Fredrick L. Kiechle, MD, PhD
Q. What is the requirement for manual body fluid cell counting, and what are the various ways it can be done? Please explain the procedural controls.
A. CAP Laboratory Accreditation Program hematology checklist question HEM.35340 (phase I) addresses quality control and says at least one cell count control specimen must be analyzed, or a procedural control used, for each eight hours of patient testing. The requirement can be met with assayed liquid control material, a previously assayed patient sample, or a procedural control, according to the accompanying note. The note provides an example of a procedural control: correlation of the cell count with the cellularity of a stained slide prepared by a standard, validated method. Liquid controls performed by a hemocytometer should be run in duplicate, it says. The evidence of compliance are records of the cell count or the procedural controls documented at a defined frequency.
How it can be done is answered in a chapter that I wrote for the CAP’s Color Atlas of Body Fluids, published in 2006. The chapter is titled “Technical Considerations”; its section on cell counts follows.
If a cell count or differential is ordered, the sample should be collected in an anticoagulant, such as EDTA or heparin. Cell counts should not be performed on clotted or partially clotted specimens, as the count may be inaccurate and small fibrin clots can clog the tubing in automated cell counters. Cell counts should be performed in a hemocytometer counting chamber or, if volume and cellularity are sufficient, in an automated cell counting instrument. Automated and manual WBC counts compare very well when collected in EDTA, but if the specimen is not anticoagulated, the counts will drop in both methods. Cerebrospinal fluids with very low counts may require a manual count. Each laboratory should establish the lower limit for counting cells on automated instruments, and hemocytometer counts should be used when counts fall below these limits. The hemocytometer chamber should be scanned at low power to determine if dilution will be necessary. Only certified pipettes or commercial dilution systems should be used. Counts should be performed in duplicate utilizing both chambers. The laboratory must also use an additional procedure beyond unstained bright field microscopic visualization of cells on the hemocytometer to reliably distinguish erythrocytes from leukocytes or other nucleated cells. Suggested techniques include acid rinsing of the fluid sample to lyse erythrocytes after initially counting all cells, addition of a stain such as methylene blue to improve recognition of nonerythrocytes, or phase microscopy.
Body fluid differential cell counts are most accurate when performed on cytospin preparations or a similar concentrating system, as these have been shown to be superior to counts from hemocytometer chambers or wedge (push) slide preparations. The cytocentrifuge preparations stained with Gram stain also show increased sensitivity for the detection of bacterial organisms over a wedge preparation. However, push smears may be useful if the fluid is grossly bloody. Highly viscous synovial fluids may require direct “pull-apart” smears or addition of hyaluronidase in phosphate buffer to the fluid prior to cytocentrifuge preparation. Automated differentials are not as accurate as manual differentials and should not be used. The hemocytometer chamber should also not be used for the differential count unless a cytocentrifuge slide is not available, in which case only a differential of mononuclear versus polymorphonuclear cells should be reported. The cytocentrifuge slide(s) should be scanned at low power to determine if clumps of cells or large ma-lignant cells are present. On high power examination, the nonerythrocytic cells should be divided into groups including neutrophils, lymphocytes, monocytes, eo-sinophils, basophils, more immature white blood cell categories, and cavity lining cells if present. One hundred cells should be counted, if possible, or all cells present if the sample is hypocellular. Microorganisms should also be reported, as well as their location (extracellular, intracellular).
If the initial hemocytometer WBC count of a cerebrospinal fluid is zero, it is still useful to concentrate the specimen by preparing a cytocentrifuge slide, increasing the chance of identifying rare abnormal cells, especially in patients with a history of central nervous system malignancy. A differential count would only be performed in this circumstance if cells were present. Although some laboratories use elevated cell counts as a decision point for preparing a cytocentrifuge slide differential count, this practice is not recommended, as malignant cells can be present in fluids with low cell counts. Some laboratories use a slide scan, instead of a differential count, in these situations. The slide is reviewed and should include statements on the general cell composition (e.g. lymphs predominate), the presence or absence of malignant cells, and the presence or absence of microorganisms.
It can be difficult to distinguish mesothelial cells, macrophages, and “other” cells in body fluids. Thus, there is considerable variation in the way cells in body fluids are counted in the differential. Some experts recommend that “other” cells, such as mesothelial cells, histiocytes, synoviocytes, and malignant cells, be enumerated separately or estimated as a percentage, either individually or under the broad category of “other.” As an alternative approach, they can be quantitated separately as 1+, 2+, or 3+.
Since macrophages and mesothelial cells are often difficult to distinguish from each other, they can be counted together, either as part of the differential or separately. Failure to include “other” cells in the differential count may convey a false impression of the relative number of leukocytes present. For example, if a patient has a low total cell count with 100 mesothelial/macrophage/monocytes for every 80 neutrophils and 20 lymphocytes, failure to include the “other” category in the differential will result in a report of 80 percent neutrophils and 20 percent lymphocytes, giving a false impression of an acute cellular reaction. An optimal reporting format identifies neutrophils, lymphocytes, and “other mononuclear cells.” Clumps of cells should not be counted, and it is critical to note separately but clearly that malignant cells are present. Each laboratory should establish criteria for pathologist review, which should include, but not necessarily be limited to, cases in which malignant cells are suspected.
Galagan KA, Blomberg D, Cornbleet PJ, Glassy EF, eds. Color Atlas of Body Fluids. An Illustrated Field Guide Based on Proficiency Testing. Northfield, Ill.: College of American Pathologists; 2006:9–11.
Katherine A. Galagan, MD
Director, Clinical Laboratories
Virginia Mason Medical Center
Q. With the laboratory workforce shrinking, autovalidation has become a big issue. What guidelines are available for setting up autovalidation criteria?
A. In an era of decreasing workforce resources and increasing pressure on laboratories to meet demands for efficiency, greater productivity, and acceptable turnaround times, many laboratories have considered autovalidation or autoverification of laboratory results as a possible solution.
The CAP provides in its 2011 laboratory general checklist the following definition for autoverification:
Autoverification is the process by which patient results are generated from interfaced instruments and sent to the laboratory information system, where the results are compared against laboratory-defined acceptance parameters. If the results fall within these defined parameters, they are automatically released to patient reporting formats without additional laboratory staff intervention. Any data that fall outside the defined parameters are reviewed by laboratory staff before they are reported.1
Guidelines for autoverification of clinical laboratory results can be found in a publication of the Clinical and Laboratory Standards Institute.2 The publication provides an outline that will enable individual laboratories to design, implement, validate, and customize rules for autoverification in which analytical results are electronically checked against certain criteria, such as reference ranges, quality control results, moving averages, instrument flagging, delta checks, maintenance and lot checks, clinician information/requests, and critical limits.2 Each medical facility should customize these processes under the supervision of its laboratory director and staff and with consideration for its patient population. Computer-based algorithms using Boolean logic, or rules consisting of a series of questions or using “and” or “or” statements, are designed to eliminate outliers and lead to a logical decision for autoverification before the laboratory result is released.
An American Association for Clinical Chemistry membership survey in 2009 found that 56 percent of laboratories do not autoverify laboratory results primarily because they lack the time to “figure it out.”3 In a four-part series in 2010, a roadmap was provided to help laboratories through the process of autoverification.4 The guide outlines how to get started (use of algorithms and rules) and discusses software options and where to locate the autoverification function (instrument, middleware, or LIS), initial testing, regulation and inspection considerations, and the go-live process and post-live aspects. The experience of a large network laboratory in Virginia may provide a practical how-to guide and insight into the pitfalls of autoverification, including early lessons learned, autoverification in hematology and chemistry, patient identification and delta checks, regulatory requirements, and a retrospective review of the impact of autoverification on the laboratory.5
1. Laboratory general checklist, CAP Laboratory Accreditation Program, July 11, 2011, Northfield, Ill.: College of American Pathologists.
2. Clinical and Laboratory Standards Institute. Autoverification of Clinical Laboratory Test Results; Approved Guideline. CLSI document AUTO 10-A, Vol. 26, No. 32, Wayne, Pa.: CLSI; 2007.
3. Leveraging autoverification for greater lab efficiency. Autoverification practices survey (audio conference). AACC; April 22, 2009.
4. Roadmap to autoverification—Simple steps to automate your lab’s total manual verification process are provided. Advance for Administrators of the Laboratory. 2010;19:parts I–IV. January–April.
5. Johnson J, Stelmach D. Autoverification of test results. Clin Lab News. 2007;33:9.
Gifford Lum, MD
Associate Chief, Clinical Pathology
Pathology and Laboratory Medicine Service
VA Boston Healthcare System
Member, CAP Chemistry
Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla. Use the reader service card to submit your inquiries, or address them to Sherrie Rice, CAP TODAY, 325 Waukegan Road, Northfield, IL 60093; firstname.lastname@example.org.