REPRODUCIBILITY OF SYNOVIAL FLUID ANALYSES

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1 770 REPRODUCIBILITY OF SYNOVIAL FLUID ANALYSES A Study Among Four Laboratories H. RALPH SCHUMACHER, JR., MARIE S. SIECK, SUSAN ROTHFUSS, GILDA M. CLAYBURNE, DOROTHY F. BAUMGARTEN, BONNIE S. MOCHAN, and JEFFREY A. KANT Aliquots from 30 synovial fluids were submitted to 4 laboratories for comparison of leukocyte counts and differential cell counts, and to 3 laboratories for a search for and identification of crystals. Leukocyte counts showed only fair correlation (coefficients of ) with the reference laboratory. In synovial fluid from 4 patients, there was sufficient difference in leukocyte counts to cause the fluids to be erroneously classified as either inflammatory or noninflammatory. In 12 of 24 fluid specimens examined, percentages of neutrophils fell outside the 95% confidence limits of the value determined by the reference laboratory. In 7 of the 11 patients with crystals reported, discrepancies were From the ArthritisImmunology Center, Veterans Administration Medical Center, and the Hematology Section, William Pepper Laboratory, Hospital of the University of Pennsylvania, Philadelphia. H. Ralph Schumacher, Jr., MD: Professor of Medicine, University of Pennsylvania School of Medicine, and Director, ArthritisImmunology Center, Veterans Administration Medical Center and Hospital of the University of Pennsylvania; Marie S. Sieck: Technician, ArthritisImmunology Center, Veterans Administration Medical Center; Susan Rothfuss: Technician, Arthritis Immunology Center, Veterans Administration Medical Center; Gilda M. Claybume: Technician, ArthritisImmunology Center, Veterans Administration Medical Center; Dorothy F. Baumgarten: Technician, ArthritisImmunology Center, Veterans Administration Medical Center; Bonnie S. Mochan, PhD: Technical Manager, Hematology Section, William Pepper Laboratory, Hospital of the University of Pennsylvania; Jeffrey A. Kant, MD, PhD: Director, Hematology Section, William Pepper Laboratory, Hospital of the University of Pennsylvania, and Established Investigator of the American Heart Association. Address reprint requests to H. Ralph Schumacher, Jr., MD, Director, ArthritisImmunology Center, Veterans Administration Medical Center, University and Woodland Avenues, Philadelphia, PA Submitted for publication September 30, 1985; accepted in revised form February 10, found between the reports from 1 or more laboratories. More attention to quality control of synovial fluid analyses is important. Analysis of synovial fluid is widely accepted as a crucial aid in the evaluation of joint disease (13). Identification of crystals or infectious agents in joint fluid establishes a definite diagnosis of these diseases, and the leukocyte count can be used to broadly group diseases as inflammatory or noninflammatory. In a recent study, we found that the identification of crystals or bacteria and the leukocyte count were the only important factors in most diagnostic and therapeutic decisions that are made after arthrocentesis (4). The analysis of synovial fluid can present technical problems because of sample viscosity, and crystal identification requires skilled training. Little is known about reliability and reproducibility of these joint fluid examination results among laboratories, and no established quality control system is available to help laboratories evaluate their performance. To examine whether significant problems in reproducibility and quality control exist, we submitted aliquots of the same synovial fluids to 4 different laboratories for analysis. A variety of discrepancies were detected in leukocyte counts, differential cell counts, and crystal identification. The largest problem we found was in the detection and identification of crystals. MATERIALS AND METHODS Thirty consecutive heparinized synovial fluids, of sufficient volume, were obtained from patients at the Hospital of the University of Pennsylvania or Philadelphia Veterans Administration Medical Center and delivered to Arthritis and Rheumatism, Vol. 29, No. 6 (June 1986)

2 SYNOVIAL FLUID ANALYSES 77 1 the reference laboratory. The fluid samples were carefully shaken, immediately divided into 2cc aliquots, and delivered within 30 minutes to the 4 participating laboratories: the Arthritis Research Laboratory at the Philadelphia Veterans Administration Medical Center (which served as the reference laboratory), a Veterans Administration clinical laboratory, a university clinical laboratory, and a commercial laboratory used by the university outpatient clinics. All participating technicians were certified and were accustomed to performing synovial fluid analyses. Except for those in the reference laboratory who performed only synovial fluid analyses, the primary duties of the technicians were in hematology. There was no crossover of training between the laboratories. The technicians, except for those on the reference laboratory staff, did not know which fluids were part of the study. A leukocyte count, 100ce11 differential count, and search for crystals were requested for each specimen. The full analysis in the reference laboratory was completed in 1 hour. Clinical information on the patients was not known by any technicians examining the specimens. Procedures used in the different laboratories included manual leukocyte count, using 0.3% saline diluent in the reference laboratory and laboratory 4, normal saline in laboratory 2, and buffered ammonium oxalate in laboratory 3. Differential counts were done on Wrightstained smears or cytocentrifuge preparations in all laboratories. Laboratories 3 and 4 reported only percentages of polymorphonuclear leukocytes. Differential cell counts were completed in 2 or more laboratories on 24 fluids. A search for crystals was performed with compensated polarized light microscopy in 3 laboratories. Statistical comparison of values found between pairs of laboratories was performed by linear regression analysis. Estimates of precision for leukocyte counts and differential leukocyte percentages in the reference laboratory were obtained by having 2 technicians perform 38 replicate analyses on the same specimen for 4 separate synovial fluids. This also permitted a comparison of variability between technicians. The samples were chosen to encompass a range of leukocyte counts from approximately 20040,000/pl and neutrophil percentages from approximately 1090%. RESULTS Leukocyte counts. The synovial fluid leukocyte counts from the 4 laboratories are shown in Table 1. Figure 1 provides a scattergram of these counts. The overall correlation among participating and reference laboratories was fairly good. Correlation coefficients, compared with laboratory I, were 0.76, 0.80, and 0.80 for laboratories 2, 3, and 4, respectively. (Figure I). However, there were a number of samples with modest to marked differences in leukocyte counts. In synovial fluid from 4 patients, the difference in the leukocyte count would have caused the fluid to be Table 1. Patient number Svnovial fluid leukocvte counts from 4 laboratories* Labora Labora Laboratory 1 tory 2 tory SO OO * Leukocyte counts are x 103/mm3. Laboratory misclassified as inflammatory (1 patient) or noninflammatory (3 patients), when the results of 1 laboratory were compared with the consensus of the others (see Figure 1). In the results of assays on several other fluids, wide differences in leukocyte counts were displayed among laboratories, but results still fell within the inflammatory range. Leukocyte differentials. Table 2 shows the percentages of polymorphonuclear leukocytes (PMN) recorded. Figure 2 is a scattergram of percentages among the 4 laboratories. In I or more laboratories, the percentage of PMN in 12 patients synovial fluids, fell outside?2 SD of the value determined by the reference laboratory. However, the deviations were usually only slightly outside of the 95% confidence intervals (5). Correlation coefficients for PMN findings in laboratories 2, 3, and 4 versus the reference laboratory were 0.73, 0.84, and 0.94, respectively. In at least 3 cases, very marked differences (e.g., reference

3 772 I 40 0 o 0, 0. (3, WBC, LAB 1 Figure 1. Scattergram comparing leukocyte counts (WBC) (x lo3) from laboratories 2 (+), 3 (a), and 4 ). ( with those of reference laboratory 1. Fluid 12, with leukocyte counts over 50,000/mm3 in 3 of the 4 laboratories, is omitted. Patients whose leukocyte counts were <l,ooo in all laboratories are not plotted. Circled points indicate patients in whom the difference in leukocyte counts, by itself, would have led to a difference in classification of inflammatory (>2,OOO WBC/mm3) or noninflammatory disease, and thus could have resulted in different clinical considerations. I SCHUMACHER ET AL Table 2. Percentages of polymorphonuclear leukocytes reported on leukocyte differential counts from 4 laboratories Patient Labora Labora Labora Laboranumber tory 1 tory 2 tory 3 tory laboratory, 8 1%, laboratory 3, 2%; reference laboratory, l%, laboratory 2, 82%) were noted. One report described 85% eosinophils not found by other laboratories. Crystal identification. Crystals identified in the 3 laboratories that performed crystal analysis are shown in Table 3. Discrepancies in results were found for 7 of 11 patients, compared with results reported by the reference laboratory. Reproducibility within the reference laboratory. Coefficients of variation (CV) for leukocyte counts >1,500/mm3 for a given sample varied from 118%. Not surprisingly, somewhat higher CV (2042%) were seen when leukocyte counts were <300/mm3. However, none of these variations would have had any clinical significance. The maximum difference in mean leukocyte counts between the 2 technicians who analyzed the same specimens was 25%; the mean percentages of neutrophils counted by these 2 technicians were similar. When a 2sided significance test for difference from the previous value was applied (5), all but 1 of 24 neutrophil percentages were within 95% confidence intervals. t P..., n... t b 166 % PMN LAB 1 Figure 2. Scattergram comparing percentages of polymorphonuclear leukocytes (PMN) reported from laboratories 2 (+), 3 (O), and 4 (m), plotted against those of reference laboratory 1. The elliptical area represents theoretic 95% confidence intervals obtained by counting 100 cells (5).

4 SYNOVIAL FLUID ANALYSES 773 Table 3. Crystals detected in synovial fluid analyzed by 3 different laboratories* Patient Labora Labora Laboranumber tory 1 tory 2 tory 4 9 CPPD None CPPD 12 MSU None CPPD 13 MSU MSU MSU 15 CPPD None None 16 None None CPPD 17 MSU MSU MSU 19 MSU Lost MSU 20 MSU MSU MSU 23 Steroids None CPPD 24 MSU None None 28 MSU None MSU * CPPD = calcium pyrophosphate dihydrate; MSU = monosodium urate. DISCUSSION Our study compared the results of analyses of aliquots of the same synovial fluids by 4 different laboratories and showed a number of differences in leukocyte counts, percentages of PMN, and crystals identified. Such variations could have major impacts on disease management. The discrepancies identified in our study suggest the urgent need for better quality control of this important diagnostic procedure. A previous report has suggested that leukocyte counts in the same joint fluid decrease sequentially with any delay in performing the count (6). We have confirmed in our own laboratory that leukocyte counts do fall after as little as 1 hour. We used the Arthritis Research Laboratory, which distributed the fluids to the other laboratories, as a reference, since we are certain that their counts were done promptly. Taking this into consideration, it is of interest that no laboratory gave consistently lower or higher counts. This suggests that variation in leukocyte count was due to factors other than delay in handling. In 4 cases, differences in the leukocyte counts would have caused fluids to be classified by different laboratories as either noninflammatory (leukocytes <2,000/mm3) or inflammatory (7,8). This variation could have affected both diagnosis and treatment. A fluid laden with lipid droplets in patient 1 seems to have accounted for 1 erroneously high leukocyte count by laboratory 2. Some of the discrepancies found in differential counts are difficult to explain. For example, percentages of PMN in 3 fluids varied from 182, 281, and 2978 among the 4 laboratories. Wright stains are somewhat more difficult to perform on joint fluids than on blood, and it is possible that some differential counts were done using inadequately stained smears that should not have been used. In addition, transcriptional errors or specimen mislabeling cannot be excluded; 1 transcriptional error on the leukocyte count of patient 13 was caught at the completion of the study. If errors had been made in initial labeling of specimens, one might have expected parallel errors to occur in leukocyte counts and differentials, but this was not the case in patients 6, 10, and 22, for whom there were the most glaring discrepancies in differential counts. Most laboratories did not attempt to differentiate the various mononuclear cells found in joint fluid. These can include synovial lining cells, lymphoblasts, and macrophages, in addition to small lymphocytes and monocytes. Changes in the percentages of these cells may be of some diagnostic value, although this has not yet been systematically tested (9). Most laboratories do not assign the testing of synovial fluid specimens to specialized technologists. Because there are a number of different aspects to the skilled handling and interpretation of synovial fluid specimens, it would probably be wise to designate certain people to handle these fluids routinely. The many discrepancies noted in the crystals identified are a major concern, since crystal identification is now considered the only way to definitively diagnose gout and calcium pyrophosphate crystal deposition diseases. Inappropriate treatments with potentially toxic uric acidlowering drugs, or failures to treat, could have resulted. Clearly, quality control measures are important for laboratories searching for crystals. We have previously noted that, even in proven gout, crystals may sometimes be very difficult to identify because of small size and other factors (10). Not included in this study were other puzzling crystals, such as anticoagulant crystals and other calcium crystals, which can also be sources of confusion (3). We are developing kits for selftesting of crystals. Unless they are certain about the handling of specimens and quality control methods used in their laboratories, physicians should interpret laboratory results of synovial fluid analyses with caution. ACKNOWLEDGMENT The authors thank Mary Ellen Maguire for typing and preparation of the manuscript.

5 774 SCHUMACHER ET AL REFERENCES 1. Cohen AS, Goldenberg D: Synovial fluid, Laboratory Diagnostic Procedures in the Rheumatic Diseases. Third edition. Edited by AS Cohen. Orlando, FL, Grune & Stratton, 1985, pp Gatter RA: A Practical Handbook of Joint Fluid Analysis. Philadelphia, Lea & Febiger, Schumacher HR: Synovial fluid analysis, Textbook of Rheumatology. Second edition. Edited by WN Kelley, ED Harris Jr, S Ruddy, CB Sledge. Philadelphia, WB Saunders, 1985, pp Eisenberg JM, Schumacher HR, Davidson PK, Kauffman L: Usefulness of synovial fluid analysis in the evaluation of joint effusions: use of threshold analysis and likelihood ratios to assess a diagnostic test. Arch Intern Med 144:715719, Kumke CL: The statistically expected variability in differential leukocyte counting, Differential Leukocyte Counting. Edited by JA Koepke. Skokie, IL, College of American Pathologists, 1977, pp Palmer DG: Total leukocyte enumeration in pathological synovial fluids. Am J Clin Pathol 49:812814, Ropes MW, Bauer W: Synovial Fluid Changes in Joint Disease. Cambridge, MA, Harvard University Press, 1953, pp Hollander JL: Examination of synovial fluid as a diagnostic aid in arthritis. Med Clin North Am 50: , Traycoff RB, Pascual E, Schumacher HR Jr: Mononuclear cells in human synovial fluid: identification of lymphoblasts in rheumatoid arthritis. Arthritis Rheum 19:743748, Schumacher HR, Jimenez SA, Gibson T, Pascual E, Traycoff R, Dorwart BB, Reginato AJ: Acute gouty arthritis without urate crystals identified on initial examination of synovial fluid: report on nine patients. Arthritis Rheum 18:603612, 1975