase Il Specimen treatment with nucleic acid dye includes treatment with RNAse if the dye is not specific for DNA FLO 31150 FLO 31200 FLO 31250 FLO 31300 FLO 31350 34 of 36 Flow Cytometry Checklist 07 11 2011 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 Evidence of Compliance Written procedure for specimen treatment with RNAse REFERENCES 1 Shapiro HA Practical flow cytometry New York NY Alan R Liss 1985 Neoplasm DNA Analysis Criteria Phase There are 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 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 Arc
4. Approved Guideline Second Edition CLS 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 Final Report Phase Il The final report includes 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 Ideally direct morphologic correlation should be done Such review is appropriate for bone marrow samples solid tissues and blood in cases of suspected hematolymphoid neoplasia 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 interpreting pathologist in difficult cases The peak channel and shapes of the curves may be helpful in identifying clonal populations REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edition CLS 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 immu
5. Evidence of Compliance v Record of review of results OR records of consistent implementation of the error detection system s defined in the procedure AND d Records of timely corrective action of identified errors FLO 20200 Supervisory Result Review Phase Il In the absence of on site supervisors the results of tests performed by personnel are reviewed by the laboratory director or general supervisor within 24 hours NOTE The CAP does NOT require supervisory review of all test results before or after reporting to patient records Rather this requirement is intended to address only that situation defined under CLIA for high complexity testing performed by trained high school graduates qualified under 42CFR493 1489 b 5 when a qualified general supervisor is not present Evidence of Compliance d Written policy defining the review process and personnel whose results require review AND v Records of result review for specified personnel REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 1992 Feb 28 7182 42CFR493 1463 a 3 and 42CFR493 1463 c 7183 42CFR493 1489 b 1 and 42CFR493 1489 b 5 SPECIMEN COLLECTION AND HANDLING Inspector Instructions e Sampling of flow cytometry specimen collection and handling policies and procedures e Sampling of specimen rejection records log 12 of 36 Flow Cyt
6. cluster analysis should be used to define the population of interest CD34 cells REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Enumeration of Immunologically Defined Cell Populations by Flow Cytometry Approved Guideline Second Edition CLSI document H42 A2 ISBN 1 56238 640 9 Clinical and Laboratory Standards Institute 940 West Valley Road Suite 1400 Wayne PA 19087 1898 USA 2007 2 Sutherland DR Anderson L Keeney M et al Towards a worldwide standard for CD34 enumeration J Hematotherapy 1997 6 85 89 Leukemia and Lymphoma Inspector Instructions e Sampling of leukemia lymphoma immunophenotyping policies and procedures e Sampling of patient reports and histograms to include abnormal cell immunophenotype interpretive comments etc e H flow leukemia lymphoma immunophenotyping is done at an outside facility how does your laboratory ensure that the testing is sufficiently comprehensive to facilitate accurate diagnosis with appropriate gating and retention of records e Under what circumstances does your laboratory measure the percentage of viable cells SERRE RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR R RR RRRRRRRRRRRRRRRnn a
7. containing reagents and patient soecimens must be monitored daily as equipment failures could affect accuracy of patient test results Items such as water baths and heat blocks used for procedures need only be checked on days of patient testing The two acceptable ways of recording temperatures are 1 recording the numerical temperature or 2 placing a mark on a graph that corresponds to a numerical temperature either manually or using a graphical recording device The identity of the individual recording the temperature s must be documented recording the initials of the individual is adequate The use of automated including remote temperature monitoring systems is acceptable providing that laboratory personnel have ongoing immediate access to the temperature data so that appropriate corrective action can be taken if a temperature is out of the acceptable range The functionality of the system must be documented daily REVISED 07 11 2011 FLO 30320 Temperature Range Phase Il Acceptable ranges have been defined for all temperature dependent equipment with documentation of corrective action when values exceed these ranges Evidence of Compliance d Temperature log or record with defined acceptable range AND d Evidence of corrective action Thermometers Inspector Instructions 24 of 36 Flow Cytometry Checklist 07 11 2011 FLO 30360 Thermometric Standard Device Phase Il An appropriate thermometric standard
8. detect and correct significant clerical and analytical errors and unusual laboratory results in a timely manner NOTE One common method is review of results by a qualified person technologist supervisor pathologist laboratory director before release from the laboratory but there is no requirement for supervisory review of all reported data for single analyte tests that do not include interpretation All tests that include an interpretation must be reviewed by the laboratory director or qualified designee before release from the laboratory In computerized laboratories there should be automatic traps for improbable results The system for detecting clerical errors significant analytical errors and unusual laboratory results must provide for timely correction of errors Le before results become available for clinical decision making For confirmed errors detected after reporting corrections must be promptly made and reported to the ordering physician or referring laboratory as applicable Each procedure must include a listing of common situations that may cause analytically inaccurate results together with a defined protocol for dealing with such analytic errors or interferences This may require alternate testing methods in some situations it may not be possible to report results for some or all of the tests requested The intent of this requirement is NOT to require verification of all results outside the reference normal range
9. fresh human samples for the instruments methods involved This checklist requirement applies only to instruments methods accredited under a single CAP number Evidence of Compliance v Written procedure for performing instrument method correlation including criteria for acceptability AND v Records of correlation studies reflecting performance at least twice per year with appropriate specimen types REFERENCES 1 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 2 Miller WG Erek A Cunningham TD et al Commutability limitations influence quality control results with different reagent lots Clin Chem 2011 57 76 83 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 procedures and schedules for instrument maintenance must be as thorough and as frequent as specified by the manufacturer Inspector Instructions e Sampling of instrument s policies and procedures e Sampling of instrument maintenance logs and repair records e Sampling of optical alignment laser output check
10. instrument correlation records TTT REEL ELELELESESEOTOTOTOTESESTOTOTOLOTESOSTOTOOTOLOTESTOTOTOTOTOTESESTOTOTOTOSeTESESTOTOTOTeTerereererererere terre rerererererirrererererererrererererererirrererererererirrerererereririrrerererereriritrererereri titre re terete titi i ete e How do you determine when quality control is unacceptable and when corrective actions are needed e How does your laboratory establish or verify acceptable QC ranges e Select several occurrences in which QC is out of range and follow documentation to DISCOVER determine if the steps taken follow the laboratory policy for corrective action FLO 23737 QC Reagents Stain Phase Il The performance of reagents and staining procedures are verified 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 requirement below For dual platform measurements of lymphocyte subsets CD4 lymphocytes one level of positive control
11. 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 When antigen positive cells are not readily available through commercial controls or patient materials then the laboratory director must implement an equivalent procedure to meet the positive control requirements e g CD1a CD103 This may include cryopreserved or fresh cell lines and patient material FLO 23800 FLO 23925 REVISED FLO 24230 18 of 36 Flow Cytometry Checklist 07 11 2011 Evidence of Compliance d Written procedure defining QC requirements for each test AND v Records of QC results QC Single Dual Platform Tests Phase Il For single platform quantitative tests e g CD4 CD34 cell concentrations and dual platform quantitation of CD34 stem cell concentrations at least 2 levels of positive cellular controls are analyzed at least daily or each time the flow cytometer is restarted to verify the performance of reag
12. 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 FLO 23250 FLO 23300 15 of 36 07 11 2011 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 Flow Cytometry Checklist Evidence of Compliance Written procedure for the verification of new lots and shipments prior to use AND d Records of verification of new reagents shipments Reagent Usage Phase Il The recommendations of the manufacturer for the proper use of reagents and controls in kit procedures are followed Evidence of Compliance Written procedure consistent with manufacturer s instructions OR records of method accuracy evaluation if alternative procedures are used REFERENCES 1 Caldwell CW Analyte specific reagents in the flow cytometry laboratory Arch Pathol Lab Med 1998 122 861 864 Reagent Kit Components Phase Il If there are multiple components of a reagent kit the laboratory only uses 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
13. same lot number unless otherwise specified by the manufacturer or accuracy equivalency is verified by the laboratory Evidence of Compliance Written documentation defining allowable exceptions for mixing kit components from different lots REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 2003 Jan 24 7164 42CFR493 1252 d RECORDS AND REPORTS Inspector Instructions READ FLO 23675 Sampling of patient reports includes disclaimer when Class ASR s are used e Record retention policy gated dot plots histograms ASR Report Phase Il If patient testing is performed using Class analyte specific reagents ASR s obtained or purchased from an outside vendor the patient report includes 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 FLO 23706 16 of 36 Flow Cytometry Checklist 07 11 2011 specimens An ASR is the active ingredient of a laboratory developed test system This checklist requirement concerns Class ASR s C
14. 121 118 121 Reference Intervals Established Phase Il The report includes 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 A3 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 Evidence of Compliance v Patient result reported with reference interval as applicable and record of completed reference range study OR records of verification of manufacturer s stated range when reference range study is not practical e g unavailable normal population OR other methods approved by the laboratory director REFERENCES 1 Knight JA Laboratory issues regarding geriatric patients Lab Med 1997 28 458 461 2 Clinical Laboratory and Standards Institute CLSI Defining Establishing and Verifying Reference Intervals in the Clinical Laboratory Approved Guideline Third Edition CLS Document C28 A3c ISBN 1 56238 682 4 CLSI 940 West Valley Road
15. EFERENCES 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 CLS 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 FLO 30550 2 of 36 Flow Cytometry Checklist 07 11 2011 Markers Cursors Phase II There is a procedure to set markers cursors to distinguish fluorescence negative and fluorescence positive cell populations NOTE Each laboratory must have a set of objective criteria to define the appropriate placement of markers cursors to delineate the population of interest Isotypic controls may not be necessary in all cases and cursor settings for the isotype control may not be appropriate for all markers Cursor settings must be determined based on the fluorescence patterns from the negative and positive populations for CD3 CD4 and CD8 REFERENCES 1 National Institute of Allergy and Infectious Diseases Division of AIDS flow cytometry guidelines sec 3 09B and 5 03A 2 Sreenan JJ et al The use of isotypic control antibodies in the analysis of CD3 and CD3 CD4 lymphocyte subsets by flow cytometry Are they really necessary Arch Pathol Lab Med 1997
16. Master Every patient deserves the GOLD STANDARD Flow Cytometry Checklist CAP Accreditation Program College of American Pathologists 325 Waukegan Road Northfield IL 60093 2750 www cap org 07 11 2011 Flow Cytometry Checklist 07 11 2011 Disclaimer and Copyright Notice If you are enrolled in the CAP s Laboratory Accreditation Program and are preparing for an inspection you must use the Checklists that were mailed in your application or reapplication packet not those posted on the Web site The Checklists undergo regular revision and Checklists may be revised after you receive your packet If a Checklist has been updated since receiving your packet you will be inspected based upon the Checklists that were mailed If you have any questions about the use of Checklists in the inspection process please e mail the CAP accred cap org or call 800 323 4040 ext 6065 The checklists used in connection with the inspection of laboratories by the Laboratory Accreditation Program of the College of American Pathologists have been created by the College and are copyrighted works of the College The College has authorized copying and use of the checklists by College inspectors in conducting laboratory inspections for the CLA and by laboratories that are preparing for such inspections Except as permitted by section 107 of the Copyright Act 17 U S C sec 107 any other use of the checklists constitutes infringement of the Col
17. O 05075 07 10 2011 FLO 10150 07 10 2011 FLO 10180 07 10 2011 FLO 10210 07 10 2011 FLO 10260 07 10 2011 FLO 13540 07 10 2011 FLO 16770 07 10 2011 FLO 20020 07 10 2011 FLO 20050 07 10 2011 FLO 21000 07 10 2011 FLO 21100 07 10 2011 FLO 21125 07 10 2011 FLO 21150 07 10 2011 FLO 21210 07 10 2011 FLO 21220 07 10 2011 FLO 21250 FLO 24100 FLO 25000 FLO 25050 FLO 30380 FLO 30390 FLO 30400 FLO 30500 FLO 30599 FLO 31450 FLO 50000 FLO 50050 FLO 50100 FLO 50150 FLO 50200 FLO 50250 FLO 50300 FLO 50350 FLO 50400 FLO 50450 FLO 50550 FLO 50600 FLO 50650 FLO 50700 FLO 50750 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 07 10 2011 Flow Cytometry Checklist Flow Cytometry Checklist 07 11 2011 UNDERSTANDING THE CAP ACCREDITATION CHECKLIST COMPONENTS To provide laboratories with a better means to engage in and meet their accreditation requirements the CAP has enhanced the checklist content and updated its design New components containing additional information for both the laboratory and inspectors include Subject Headers Declarative Statements and Evidence of Compliance See below for a definition of each new feature as an example of how they appear in the checklists Subject Header D
18. Suite 1400 Wayne PA 19087 1898 USA 2008 CD34 Stem Cell Enumeration Inspector Instructions e Sampling of CD34 analysis policies and procedures includes procedure for apheresis sample handling e Sampling of CD34 records events counted e How does your laboratory document CD34 cellular viability How does your laboratory gate to define the population of CD34 cells e What class of anti CD34 monoclonal antibodies does your laboratory use and how are they conjugated FLO 30564 FLO 30571 FLO 30578 28 of 36 Flow Cytometry Checklist 07 11 2011 CD34 Cellular Viability Phase There is 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 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
19. aks are demonstrated 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 PERSONNEL Inspector Instructions FLO 40000 Personnel Technical Operations Phase Il The person in charge of technical operations in flow cytometry has education equivalent to that of an MT ASCP and at least 4 years experience one of which is in flow cytometry under a qualified director Evidence of Compliance v Records of qualifications including degree or transcript certification registration current license if required and work history in related field 36 of 36 Flow Cytometry Checklist LABORATORY SAFETY 07 11 2011 The inspector should review relevant requirements 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 Inspector Instructions e Sampling of procedures for safety information READ FLO 50850 Safety Manual Phase Il 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
20. 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 Evidence of Compliance Y Records reflecting that QC is run by the same personnel performing patient testing REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 2003 Jan 24 7166 42CFR493 1256 d 8 QC Verification Phase Il The results of controls are 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 Evidence of Compliance v Defined QC tolerance limits and records of verification of acceptable QC results REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 2003 Jan 24 7166 42CFR493 1256 f 07 11 2011 Monthly QC Review Phase Il Quality control data are reviewed and assessed at least monthly by the laboratory di
21. antibodies REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edition CLS 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 30670 FLO 30720 FLO 30730 31 of 36 07 11 2011 2 Rimsza LM etal 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 6 Wood BL et al 2006 Bethesda International Consensus Recommendations on the Immunophenotypic Analysis of Hematolymphoid Neoplasia by Flow Cytometry Optimal Reagents and Reporting for the Flow Cytometric Diagnosis of Hematopoietic Neoplasia Cytometry Part B Clinical Cytometry 2007 72B S12 S22 Flow Cytometry Che
22. areas throughout the Inspector Checklists Please note that all four R O A D elements are not always applicable for each grouping or sections of related requirements Inspector Instructions BLL GELTZ D D D READ review a sampling of laboratory documents Information obtained from this review will be useful as you observe processes and engage in dialogue with the laboratory staff Example of the complimentary inspector instructions for Quality Management Quality Control General Issues section appearing across checklists e Sampling of QM QC policies and procedures e Incident error log and corrective action OBSERVE laboratory practices by looking at what the laboratory personnel are actually doing and OBSERVE note if practice deviates from the documented policies procedures Example e Observe the settings QC range limits established in the laboratory LIS HIS to ensure that the laboratory s stated ranges are accurately reflected D bebhdelkelkelkelbelkelkelkelbebelkelkelkelbelbelkelkelkelbelbelelkelkelbelblellellele AAL ALLAN ASK open ended probing questions that start with phrases such as tell me about or what would you do if This approach can be a means to corroborate inspection findings that were examined by other techniques such as Read amp Observe Ask follow up questions for clarification Include a variety of staff levels in your communication process Example e As a staff member what is your inv
23. cal and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edition CLS 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 Abnormal Cell Distinction Phase Il There are 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 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 etal 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 C
24. cklist Cell Concentrations Phase Il Cell concentrations are adjusted for optimal antibody staining Evidence of Compliance d Written procedure for adjusting cell concentrations to ensure optimal antibody staining REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edition CLS 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 Immunoglobulin Staining Phase Il Methods are established to ensure that immunoglobulin staining is intrinsic and not extrinsic cytophilic NOTE The requirement 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 Evidence of Compliance v Written procedure defining method to ensure intrinsic immunoglobulin staining REFERENCES 1 Clini
25. d by laboratory REVISED FLO 23050 FLO 23100 FLO 23150 14 of 36 Flow Cytometry Checklist 07 11 2011 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 Evidence of Compliance Written policy defining elements required for reagent labeling REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 2003 Jan 24 7164 42CFR493 1252 c 07 11 2011 Reagent Expiration Date Phase Il All reagents are used within their indicated expiration date NOTE The laboratory must assign an expiration date to any reagents that do not have a manufacturer provided expiration date The assigned expiration date should be based on known Stability frequency of use storage conditions and risk of deterioration For laboratories not subject to US regulations expired reagents may be used only under the following circumstances 1 The rea
26. device of known accuracy is available guaranteed by manufacturer to meet NIST Standards NOTE Thermometers should be present on all temperature controlled instruments and environments and checked daily Thermometric standard devices should be recalibrated or recertified prior to the date of expiration of the guarantee of calibration Evidence of Compliance Thermometer certificate of accuracy FLO 30370 Non Certified Thermometers Phase II All non certified thermometers in use are checked against an appropriate thermometric standard device before use Evidence of Compliance Written procedure defining criteria for verification of non certified thermometers AND v Records of verification prior to being placed in service Automatic Pipetting Devices Inspector Instructions Automatic pipette calibration procedure e Sampling of pipette dilutor checks FLO 30415 Automatic Pipettes Phase Il Automatic pipettes used for quantitative dispensing are checked for accuracy and reproducibility before being placed in service and at least annually with documentation of results NOTE Such checks are most simply done gravimetrically Alternative approaches include spectrophotometry and the use of commercial kits The frequency of checks depends on how the pipettor is used for materials requiring high precision and accuracy such as internal standards quarterly checks are appropriate Less frequent checks may be appropriate for other mater
27. eclarative Statement A phrase that provides Checklist questions are reworded the key concept of the as declarative statements to requirement better convey the regulatory nature of requirements HEM 20050 Numeric QC Phase Il For numeric QC data Gaussian or other quality control statistics e g SD and CV are calculated monthly to define analytic imprecision NOTE For CBC data where stabilized whole blood is not used for quality control such Statistics may be generated from previous patient samples using the standard deviation of duplicate pairs Evidence of Compliance d Written procedure for monitoring analytic imprecision including statistical analysis of data AND d QC records showing monthly monitoring of imprecision Evidence of Compliance Information that highlights what is needed to prove that a laboratory is in compliance with the requirement Using Evidence of Compliance EOC This component which appears with several checklist requirements is intended to 1 Assist a laboratory in preparing for an inspection and managing ongoing compliance 2 Drive consistent understanding of requirements between the laboratory and the inspector 3 Provide specific examples of acceptable documentation policies procedures records reports charts etc In addition to the Evidence of Compliance listed in the checklist other tyoes of documentation may be acceptable Whenever a policy procedure process is referenced wit
28. ed devices analyte specific reagents Final rule Fed Register 1997 Nov 21 62243 21CFR809 and 864 Caldwell CW Analyte specific reagents in the flow cytometry laboratory Arch Pathol Lab Med 1998 122 861 864 Graziano Disclaimer now needed for analyte specific reagents CAP Today 1998 12 11 5 11 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 E Record Retention Phase Il Gated dot plots and histograms are retained for at least 10 years NOTE The intent of this checklist requirement is retention of gated dot plots and histograms of hematolymphoid neoplasias CD34 stem cell records and congenital immunodeficiency evaluations for 10 years Paper copies of gated dot plots and histograms are not required as long as the information is available electronically e g pdf tiff joeg files List mode data files with embedded gates and analysis regions are also acceptable REFERENCES 1 CAP Policy PP Retention of Laboratory Records and Materials CONTROLS AND STANDARDS Controls are samples that act as surrogates for patient soecimens They are periodically processed like a patient sample to monitor the ongoing performa
29. eeeeeeessaaaaaeees 25 CD34 Stem Cell Enumeratio assoni ien EEEE EEEE EEEO nE DAETA nE 27 Leukemia and Lymphoma ME 29 DNA CONTENT AND CELL e ee E EE 32 PERSONNEL ee 35 ETA OTA E HR EE 35 Flow Cytometry Checklist 07 11 2011 SUMMARY OF CHECKLIST EDITION CHANGES Flow Cytometry Checklist 07 11 2011 Edition The following requirements have been added revised or deleted in this edition of the checklist or in the two editions immediately previous to this one lf this checklist was created for a reapplication on site inspection or self evaluation it has been customized based on the laboratory s activity menu The listing below is comprehensive therefore some of the requirements included may not appear in the customized checklist Such requirements are not applicable to the testing performed by the laboratory Note For revised checklist requirements a comparison of the previous and current text may be found on the CAP website Click on Laboratory Accreditation Checklists and then click the column marked Changes for the particular checklist of interest NEW Checklist Requirements Requirement Effective Date FLO 30605 06 17 2010 REVISED Checklist Requirements Requirement Effective Date FLO 20100 07 11 2011 FLO 22050 07 11 2011 FLO 23050 07 11 2011 FLO 24230 07 11 2011 FLO 24475 07 11 2011 FLO 24650 07 11 2011 FLO 30290 06 17 2010 FLO 30320 07 11 2011 DELETED Checklist Requirements Requirement Effective Date FL
30. ells Approved Guideline Second Edition CLS 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 De nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn eee Pee eee eee eee eee EE EE ERR eee eee eee eee eee eee eee eee RER EIERE EIERE EE EE EE EE eee eee eee eee 1 FLO 30760 FLO 30790 32 of 36 07 11 2011 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 Flow Cytometry Checklist Cell Population Distinction Phase Il There is a procedure to distinguish fluorescence negative and fluorescence positive cell populations NOTE This does not imply that a separate negative control sample must be run It is possible to coordinate panels of monoclonal antibodies to compare the binding of monoclonal antibodies of the same subclass that typically have mutually exclusive patterns of reactivity of subsets of hematopoietic cells In this way test antibodies may also double as control reagents REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells
31. ents 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 Evidence of Compliance Written procedure defining QC requirements for each test OR records of validation of an alternate equivalent procedure when commercial controls are not available v Records of QC results REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 1992 Feb 28 7146 42CFR493 1256 QC Range Validation Phase Il A statistically valid target mean and range are 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 laborator
32. find an abnormality should be interpreted with caution If specimen viability is below the established laboratory minimum test results may not be reliable and this should be noted in the test report Routine viability testing may not be necessary However viability testing of soecimens with a high risk of loss of viability such as disaggregated lymph node specimens is required REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edition CLS 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 Appropriate Antibodies Phase Il The laboratory uses 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 Evidence of Compliance Written procedure defining use of appropriate monoclonal
33. gents are unique rare or difficult to obtain or 2 Delivery of new shipments of reagents is delayed through causes not under control of the laboratory The laboratory must document validation of the performance of expired reagents in accordance with written laboratory policy Laboratories subject to US regulations must not use expired reagents Evidence of Compliance Written policy for evaluating reagents lacking manufacturer s expiration date REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 2003 Jan 24 7164 42CFR493 1252 d Reagent Storage Phase Il Reagents are 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 Evidence of Compliance v Records of reagent storage consistent with manufacturer s instructions including refrigerator freezer and room temperature monitoring as applicable New Reagent Lot Verification Phase Il New reagent lots and or shipments are checked against old reagent lots or with suitable reference material before or concurrently with being placed in service NOTE The
34. h Pathol Lab Med 1995 119 1109 1112 Histogram Acceptability Criteria Phase Il There are documented criteria for acceptability of histograms for interpretation Specimen Rejection Criteria Phase Il There are documented criteria for specimen rejection for DNA content analysis Evidence of Compliance Written procedure defining criteria for specimen rejection and handling of sub optimal specimens AND v Records of specimen rejection AND v Records noting the conditions of sub optimal specimens Nucleic Acid Specific Dye Concentration Phase Il The concentration of nucleic acid specific dye has 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 Evidence of Compliance Written procedure to determine the nucleic acid specific stain concentration REFERENCES 1 Shapiro HA Practical flow cytometry New York NY Alan R Liss 1985 G0 G1 Peak Phase Il Control cells of known DNA content are run with each specimen or batch of specimens to establish an acceptable CV for the G0 G1 peak and to determine the DNA index 35 of 36 07 11 2011 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 Flow Cytometr
35. he same conditions used to run test samples REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edition CLS 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 Instrument Troubleshooting Phase Il Instructions are provided for minor troubleshooting and repairs of instruments Such as manufacturer s service manual Instrument Function Checks Phase Il Instrument maintenance service and repair records or copies are 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 Instrument Maintenance Review Phase Il Records of maintenance are kept and reviewed at least monthly by the person in charge of technical operations of the flow cytometry laboratory Evidence of Compliance d Instrumentation records includ
36. hin a requirement it is only repeated in the Evidence of Compliance if such statement adds clarity All policies orocedures processes covered in the CAP checklists must be documented A separate policy is not needed for each item listed in EOC as it may be referenced in an overarching policy Flow Cytometry Checklist 07 11 2011 HOW TO INSPECT USING R O A D INSPECTION TECHNIQUES Read Observe Ask Discover CAP has streamlined the inspection approach used during onsite inspections and is now offering guidance to inspectors by providing assessment techniques to facilitate a more efficient consistent and effective inspection process Specific inspector instructions are listed at the beginning of a grouping of related requirements Rather than reviewing each individual requirement CAP inspectors are encouraged to focus on the Inspector Instructions for a grouping of related requirements Once an area of concern has been identified through Read Observe Ask Discover or a combination thereof inspectors are encouraged to drill down to more specific requirements when necessary and review more details outlined in the Evidence of Compliance statements If a requirement is non compliant circle the requirement number to later list on the Inspector Summation Report Inspectors may also make notes in the margins of the checklist document Inspector Instructions and Icons used to evaluate a laboratory s performance now appear in several
37. ials Computer software is useful where there are many pipettes and provide convenient documentation For analytic instruments with integral automatic pipettors this checklist requirement applies unless such checks are not practical for the end user laboratory Manufacturers recommendations should be followed REFERENCES 1 Curtis RH Performance verification of manual action pipets Part Am Clin Lab 1994 12 7 8 9 25 of 36 Flow Cytometry Checklist 07 11 2011 2 Curtis RH Performance verification of manual action pipets Part Il Am Clin Lab 1994 12 9 16 17 3 Perrier S et al Micro pipette calibration using a ratiometric photometer reagent system as compared to the gravimetric method Clin Chem 1995 41 S183 4 Bray W Software for the gravimetric calibration testing of pipets Am Clin Lab Oct 1995 5 CLSI Laboratory Instrument Implementation Verification and Maintenance Approved Guideline CLSI Document GP31 A ISBN 1 56238 697 2 CLSI 940 West Valley Road Suite 1400 Wayne PA 19087 1898 USA 2009 Johnson B Calibration to dye for Artel s new pipette calibration system Scientist 1999 13 12 14 Connors M Curtis R Pipetting error a real problem with a simple solution Parts and Il Am Lab News 1999 31 13 20 22 Skeen GA Ashwood ER Using spectrophotometry to evaluate volumetric devices Lab Med 2000 31 478 479 E PROCEDURES AND TEST SYSTEMS NOTE Reticulocyte quantification by flow cytometry is
38. ing documentation of review and corrective action 22 of 36 Flow Cytometry Checklist 07 11 2011 FLO 30250 Fluorochrome Standards Phase II Appropriate standards for each fluorochrome e g fluorescent beads are run each day that the instrument is used as part of the calibration process and the results are recorded for quality control purposes NOTE These steps are necessary to optimize the flow system and the optics of the instrument Evidence of Compliance Written procedure for calibration using appropriate fluorochrome standards with documentation of results REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Enumeration of Immunologically Defined Cell Populations by Flow Cytometry Approved Guideline Second Edition CLS 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 Color Compensation Settings Phase Il Procedures are established for determining appropriate color compensation settings NOTE For two or more color analysis there must be a procedure to ensure that cells co labeled with more than one fluorescent reagent can be accurately distinguished from cells labeled only with one reagent Cells stained with mutually exclusive antibodies bearing the relevant fluorochromes or singly stained cell samples for each fluorochrome are the proper reference material for establishing appropriate c
39. lass ASR s are not subject to preclearance by the US Food and Drug Administration FDA or to special controls by FDA Most ASR s are Class 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 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 US Food and Drug Administration The CAP recommends additional language such as FDA does not require this test to go through premarket FDA review 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 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 ASR s REFERENCES 1 Department of Health and Human Services Food and Drug Administration Medical devices classification reclassification restrict
40. lege s copyrights in the checklists The College will take appropriate legal action to protect these copyrights All Checklists are 2011 College of American Pathologists All rights reserved Flow Cytometry Checklist 07 11 2011 Flow Cytometry Checklist TABLE OF CONTENTS SUMMARY Eeer 5 UNDERSTANDING THE 2010 CAP ACCREDITATION CHECKLIST CGOMPRONENTE 7 HOW TO INSPECT USING R O A D INSPECTION TECHNIQUES cc cccccccceceeeeseeeeeeeeeeseeeeaeaeeeeeeesaaeeeees 8 Igel let e CHE 9 PROFICIENCY TESTING occcstcaacsccecscsschaapacenctdeosyaheatisenidtesocnahdazedsvess uses aneahensacsatacasbcapndindsisacuesentossastasacenbiapadsussatads 9 QUALITY MANAGEMENT AND QUALITY CGONTROL 10 EI TEE 10 SPECIMEN COLLECTION AND HANDLING aaaannnnnnnnnnannnnnnnnnnnnnnnnnenssnnnnnnrnonsnnnnrnrensssnnrrnrrensnnnnrrrrensennnrernee 11 EISE MK 13 RECORDS AND REPOR TO EE 15 CONTROLS AND STANDARDS icccccc ccccccescctensceenssedeccceenscotedeenesedeeneceessvecereessnceesdssecetedsccesseeseecescetecesseeesees 16 INSTRUMENTS AND EQUIPMENT 1 00 cccecccccccccccseeseeeceeeeeeeeeeeesceceeeeseeeeeseeeeeeeseeeeeseeeeeeessssaaaeeseeeeessssaageses 20 Flow Rue EE 20 Temperature Dependent Eoupment reenn 22 ThermomeololS sssr otras peated ren Eege eng 23 Automatic Pipetting Uevces AA 24 PROCEDURES AND TEST SY EA 25 IMMUNOP RENO TY TUN Te 25 Blood Lymphocyte Subset Enumeration ccccceccsccccceecceeeeeeceeeeeeeeueeeeceeeeeessaeaaeseeeeeeessaease
41. lid on such a specimen The laboratory may wish to record that a dialogue was held with the physician when such occurs Evidence of Compliance v Defined specimen acceptability criteria AND v Records of rejected unacceptable specimens REFERENCES 1 Department of Health and Human Services Centers for Medicare and Medicaid Services Clinical laboratory improvement amendments of 1988 final rule Fed Register 2003 Jan 24 7183 42CFR493 1249 a and bi 2 National Institute for Allergy and Infectious Diseases Division of AIDS guidelines for flow cytometric immunophenotyping version 1 0 Jan 1993 sec 1 06 13 of 36 Flow Cytometry Checklist 07 11 2011 FLO 22100 Disposition of Unacceptable Specimens Phase Il The disposition of all unacceptable specimens is documented in the patient report and or quality management records NOTE This information is essential to proper patient test management and to the laboratory quality management program 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 requirements is for new reagents to be checked b
42. linical decision points Precision is most important at clinical decision thresholds and laboratories should verify their precision at such decision points Evidence of Compliance Written procedure defining minimum number of CD34 events for analysis AND v Records of number of events counted REFERENCES 1 Sutherland DR Anderson L Keeney M et al The ISHAGE Guidelines for CD34 Cell Determination by Flow Cytometry J Hematotherapy 1996 3 213 226 2 Gratama JW Orfao A Barnett D et al Flow cytometric enumeration of CD34 hematopoietic stem and progenitor cells European Working Group on Clinical Cell Analysis Cytometry 1998 34 128 142 FLO 30592 Sequential Gating Techniques Phase Il Sequential Boolean gating techniques are used to define the CD34 stem cells NOTE Negative reagent controls isotypic isoclonic are of limited if any utility in the enumeration of rare events such as CD34 cells Some isotype controls can stain more cells nonspecifically than are stained specifically by a CD34 conjugate Studies of a large number of normal hematopoietic samples have shown that the sequential gating approach best delineates specific from nonspecific staining and that traditional isotype controls provide no useful information regarding the levels of nonspecific staining in the flow cytometric analysis of rare events For this reason the use of isotypic isoclonic controls is not recommended In their place sequential Boolean gating and
43. nce 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 17 of 36 Flow Cytometry Checklist 07 11 2011 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 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 e Sampling of QC policies and procedures includes acceptable control type frequency for each flow cytometric application e Sampling of QC records e Biannual
44. nchmark against which laboratory performance can be evaluated Evidence of Compliance 10 of 36 Flow Cytometry Checklist 07 11 2011 v Records of enrollment participation in an educational peer comparison program for leukemia lymphoma interpretive flow cytometry OR records for participation in a laboratory developed program circulating cases with other laboratories or within the laboratory s own practice with documentation of peer review REFERENCES 1 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 QUALITY MANAGEMENT AND QUALITY CONTROL GENERAL ISSUES Inspector Instructions e Sampling of QM policies and procedures e QM QC program including pre analytic analytic and post analytic monitor records and corrective action when indicators do not meet threshold e Incident error log and corrective action e Records of high school graduate high complexity test review by supervisor e How do you evaluate data on the incident error log How do you determine appropriate corrective action e As a staff member what is your involvement with quality management e How do you detect and correct laboratory errors e Follow an incident identified on the incident error log and follow actions i
45. ncluding notification and resolution e Select several problems identified by the QM plan and follow tracking and corrective action Determine if the methods used led to discovery and effective correction of the problem FLO 20000 Documented QM QC Plan Phase II The flow cytometry laboratory has a written quality management quality control QM QC program NOTE 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 requirements in the Laboratory General Checklist pertain to the flow cytometry laboratory 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 11 of 36 Flow Cytometry Checklist 07 11 2011 REVISED 07 11 2011 FLO 20100 Unusual Laboratory Results Phase Il There is a documented system in operation to
46. nophenotyping by flow cytometry Am J Clin Pathol 2000 113 95 106 3 Wood BL et al 2006 Bethesda International Consensus Recommendations on the Immunophenotypic Analysis of Hematolymphoid Neoplasia by Flow Cytometry Optimal Reagents and Reporting for the Flow Cytometric Diagnosis of Hematopoietic Neoplasia Cytometry Part B Clinical Cytometry 2007 72B S12 S22 DNA CONTENT AND CELL CYCLE ANALYSIS e Sampling of DNA analysis policies and procedures includes reference to established methodology and list of acceptable neoplasms for DNA analysis e Sampling of specimen evaluation records e Sampling of DNA analysis linearity and QC records e Sampling of sub optimal specimen rejection records log e What is your laboratory s course of action when unacceptable or sub optimal specimens are received e How does your laboratory ensure debris and aggregates are excluded from consideration 33 of 36 Flow Cytometry Checklist 07 11 2011 e How does your laboratory ensure that the analysis contains neoplastic cells of interest e How does your laboratory ensure detection of DNA aneuploidy FLO 31000 Neoplastic Cell Content Phase Il There are 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 pop
47. olvement with quality management e How do you detect and correct laboratory errors DISCOVER is a technique that can be used to drill down or further evaluate areas of concern DISCOVER uncovered by the inspector Follow the specimen and teach me are two examples of Discovery Utilizing this technique will allow for the discovery of pre analytic analytic and post analytic processes while reviewing multiple requirements simultaneously Example e Select several occurrences in which QC is out of range and follow documentation to determine if the steps taken follow the laboratory policy for corrective action Flow Cytometry Checklist 07 11 2011 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 requirements Inspectors preferably should have participated in a recent CAP Inspector Training activity Inspectors should to the greatest extent possible be peers of the laboratory being inspected An inspection of a laboratory section or department will include the discipline specific checklist s the Laboratory General Checklist and the All Common Checklist COM In response to the ongoing request to reduce the redundancy within the Accreditation Checklists the CAP accreditation program is introd
48. ometry Checklist 07 11 2011 Sampling of flow cytometry specimens labeling OBSERVE KaeeeeeeereeeeeeeegeeEreeeen OCC e What is your course of action when you receive unacceptable sub optimal flow cytometry specimens FLO 20500 Specimen Collection Manual Phase Il There is a documented procedure describing methods for patient identification patient preparation specimen collection and labeling specimen preservation and conditions for transportation and storage before testing consistent with good laboratory practice FLO 22000 Specimen Identity Integrity Phase Il Procedures are adequate to verify sample identity and integrity includes specimens of blood body fluids and tissues Evidence of Compliance Specimen collection and handling procedure AND v Patient collection and processing records REVISED 07 11 2011 FLO 22050 Specimen Rejection Criteria Phase Il There are documented criteria for the rejection of unacceptable specimens or the special handling of sub optimal specimens NOTE This requirement does not imply that all unsuitable soecimens are discarded or not analyzed If for example improper storage hemolyzes a sample and hemolysis interferes with testing there must be a mechanism to notify clinical personnel responsible for patient care If the treating physician desires the result then the laboratory must note the condition of the sample on the report Some or all tests may not be analytically va
49. ompensation settings REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edition CLS 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 Laser Current Phase For laser instruments there are procedures in place to ensure acceptable and constant laser current NOTE For some instruments current is a better gauge of laser performance than is power output which may be relatively constant FLO 30280 Calibration Laser Review Phase Il The results of instrument calibration and laser output checks where appropriate are reviewed monthly by the person in charge of technical operations of the flow cytometry laboratory Evidence of Compliance Y Records of follow up for any outliers or trends as applicable Temperature Dependent Equipment Inspector Instructions e Sampling of temperature logs refrigerator freezer water bath heat blocks 23 of 36 Flow Cytometry Checklist 07 11 2011 REVISED 06 17 2010 FLO 30290 Temperature Checks Phase Il Temperatures are checked and recorded daily 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
50. ould 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 Evidence of Compliance Written procedure for apheresis specimen handling REFERENCES 1 Keeney M et al Single platform flow cytometry absolute CD34 cell counts based on the ISHAGE guidelines Cytometry 1998 34 61 70 Monoclonal Antibodies Reagent Class Phase Il Appropriately conjugated Class Il or Class III anti CD34 monoclonal antibodies are used NOTE Class reagents are not recommended Class II reagents conjugated to FITC are not recommended Evidence of Compliance Written procedure defining use of appropriate class of monoclonal antibodies AND d Reagent logs 29 of 36 Flow Cytometry Checklist 07 11 2011 FLO 30585 CD34 Events Phase Il A statistically valid number of CD34 events are 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 c
51. rector or designee NOTE The QC data for tests performed less frequently than once per month should be reviewed when the tests are performed Evidence of Compliance Y Records of QC review with documented follow up for outliers trends or omissions 07 11 2011 Comparability of Instrument Method Phase Il If the laboratory uses more than one instrument method to test for a given analyte the instruments methods are checked against each other at least twice a year for correlation of results NOTE This requirement applies to tests performed on the same or different instrument makes models or by different methods This comparison must include all nonwaived instruments methods The laboratory director must establish a protocol for this check 20 of 36 Flow Cytometry Checklist 07 11 2011 Quality control data may be used for this comparison for tests performed on the same instrument platform with both control materials and reagents of the same manufacturer and lot number Otherwise the use of human samples rather than stabilized commercial controls is preferred to avoid potential matrix effects The use of pooled patient samples is acceptable since there is no change in matrix In cases when availability or 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 when applicable to have the same response as
52. s e Instrument records promptly retrievable OBSERVE e How does your laboratory monitor instrument reproducibility e How does your laboratory ensure each fluorochrome is appropriately calibrated e How does your laboratory determine appropriate color compensation settings FLO 25100 FLO 25150 FLO 25200 FLO 25250 FLO 25300 21 of 36 Flow Cytometry Checklist 07 11 2011 Function Checks Phase Il Appropriate function checks are performed for all instruments prior to testing patient samples NOTE There must be a schedule and procedure at the instrument for appropriate function checks These may include but are not limited to electronic mechanical and operational checks The procedure and schedule must be as thorough and as frequent as specified by the manufacturer Function checks should be designed to check the critical operating characteristics to detect drift instability or malfunction before the problem is allowed to affect test results All servicing and repairs must be documented Optical Alignment Phase Il There are procedures for monitoring of optical alignment where applicable and instrument reproducibility at least daily or after each time the flow cytometer is restarted and there is 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 t
53. separately covered in the Hematology and Coagulation Checklist IMMUNOPHENOTYPING select a representative assay and follow the entire process from specimen receipt to final result reporting e lf problems are identified during the review of immunophenotyping procedures further evaluate the laboratory s responses corrective actions and resolutions DISCOVER Blood Lymphocyte Subset Enumeration Inspector Instructions e Sampling of lymphocyte subset analysis policies and procedures includes procedure describing method to set markers cursors to distinguish between negative and positive fluorescence cell populations e How have you established or verified reference ranges e How does your laboratory ensure specimen integrity e How are specimens stored after initial processing e How does your laboratory validate lymphocyte gates e How are results of lymphocyte subset analysis corrected for gate purity FLO 30430 Specimen Integrity Phase Il There is 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
54. tion CLS 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 Gating Technique Phase Il Appropriate gating techniques are used to select the cell population for analysis NOTE This may involve a combination of light scatter and or fluorescence measurements This is particularly important if the cell samples have a low lymphocyte count and or a relatively high monocyte granulocyte count Lymphocyte gates may be validated using linear forward angle light scatter and 90 degree side scatter or using CD45 FITC and CD14 PE monoclonal antibodies REFERENCES 1 National Institute for Allergy and Infectious Diseases Division of AIDS guidelines for flow cytometric immunophenotyping ver 1 0 Jan 1993 Gate Purity Phase Il Results of lymphocyte subset analysis are corrected for gate purity as appropriate NOTE When gt 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 Evidence of Compliance Written procedure defining method for correction of results for gate purity R
55. ucing the All Common Checklist The purpose of the All Common Checklist is to group together requirements that were redundant in Laboratory General and the discipline specific checklists Therefore the CAP centralized all requirements regarding proficiency testing procedure manuals test method validation and critical results into one checklist the COM checklist Note for non US laboratories Checklist requirements apply to non US laboratories unless the checklist items contain a specific disclaimer of exclusion PROFICIENCY TESTING Inspector Instructions e Sampling of peer education records SER FLO 18385 Peer Education Program Phase For laboratories that perform only interpretations of flow immunophenotyping data for leukemias and lymphomas the laboratory participates 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 be
56. ulation 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 Evidence of Compliance Written procedure defining method for verifying the presence of neoplastic cells AND v Records of specimen evaluation FLO 31010 Cellular Debris Phase Il There are 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 Evidence of Compliance Written procedure defining method to account for debris and aggregates FLO 31020 DNA Content Linearity Phase Il Criteria are established for determining acceptable linearity for DNA content measurement using cells or particles of known relative fluorescence FLO 31050 Procedure Manual Phase II The staining and analytical procedures described in the procedure manual are based upon established methodology reference cited
57. whole blood that are analyzed within 24 hours of drawing Analyses on older samples are possible if the laboratory has verified FLO 30450 FLO 30460 FLO 30470 26 of 36 07 11 2011 the absence of statistical differences between the fresh and aged specimen phenotype fractions being evaluated Flow Cytometry Checklist Evidence of Compliance v Records of specimen evaluation e g viability results as applicable REFERENCES 1 Clinical and Laboratory Standards Institute CLSI Enumeration of Immunologically Defined Cell Populations by Flow Cytometry Approved Guideline Second Edition CLS 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 Specimen Storage Phase Il Specimens are 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 Evidence of Compliance Written procedure for specimen storage REFERENCES 1 American Society for Microbiology Manual of clinical immunology 4th ed Washington DC ASM 1992 940 2 Clinical and Laboratory Standards Institute CLSI Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells Approved Guideline Second Edi
58. y Checklist Evidence of Compliance Written procedure defining controls used for DNA analysis AND v Records of QC results 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 Aneuploid Cell Population ID Phase Il Analytical criteria are 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 GO 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 GO G1 pe
59. y 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 Inspector Instructions BLL GELTZ D D D ie Sampling of test procedures for reagent handling e Sampling of new reagent shipment verification records e Sampling of ambient temperature logs if reagents stored at ambient temperature e Sampling of reagents expiration date labeling storage OBSERVE e How do you store reagents and controls used in test procedures e How do you verify new reagent lots e What process does your laboratory follow to ensure manufacturer s recommendations are followed regarding the use of reagents controls in kit procedures e What are your laboratory s criteria for mixing components from one lot number of reagent kit with components from another lot number of kit e How does your laboratory manage and control reagent inventory FLO 23000 Reagent Labeling Phase Il Reagents calibrators cellular controls and solutions are properly labeled as applicable and appropriate with the following elements 1 Content and quantity concentration or titer 2 Storage requirements 3 Date prepared or reconstitute
60. y must verity the recovery ranges supplied by the manufacturer Evidence of Compliance Written procedure defining methods used to establish or verify control ranges AND v Records for control range verification of each lot 07 11 2011 QC Corrective Action Phase Il There is documentation of corrective action taken when 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 FLO 24250 FLO 24300 REVISED FLO 24475 REVISED FLO 24650 19 of 36 07 11 2011 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 Flow Cytometry Checklist QC Handling Phase Il Control specimens are tested in the same manner and by the same personnel as patient samples NOTE QC specimens must be

Master Flow Cytometry Checklist - College of American Pathologists

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