Evaluation of cell counting and leukocyte differentiation in cerebrospinal fluid controls using hematology analyzers by the German Society for Clinical Chemistry and Laboratory Medicine.

BACKGROUND: Manual cell counting in cerebrospinal fluid (CSF) is technique-dependent, time-consuming, and thus costly and prone to inter-operator variability and low precision. Flow cytometry (FCM) with fast hematology analyzers (HAs) appears to improve accuracy and precision of CSF cell analysis; rapid CSF cell analysis is especially needed in emergency laboratories. Ten external trials of the German Society for Clinical Chemistry and Laboratory Medicine evaluated FCM with Coulter (LH750, 755), Abbott CD3200, CD3500, CD3700, CD4000, Sapphire, ADVIA120 CSF assay, and Sysmex XE-2100 single platform analyzers. METHODS: CSF controls were produced using native blood leukocytes and erythrocytes, resembling CSF and thus rendering the trials feasible and allowing comparison with native manual counting in a Fuchs-Rosenthal chamber and FACScan-CD45-CD14 dual platform analysis, which was used as the reference method. Statistical evaluation was performed using Passing/Bablok regression analysis. RESULTS: Our evaluation revealed significant differences with respect to target values in leukocyte and erythrocyte counts, as well as leukocyte differentiation. These differences were attributed to inaccuracies produced by the HAs, due to blank correction in connection with impedance analysis, leukocyte loss, especially through monocyte injury due to the erythrocyte lysing agent, incomplete erythrocyte lysis, ADVIA cell sphering, cell differentiation using algorithms and peroxidase activity. Erythrocyte counting in the CSF controls was inaccurate with the Coulter and ADVIA analyzers. CONCLUSIONS: Evaluation of HAs by means of the CSF controls revealed inaccuracies in cell counting and leukocyte differentiation. Analyzer techniques, used for CSF cell assays, therefore need to be improved.

Modifications of haematology analyzers to improve cell counting and leukocyte differentiating in cerebrospinal fluid controls of the Joint German Society for Clinical Chemistry and Laboratory Medicine.

Flow cytometry (FCM) is used with haematology analyzers (HAs) to count cells and differentiate leukocytes in cerebrospinal fluid (CSF). To evaluate the FCM techniques of HAs, 10 external DGKL trials with CSF controls were carried out in 2004 to 2008. Eight single platform HAs with and without CSF equipment were evaluated with living blood leukocytes and erythrocytes in CSF like DGKL controls: Coulter (LH750,755), Abbott CD3200, CD3500, CD3700, CD4000, Sapphire, ADVIA 120(R) CSF assay, and Sysmex XE-2100(R). Results were compared with visual counting of native cells in Fuchs-Rosenthal chamber, unstained, and absolute values of leukocyte differentiation, assayed by dual platform analysis with immune-FCM (FACSCalibur, CD45, CD14) and the chamber counts. Reference values X were compared with HA values Y by statistical evaluation with Passing/Bablock (P/B) linear regression analysis to reveal conformity of both methods. The HAs, studied, produced no valid results with DGKL CSF controls, because P/B regression revealed no conformity with the reference values due to:-blank problems with impedance analysis,-leukocyte loss with preanalytical erythrocyte lysis procedures, especially of monocytes,-inaccurate results with ADVIA cell sphering and cell differentiation with algorithms and enzyme activities (e.g., peroxidase). HA techniques have to be improved, e.g., using no erythrocyte lysis and CSF adequate techniques, to examine CSF samples precise and accurate.

Granulocyte-macrophage colony-stimulating factor-induced vessel growth restores cerebral blood supply after bilateral carotid artery occlusion.

BACKGROUND AND PURPOSE: Hemodynamic compromise due to occlusive cerebrovascular disease is associated with an increased stroke risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to stimulate collateral blood vessel growth in various models of hemodynamic compromise. The purpose of this study was to investigate the effects of GM-CSF on cerebral hemodynamics and vessel growth in a rat model of chronically impaired cerebral blood flow (CBF). METHODS: Male Sprague-Dawley rats underwent sequential bilateral carotid artery occlusion (BCO) and were treated with GM-CSF or saline for 6 weeks. Sham-occluded animals served as a control group. Baseline CBF was measured by iodo[(14)C]antipyrine autoradiography, and cerebrovascular reserve capacity was assessed by laser-Doppler flowmetry after application of 20 mg/kg body weight acetazolamide. The capillary density and arterioles immunopositive for alpha-smooth muscle actin were counted on brain sections. The cerebral angioarchitecture was visualized with a latex perfusion technique. RESULTS: Baseline CBF as measured by iodo[(14)C]antipyrine autoradiography was not affected by BCO. The cerebrovascular reserve capacity, however, was significantly impaired 1 week after BCO. CBF and cerebrovascular reserve capacity recovered completely in GM-CSF-treated animals but not in solvent-treated animals. Histologic analysis of the hippocampus revealed integrity of the hypoxia-vulnerable neurons in all animals. The capillary density showed a very mild increase in GM-CSF-treated animals. However, the number of intraparenchymal and leptomeningeal arterioles was significantly higher in GM-CSF-treated animals than in both other groups. CONCLUSIONS: Long-term GM-CSF treatment in a BCO model in rats leads to restoration of impaired cerebral hemodynamics and accompanies structural changes in the resistance-vessel network.

Neutrophil chemotaxis in cord blood of term and preterm neonates is reduced in preterm neonates and influenced by the mode of delivery and anaesthesia.

Bacterial infections, even without any perinatal risk factors, are common in newborns, especially in preterm neonates. The aim of this study was to evaluate possible impairment of neutrophil chemotaxis in term and preterm neonates compared with adults as well as neonates with different modes of delivery and anaesthesia. We analysed the expression of the adhesion molecule L-Selectin as well as shape change, spontaneous and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced transmigration of neutrophils in a flow cytometric assay of chemotaxis after spontaneous delivery with Cesarian Section (CS) under spinal anaesthesia (mepivacaine, sufentanil), epidural anaesthesia (ropivacaine or bupivacaine, sufentanil) or general anaesthesia (ketamine, thiopental, succinylcholine). Chemokinesis was higher (p=0.008) in cord blood neutrophils than in the adult ones, whereas those could be more stimulated by fMLP (p=0.02). After vaginal delivery neutrophils showed a higher spontaneous and fMLP-stimulated chemotactic response compared to neonates after CS without labor. Comparing different types of anaesthesia for CS, spinal anaesthesia resulted in less impairment on chemotaxis than general anaesthesia or epidural anaesthesia. The new flow cytometric assay of neutrophil chemotaxis is an appropriate and objective method to analyse functional differences even in very small volumes of blood, essential in neonatology. Term neonates do not show reduced chemotaxis compared to adults. Preterm neonates present with reduced chemotaxis and chemokinesis, confirming the well known deficits in their neutrophil function. The side effects of maternal drugs on the neonatal immune system have to be considered especially when the immune response is already impaired, as in preterm infants.

Age-related low expression of HLA-DR molecules on monocytes of term and preterm newborns with and without signs of infection.

OBJECTIVES: Presentation of human leucocyte antigen (HLA) molecules is an important part of an efficient immune response. Since bacterial infections are more common in newborns, we hypothesized that the level of HLA-DR expression may influence the host defense system. STUDY DESIGN: HLA-DR expression on monocytes was examined by flow cytometry during the first week of life of term and preterm neonates with and without signs of infection and of adults. RESULTS: HLA-DR expression of term and preterm newborns with or without signs of infection was lower compared with adults during the first day of life (p<0.0001). Prematurity correlates with lower expression in neonates with gestational age less than 32 weeks (p=0.0008). HLA-DR expression in neonates with signs of infection was decreased compared to healthy neonates (p=0.0196). Maternal conditions such as preeclampsia, prenatal treatment with steroids and mode of delivery had no influence on the expression of HLA-DR. In contrast, newborns with respiratory distress syndrome but without signs of infection showed reduced HLA-DR expression (p=0.0370). CONCLUSION: Low HLA-DR expression on monocytes contributes to impaired neonatal host defense, especially in preterm neonates.

A variable immunoreceptor in a subpopulation of human neutrophils.

Neutrophils are thought to rely solely on nonspecific immune mechanisms. Here we provide molecular biological, immunological, ultrastructural, and functional evidence for the presence of a T cell receptor (TCR)-based variable immunoreceptor in a 5-8% subpopulation of human neutrophils. We demonstrate that these peripheral blood neutrophils express variable and individual-specific TCRalphabeta repertoires and the RAG1/RAG2 recombinase complex. The proinflammatory cytokine granulocyte colony-stimulating factor regulates expression of the neutrophil immunoreceptor and RAG1/RAG2 in vivo. Specific engagement of the neutrophil TCR complex protects from apoptosis and stimulates secretion of the neutrophil-activating chemokine IL-8. Our results, which also demonstrate the presence of the TCR in murine neutrophils, suggest the coexistence of a variable and an innate host defense system in mammalian neutrophils.

Detection of Chlamydophila pneumoniae in the bone marrow of two patients with unexplained chronic anaemia.

Anaemia of chronic disease (ACD) is a common finding involving iron deficiency and signs of inflammation. Here, we report on two patients with ACD where a persistent infection with Chlamydophila (Chlamydia) pneumoniae (CP) was detected in bone marrow (BM) biopsies. Infection was suspected by routine cytology and confirmed by immunofluorescence, electron microscopy, polymerase chain reaction (PCR) including different primer sets and laboratories and sequencing of the PCR product. This is a first report of chlamydial presence in the BM of anaemic patients. The cases are presented because persistent chlamydial infection may contribute more frequently to chronic refractory anaemia than previously suspected.