Interlaboratory Reproducibility of Blood Morphology Using the Digital Microscope.

Differential counting of peripheral blood cells is an important diagnostic tool. However, manual morphological analysis using the microscope is time-consuming and requires highly trained personnel. The digital microscope is capable of performing an automated peripheral blood cell differential, which is as reliable as manual classification by experienced laboratory technicians. To date, information concerning the interlaboratory variation and quality of cell classification by independently operated digital microscopy systems is limited. We compared four independently operated digital microscope systems for their ability in classifying the five main peripheral blood cell classes and detection of blast cells in 200 randomly selected samples. Set against the averaged results, the R(2) values for neutrophils ranged between 0.90 and 0.96, for lymphocytes between 0.83 and 0.94, for monocytes between 0.77 and 0.82, for eosinophils between 0.70 and 0.78, and for blast cells between 0.94 and 0.99. The R(2) values for the basophils were between 0.28 and 0.34. This study shows that independently operated digital microscopy systems yield reproducible preclassification results when determining the percentages of neutrophils, eosinophils, lymphocytes, monocytes, and blast cells in a peripheral blood smear. Detection of basophils was hampered by the low incidence of this cell class in the samples.

Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation.

BACKGROUND: Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches. METHODS: Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects. RESULTS: AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS. CONCLUSIONS: Our findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.

Flow cytometric characterization of cerebrospinal fluid cells.

Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately.

Addition of serum-containing medium to cerebrospinal fluid prevents cellular loss over time.

Immediately after sampling, leukocyte counts in native cerebrospinal fluid (CSF) start to decrease rapidly. As the time lapse between CSF collection to analysis is not routinely registered, the clinical significance of decreasing cell counts in native CSF is not known. Earlier data suggest that addition of serum-containing medium to CSF directly after sampling prevents this rapid decrease in leukocyte counts and, thus, may improve the accuracy of CSF cell counting and cell characterization. Here, we prospectively examined the effect of storage time after lumbar puncture on counts of leukocytes and their major subsets in both native CSF and after immediate addition of serum-containing medium, measured by flow cytometry and microscopy. We collected CSF samples of 69 patients in tubes with and tubes without serum-containing medium and determined counts of leukocytes and subsets at 30 minutes, 1 hour, and 5 hours after sampling. Compared to cell counts at 30 minutes, no significant decrease in cell number was observed in CSF with serum-containing medium 1 and 5 hours after sampling, except for the granulocytes at 1 hour. In native CSF, approximately 50% of leukocytes and all their subsets were lost after 1 hour, both in flow cytometric and microscopic counting. In 6/7 (86%) samples with mild pleocytosis (5-15 × 10(6) leukocytes/l), native CSF at 1 hour was incorrectly diagnosed as normocellular. In conclusion, addition of serum-containing medium to CSF directly after sampling prevents cell loss and allows longer preservation of CSF cells prior to analysis, both for microscopic and flow cytometric enumeration. We suggest that this protocol results in more accurate CSF cell counts and may prevent incorrect conclusions based on underestimated CSF cell counts.

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.

[Application of tumour markers in clinical practice]. / Toepassing van tumormarkers in de klinische praktijk.

Usefully requesting and applying serum tumour markers in diagnosis and treatment can be difficult. It should be noted that tumour markers are used for varying purposes: screening, diagnosis, staging and prognostic evaluation, detection of recurrence and treatment monitoring. Due to the poor sensitivity and specificity of current tumour markers, most are not suitable for screening an asymptomatic population. Further, the benefits of an improved prognosis by early detection should be weighed against a poorer quality of life and the cost of substantial over-diagnosis and over-treatment. Serum tumour markers are particularly applicable in treatment monitoring and detection of recurrence. Sometimes they can be used to support the diagnostic process and give useful prognostic information.

MUC1 568 A/G genotype-dependent cancer antigen 15-3 levels in breast cancer patients.

OBJECTIVES: CA 15-3 is a widely used tumor marker for breast cancer. We have investigated whether the MUC1 568 A/G polymorphism can influence CA 15-3 levels in healthy women and patients with breast tumors. DESIGN AND METHODS: CA 15-3 was measured in 208 healthy women, in 67 with benign disease, and in 162 women with breast cancer. All subjects were genotyped for the MUC1 568 A/G polymorphism. RESULTS: Significant differences were observed between mean CA 15-3 levels of control subjects grouped according to the MUC1 568 genotype (mean+/-SD): AA (10.3+/-3.8), AG (15.9+/-5.0) and GG (19.0+/-5.6) U/mL, p<0.0001. Similar (median) results were observed in women with benign breast disease: AA (10.2), AG (14.2) and GG (16.6) U/mL, p<0.0001, and those with breast cancer: AA (10.4), AG (17.1) and GG (23.9) U/mL, p<0.0001. CONCLUSIONS: The MUC1 568 A/G polymorphism strongly influences CA 15-3 levels in healthy women and women with either benign or malignant breast tumors.

CD38 as a prognostic factor in B cell chronic lymphocytic leukaemia (B-CLL): comparison of three approaches to analyze its expression.

BACKGROUND: Increased CD38 expression by leukemic cells has been suggested as an adverse prognostic factor in B-CLL. Several approaches have been proposed to quantify its level of expression by flow cytometry. METHODS: We compared the use of (i) the percentage of CD38 positive cells, (ii) CD38 antibodies bound per cell (ABC), and (iii) a semi-quantitative method based on the shape of the CD38 histogram, within a cohort of 78 B-CLL patients. RESULTS: A decreased overall survival was seen with >30% CD38 positivity among B-CLL cells, with CD38 ABC >100, and with bimodal or unimodal, strongly positive CD38 histograms. However, patients with unimodal weakly positive CD38 histograms also showed a significantly reduced survival as did patients with intermediate proportions (i.e. 5-30%) of CD38+ cells. Furthermore, within the group with <5% CD38 positivity among their B-CLL cells, 84% of patients showed prognostically favourable mutated IGVH gene segments and 100% had low ZAP70 gene expression. For 5-30% CD38 positivity, these proportions were 50 and 83%, while for >30% CD38 positivity, these proportion were only 28 and 56%, respectively. CONCLUSIONS: We found a simple method of quantitation of CD38 expression (i.e., >5% CD38 positivity among B-CLL cells) to be sufficient to identify patients with an unfavourable prognosis. The level of CD38 expression as defined with this method correlated well with the IGVH mutation status and ZAP70 gene expression.

Clinical pharmacokinetics of unbound docetaxel: role of polysorbate 80 and serum proteins.

OBJECTIVE: Our objectives were to study the extent of docetaxel binding to plasma in the presence and absence of its excipient, polysorbate 80 (Tween 80; Imperial Chemical Industries PLC, London, United Kingdom), in vitro and to evaluate the pharmacokinetics of unbound docetaxel in vivo. METHODS: Equilibrium dialysis was used for determination of the fraction unbound (f(u)) docetaxel and was applied to study the pharmacokinetic behavior of unbound docetaxel in 23 patients with cancer receiving an intravenous infusion of the drug formulated in polysorbate 80 (Taxotere; Aventis Pharma SA, Vitry-sur-Seine Cedex, France). RESULTS: Polysorbate 80, added at clinically relevant concentrations (up to 1.0 microL/mL), increased f(u) in vitro by 13% (7.84% +/- 0.0752% versus 6.95% +/- 0.0678%, P <.00001). Similarly, f(u) calculated on the basis of the observed area under the plasma concentration-time curve (AUC) values [f(u)(AUC)] in vivo was 12% higher than f(u) in pretreatment samples [f(u)(pre)] (6.00% +/- 1.03% versus 5.49% +/- 1.01%, P =.038). Of various serum proteins evaluated, only alpha(1)-acid glycoprotein was significantly related to f(u) (P <.0018), with higher f(u) in the presence of lower protein levels. Total docetaxel clearance was related to alpha(1)-acid glycoprotein (R(2) = 0.13, P =.058), f(u)(pre) (R(2) = 0.15, P =.039), and f(u)(AUC) (R(2) = 0.29, P =.0048). CONCLUSION: This study demonstrates that the plasma binding of docetaxel is influenced by both alpha(1)-acid glycoprotein and its formulation vehicle. Further investigation is required to resolve the potential clinical significance of these observations.

Interleukin-17 and CD40-ligand synergistically enhance cytokine and chemokine production by renal epithelial cells.

Renal allograft rejection is characterized by an influx of inflammatory cells. Interaction between infiltrating T cells and resident parenchymal cells might play an important role in the ongoing inflammatory response. The present study demonstrates that CD40L, a product of activated T cells, is locally expressed in kidneys undergoing rejection. Furthermore, during rejection, CD40 expression not only is present on most graft infiltrating cells but also is increased on resident tubular epithelial cells (TEC). To obtain more detailed insight in the consequences of T cell/TEC interaction, we analyzed the production of chemokines, including interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T cell expressed and secreted (RANTES), and the production of IL-6 by cultured human primary TEC in response to activation with CD40L in vitro. In addition, we studied the interaction with IL-17, a T-cell-specific cytokine previously demonstrated to be present during renal allograft rejection. The results, obtained by enzyme-linked immunosorbent assay, indicate that simultaneous activation of TEC with IL-17 and CD40L synergistically enhances production of IL-6 (2.1-fold higher than sum of single stimulations) and the chemokines IL-8 (15-fold) and RANTES (5.8-fold) as demonstrated by statistical analysis (P: < 0.05), whereas effects on MCP-1 (1.4-fold) are additive. Part of the synergy can be explained by increased CD40 expression on TEC upon IL-17 stimulation. The synergy is not unique for TEC, because similar responses were found with human synoviocytes and a foreskin fibroblast cell line (FS4). Stimulation of TEC with CD40L results in activation of NF-kappaB and induction of cytokine production by IL-17 and CD40L is prevented by addition of the NF-kappaB inhibitor pyrrolidine dithiocarbamate. These data suggest an important role for T cells in renal allograft rejection by acting on parenchymal cells via both soluble mediators and direct cellular contact.