Feature Story

Flow cytometry’s ’10-most-wanted’ checklist items

December 2003
Anne Ford

Billboard’s Top 40. The Forbes 400.

With all the snazzy lists floating around, isn’t it time flow cytometry laboratories got their own? Here, then, are 10 key flow cytometry checklist items, as detailed by R. Bruce Williams, MD, in the Sept. 17 Laboratory Accreditation Program flow cytometry lab inspection audioconference.

» Checklist question FLO.20010 asks whether the laboratory has documented procedures for patient identification and preparation, specimen collection and labeling, specimen preservation and conditions for transportation, and storage before testing. "This question involves evaluation of many different aspects of preanalytical specimen handling," Dr. Williams said. He advised laboratories to make sure their specimen labeling is accurate and the sample absolutely identified.

The laboratory must identify the type of anticoagulant it will accept-EDTA, heparin, or ACD. For stem cell collection, addressed in FLO.22000, FLO.22050, and FLO.22100, "the anticoagulant and nature of collection must be specified," Dr. Williams said. Labs must also detail specimen transportation and storage requirements, which must mandate transportation at room temperature and prohibit refrigeration. Quantitative tests cannot use ACD because of its dilutive effect. For CD4 testing, Dr. Williams referred laboratories to the CDC guidelines published in the Jan. 31, 2003 Morbidity and Mortality Weekly Report. It addresses many specific details of CD4 testing, such as the fact that samples are good for 72 hours.

» Checklist item FLO.23800 reads: For quantitative tests (e.g., CD4+, CD34+ cell concentrations), are at least 2 levels of positive cellular controls analyzed daily to verify the performance of reagents, preparation methods, and staining procedures? Note: One of the levels of these controls should be at (or near) clinical decision levels (e.g., low CD34). The CAP added this question to comply with CLIA requirements. Dr. Williams said the next checklist update will make it clear that the requirement applies to CD4 and CD34 only.

"The laboratory needs to determine its own acceptable range," Dr. Williams pointed out. "It’s common practice to use a control with a low CD34 positivity because precision can be an issue in this case."

» Another checklist item concerned with quality control, FLO.23925, requires that when performing two levels of controls, laboratories establish a statistically valid target mean and acceptance range through repetitive analysis that includes previously tested control materials. For assayed commercial controls, the manufacturer’s range can be used for initial verification that the lot or shipment is good until the laboratory has sufficient data to calculate in-house statistical limits.

» Where applicable, labs must establish procedures for monitoring optical alignment and instrument reproducibility on each day of use, item FLO.25150 stipulates. They must document this monitoring as well. Inspectors should make sure the instruments routinely pass the vendor’s acceptance criteria for daily bead testing and look for records that monitor daily compensation and daily voltage.

"The laboratory needs to check to see if there’s any significant variation," said Dr. Williams. "If there is, they need to record on the daily worksheet or on a different log why this variation occurred and what they did to take corrective action-and to make sure the corrective action actually solved the problem."

» Are appropriate standards for each fluorochrome (that is, fluorescent beads) run each day that the instrument is used as part of the calibration process? And are the results recorded for quality control purposes? asks item FLO.30250. It mandates that fluorescent beads, which are used in daily calibration, fall within the expected range. As Dr. Williams noted, beads can be used to examine color compensation and adequacy of separation as well.

» Laboratories must establish procedures for determining appropriate color compensation settings, says checklist item FLO.30260. Defaults to the standard range setting are stored in the instrument, and beads are run first. The newer instruments, Dr. Williams said, perform color compensation automatically.

"There must be some control performed using fresh samples," he added. "There are numerous ways to do this. One way would be to have normal peripheral blood lymphocytes that are drawn from a willing donor on a daily basis. Some laboratories use fresh patient samples from the hematology laboratory that are among the normal CBCs for that morning run." The laboratory should then evaluate the expression of mutually exclusive cell populations on a single tube (that is, the tube which contains the combination CD8-CD4). The laboratory should examine the result not only for the correct positive expression of these antigens on mature lymphocytes, but also for the fluorescent staining intensity and the double negatively staining cells in the target population.

» Checklist item FLO.30430-Is there a procedure in place to document specimen integrity?-pertains to blood lymphocyte subset enumeration and stipulates that laboratories aren’t required to perform viability testing on specimens less than 24 hours old. So "most laboratories only run specimens that are less than 24 hours old and don’t need to perform viability testing routinely," Dr. Williams said.

Labs that do run specimens more than 24 hours old usually use trypan blue to assess viability. If there is decreased cell viability, loss of cell subpopulations may occur, or the presence of dead cells may lead to spurious results. The checklist doesn’t require labs to routinely report the percentage of viability, but some report viability rates below 80 percent and allow the physician to decide whether to repeat the test on a new sample. "Even if the viability is less than 80 percent," Dr. Williams said, "this doesn’t necessarily lead to the rejection of the specimen because of the difficulty of obtaining the specimen-and the fact that useful information can be obtained, in many cases, even on less-than-ideal specimens."

» Viability testing also comes up in FLO.30564 in the CD34+ stem cell enumeration section of the checklist: Is there a procedure in place to document CD34 cellular viability, where applicable? Viability testing isn’t required on specimens that are stained and analyzed within four hours of drawing. Again, most laboratories don’t perform this viability testing routinely. Specimens are often drawn and tested within the same institution, so labs that perform CD34 testing generally have no trouble testing the specimen within four hours.

If the testing falls outside the four-hour time frame, however, laboratories must verify viability. They can do this with a fluorescent dye that would normally be excluded from viable cells, such as a 7AAD. "Gating would then be done on the CD34-positive, 7AAD-negative cells-which would be the viable cells-and the CD34-positive, 7AAD-positive cells, which are the dead cells," Dr. Williams said. Criteria for using a sample (the percentage of viable cells) for stem cell transplantation need to be developed by the laboratory in conjunction with the transplant team.

» Checklist item FLO.30610 pertains to samples submitted for phenotyping or potential leukemias or lymphomas. Laboratories must have a policy for determining when the percentage of viable cells in each test specimen should be measured, the item stipulates. Again, in most cases, if the sample is run within 24 hours of drawing, laboratories aren’t required to perform viability testing routinely. Otherwise, they use trypan blue and report the viability. "Many laboratories use a benchmark of 80 or 85 percent viability in order to report an accurate result," Dr. Williams noted. Whatever benchmark a laboratory uses, it should record the actual viability if it falls below that benchmark. "If a clonal population is not identified, a disclaimer should be made that the viability was low and that this might hinder the detection of an abnormal clone."

» FLO.30585 asks: Are a statistically valid number of CD34+ events collected to ensure clinically relevant precision and accuracy? The item notes that a minimum of 100 CD34+ events collected will provide a coefficient of variation of 10 percent. What if fewer than 100 events are recorded? "The specimen most likely shouldn’t be used," Dr. Williams said. Furthermore, "that sample will likely be rejected for a stem cell transfusion" because of the low number of stem cells available.

Dr. Williams would like to thank Charles W. Caldwell, MD, PhD, John L. Carey, MD, MS, and Fred R. Cox, PhD, for their assistance with the information in this article. Anne Ford is CAP TODAY senior editor.