Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer.

Björnsson S, Wahlström S, Norström E, Bernevi I, O'Neill U, Johansson E, Runström H, Simonsson P.

Department of Clinical Chemistry, University Hospital MAS, S 205 02 Malmö, Sweden.

BACKGROUND:: Flow cytometry allows the use of several antibodies in addition to light scatter, and most flow cytometers will provide at least seven measurements on each cell passing through the laser beam. A skilled microscopist will classify at least 14 cell classes in bone marrow or blood. Our goal was to use the seven parameters available in our flow cytometer to provide a reliable differential count using only one tube. METHODS:: Peripheral blood samples were analyzed on the Beckman Coulter LH750 cell counter, and the flagging and messages from the cell counter were used to select normal or pathological samples. Samples without flags (N = 50), with >2% erythroblasts (N = 80), or with "Blast" or "Verify diff" flags (N = 54) were investigated. We used a lyse-no-wash method to ensure minimal loss of fragile cells with live gating on DRAQ5-positive cells to acquire only nucleated cells. The FL-1 to FL-4 channels were used for the antibodies CD36-FITC, CD203-PE, CD138-PE, CD45-ECD, CD16-Pcy5, and CD56-Pcy5. FL-5 was used for the DNA-stain DRAQ5. RESULTS:: Using live gate acquisition on DRAQ5, we were able to classify total nucleated cells into 10 classes. We were unable to identify megakaryocytes, but platelets could be studied by rerunning the sample after dilution and gating on DRAQ5-negative CD36-posive events. Validation against digitized microscopy and cell counter showed linear correlations within each cell class with correlation coefficients that seem reasonable for cellular classification. The lowest correlation was found for basophil granulocytes. Flow cytometry detected twice as many immature neutrophils compared to microscopy. CONCLUSIONS:: We have designed a one-tube immunophenotyping panel for classification of total nucleated cells and platelets in blood or bone marrow. The seven parameters available in one single tube in our cytometer seem to be enough for reliable differential count even in difficult pathological samples. The analytical imprecision of the flow cytometer differential was much lower than that obtained with microscopy or cell counter differentials. (c) 2007 Clinical Cytometry Society.


Maturation and Localization of Macrophages and Microglia During Infection with a Neurotropic Murine Coronavirus.

Templeton SP, Kim TS, O'malley K, Perlman S.

Interdisciplinary Program in Immunology, University of Iowa, Iowa City, Iowa, USA.

Macrophages and microglia are critical in the acute inflammatory response and act as final effector cells of demyelination during chronic infection with the neutrotropic MHV-JHM strain of mouse hepatitis virus (MHV-JHM). Herein, we show that "immature" F4/80(+)Ly-6C(hi) monocytes are the first cells, along with neutrophils, to enter the MHV-JHM-infected central nervous system (CNS). As the infection progresses, macrophages in the CNS down-regulate expression of Ly-6C and CD62L, consistent with maturation, and a higher frequency express CD11c, a marker for dendritic cells (DCs). Microglia also express CD11c during this phase of the infection. CD11c(+) macrophages in the infected CNS exhibit variable properties of immature antigen-presenting cells (APCs), with modestly increased CD40 and MHC expression, and equivalent potent antigen uptake when compared with CD11c(-) macrophages. Furthermore, CDllc(+) and F4/80(+) macrophages and microglia are localized to areas of demyelination, in some instances directly associated with damaged axons. These results suggest that chronic CNS infection results in the appearance of CD11c-expressing macrophages from the blood that exhibit properties of immature APCs, are closely associated with areas of demyelination, and may act as final effectors of myelin destruction.

PMID: 17935605 [PubMed - as supplied by publisher]

Neutrophil elastase converts human immature dendritic cells into transforming growth factor-beta1-secreting cells and reduces allostimulatory ability.

Maffia PC, Zittermann SE, Scimone ML, Tateosian N, Amiano N, Guerrieri D, Lutzky V, Rosso D, Romeo HE, Garcia VE, Issekutz AC, Chuluyan HE.

Lanais de la Facultad de Medicina, Universidad de Buenos Aires, Avenida Córdoba 2351, C.P. 1120, Buenos Aires, Argentina.

During microbial infection, neutrophils (polymorphonuclear leukocytes; PMNs) activate dendritic cells (DCs). However, early reports illustrated that neutrophil-derived mediators may suppress responses to mitogens. In the present study, we investigated the mechanism used by PMNs to modulate the immunostimulatory ability of DCs. Autologous syngeneic PMNs decreased T-cell proliferation induced by allogeneic DCs. Culture supernatant (CS) derived from PMNs also decreased allostimulation ability of immature DCs and increased the expression of transforming growth factor (TGF)-beta1 on DCs. A TGF-beta1 monoclonal antibody, a CD40 monoclonal antibody, or a serine protease inhibitor reversed the effect of PMN CS on DC allostimulatory ability. Furthermore, elastase reproduced the inhibitory effect of PMN CS on DC allostimulatory ability and the TGF-beta1 production. The role of elastase was confirmed by examining PMN CS from two patients with cyclic neutropenia, a disease due to mutations in the neutrophil elastase gene. These PMN CS samples had reduced elastase activity and were unable to increase DC TGF-beta1 production. Moreover, elastase and PMN CS induced IkappaBalpha degradation in DCs. We conclude that PMNs decrease DC allostimulatory ability via production of elastase leading to a switch of immature DCs into TGF-beta1-secreting cells.

PMID: 17690184 [PubMed - indexed for MEDLINE]

Tissue expression of copines and isolation of copines I and III from the cytosol of human neutrophils.

Cowland JB, Carter D, Bjerregaard MD, Johnsen AH, Borregaard N, Lollike K.

Department of Hematology, Rigshospitalet, Copenhagen, Denmark.

Copines are a recently identified group of proteins characterized by two Ca(2+)-binding C2-domains at the N terminus and an A-domain at the C terminus. Although pEST sequences indicate the existence of at least seven copines in man, only copines I, III, and VI have been identified at protein level. Here, we describe the isolation of copines I and III in the cytosol of human neutrophils by use of Ca(2+)-induced hydrophobic chromatography. This is the first demonstration that copines are coexpressed in the same cell. We found that copine III exists in the cytosol of human neutrophils as a monomer with a blocked N terminus. Copines I and III undergo conformational changes upon Ca(2+) binding that lead to exposure of hydrophobic patches. Examination of RNA from 68 human tissues demonstrated that copines I-III are ubiquitously expressed whereas copines IV-VII each has a more restricted and individual expression profile. Expression of copines I-III was also demonstrated in neutrophil precursors from bone marrow. Copine I was uniformly expressed at all stages of neutrophil differentiation, whereas copine II and even more so, copine III were expressed in the more immature neutrophil precursors, which indicates an individual function of these copines.

PMID: 12949241 [PubMed - indexed for MEDLINE]

test: neutrophilic promyelocyte