The infections of the central nervous system (CNS) are conventionally classified as meningitis or encephalitis. The analysis of white blood cells (WBC) in cerebrospinal fluid (CSF) is a key diagnostic element for the different diagnosis of a kaleidoscope of diseases, thus including inflammatory conditions and infectious or non-infectious disorders involving the CNS (1-3).
The CSF normally contains a very low number of WBC. In adults, the number of nucleated cells in the CSF is conventionally <5.0×106 cells/L, whilst their count should remain below 7.0×106 cells/L in the CSF of children and below 30.0×106 cells/L in that of neonates, respectively (4). The presence of a high number of WBC may hence be suggestive, or even diagnostic, for an infection, for diseases such as meningitis and encephalitis, or for other neurological disorders (1,5). The normal cellular components of CSF include mainly lymphocytes (i.e., approximately 70%, mostly T-cells) and monocytes (i.e., approximately 30%). Neutrophils are not normally present in CSF, and their occasional observation in non-pathological CSF samples can be attributed to a microscopic contamination during lumbar puncture, which is then magnified by cytocentrifugation. Therefore, occasional finding of rare neutrophils in CSF is not necessarily a hallmark of a pathological condition (4).
Optical microscopy is still regarded as the “gold standard” for identification and enumeration of WBC in CSF (4,6). Nevertheless, this technique has some well-known drawbacks, such as the relatively high inaccuracy, low imprecision, lengthy turnaround time (TAT) and the need to educate and train specialized personnel for this type of manual analysis (7). A new generation of fully-automatic hematologic analyzers has been developed and commercialized in recent years. In 2006, the Sysmex (Kobe, Japan) in vitro diagnostic (IVD) company has integrated a specific body fluid (BF) mode on its XE-5000 analyzer, which is hence mainly aimed to be used for analysis of biological fluids other than blood, so mainly including CSF, ascites, synovial, pericardial and pleural fluids. This mode not only exhibits acceptable analytical performance, but also displays many potential advantages for laboratory workout and organization, mostly attributable to the faster TAT and the more cost-effective management of specimens than using optical microscopy (8-10). Nevertheless, high imprecision in CSF samples with a number of WBC ≤20×106/L, probably due to overestimation of polymorphonuclear cell (PMN) counts, was an important shortcoming that has been highlighted in some previous investigations (11-13).
Therefore, the aim of study was to assess the analytical performance of the novel and fully automated Sysmex XN-9000 BF mode for identification and enumeration of WBC in CSF, as well as the evaluation of its basic analytical performance.
The new automated haematology analyser Sysmex XN-9000 (Sysmex, Kobe, Japan) can operate either in “whole blood” mode, or with a second mode dedicated to BF analysis (i.e., BF mode). The analytical performance of the whole blood mode has been recently and exhaustively evaluated (14,15). The second mode, specifically dedicated to BF analysis, entails the use of an innovative software, which allows enumeration and classification of WBC, PMN, mononuclear (MN) and high fluorescent BF (HF-BF) cells in the “DIFF” channel according to their side scatter (so reflecting internal complexity) and fluorescence intensity (i.e., mirroring DNA/RNA content) (4,16,17). The analyzer only uses 88 µL for each BF sample analysis. A rinsing cycle is also activated before starting a new test, thus preventing spurious contamination from material of the previous sample.
The comparison study was carried out using 44 CSF samples referred to the local laboratory for routine analysis. All samples were tested within 1 h from arrival in the laboratory. CSF collection and analysis of cellular composition were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) document H56-A (4). The results of total WBC count were directly compared with those obtained on the same CSF sample by optical microscopy on Nageotte chamber, after staining with Turk’s solution. The slides for differential WBC count were prepared with cytospin (Shandon Cytospin 3 centrifuge; Thermo Shandon, Cergy Pontoise, France), starting from 200 µL of CSF. The slides were then stained whit May-Grunwald-Giemsa, using a Sysmex SP-10 slide-stainer unit (Sysmex, Etten-Leur, The Netherlands). Microscopic analysis was performed with light microscopy under oil immersion, at 50× magnification. Two independent experts performed all the microscopic analyses, and the individual results were then averaged to obtain the final count. The bias [and its 95% confidence interval (CI)] between data obtained with either optical microscopy or Sysmex XN-9000 BF mode was estimated with Bland-Altman plot analysis.
The within-run precision of the BF mode on XN-9000 was evaluated with 20 consecutive measurements of two CSF samples with low (mean value, 36×106 cells/L) and high (mean value, 1,346×106 cells/L) WBC counts. The imprecision was finally expressed in percentage, as coefficient of variation (CV), according to the CLSI document EP5-A2 (18).
Carry-over was assessed by measuring a CSF sample with high cell count in triplicate (H1, H2, H3), followed by three consecutive measurements of blank solution (Cellpack; L1, L2, L3). The final result was calculated as follows: [(L1−L3)/(H3−L3)] ×100, as currently recommended by the International Council for Standardization in Haematology (ICSH) (19).
The linearity was assessed measuring two CSF samples with different cell counts (sample A: WBC, 3,197×106 cells/L; sample B: WBC, 85×106 cells/L). Each sample was serially diluted with Cellpack to obtain scalar values, which were then measured three consecutive times each. Results were then compared with expected values, as for indications of CLSI document EP06-A (20).
The statistical analysis was performed using MedCalc statistical package (version 17.6, MedCalc Software, Mariakerke, Belgium).
The main results of this study are shown in Table 1. The overall bias between the Sysmex XN-9000 BF mode and optical microscopy was always clinically meaningless. In particular, the Bland-Altman plot analysis allowed to estimate a mean bias with optical microscopy of 1.6×106 cells/L (95% CI: −21.8×106 to 25.1×106 cells/L) for total WBC, 1.3×106 cells/L (95% CI: −13.9×106 to 16.5×106 cells/L) for PMN, and −0.6×106 cells/L (95% CI: −21.5×106 to 20.3×106 cells/L) for MN, respectively (Figure 1).
The XN-9000 boy fluid mode also displayed excellent linearity throughout a broad range of clinically significant values (i.e., between 85×106–3,197×106 cells/L), with all correlation coefficients for WBC, PMN and MN equal to 1.00 (P<0.001). The carryover was negligible (i.e., <0.01%), and the imprecision excellent, with CVs <5% and <4% for CSF samples with mean values of 36×106 WBC/L and 1,346 ×106 WBC/L, respectively.
The accurate identification and enumeration of cells in CSF are essential needs for rapid diagnosis and appropriate therapeutic treatment of patients with CSF disorders. Optical microscopy, using the so-called “counting chambers” (i.e., Fuchs Rosenthal, Burker, Neubauer or Nageotte), remains “gold standard” for total WBC and erythrocyte counts in this BF, whilst stained cytocentrifuged slides are typically used for differentiating WBC into MN and PMN (8,13).
The considerable technological advancements occurred in recent years have allowed to introduce a new generation of automated hematological analyzers in routine clinical laboratories, which are now increasingly used for cell identification and counting in samples other than whole blood. For certain types of BF, 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), even if some doubts remain about their reliability in BF samples with low cellularity (9,10,23). In a previous evaluation of the Sysmex XE-5000 BF mode, Fleming et al. found a good agreement with optical microscopy for WBC (r=0.97) and MN (r=0.93) counts, whereas a less satisfactory concordance was observed for the PMN count (r=0.87) (5). Paris et al. (6) also showed a good correlation for total WBC (r=0.96) and MN counts (r=098) between XE-5000 BF mode and optical microscopy using 81 CSF samples, which were obtained from oncology, pediatric and adult hematological patients. Notably, the poor correlation between XE-5000 BF mode and optical microscopy was confirmed in their study (r=0.58) (6), whilst it was also highlighted that automated testing was not efficient enough to identify blast cells, since the XE-5000 BF mode overlaps blast cells with MN, so that optical microscopy would remain mandatory in these cases (6).
Our study was mainly aimed to assess the analytical performance of the new BF mode on the Sysmex XN-9000 using CSF samples, and comparing data with those obtained with the reference technique (i.e., optical microscopy). Taken together, our results suggest that this BF mode displays excellent analytical performance in terms of imprecision, linearity and carry-over throughout a broad range of cellularity in CSF samples. This data, combined with the excellent correlation with optical microscopy observed from the analysis of 44 routine CSF samples displaying heterogeneous cellularity, suggests that the Sysmex XN-9000 BF mode may be used as a first-line, screening technique for rapid analysis of CSF samples either referred for routine or, especially, for urgent testing.
Conflicts of Interest: The authors have no conflicts of interest to declare.
Ethical Statement: The investigation was entirely based on the use of routine samples refereed to the local laboratory for CSF analysis, all samples were anonymized before testing, test results did not impact the clinical management of patients and, therefore, ethical approval or patient’s permission to use the samples for this study were cleared by the local institutional review board. The study was performed in accordance with the Declaration of Helsinki and under the terms of all relevant local legislations.
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Cite this article as: Roccaforte V, Daves M, Proserpio V, Sciarini F, Sangiorgio R, Costanzo A, Di Pierro AM, Ugo S, Lippi G, Bonato C. Evaluation of body fluid mode of Sysmex XN-9000 for white blood cell counts in cerebrospinal fluid. J Lab Precis Med 2018;3:22.