Method comparison of four clinically available assays for serum free light chain analysis.

Background Serum free light chain (sFLC) measurements are increasingly important in the context of screening for monoclonal gammopathies, prognostic stratification and monitoring of therapy responses. In this study we have performed a method comparison of four sFLC assays that are currently available for routine clinical use. Methods In a retrospective study, sFLC analyses were performed on a cohort that included 139 patients with various monoclonal gammopathies and 54 control sera without an M-protein. Method comparisons of the following four FLC assays were performed: Freelite (Binding Site), N-Latex FLC (Siemens), Seralite (Abingdon Health) and Sebia FLC (Sebia). Results Bland-Altman agreement analysis showed biases varying between -0.1 and 16.2 mg/L for κFLC, -6.0 and 6.8 mg/L for λFLC and -0.04 and 0.38 for the ratio of the involved to uninvolved FLC. Strong agreements were observed for FLC-concentrations below 100 mg/L. The clinical concordance of the κ/λFLC-ratio of the four methods varied between 86% and 92%. Significant quantitative differences were observed between the different methods, mainly in sera with high FLC concentrations. Most assays consistently overestimated FLC concentrations compared to SPE. Conclusions Good overall clinical concordances were observed between the four sFLC assays that were compared in this study. Although good agreements were observed between the FLC assays, significant absolute differences in FLC concentrations in individual patients can be seen, particularly at higher FLC concentrations. Because of inequivalent absolute sFLC values between the methods in individual patients, none of the four sFLC assays can be used interchangeably.

Evaluation of Sysmex XN-1000 High-Sensitive Analysis (hsA) Research Mode for Counting and Differentiating Cells in Cerebrospinal Fluid.

OBJECTIVES: Counting cells in cerebrospinal fluid (CSF) using automated analyzers is generally problematic due to low precision at low cell numbers. To overcome this limitation, Sysmex (Kobe, Japan) developed the high-sensitive analysis (hsA) research mode specifically for counting cells in fluids that contain low cell counts. We evaluated this mode by counting RBCs, WBCs, and differentiated WBCs in CSF samples. METHODS: We analyzed 248 CSF samples using the hsA mode and compared these results with those obtained using the manual counting method. We also evaluated the linearity, detection limits, carryover, and precision of the hsA mode. RESULTS: Using the hsA mode, the lower limit of quantification for RBCs and WBCs was 10 and 2 cells/µL, respectively. Comparing the two methods revealed good agreement with respect to WBCs (y = 1.08x + 0.52), RBCs (y = 1.07x + 0.00), lymphocytes (y = 1.00x + 0.00), neutrophils (y = 1.05x + 0.00), and monocytes (y = 0.88x + 0.07). Regression analysis for samples containing low WBCs (<10 cells/µL) and low RBCs (<50 cells/µL) also had good agreement, although a slight positive bias was found for RBCs. Linearity was good (r(2) ≥ 0.99) for all parameters evaluated. Carryover was negligible and never exceeded 0.04%. CONCLUSIONS: The XN hsA research mode provides reliable cell counts in CSF samples, even in samples containing low numbers of WBCs and RBCs.

Clinical relevance and contemporary methods for counting blood cells in body fluids suspected of inflammatory disease.

In many inflammatory diseases, the cellular components in body fluids [cerebrospinal fluid (CSF), serous fluids] are increased, rendering essential diagnostic information. The diagnostic value of the total white blood cell count (WBC) and differential count has been evaluated extensively over the years, and a remarkable amount of knowledge has been gained; yet, there is a great deal of clinical uncertainty whether the diagnosis should be based solely on these variables. In some diseases, such as peritonitis, the total WBC and differential count has high sensitivity; whereas, in differentiating pleural effusions, it lacks the sensitivity required to be clinically useful. Nevertheless, many guidelines consider these tests as cornerstone parameters, and in combination with clinical variables, they can successfully guide clinical decision making in initiating or postponing a treatment course for infection and/or inflammatory diseases while awaiting culture results. Although other methods are available for detecting and differentiating WBCs in body fluids, manual microscopy is still considered the gold standard despite its many limitations. During the last decade, automated analyzers have become a popular method for first line screening. Continued progress in their design has led to major improvements including their speed, improved accuracy and lower variability compared with microscopy. Disadvantages of this method include high imprecision in low ranges (depending on the method) and interfering factors. In a time where automation is at the front line in clinical laboratories, it is essential the results obtained are precise, accurate and reproducible. This review provides an overview of the relevance for cell counting in a variety of diagnostic body fluids, and highlights the current technologies used.

UF-1000i: validation of the body fluid mode for counting cells in body fluids.

BACKGROUND: We evaluated the new body fluid mode on the UF-1000i urinalysis analyzer for counting total white blood cells (WBC) and red blood cells (RBC) in continuous ambulatory peritoneal dialysis (CAPD), ascites and pleural fluids. METHODS: We collected 154 body fluid samples, and compared the results of the UF-1000i BF mode with the Fuchs-Rosenthal counting chamber and the XN-1000 BF mode. Linearity, carry over and precision were also assessed. RESULTS: Method comparison results showed acceptable WBC agreement between UF-1000i and chamber (y=1.27x+3.13, n=135, r=0.99) and between UF-1000i and XN (y=1.15x+0.31, n=135, r=1.00). Comparison between the UF-1000i and both comparison methods showed good agreement for RBC counts. Overall results were better when UF-1000i was compared with the XN-1000 than with the Fuchs-Rosenthal chamber. The lower limit of quantitation was defined at 9×106 WBC/L and at 25×106 RBC/L. Linearity for both WBC (r=1.00) and RBC (r=0.99) was good. Carry over was negligible, and it never exceeded 0.01%. In one sample, a high discrepancy was observed between WBC results for both automated analyzers and the counting chamber. This discrepancy was due to interfering factors, such as bacteria and yeast cells, and it led to a false increased WBC count on both automated systems. CONCLUSIONS: The UF-1000i BF mode offers rapid and reliable total WBC and RBC counts for initial screening of CAPD, ascites and pleural fluid, and can improve the workflow in a routine laboratory; however, when using automated analyzers, the inspection of scattergrams is required to ensure the most accurate results are obtained.

Validation of the body fluid module on the new Sysmex XN-1000 for counting blood cells in cerebrospinal fluid and other body fluids.

BACKGROUND: We evaluated the body fluid (BF) module on the new Sysmex XN-1000 for counting blood cells. METHODS: One hundred and eighty-seven BF samples [73 cerebrospinal fluid (CSF), 48 continuous ambulatory peritoneal dialysis (CAPD), 46 ascites, and 20 pleural fluid] were used for method comparison between the XN-1000 and manual microscopy (Fuchs-Rosenthal chamber and stained cytospin slides) for counting red blood cells (RBCs) and white blood cells (WBCs) (differential). RESULTS: Good agreement was found for counting WBCs (y=1.06x+0.09, n=67, R2=0.96) and mononuclear cells (MNs) (y=1.04x-0.01, n=40, R2=0.93) in CSF. However, the XN-1000 systematically counted more polymorphonuclear cells (PMNs) (y=1.48x+0.18, n=40, R2=0.99) compared to manual microscopy. Excellent correlation for RBCs >1×109/L (y=0.99x+116.56, n=26, R2=0.99) in CSF was found. For other fluids (CAPD, ascites and pleural fluid) excellent agreement was found for counting WBCs (y=1.06x+0.26, n=109, R2=0.98), MNs (y=1.06x-0.41, n=93, R2=0.96), PMNs (y=1.06x+0.81, n=93, R2=0.98) and RBCs (y=1.04x+110.04, n=43, R2=0.98). By using BF XN-check, the lower limit of quantitation (LLoQ) for WBC was defined at 5×106/L. Linearity was excellent for both the WBCs (R2=0.99) and RBCs (R2=0.99) and carry-over never exceeded 0.05%. CONCLUSIONS: The BF module on the XN-1000 is a suitable tool for fast and accurate quantification of WBC (differential) and RBC counts in CSF and other BFs in a diagnostic setting.

Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids.

BACKGROUND: We evaluated the body fluid (BF) mode on the new Sysmex XE-5000 analyzer. METHODS: Red (RBC) and white blood cell (WBC) (differential) counts of BFs (139 patient samples and 87 normal samples) were measured and compared to the Fuchs-Rosenthal chamber and stained cytospin slides. RESULTS: Extended cell counting using the BF mode was noted to have an improved WBC detection limit (CV(20)%) of 10 x 10(6)/L. Excellent agreement with the manual method was observed for most BFs [mean bias +2 to 6 x 10(6)/L for cerebrospinal fluid (CSF) and -1 to 12 x 10(6)/L for other fluids]. In CSF, the BF-mode counted more WBC (polymorphic nuclear cells) compared with the manual method (mean bias +5 to 6 x 10(6)/L), especially in samples with low cell counts (<20 x 10(6)/L). Carry over was negligible (mostly <0.17%) and linearity was excellent (mean bias <5%). The reference ranges for CSF (n=87) were RBC 0 x 10(6)/L, WBC and mononuclear <7 x 10(6)/L, and polymorph nucleated cells <3 x 10(6)/L. CONCLUSIONS: The BF mode on the Sysmex XE-5000 offers rapid and accurate RBC and WBC (differential) counts in clinically relevant concentration ranges in CSF and other fluids. In addition, the exclusion of high fluorescent cells, such as mesothelial cells and macrophages from WBC counting may reduce the number of manual analyses in pleural fluids and ascites.

Transcriptional expression levels of chicken collectins are affected by avian influenza A virus inoculation.

Mammalian collectins have been found to play an important role in the defense against influenza A virus H9N2 inoculation, but for chicken collectins this has not yet been clarified. The aim of this study was to determine the effect of avian influenza A virus (AIV) inoculation on collectin gene expression in the respiratory tract of chickens and whether this was affected by age. For this purpose 1- and 4-week-old chickens were inoculated intratracheally with PBS or H9N2 AIV. Chickens were killed at 0, 8, 16 and 24h post-inoculation and trachea and lung were harvested for analysis. Viral RNA expression and mRNA expression of chicken collectins 1 and 2 (cCL-1 and cCL-2), chicken lung lectin (cLL) and chicken surfactant protein A (cSP-A) were determined using real-time quantitative RT-PCR. In lung, a decrease in mRNA expression of cCL-2, cLL and cSP-A after inoculation with H9N2 was seen in both 1- and 4-week-old birds, although at different time points, while in trachea changes were only seen in 4-week-old birds and expression was increased. Moreover, collectin expression correlated with viral RNA expression in lung of 1-week-old birds. These results suggest that both age and location in the respiratory tract affect changes in collectin mRNA expression after inoculation with H9N2 and indicate a possible role for collectins in the host response to AIV in the respiratory tract of chickens.