Identification and quantification of high fluorescence-stained lymphocytes as antibody synthesizing/secreting cells using the automated routine hematology analyzer XE-2100.

OBJECTIVES: The aim of this study was to classify and quantify the high fluorescence lymphocytes area (HFL-count) from the SYSMEX XE-2100 leucocyte differential channel as antibody-synthesizing or -secreting cells (ASC, plasma cells or lymphoplasmacytoid cells) in reactive diseases. To unequivocally identify the HFL cells, all possibly eligible cell populations have been investigated: activated B-lymphocytes, activated T-lymphocytes, large granular lymphocytes (LGL), activated monocytes, and immature granulocytes. METHODS: In total, 85 patients were analyzed on the XE-2100 and compared with the automated image analysis system Cellavision Diffmaster 96 based on artificial neural network and immunophenotyping method with the BD FACSCalibur. RESULTS: Reproducibility tests for HFL demonstrated a mean coefficient of variation of 13.9% for very low results and 1.5% for high results. The linearity data showed a good correlation (R(2) = 0.99) between expected and measured HFL. The comparison with possibly eligible cell populations showed no significant correlation between activated monocytes and immature granulocytes, with most immature granulocytes (promyelocyte I or II), natural killer cells or LGLs, activated T-lymphocytes, and sub-T-lymphocytes populations. However, for activated B-lymphocytes an excellent significant correlation with the peripheral blood smear, and the immunophenotyping method has been found with R(2) = 0.900, P < 0.001 and R(2) = 0.897, P < 0.001, respectively. The slope of 1.1 and intercept of minus 5 cells/microL of the regression equation between HFL-count and ASC (smear) do indicate an excellent quantification of the HFL-count, as well. CONCLUSION: The fully automated SYSMEX XE-2100 HFL-count identifies and quantifies the ASC cells (activated B-lymphocytes) with high precision and reliability in patients without hematology system diseases, thus providing a potential screening and monitoring tool for any patient with suspected infection. Additional studies are required to comprehend in more detail the full clinical utility of an HFL (ASC) count as a potential diagnostic indicator of inflammation, infection, or sepsis.

Relation of ex vivo stimulated blood cytokine synthesis to post-traumatic sepsis.

The cytokine production in endotoxin stimulated blood of patients immediately after polytrauma with high risk for developing sepsis or multi organ failure was analysed. Forty patients sustaining traumatic injury with >/=317 pts according to the Injury Severity Score (ISS), 10 of whom developed severe sepsis (ACCP/SCCM conference 1992) were included in the study. Levels of interleukin 8 (IL-8), IL-6 and tumour necrosis factor (TNF) were measured by ELISA in endotoxin-stimulated whole blood and IL-10 and IL-6 in serum. The allotype for the bi-allelic Nco I restriction length polymorphism in the TNF locus was determined for each patient.Two to four hours after polytrauma endotoxin-stimulated synthesis of TNF and IL-6 was found to be reduced in whole blood from patients compared to healthy donors, whereas no such differences were found for IL-8 synthesis. At this time, however, the patients who developed sepsis at a later stage (day 4-6) showed significantly (P<0.05) enhanced IL-8 synthesis in endotoxin stimulated whole blood in comparison to healthy donors. The IL-6 and TNF production of their blood was significantly enhanced compared to patients with uncomplicated recovery. Ninety per cent of the patients developing sepsis were of the TNFB2/TNFB2 allotype, whereas this was the case for only 30% of the non-septic group. Assessment of endotoxin-stimulated cytokine synthesis may provide a prognostic indicator for patients at high risk for developing a sepsis syndrome.

Regulation of whole blood tumor necrosis factor production upon endotoxin stimulation after severe blunt trauma.

BACKGROUND: Trauma has been recognized to be accompanied by alterations of leukocyte functions such as cytokine release. The regulatory principles involved in these changes are still poorly defined. To further characterize leukocyte function after multiple trauma, endotoxin-stimulated tumor necrosis factor (TNF) production of trauma patients' whole blood and a possible regulatory mechanism were studied. METHODS: Endotoxin responsiveness in trauma patients (n = 18, Injury Severity Score = 24 +/- 7) was assayed ex vivo using a whole blood model. TNF release and TNFalpha mRNA levels were determined during a 14-day period. Furthermore, the influence of patients' sera on whole blood TNF production was evaluated. MAIN RESULTS: The capacity of trauma patients' whole blood to produce TNF was reduced for 2 to 6 days after trauma and was equally evident for both TNF release and TNFalpha mRNA levels. The reduction of TNF coincides with the appearance of an inhibitory activity for TNF production in trauma patients' sera. No correlation was found between the inhibitory activity and soluble TNF receptors, endotoxin-neutralizing molecules, inhibitory cytokines (interleukin 10 and transforming growth factor beta), or prostaglandins. CONCLUSIONS: Major trauma leads to the appearance of a circulating inhibitory activity for TNF synthesis that may potentially contribute to an anti-inflammatory response in patients with multiple trauma. The elucidation of its structural and functional properties may contribute to the understanding of the pathogenesis of severely injured patients.

The extent of traumatic damage determines a graded depression of the endotoxin responsiveness of peripheral blood mononuclear cells from patients with blunt injuries.

OBJECTIVE: To study whether the endotoxin responsiveness of peripheral blood mononuclear cells correlates with the severity of injury in trauma patients. DESIGN: Prospective, observational study. SETTING: University trauma center. PATIENTS: Fifty-nine patients with blunt trauma (Injury Severity Score [ISS] 4 to 57 points). INTERVENTIONS: Standard emergency department care, surgical care, and postoperative intensive care unit treatment. MEASUREMENTS AND MAIN RESULTS: Whole blood and serum were obtained 94+/-89 (SD) mins post trauma (day 0) and during a 14-day period postinjury. Endotoxin-induced tumor necrosis factor-alpha (TNF-alpha) synthesis of peripheral blood mononuclear cells ex vivo was tested using a whole blood assay. Serum samples were assayed for TNF-alpha concentrations. A reduced capacity of whole blood to produce TNF-alpha ex vivo with endotoxin treatment was found to be closely correlated with the ISS. The capacity to produce TNF-alpha on endotoxin stimulation of whole blood from patients with an ISS > or =16 points was depressed immediately after trauma and did not reach normal values during the observation period. In patients with an ISS >22 points, maximum depression of the capacity of whole blood to produce TNF-alpha occurs within 100 mins post injury. In contrast, in patients with an ISS <22 points, maximal depression of whole blood TNF-alpha production occurs with a delay of 24 to 48 hrs after trauma. Based on pre- and postoperative values, primary surgical intervention caused a decrease of the endotoxin-stimulated TNF-alpha production of whole blood in the latter patient subgroup, as well as in the entire patient population (ISS 4 to 57) when secondary surgical treatment was necessary 5 to 13 days after trauma. CONCLUSIONS: The extent of traumatic tissue damage leads to a graded depression of immunocyte function and appears to be amplified by surgical treatment. The endotoxin responsiveness of peripheral blood mononuclear cells displays a functional marker of the anatomically defined severity of injury and gives insights into the regulation of immunocyte function after severe blunt trauma.