measurement of total and differential white blood cell counts in synovial fluid by means of an...
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easurement of total and differential white bloodell counts in synovial fluid by means of anutomated hematology analyzer
IROYUKI SUGIUCHI, YUKIO ANDO, MASAHIRO MANABE, EIICHI NAKAMURA,IROSHI MIZUTA, SHIRO NAGATA, and HIROAKI OKABE
UMAMOTO, JAPAN
We developed a rapid and accurate method for quantifying total and differentialwhite blood cell (WBC) counts by pretreating synovial fluid with hyaluronidase andusing an automated hematology analyzer. Forty-seven samples of synovial fluidthat had been placed in blood-collection tubes containing ethylenediamine-N,N,N=,N=-tetraacetic acid as an anticoagulant were treated with hyaluronidase at37°C for 10 minutes, and then the total and differential WBC counts were determinedby means of the automated hematology analyzer. Results were compared withthose achieved by traditional manual counting methods. For the automatedmethod, the coefficient of variation values for within-run precision of the WBC countwere 5.27%, 3.56%, and 3.01% at 0.54, 1.12, and 2.05 � 109/L, respectively; therun-to-run coefficient of variation values was less than 10.0%. The total and differ-ential WBC counts obtained by this automated method showed good correlationwith those obtained by the hemocytometer method (r � .998; P < .0001; regressionformula, y � 0.986x � 0.072). Bland-Altman plots indicated no significant discrep-ancy between the methods. Our evaluation supports the use of this automatedhematology analyzer method to measure total and differential WBC counts, whichshould aid clinical diagnosis. (J Lab Clin Med 2005;146:36–42)
Abbreviations: WBC � white blood cell; PMN � polymorphonuclear neutrophil leukocyte;
CV � coefficient of variation; DIFF � differential: BASO � basophilbiaflimms1(pitmc8d
he main components of synovial fluid are viscousmucopolysaccharides such as hyaluronate andchondroitin sulfate.1 When inflammation occurs
n synovial fluid, the free lysozymes within a joint
rom the Department of Laboratory Medicine, Kumamoto Universityedical School; Department of Diagnostic Medicine, Graduate
chool of Medical Sciences; and Department of Orthopedic Surgery,raduate School of Medical Sciences, Kumamoto University, Kum-
moto, Japan.
ubmitted for publication September 10, 2004; accepted for publica-ion April 3, 2005.
eprint requests: Yukio Ando, MD, PhD, Department of Diagnosticedicine, Graduate School of Medical Sciences, Kumamoto Univer-
ity, 1-1-1 Honjo, Kumamoto 860-0811, Japan; e-mail: [email protected]
022-2143/$ – see front matter
2005 Mosby, Inc. All rights reserved.
oi:10.1016/j.lab.2005.04.004
6
reak down hyaluronate. The synovial fluid volumencreases, and fluid viscosity decreases. Counts of totalnd differential white blood cells (WBCs) in synovialuid may allow clinicians to distinguish between non-
nflammatory conditions (eg, osteoarthritis and trau-atic arthritis) and inflammatory conditions (eg, rheu-atoid arthritis, gout, and septic arthritis). In normal
ynovial fluid, the total WBC count is less than 0.2 �09/L, with polymorphonuclear neutrophil leukocytesPMNs) contributing approximately 10% of the WBCopulation. In synovial fluid from patients with acutenflammation, however, the total WBC count increaseso more than 2 � 109/L, with PMNs accounting forore than 75% of the total WBCs.1,2A total WBC
ount in synovial fluid of more than 50 � 109/L, with5% of these cells PMNs, often has an important role iniagnosis of septic arthritis.1
Total and differential WBC counts are traditionally
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J Lab Clin MedVolume 146, Number 1 Sugiuchi et al 37
etermined by using a hemocytometer or by examiningiemsa-stained smears under a microscope.3–5 Although
hese techniques have the advantage of low cost and doot require much space, they are time-consuming,abor-intensive, and unsuitable for rapid analysis.
oreover, WBC counts that are determined by use of aemocytometer are often inaccurate: Acetic acid in theiluting solution and hyaluronate react to form aggre-ates of synovial fluid components.6 Although auto-ated electrical impedance technology has also been
sed to establish synovial fluid WBC counts, reports ofegative discrepancies, such as improper sample aspi-ation or poor mixing (because synovial fluid is highlyiscous) causing low WBC counts, and positive dis-repancies, such as noncellular entities including fatlobules, crystals, joint cells, and extraneous debriseing counted as WBCs, have been published.7,8 Re-ently, the demand for rapid analysis of synovial fluidn clinical practice has been growing, but there haveeen no reports thus far of an assay that can simulta-eously measure total and differential WBC counts. Inhis article, we present a novel method that can rapidlynd accurately determine total and differential WBCounts in synovial fluid; the technique involves pre-reating the samples of synovial fluid, which containsyaluronate, with hyaluronidase and processing theamples with an automated hematology analyzer thatakes use of flow cytometry with a semiconductor
aser.
ETHODS
Materials and blood collection. Hyaluronidase (EC.2.1.35; from sheep testes, 3320 U/mg type A, Lot83K1255) was obtained from Sigma Chemical Co. (St.ouis, Mo). Sodium dihydrogen phosphate dihydrate
NaH2PO4·2H2O), and disodium hydrogen phosphate do-ecahydrate (Na2HPO4·12H2O) were purchased from Wakoure Chemical Industries (Osaka, Japan). May-Grünwaldtain solution was obtained from Muto Pure Chemical Co.Tokyo, Japan). Giemsa stain solution was provided by Sys-ex Co. (Kobe, Japan). All other materials and solvents usedere of analytic grade.All 47 synovial fluid samples (Table I) from inpatients and
utpatients with arthritis of the knee who had undergoneperations at the Department of Orthopedic Surgery, Kum-moto University Hospital, were placed in blood collectionubes. Total and differential WBC counts were determinedithin 24 hours after sample collection. As an anticoagu-
ant, ethylenediamine-N,N,N=,N=-tetraacetic acid dipotas-ium, which is suitable for crystal tests and is used in con-entional peripheral blood testing, was used instead ofeparin.9
Analytic procedures - Pretreatment. Hyaluronate in theynovial fluid samples was digested by adding hyaluronidase.
500 U/mL hyaluronidase solution was prepared by dissolv-
ng 10 mg of hyaluronidase in 60 mL of a 0.1 mol/L phos- chate-buffered saline buffer (pH 7.4). Next, 200 �L of theyaluronidase solution was mixed with 200 �L of a synovialuid sample, and the mixture was incubated at 37°C for 10inutes.Within 1 hour after this enzymatic treatment, total WBC
ounts were determined by means of the XE-2100 automatedematology analyzer (Sysmex Co.) and a Bürker-Türk hemo-ytometer (Erma, Tokyo, Japan).
Total and differential white blood cell counts obtainedith the XE-2100 automated hematology analyzer. By us-
ng flow cytometry with a semiconductor laser, the XE-2100etects three kinds of optical information: forward-scatteredight intensity, which provides information on microparticleize and material; side-scattered light intensity, which de-ends on intracellular structural complexity, such as nucleus,hape, density, and granular content; and side fluorescencentensity, which contributes information on the degree ofuorescent staining of the blood cells stained with fluorescentye. Samples in the WBC/basophil (WBC/BASO) and dif-erential (DIFF) channels were diluted 1:50 and 1:51, respec-ively, with hemolytic reagents and staining solutions. Theiluted samples were then delivered to the detector, and theumber of cells in 40 �L was measured by flow cytometry.he results are shown in the form of two-dimensionalcattergrams.
The WBC/BASO channel is used to determine total WBCounts (x-axis: side-scattered light; y-axis: forward-scatteredight), and the DIFF channel is used to determine the numbersf neutrophils, lymphocytes, monocytes, and eosinophils (x-xis: side-scattered light; y-axis: side fluorescence).10 In theervice screen of the DIFF channel of the data presentationenu, differential WBC counts are presented as the DIFF/BC ratio. When the DIFF/WBC ratio is not close to 1, the
esult of the differential WBC analysis is not displayed. TheBC/BASO channel is the standard for reporting total WBC
ounts.11 After samples were mixed by inverting more thanve times, 130 �L of a sample was used to obtain the totalnd differential WBC counts within 24 seconds. The accuracyf the data was confirmed and corrected with e-CHECKontrol (low and high levels; Sysmex Co.) of human bloodpecimens. When WBC counts in treated synovial samplesere measured, the results were corrected for the dilution
able I. Clinical diagnoses for 47 samples ofynovial fluid
iseaseNumber of
samples
oninflammatory diseaseOsteoarthritis 18Traumatic arthritis 6
nflammatory diseaseRheumatoid arthritis 10Gout 5Septic arthritis 1
iagnosis unknown 7ll diseases 47
aused by the added hyaluronidase solution.
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J Lab Clin Med38 Sugiuchi et al July 2005
Total white blood cell counts obtained by the hemocy-ometer-microscopic method. To count PMNs, synovialuid samples were diluted (1:10) with Turk’s solution, whichonsisted of a 2% to 3% aqueous solution of acetic acid andentian violet. After a synovial fluid sample was mixed withhis solution, the mixture was placed in a hemocytometer, and
BCs were counted under low magnification. The sampleas first treated with hyaluronidase to prevent coagulation
aused by the presence of acetic acid in the Turk solution.Differential white blood cell counts as determined byicroscopy. To obtain smears of synovial fluid, samplesere centrifuged at 1000 rpm for 5 minutes. DifferentialBC counts were determined in hyaluronidase-treated syno-
ial fluid smears by means of May-Giemsa staining. The first00 nucleated cells counted in random fields were subclassi-ed as neutrophils, lymphocytes, and monocytes. These dif-erential WBC counts were compared with those obtainedhrough the DIFF channel of the XE-2100.
Agreement between methods. Agreement between ourroposed method and the hemocytometer-microscopic methodas evaluated by using Pearson’s correlation coefficient andassing-Bablok regression analysis. The resulting valuesere analyzed by means of the Bland-Altman method, inhich the difference against the mean of both methods wasraphically plotted, after which the mean difference between theethods and the standard deviation of the differences were
stimated.
ESULTS
White blood cell counts in synovial fluid with and withoutyaluronidase pretreatment. XE-2100 analysis of all 47ynovial fluid samples that had had no hyaluronidaseretreatment resulted in total WBC counts being mostlynderestimated because of aggregation of the WBCs inhe fluid. In untreated synovial fluid, the total WBCount determined through the DIFF channel was muchigher (Fig 1A) than that determined through the WBC/ASO channel (Fig 1B). Furthermore, differentialBC counts could not be determined because theIFF/WBC ratio was extremely high. In contrast, for ayaluronidase-pretreated sample, the plots of the WBCounts in two-dimensional scattergrams for the WBC/ASO and DIFF channels showed an approximatelyqual distribution in both scattergrams (Fig 1C and D),nd the result of the differential WBC analysis wasisplayed on the graph page of the data presentationenu because the DIFF/WBC ratio was close to 1.Determination of hyaluronidase concentration. Solu-
ions containing different concentrations of hyaluroni-ase (100, 200, 400, 600, 800, 1000, and 2000 U/mL)ere prepared, and synovial fluid solutions having
hree different viscosities were mixed 1:1 with theseyaluronidase solutions. The samples that contain hy-luronate were treated with hyaluronidase at 37°C for0 minutes. The DIFF and WBC/BASO channels of the
E-2100 permitted analysis of these samples and cal- wulation of the WBC counts and the DIFF/WBC ratio.hen final concentrations of hyaluronidase exceeded
00 U/mL, the DIFF/WBC ratio was close to 1.0 (Fig 2).Reactivity of synovial fluid with XE-2100 reagents. He-olytic reagents used for the WBC/BASO and DIFF
hannels were mixed with synovial fluid samples ineparate test tubes, and smears were obtained afterentrifugation as described previously. The resultingmears were subjected to May-Giemsa staining andnalyzed under the microscope. With the DIFF channeleagent, the WBCs were distributed evenly and coulde analyzed and measured, but the red blood cells wereysed (Fig 3A and B). In contrast, with the WBC/BASOhannel reagent, total WBC counts were mostly under-stimated because of WBC aggregation, and fibrinoidggregates trapped many WBCs (Fig 3C and D). How-ver, when synovial fluid was treated with 500 U/mLyaluronidase, these aggregates were not seen underhe microscope, and WBCs were diffusely distributeddata not shown). In hyaluronidase-treated samples,
ig 1. Scattergrams for total white blood cell (WBC) counts inynovial fluid. Synovial fluid was collected from the knee of a4-year-old female patient with rheumatoid arthritis. (A and B) WBCounts for nonenzyme-treated synovial fluid. (C and D) WBC countsor hyaluronidase-treated synovial fluid. The total WBC count ofntreated synovial fluid as determined through the WBC/basophilBASO) channel was 0.13 � 109/L (B), whereas that determinedhrough the differential (DIFF) channel was 6 � 109/L (A). When theample was pretreated with hyaluronidase, the plots of total WBCounts showed an approximately equal distribution in both scatter-rams (C and D).
hen processed by the XE-2100, the results had ac-
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J Lab Clin MedVolume 146, Number 1 Sugiuchi et al 39
eptable correlation with those calculated by means oficroscopy.Accuracy of studies. Repeatability and reproducibil-
ty of the automated method are shown in Table II.hree different samples of synovial fluid from thenees of arthritic patients were used to assess within-un and run-to-run precision. Samples contained from.49 to 2.52 � 109/L WBCs. The coefficient of varia-ion (CV) values of within-run and run-to-run precisionf automated total WBC counts were 3.01% to 5.27%nd 5.95% to 9.83%, respectively, whereas the totalBC counts for the hemocytometer were 5.94% to
2.24% and 10.16% to 19.61%, respectively. Corre-ponding CV values for the automated differential
BC counts were 0.85% to 1.85% and 1.21% to.23%, respectively.Correlation of results obtained by the automatedethod with those determined by means of light micros-
opy. Figures 4 to 6 show that WBC counts obtainedor treated samples by means of the WBC/BASO chan-el were highly correlated with those obtained for un-reated and treated samples by the hemocytometer andhe DIFF channel (r � .99). The mean differencesetween WBC counts obtained by the hemocytometerethod and the WBC/BASO channel for treated sam-
les was 0.01 � 109/L (0.1%), with a 95% confidencenterval of �1.31 � 109/L to 1.22 � 109/L (�20%–9%) (Fig 4B). The mean differences between WBCounts determined by the DIFF channel for untreatedamples and by the WBC/BASO channel for treatedamples was �0.15 � 109/L (�2%), with a 95% con-
ig 2. Effects of hyaluronidase on WBC counts for synovial fluidamples with three different viscosities. Synovial fluid samples wereretreated with hyaluronidase in phosphate-buffered saline bufferpH 7.4) at 37°C for 10 minutes. Osteoarthritis (closed circles);raumatic arthritis (closed squares); rheumatoid arthritis (closedriangles).
dence interval from �1.49 to 1.19 � 109/L (�21%– s
7%) (Fig 5B). The corresponding value for the WBC/ASO channel and DIFF channel for treated samplesas 0.01 � 109/L (0.1%), with a 95% confidence
nterval from �0.65 to 0.67 � 109/L (�9%–9%) (FigB).The correlation between the proposed method and
he microscopic method for PMNs in the differentialBC counts was r� .989 (P � .0001), with a regres-
ion formula of y � 1.003x � 0.824, and for lymphoidells, r � .985 (P � .0001), with a regression formulaf y � 1.070x � 2.145. As shown in Figure 7, Bland-ltman plots revealed no significant discrepancy be-
ween the proposed automated method and the micro-copic method.
ISCUSSION
We presented here a novel method for rapid and
ig 3. May-Giemsa–stained smears showing WBC distribution. (And B) Distribution of WBCs as assessed through the DIFF channel.he hemolytic solution (pH 7.3) used for the DIFF channel and aynovial fluid sample were mixed at a 1:51 ratio to prepare a smear.
BCs were evenly distributed. Magnification: �10 (A) and �20 (B).C and D) Distribution of WBCs as assessed through the WBC/ASO channel. The hemolytic solution (pH 3.4) used for the WBC/ASO channel and a synovial fluid sample were mixed at a 1:50 ratio
o prepare a smear. WBCs were scattered around aggregates ofynovial fluid components. Magnification: �5 (C) and �10 (D).
imultaneous determination of total and differential
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J Lab Clin Med40 Sugiuchi et al July 2005
BC counts in synovial fluid by pretreating samples ofynovial fluid (which contains hyaluronate) with hyal-ronidase and then using an automated hematologynalyzer. A recent report showed that WBC counts inntreated synovial fluid samples could reliably be de-ermined by using the DIFF channel of an automatedematology analyzer; the results obtained by the WBC/
able II. Accuracy of the two methods used to me
ample
Automated hematolog
Mean � SD � 109/L
ithin-run precision*A 0.54 � 0.03B 1.12 � 0.04C 2.05 � 0.06
un-to-run precision†
A 0.58 � 0.06B 1.25 � 0.11C 2.52 � 0.15
Ten aliquots of each sample were analyzed simultaneously.Three samples were analyzed in duplicate for 5 days with a singarget values).
ig 4. Correlation between WBC counts obtained through theBC/BASO channel and those measured by the hemocytometer for
7 treated samples (dashed line: y � x) (A). Bias plots of relativeifferences between these WBC counts versus the average counts ofhe two methods, with the mean of the differences (solid line) and �
standard deviation (SD) limits (dashed lines) shown (B).
ASO channel were underestimates.11 However, the m
ethod described in that report had a major disadvan-age in that the differential counts from the DIFF chan-el were not reported because the DIFF/WBC ratioeviated substantially from 1.0. For our untreated sam-les, WBC counts obtained by the WBC/BASO chan-el with the automated hematology analyzer were
otal white blood cell counts in synovial fluid
r Hemocytometer
V (%) Mean � SD � 109/L CV (%)
5.27 0.49 � 0.03 12.243.56 1.27 � 0.09 7.093.01 2.02 � 0.11 5.94
9.83 0.51 � 0.10 10.618.80 1.21 � 0.16 13.225.95 2.46 � 0.25 10.16
nt lot. Underlined values are �10% (permissible limits: �10% from
ig 5. Correlation between WBC counts obtained through theBC/BASO channel for 47 treated samples and those obtained
hrough the DIFF channel for 47 untreated samples (dashed line: y �) (A). Bias plots of relative differences between these WBC countsersus the average counts of the two methods, with the mean of theifferences (solid line) and � 2 SD limits (dashed lines) shownB).
asure t
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arkedly lower compared with those from the DIFF, in
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J Lab Clin MedVolume 146, Number 1 Sugiuchi et al 41
greement with values reported before.11 This discrep-ncy may be because the pH of the hemolytic reagentsed for the WBC/BASO channel is lower (pH ofnorganic surfactant: 3.4) than that of the reagent usedor the DIFF channel (pH of nonionic surfactant: 7.3).
hen total WBC counts in untreated synovial fluid areetermined by the WBC/BASO channel, interactionetween the acidic hemolytic agent and hyaluronateay induce aggregation of cells in the solution, whichould result in low WBC counts compared with DIFF
hannel results. In contrast, aggregation of cells did notccur in the DIFF channel because of the neutral pH ofhe hemolytic reagent.
On the basis of these findings, we used hyaluronidaseo avoid aggregation of cells in WBC/BASO channel.cid mucins in synovial fluid, such as hyaluronate and
hondroitin sulfate, were completely degraded by treat-ent with 500 U/mL hyaluronidase at 37°C for 10inutes. This pretreatment did not affect cell numbers
nd morphology when May-Giemsa–stained samplesere examined microscopically. Hyaluronidase treat-ent allowed the DIFF/WBC ratio to approach 1.0, and
he result of the differential WBC analysis was dis-layed on the graph page of the data presentation menu.
ig 6. Correlation between WBC counts obtained through theBC/BASO and DIFF channels for 47 treated samples (dashed line:
�x) (A). Bias plots of relative differences between these WBCounts versus the average counts of the two methods, with the meanf the differences (solid line) and � 2 SD limits (dashed lines) shownB).
urthermore, the differential WBC counts obtained by (
he DIFF channel of the automated analyzer were con-istent with those obtained by the microscopic method.
The new method produced smaller (�10%) within-un and between-run CV values for three differentynovial fluid samples compared with values deter-ined by the hemocytometer (�20%), in agreementith values reported before.11 Therefore, the repeat-
bility and reproducibility of the proposed method weret least two times better than those of the hemocytom-ter method, which required great skill to count cellsccurately (Table II).WBC/BASO channel WBC counts for treated sam-
les were highly correlated with those obtained by theemocytometer (Figs 4–6). However, although theimits of the differences seemed to be numerically largevariation from �20% to 19%), both automated andemocytometer results wereweeee were small enougho be used for diagnosis of inflammation in synovialuid. However, the mean differences between the DIFFhannel result for untreated samples and the WBC/ASO channel result for treated samples showed a
light negative bias (�2%), with a 95% confidencenterval from �21% to 17%. Moreover, the differenceas numerically large compared with the differences
ig 7. Bland-Altman plots comparing the automated and micro-copic methods for obtaining differential polymorphonuclear neutro-hil leukocyte (PMN) counts for 20 synovial fluid samples: (A)eutrophils and (B) lymphocytes. Automated neutrophil counts wereetween �5.02% and �4.04%, and lymphocyte counts were between2.91% and �5.02% (solid line: the mean of the differences, dashed
ines: � 2 SD limits).
limits of agreement: �9% to 9%) between the WBC
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J Lab Clin Med42 Sugiuchi et al July 2005
ounts by the WBC/BASO channel and those by theIFF channel for treated samples (Figs 5B and 6B).herefore, it was assumed that the WBC counts by theIFF channel varied considerably for untreated sam-les. The lower DIFF channel WBC counts for somentreated samples may be because of improper mixingf the aspirate samples with reagents, whereas theigher DIFF channel WBC counts may derive fromontamination with extraneous debris or other compo-ents of synovial fluid that registered as nucleated cells.hese factors may thus cause positive or negative dis-repancies between DIFF and WBC/BASO channelBC counts.Unlike the automated method for determining WBC
ounts that does not make use of any pretreatment (11),ur proposed method requires pretreatment of synovialuid (containing hyaluronate) with hyaluronidase;ithout such treatment, the method cannot determine
otal and differential WBC counts simultaneously be-ause the DIFF/WBC ratio would deviate markedlyrom 1.0. When we analyzed WBC counts in untreatedynovial fluid by the automated analyzer, insolubleggregates that formed in some samples clogged theow cell of the analyzer and caused malfunction.The substrate specificity of hyaluronidase varies de-
ending on its origin. Bacterial hyaluronidase reactsnly with hyaluronate, whereas testicular hyaluronidaseeacts with hyaluronate, chondroitin sulfate, and kera-osulfate.12–14 In our study, we found that total andifferential WBC counts in synovial fluid could beapidly and accurately measured in a convenient man-er by using testicular hyaluronidase.Although the method that we propose may not re-
lace the microscopic method for all clinical arthritisnvestigations because of the need to detect crystal
ormation in several types of metabolic arthritis, itlearly holds promise as a rapid screening tool forommon arthritis.
EFERENCES
1. Tercic D, Bozic B. The basis of the synovial fluid analysis. ClinChem Lab Med 2001;39:1221–6.
2. Shmerling RH, Delbanco DL, Tosteson AN, Trentham DE. Sy-novial fluid tests. What should be ordered? JAMA 1990;264:1009–14.
3. Pascual E. Persistence of monosodium urate crystals and low-grade inflammation in the synovial fluid of patients with un-treated gout. Arthritis Rheum 1991;34:141–5.
4. Moreno MJ, Claybume G, Schumacher HR Jr. Processing ofnoninflammatory synovial fluids with hyaluronidase for cytospinpreparations improves the accuracy of differential counts. DiagnCytopathol 2000;22:256–8.
5. Serre G, Vincent C, Mauduyt MA, Soleilhavoup JP, Planel H.Morphologic quantitative and cytoenzymologic studies of syno-viocytic and monocytic cells in synovial fluid. Anal Quant Cytol1984;6:227–37.
6. Hasselbacher P. Variation in synovial fluid analysis by hospitallaboratories. Arthritis Rheum 1987;30:637–42.
7. Vincent J, Korn JH, Podewell C, Tully E. Synovial fluid pseudo-leukocytosis. Arthritis Rheum 1980;23:1399–400.
8. Atilola MA, Lumsden JH, Rooke F. A comparison of manual andelectronic counting for total nucleated cell counts on synovialfluid from canine stifle joints. Can J Vet Res 1986;50:282–4.
9. Salinas M, Rosas J, Iborra J, Manero H, Pascual E. Comparisonof manual and automated cell counts in EDTA preserved syno-vial fluids. Storage has little influence on the results. Ann RheumDis 1997;56:622–6.
0. Inoue H. Overview of automated hematology analyzer XE-2100.Sysmex J Int 1999;9:58–64.
1. de Jonge R, Brouwer R, Smit M, et al. Automated counting ofwhite blood cells in synovial fluid. Rheumatology 2004;43:170–3.
2. Kreil G. Hyaluronidases—a group of neglected enzymes. ProteinSci 1995;4:1666–9.
3. Frost GI, Csoka TB, Stem R. The hyaluronidases: a chemical,biological and clinical overview. Trends Glycosci Glycotechnol1996;8:419–34.
4. Csoka TB, Frost GI, Stem R. Hyaluronidases in tissue invasion.
Invasion Metastasis 1997;17:297–311.