Kit Contents FL-10 FL-12 FL-07 FL-09. Important: Before You Begin. New for this Mailing. Reporting Code Changes. Critical Reporting Information

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1 FL-B 2014 Kit Instructions CAP 2014 Flow Cytometry Survey FL FL1 FL2 Table of Contents Kit Contents Kit Contents... 1 Important: Before You Begin... 1 Detailed Testing Instructions... 2 FL FL1 FL2 FL-07 FL-12 FL-07 FL-09 FL-10 FL-12 Reporting Your Results... 3 Biohazard Warning... 4 For Assistance... 5 Master Lists... 5 Important: Before You Begin New for this Mailing Reporting Code Changes The following manufacturers have either deleted or updated codes for this mailing: Critical Reporting Information Due to the nature of the proficiency testing (PT) specimens, laboratories may need to visually inspect their analyses and manually regate their specimens. Please refer to the instructions provided by the manufacturer of your instrument to perform manual gating by an approved method. Storage and Stability Instructions 1. Store specimens upright, tightly capped, at 2-8ºC when not in use. Avoid unnecessary cycles of warming and cooling. Protect from freezing, from temperatures above 30ºC, and from prolonged time at room temperature (18-26ºC). 2. Supernatant that is reddish in color is normal and does not indicate deterioration. Other discoloration (eg, very dark red supernatant) or unacceptable results may indicate deterioration. 3. Stability: Survey FL1: Opened stability: Test immediately Unopened stability: 7 days Survey FL2: Opened stability: Test immediately Unopened stability: 7 days Important: See the Biohazard Warning at the end of these instructions. Customer Contact Center option 1 (domestic) or option 1 (international) 1

2 Detailed Testing Instructions This Survey is divided into 2 modules, each with its own set of instructions: Part A - Lymphocyte Immunophenotyping (FL, FL1) Part B - DNA Content and Cell Cycle Analysis (FL, FL2) Part A - Lymphocyte Immunophenotyping (FL, FL1) Part A1 Preanalytic Variables Complete the questions on the result form at the time of specimen receipt. Parts A2 and A3 Testing Information 1. Specimens FL-07 through FL-09 contain 1.5 ml stabilized whole blood. In accordance with current recommendations by the CDC and the CLSI, we recommend the use of CD3/CD4 and CD3/CD8 in multi-color staining combinations. Report an average CD3+ value from the various CD3 results obtained from multiple tubes. 2. If your laboratory identifies lymphocytes using no gating or by light scatter gating, you must provide CD45 and CD14 results. Record the percent of bright CD45+/CD14- cells and correct data for lymphocyte gate purity as shown below. Raw % (% bright CD45+, CD14-) 100 eg, CD4+ value of 45% bright CD45+, CD14- value of 92% CD4+ % corrected = 49% Note: Laboratories using light scatter gating to define lymphocytes should define internal guidelines for correction of lymphocyte subset values derived from light scatter gates that contain less than 95% CD45+ lymphs. Laboratories may reference CLSI document H42-A2, Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline, 2nd Edition. 3. If your laboratory uses CD45 vs. side scatter gating, you do not need to report CD45 and/or CD14 results. Dual-Platform Users 1. Only report percent positive results (Part A2). 2. Absolute counts should not be reported off of dual-platform systems (ie, systems that require results from a hematology analyzer to perform absolute counts) as the material is not appropriate for hematology analyzers. Single-Platform Users 1. Report percent positive and absolute counts (Parts A2 and A3). 2. Absolute counts should only be reported if your laboratory uses an instrument/ method that directly performs absolute counts. Note: You must fill the bubble indicating dual- or single-platform and enter an instrument code on the result form in order to be graded correctly. Leaving either field blank may result in a penalty on your evaluation. Part B - DNA Content and Cell Cycle Analysis (FL, FL2) Part B1 Preanalytic and Specimen Information Complete the questions on the result form at the time of specimen receipt. Parts B2 and B3 Instrumentation/Software Fill the appropriate bubble and/or fill the boxes. 2 Flow Cytometry 2014

3 Part B4 DNA Content Results 1. Fill the appropriate bubble or fill the boxes. 2. Specimens FL-10 through FL-12 contain approximately cells per 1.1 ml fixed in an alcohol mixture. 3. FL-10 (calibrator) contains normal cells with a normal (diploid) DNA content. FL-11 and FL-12 are mixtures of FL-10 (normal cells) and cell lines that may or may not have an aneuploid DNA content. Do not add aliquots of FL-10 or normal cells to FL-11 or FL-12. Thoroughly wash cells twice with tissue culture media before processing and staining with your laboratory s usual DNA preparation procedure. During the wash step, cells must be gently vortexed or disaggregated by aspiration using a syringe with a large bore needle to disrupt clumps. If clumps are not disassociated, incorrect results will occur. Exclude any fixation steps in your procedure; the cells are already fixed. The DNA index (D.I.) is defined as: D.I. = G 0 /G 1 peak fluorescence channel number for unknown cells G 0 /G 1 peak fluorescence channel number for normal diploid cells By International Convention established by the Committee on Nomenclature of the Society of Analytical Cytology, the test specimen should be called DNA aneuploid when at least 2 separate G 0 /G 1 peaks are demonstrated (Hiddemann W, et al. Convention on nomenclature for DNA cytometry. Cytometry. 1984;5: ). Reporting Your Results General Reporting Instructions 1. Each mailing, verify the accuracy of your reporting codes (eg, manufacturer, method, instrument, reagent) by reviewing the online result form or the Method Summary Page attached to the front of your result form. 2. For any testing that you do not routinely perform in your laboratory, leave all reporting areas for that test blank unless otherwise noted. 3. Exception Codes: If you must report an analytical problem for a test or individual analyte, leave the result area for that section blank and fill one of the following bubbles on the result form. The exception code bubble that you fill in will only apply to the result area(s) left blank. Documentation on the use of these codes is the responsibility of the laboratory and should be kept internally. 11 Unable to analyze Use code 11 to indicate why specimens were not analyzed; for example, instrument not functioning, reagents not available, etc. 22 Result is outside the method/instrument reportable range Use code 22 if you obtain a high or low result outside the reportable range of your method or instrument. Do not use this code if there is an option to fill a bubble for a greater than or less than result. 33 Specimen unsatisfactory To use code 33, you must contact the CAP. 4. Corrections can be made at any time prior to the due date printed on the result form. Review all entries for accuracy prior to online approval or before sending by fax or mail. For results that are approved online, corrections must also be done online. Faxed or mailed corrections will not be accepted. Customer Contact Center option 1 (domestic) or option 1 (international) 3

4 Per the Federal Register Proficiency Testing (PT) specimens must be tested with the laboratory s regular workload, using routine methods, and testing the PT specimens the same number of times it routinely tests patient specimens. If referral for testing is routinely performed for patient specimens, the practice cannot be followed for PT specimens. Referral is considered to be movement of the specimen from a laboratory with a CLIA identification number to another laboratory that has a different CLIA identification number. Laboratories must ensure that personnel do not share results or refer PT specimens for any reflex or testing outside their CLIA identification number. Disclaimer Survey samples, their progeny, unmodified derivatives, or modifications thereof may not be transferred or incorporated into a program intended for sale. Survey samples, their progeny, unmodified derivatives, or modifications thereof, reagents, and disposable equipment used in proficiency testing, when disposed of, should be autoclaved or incinerated and disposed of as hazardous waste. Submitting Results 1. Results must be received at the CAP no later than midnight, Central Time by the due date on the result form. Results cannot be accepted if received after the due date. 2. Your laboratory must establish a laboratory web account, referred to as Opting In, to submit results online. Information about opting in and a unique PIN was mailed to all laboratory directors. If your laboratory director does not have this information, please contact the CAP for a replacement letter. 3. Laboratory staff who will enter results online must first establish a personal web account. Once a personal web account is established, laboratory staff can request access to their laboratory s information. Biohazard Warning All Survey samples should be treated as if potentially infectious and should be handled as if they are capable of transmitting disease. Survey samples are prepared from blood or other source material obtained from human donors or animals. When working with Survey samples, precautions should be taken to protect yourself and others from accidental exposure to infectious agents such as HIV, HBV, and HCV. HIV can be transmitted through accidental parenteral inoculation, mucous membranes, or non-intact skin contact with HIV infected blood or body fluids. HBV and HCV can be transmitted through accidental parenteral inoculation, mucous membranes, non-intact skin contact, aerosolization or ingestion. Precautions described in CDC and FDA recommendations and OSHA blood borne pathogen rules should be followed at all times when handling Survey samples and reagents. Such precautions include the following: Gloves should be put on before opening the container and should be kept on throughout the period samples are handled. Replace gloves if contaminated, or if their ability to function as a barrier is compromised. At high altitudes, samples should be opened in a hood or biologic safety cabinet. There should be no eating, drinking, or smoking in the laboratory. Hands should be washed after removing gloves and before leaving the testing area. Survey samples and reagents should be kept in separate refrigerators from those containing blood or blood components for transfusion. Survey samples, reagents, and disposable equipment used in testing should be autoclaved or incinerated and disposed of as hazardous waste. If there has been an accident in which you have been exposed to the Survey s materials, please call the CAP Hot Line at (domestic) or (international) at any time. You can access Safety Data Sheets (SDS) (formerly known as MSDS) by logging on to cap.org and clicking on the Accreditation and Laboratory Improvement tab. 4 Flow Cytometry 2014

5 For Assistance For replacement materials, please contact the CAP within 10 calendar days of the ship date for information. Provide your CAP number and contact information with all correspondence. Telephone: Website: Address: option 1 (Monday - Friday, 7:00 am 5:30 pm Central Time) International Participants: option 1 contactcenter@cap.org cap.org CAP Surveys Program 325 Waukegan Road Northfield, IL Instrumentation Master List Deleted codes New/Updated codes 1042 BD FACSCalibur 2074 BD FACSCanto 1917 BD FACSCanto II 1271 BD FACSCount 1868 BD FACScan 1295 BD FACSort 1756 Coulter Cytomics FC Coulter Epics Altra 1190 Coulter Epics Elite (ALL models) 1228 Coulter Epics XL (ALL models) 1374 Coulter Gallios 1780 Coulter Navios 1869 Coulter Profile 2103 IMAGN Ortho Cytoron Absolute 0010 Other, specify on result form Inclusion on this master list does not imply FDA approval. Lymphocyte Immunophenotyping Master List Deleted codes New/Updated codes Method of Preparation 122 Ammonium chloride 120 BD lyse 395 Beckman Coulter OptiLyse C 118 Coulter Immunolyse 119 Coulter Q-Prep, TQ-Prep 407 DAKO Uti-Lyse 121 Ortho lyse 010 Other, specify on result form Gating Information* 196 No gating 197 Lymphocytes by light scatter 199 Lymphocyte gating by CD45 and side light scatter 010 Other, specify on result form Reagent Manufacturer 2335 BD Biosciences 1331 BD FACSCount Reagent 2777 BD MultiTest 1787 BD MultiTest/TruCount 1131 BD Simultest 2780 BD TriTest 1786 BD TriTest/TruCount 1132 Caltag 1734 Coulter Clone 1133 Cyto-Stat/Coulter Clone 2477 Cyto-Stat tetrachrome 2478 Cyto-Stat trichrome 2957 Dako 1732 GenTrak 1130 Immunotech/Coulter IOTest 2399 Ortho 0010 Other, specify on result form Fluorochrome 57 APC 54 APC-Cy7 24 Cy-Chrome 23 ECD 17 FITC 53 PE or RD1 58 PE-Cy5 or PC5 55 PE-Cy7 or PC7 22 PerCP 56 PerCP-Cy TC (Tri-Color) 01 Other, specify on result form Inclusion on this master list does not imply FDA approval. * If your laboratory uses no gating or light scatter gating, you must provide CD45 and CD14 results. If your laboratory uses CD45 vs. side scatter gating, you do not need to report CD45 and/or CD14 results. Customer Contact Center option 1 (domestic) or option 1 (international) 5

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