The term “ascites” is derived from the Greek word “Askitos” meaning bladder or bag. The main conditions in which ascites may significantly increase include the cirrhosis, hepatic venous outflow obstruction, hepatic cancer, nephrotic syndrome, congestive cardiac failure and constrictive pericarditis, infection as tuberculous peritonitis, bacterial peritonitis, malignancy (primary peritoneal cancer, pancreatic cancer, hepatobiliary cancer, etc.) (1). For the identification and classification of ascites is crucial the chemical and morphological analysis of the peritoneal fluid. Patients with a total cell counts (TC) ≥500/µL and absolute neutrophil count ≥250/µL are the standards for establishing a diagnosis of spontaneous bacterial peritonitis (2). In tuberculous peritonitis, cell counts are typically greater than >1,000 and lymphocytes predominate (i.e., 50%) (2-4).
For identification and enumeration of white blood cell (WBC) in ascitic fluid (AF), optical microscopy (OM) is traditionally used. This technique is still regarded as the “gold standard”, though it presents lengthy turnaround time (TAT), needs to educate and train specialized personnel for this type of manual analysis and has a high inter and intra-assay imprecision (2,5,6).
From 2006, the Sysmex (Kobe, Japan) has integrated a specific body fluid (BF) mode in its automatic hematologic analyzers, which is hence mainly aimed to be used for analysis of pericardial, ascites, synovial, pleural and cerebrospinal fluids. Automated counting has several advantages: rapid TAT, it doesn’t need for highly qualified personnel and management of specimens more cost-effective than using OM. Besides, the use of a larger sample volume as compared to counting chamber leads to more cells being counted enhancing precision and accuracy (7-9).
The Sysmex XN-9000, in addition to the default parameters—total nucleated cells (TC), WBC counts, differential cell count for mononuclear cells (MN) and polymorphonuclear cells (PMN)—is equipped with a series of research parameters including neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO) and cells with high fluorescence (HF).
The performance of XN-BF for analysis of AF default parameters has been evaluated in other studies (6,7,9). However, the analytical performance of research parameters has not been evaluated. The aim of this study was, in the first instance, to verify the basic performance of the fully automated XN-BF and then to compare data obtained using manual microscopy with those ones obtained using XN-BF not only for default parameters but also for new research parameters, in the analysis of AF samples.
This comparison study was carried using 66 AFs samples received to the local laboratory from various clinical departments for routine analysis. All samples were collected in 2.0 mL, K2EDTA anticoagulated tubes and tested within 2 h from arrival in the laboratory (2). The results of total WBC count were directly compared with those obtained on the same AF sample by OM on Nageotte chamber, after diluted (1:20 or 1:200) with Turk’s solution. The slides for differential WBC count were prepared with cytospin (Cytospin 3 Thermo Shandon, France), and subsequently stained whit May-Grunwald-Giemsa (Carlo Erba, Italy). Microscopic analysis was performed with light microscopy under oil immersion, at 50× magnification. The differential WBC count included the following classes: NE, LY, monocytes/macrophages (MN/MACRO), EO, and other cells (mesothelial cell and tumor cells).
Analytical performance assessment
The linearity was assessed using 2 AF samples with low cell counts (sample 1: WBC, 65×106 cells/L) and a high cell counts (sample 2: WBC, 4,750×106 cells/L). Each sample was serially diluted with Cellpack to get scalar values, which have been then measured three consecutive times each. Results were plotted against with expected values, as according of CLSI document EP06-A (10).
The imprecision of the XN-BF was evaluated by analyzing in 20 measurements of 2 AF samples with low (sample 1: mean value, 26×106 cells/L) and high (sample 2: mean value, 2,340×106 cells/L) WBC counts, expressed in percentage by the coefficient of variation (CV), as according to the Clinical and Laboratory Standards Institute (CLSI) document EP5-A29 (11).
Carryover was performed according to the ICSH guideline: on one AF samples with high cell count in triplicate (AF1, AF2, AF3), followed by three measurements of a blank (Cellpack; B1, B2, B3) (12). Percentage of carryover was calculated as follows: [(B1 − B3)/(A3 − B3)] ×100.
The study was carried out in accordance with the Declaration of Helsinki, under the terms of all relevant local legislation.
The bias between XN-BF and OM was estimated with Bland-Altman plot analysis, while the agreement was assessed with Pearson’s correlation coefficient (r) and Passing-Bablok regression. Statistical analysis was performed using Analyse-it software version 3.90.1.
For our study were used 66 samples, the mean cellularity value was 461.8×106 cells/L (95% CI: 244.8–678.8) by OM and 472.2×106 cells/L (95% CI: 253.2–691.2) with Sysmex XN-9000 BF mode. The main results of this study are shown in Table 1.
Bland-Altman bias was −10.5×106 cells/L (95% CI: −17.5 to −3.30), −21.4×106 cells/L (95% CI: −51.9 to 9.27), 3.30×106 cells/L (95% CI: −7.3 to 13.9) and 10.3×106 cells/L (95% CI: −6.55 to 27.3) respectively, for TC-BF, WBC-BF, PMN-BF and MN-BF in all 66 samples (Table 2). The bias for WBCs subpopulations and cells with HF is shown in Table 2. The overall bias between the XN-BF and OM was always clinically meaningless.
A good correlation between Sysmex XN-9000 BF mode and OM counts has been found for all considered parameters except for HF cells (r=0.67) (Table 2).
The XN-9000 BF mode showed excellent linearity, with all correlation coefficients for TC, WBC, PMN and MN equal to 1.00 (P<0.05), in a wide range of values comprised between 65×106 and 4,750×106 cells/L. The imprecision was excellent, with CV <5% for AF samples with mean values of 26×106 cells/L and <4% for AF samples with mean values of 2,340×106 cells/L. The carryover was negligible for all parameters (<0.01).
The accurate classification and counting of cells in AF are fundamental needs for faster diagnosis and appropriate therapeutic treatment of patients with ascites. OM remains even today “gold standard” for total WBC counting and for differentiating WBCs subpopulations in this BF (13-15). However, the OM presents a high intra-operator inaccuracy, requires qualified technical personnel and requires longer analytical times (16-18). For these reasons, the use of automatic analyzers is increasing more and more in routine clinical laboratories.
In the last years the introduction of a new generation of automated hematological analyzers has allowed to overcome the main methodological problems for their use in the analysis of cavitary liquids, represented by the presence of macrophages or neoplastic cells. Furthermore, the count in automation can increase the level of analytical standardization even in the case of personnel who are not highly qualified for reading in OM (7,19,20).
For certain types of cavitary liquids, automated cell count has hence allowed to achieve a high degree of accuracy and precision, concomitantly reducing both inter-observer variability and TAT (21,22). In a previous investigation, Paris et al. (23) found an optimal agreement for PMN (r=0.99) and MONO (r=0.98) counts between the manual method and XE-5000 automated count on 81 AF samples. In another study (6), also showed a good correlation for TC (r=0.99), WBC (r=0.98), PMN (r=0.93) and MN (r=0.96) between XN-BF mode and OM. Furthermore, a satisfactory agreement was found between XN-BF and OM for the different WBC subpopulations, with correlation coefficients comprised between 0.84 and 0.93.
Our study was mainly aimed to assess the analytical performance of the new BF mode on the Sysmex XN-9000 using AF samples and comparing data with those obtained with the reference technique (i.e., OM) including neutrophils, lymphocytes, monocytes, eosinophils and cells with HF. The results of our investigation attest that the novel XN-9000 hemocytometer exhibits excellent analytical performance in terms of carryover, imprecision, linearity and throughout a broad range of cellularity in AF samples. In agreement with others studies, BF mode on the Sysmex XN-9000 confirmed a good agreement with default parameters as well as with new research parameters, as shown in Table 2.
At last, comparison between XN-BF and OM about HF cells parameter showed that the different cells instrument counts aren’t overlapped with WBC (i.e., mesothelial cell and tumor cells). The Sysmex XN-9000 BF mode not only exhibits acceptable analytical performance, but it may be used as an alternative to OM, as a first-line screening technique for rapid analysis of AF samples either referred for routine or, especially, for urgent testing. Instead, in cases where an abnormal scattergram or difference between WBC and total cells count with consequent increase of cells with HF are present, the OM revision is fundamental (6,20).
Conflicts of Interest: The authors have no conflicts of interest to declare.
Ethical Statement: The study was carried out in accordance with the Declaration of Helsinki, under the terms of all relevant local legislation. All samples were anonymized before testing, and test results did not impact the clinical management of patients, patient’s permission to use the samples for this study were cleared by the local institutional review board.
- Huang LL, Xia HH, Zhu SL. Ascitic Fluid Analysis in the Differential Diagnosis of Ascites: Focus on Cirrhotic Ascites. J Clin Transl Hepatol 2014;2:58-64. [PubMed]
- Clinical and Laboratory Standards Institute. Body fluid analysis for cellular composition; approved guidelines. CLSI document H56-A. Wayne, PA, 2006.
- Senousy BE, Dragnov PV. Evaluation and management of patients with refractory ascites. World J Gastroenterol. 2009;15:67-80. [Crossref] [PubMed]
- Kjeldsberg CL, Knight JA. Peritoneal Fluid. In: Body Fluids, 3rd ed. Chicago: ASCP Press, 1993:223-53.
- Moore KP, Wong F, Gines P, et al. The management of ascites in cirrhosis: report on the consensus conference of the International Ascites Club. Hepatology 2003;38:258-66. [Crossref] [PubMed]
- Buoro S, Mecca T, Azzarà G, et al. Cell Population Data and reflex testing rules of cell analysis in pleural and ascitic fluids using body fluid mode on Sysmex XN-9000. Clin Chim Acta 2016;452:92-8. [Crossref] [PubMed]
- Briggs C, Longair I, Kumar P, et al. Performance evaluation of the Sysmex haematology XN modular system. J Clin Pathol 2012;65:1024-30. [Crossref] [PubMed]
- Tanaka Y, Tanaka Y, Gondo K, et al. Performance evaluation of platelet counting by novel hematology analyzers. J Clin Lab Anal 2014;28:341-8. [Crossref] [PubMed]
- Lippi G, Cattabiani C, Benegiamo A, et al. Evaluation of white blood cell count in peritoneal fluid with five different hemocytometres. Clin Biochem 2013;46:173-6. [Crossref] [PubMed]
- Clinical and Laboratory Standards Institute (CLSI). Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach, 1st Edition. Approved Guideline. CLSI document EP6-A. Wayne, PA, 2003.
- Clinical and Laboratory Standards Institute (CLSI). Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline. 2nd edition. CLSI document EP5-A2. Wayne, PA, 2004.
- Guidelines for the evaluation of blood cell analysers including those used for differential leucocyte and reticulocyte counting and cell marker applications. International Council for Standardization in Haematology: prepared by the ICSH Expert Panel on Cytometry. Clin Lab Haematol 1994;16:157-74. [PubMed]
- de Jonge R, Brouwer R, de Graaf MT, et al. Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids. Clin Chem Lab Med 2010;48:665-75. [Crossref] [PubMed]
- Dux R, Kindler-Rohrborn A, Annas M, et al. A standardized protocol for flow cytometric analysis of cells isolated from cerebrospinal fluid. J Neurol Sci 1994;121:74-8. [Crossref] [PubMed]
- Fleming C, Russcher H, Lindemans J, et al. Clinical relevance and contemporary methods for counting blood cells in body fluids suspected of inflammatory disease. Clin Chem Lab Med 2015;53:1689-706. [Crossref] [PubMed]
- Zimmermann M, Ruprecht K, Kainzinger F, et al. Automated vs. manual cerebrospinal fluid cell counts: a work and cost analysis comparing the Sysmex XE-5000 and the Fuchs-Rosenthal manual counting chamber. Int J Lab Hematol 2011;33:629-37. [Crossref] [PubMed]
- Bignardi GE. Flow cytometry for the microscopy of body fluids in patients with suspected infection. J Clin Pathol 2015;68:870-8. [Crossref] [PubMed]
- Mahieu S, Vertessen F, Van der Planken M. Evaluation of ADVIA 120 CSF assay (Bayer) vs. chamber counting of cerebrospinal fluid specimens. Clin Lab Haematol 2004;26:195-9. [Crossref] [PubMed]
- Barnes PW, Eby CS, Shimer G. An evaluation of the utility of performing body fluid counts on the coulter LH 750. Lab Hematol 2004;10:127-31. [Crossref] [PubMed]
- Roccaforte V, Daves M, Proserpio V, et al. Evaluation of body fluid mode of Sysmex XN-9000 for white blood cell counts in cerebrospinal fluid. J Lab Precis Med 2018;3:22.
- Nanos NE, Delanghe JR. Evaluation of Sysmex UF-1000i for use in cerebrospinal fluid analysis. Clin Chim Acta 2008;392:30-3. [Crossref] [PubMed]
- De Smet D, Van Moer G, Martens GA, et al. Use of the Cell-Dyn Sapphire hematology analyzer for automated counting of blood cells in body fluids. Am J Clin Pathol 2010;133:291-9. [Crossref] [PubMed]
- Paris A, Nhan T, Cornet E, et al. Performance Evaluation of the Body Fluid Mode on the Platform Sysmex XE-5000 Series Automated Hematology Analyzer. Int J Lab Hematol 2010;32:539-47. [Crossref] [PubMed]
Cite this article as: Roccaforte V, Zavaroni E, Russo RM, Porreca WP, Proserpio V, Liuzzi G, Sciarini F, Bonato C, Perno CF, Pastori S. Comparison between body fluid mode of Sysmex XN-9000 and optical microscopy for the counting of cells in ascitic fluid. J Lab Precis Med 2018;3:86.