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The absolute basophil count (cells/L) can be determined by manual counting of peripheral blood smears or using cell counting chambers as well as by automated hematology analyzers and fluorescence flow cytometry. Manual basophil counting of peripheral blood smears is currently regarded as the reference method, although the limitations of this method (distribution, observer, and statistical errors) are widely recognized. Automated hematology analyzers offer an advantage of larger numbers of counted cells and high throughput but are characterized by inconsistent analytical performance for basophil enumeration. Flow cytometric enumeration of circulating basophils using panels of monoclonal antibodies is being developed as novel candidate reference method for the absolute basophil count in peripheral blood. Basophil counting using fluorescence flow cytometry is characterized by high precision and statistical superiority. Emerging innovative technologies for absolute cell counts include imaging flow cytometry, mass cytometry, and on-chip blood counting, but their analytical performance for absolute basophil counts is yet to be established. Here, we describe various techniques for absolute basophil counting in peripheral blood including manual basophil counts in smears and hemocytometers and flow cytometric methodologies using double-platform, bead-based, and volumetric approaches.

The purification of basophils from peripheral blood has represented a formidable challenge for researchers since they were discovered by Paul Ehrlich in 1879. From the first published attempts in the late 1960s, it took half a century to develop robust protocols able to give sufficient numbers of pure, functionally unimpaired basophils. The existing protocols for basophil purification exploit those properties of basophils which distinguish them from other cell types such as their localization in blood, density, and the presence or absence of surface markers. Purification techniques have been used in various combinations and variations to achieve a common goal in mind: to obtain a pure population of human basophils in sufficient numbers for downstream studies. The arduous way leading up to the modern protocols is summarized in this historical retrospective. A fast protocol for purification of basophils to near homogeneity is also described.

The molecular characterization of immune subsets is important for designing effective strategies to understand and treat diseases. We characterized 29 immune cell types within the peripheral blood mononuclear cell (PBMC) fraction of healthy donors using RNA-seq (RNA sequencing) and flow cytometry. Our dataset was used, first, to identify sets of genes that are specific, are co-expressed, and have housekeeping roles across the 29 cell types. Then, we examined differences in mRNA heterogeneity and mRNA abundance revealing cell type specificity. Last, we performed absolute deconvolution on a suitable set of immune cell types using transcriptomics signatures normalized by mRNA abundance. Absolute deconvolution is ready to use for PBMC transcriptomic data using our Shiny app (https://github.com/giannimonaco/ABIS). We benchmarked different deconvolution and normalization methods and validated the resources in independent cohorts. Our work has research, clinical, and diagnostic value by making it possible to effectively associate observations in bulk transcriptomics data to specific immune subsets., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Percentiles 10th, 25th, 50th, 75th and 90th are presented for circulating white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils and basophils in healthy European adolescents (12.5-17.5 years, n = 405, 48.9% boys), considering age, sex, puberty and body mass index (BMI). CD3(+) (mature T cells), CD4(+) (T helper), CD8(+) (T cytotoxic), CD16(+)56(+) (natural killer), CD19(+) (B cells), CD3(+)CD45RA(+), CD4(+)CD45RA(+), CD8(+)CD45RA(+) (naïve), CD3(+)CD45RO(+), CD4(+)CD45RO(+) and CD8(+)CD45RO(+) (memory) lymphocytes were also analysed by immunophenotyping. Girls presented higher WBC, neutrophil, CD3(+)CD45RO(+) and CD4(+)CD45RO(+) cell counts and CD3(+)/CD19(+) ratio, and lower CD3(+)CD45RA(+) and CD4(+)CD45RA(+) counts than boys. Age was associated with higher neutrophil counts and CD3(+)/CD19(+), and lower CD19(+) counts; in boys, with lower CD3(+)CD45RA(+), CD4(+)CD45RA(+) and CD8(+)CD45RA(+) counts as well; in girls, with higher WBC, CD3(+)CD45RO(+) and CD4(+)CD45RO(+) counts. Pubertal maturation in boys was associated with lower WBC and lymphocyte counts; in girls, with higher basophil, CD3(+)CD45RO(+) and CD4(+)CD45RO(+) values. BMI was associated with higher WBC counts; in boys, also with higher lymphocyte counts; in girls, with higher neutrophil, CD4(+), CD3(+)CD45RO(+) and CD4(+)CD45RO(+) counts., Conclusion: Our study provides normative values for circulating immune cells in adolescents, highlighting the importance of considering sex, age, pubertal maturation and BMI when establishing reference ranges for WBC in paediatric populations.

Here, we describe two new surface antigens, named 6D10 and 2B2, whose expression is restricted to porcine granulocytes. 6D10 is only detected in neutrophils and its expression decreases from promyelocytes to mature cells. By contrast, 2B2 antigen is selectively expressed in mature neutrophils, eosinophils and basophils. The expression of these antigens along granulocyte maturation allows the discrimination of several developmental stages of granulocytes based on phenotypic, morphological and functional characteristics previously established. Moreover, these new markers are useful tools to easily characterize the different granulocytes lineages (neutrophils, eosinophils and basophils). By using multiparameter flow cytometric analysis, we have performed a phenotypic and functional characterization of the granulocyte subsets identified by the combination of 6D10 and 2B2 antigens.

Chronic allergic inflammatory reactions involve the infiltration and participation of many different cell types. Although it has been evident for many years that tissue specific mast cells have a primary role in the early stages of these reactions, recent studies indicated that, in addition to mast cells response, a later reaction which selectively recruits the circulating lymphocytes, eosinophils and basophils to the site of inflammation, is the hallmark for the progression of allergic diseases.

Mast cells (MC), blood basophils (Ba) and monocytes (Mo) are of haemopoietic origin. Lineage-relationships and transdifferentiation between MC and Mo, or MC and Ba, have been considered, based on common expression of antigens. In this study, comparative phenotypic analyses on MC, Ba and Mo and on respective cell lines were performed using monoclonal antibodies (mAb) to previously defined and novel CD antigens (CD1-130). By cluster analysis, the overall (all 130 CD) phenotypic relationships (given as similarity indices, SI), between primary cells (MC, Ba and Mo) and corresponding cell lines (HMC-1, KU-812, U937) were 0.716, 0.779 and 0.757, respectively. When primary cells were compared, lower SI values were found (MC versus Ba, 0.509; MC versus Mo, 0.625; Mo versus Ba, 0.698). More distant relationships were found between MC versus Ba and MC versus Mo, compared with Ba versus Mo, for adhesion receptor (R)-, complement R- and cytokine R profiles. Analysis of cytokine R revealed most significant dissimilarities between MC versus Ba and MC versus Mo (SI < 0.2). Moreover, in contrast to other CD subgroups and other lineages, MC and HMC-1 differed from each other in cytokine R expression (SI = 0.286). Cytokine R detectable on HMC-1 but not MC were granulocyte-macrophage colony-stimulating factor (GM-CSFR)alpha(CD116), CD40, Apo-1/FAS(CD95) and gp130(CD130). Cytokine R detectable on Ba but not MC, were interleukin-3 (IL-3)R alpha(CD123), IL-1RII(CD121b), IL-2R alpha(CD25) and CD40. In summary, MC, Ba and Mo display a unique CD profile with MC being the most distantly related cell. The most significant mismatch within a given lineage is the loss of cytokine R on mature MC as compared with normal myeloid progenitors and HMC-1 cells.

The counting of particles and holes in white blood cells during thresholding at different gray levels results in a histogram, the counting densitogram, which contains information about granularity. Parameters describing the shape of this histogram appear to be sufficiently characteristic for the five normal white blood cell classes to allow a 84% correct discrimination between them. This method to quantitate granularity could be valuable for the analysis of cell texture and therefore, when used in combination with geometrical and density histogram parameters, could contribute to the results of morphometric discrimination between other than normal white blood cells.

Thirty four out of 750 patients entered into the Medical Research Council's (MRC) 9th Acute Myeloid Leukaemia Trial had more than 1% basophils (range 1-27%) often with bizarre granulation and primitive forms, a rare finding in this disease. Both normal and abnormal karyotypes were present including abnormalities of 6p, 12p, and the Philadelphia chromosome. Basophilia was found in both "monolineage" and "multilineage" leukaemias and the commonest French-American-British (FAB) classification group was M2, followed by M4. Basophilia did not seem to be associated with a worse prognosis, although cases with abnormalities of 6p died of disease that was resistant to first line conventional chemotherapy.

In a previous study, human blood basophils were separated by centrifugation on Percoll gradients into two fractions, band 1 and band 2, that differed in density and histamine content. In this study, the change in circulating numbers of band 1 and band 2 basophils, as well as other leukocytes, was measured after a single oral dose of prednisone. Three hours after ingestion of 50 mg of prednisone, the circulating number of band 1 basophils was 19 +/- 4% of the preprednisone value, whereas the band 2 level was 87 +/- 18% (SEM for five subjects). At 6 hours, values were 10 +/- 1% and 41 +/- 7% for band 1 and band 2, respectively. Circulating numbers of both basophil populations returned to near normal at 24 hours. The 3-hour response to a 10 mg oral dose in seven subjects was 28 +/- 5% and 89 +/- 10% of preprednisone levels for band 1 and band 2; the 6-hour responses were 28 +/- 7% and 84 +/- 7%. The 3-hour responses of other leukocytes in four of these subjects, expressed as a percentage of preprednisone counts were neutrophils, 171 +/- 27%; lymphocytes, 47 +/- 6%; monocytes, 36 +/- 9%; and eosinophils, 26 +/- 11%. The results demonstrate that band 2 basophils have a lower sensitivity to glucocorticoid action than do band 1 basophils or other types of circulating leukocytes.

Relationships among mature blood basophils, blood basophil colony-forming units in culture (CFU-c), and nasal metachromatic cells (NMC) were investigated using hemopoietic and histochemical techniques in 29 patients with allergic rhinitis and in 7 nonatopic control subjects. Total blood granulocyte and basophil CFU-c were significantly elevated in atopy; the highest levels of basophil CFU-c were found in patients with low total NMC counts in nasal scrapings (Group I), compared with those with intermediate (Group II) or high (Group III) counts: 10 +/- 3 basophil CFU-c per 10(6) cells in 10 Group I patients, 4 +/- 2 in 10 Group II patients, 3 +/- 1 in 9 Group III patients, and 0.1 +/- 0.1 in nonatopic control subjects (p less than 0.05). Mean histamine content and frequency of histamine-positive granulocyte colonies correlated with counts of basophils in colonies (r = 0.864, p less than 0.001). Peripheral blood basophils, which stained metachromatically with toluidine blue at pH 0.5 after Mota's lead acetate but not after formalin fixation, were highest in atopic Group III and lowest in Group I; a similar relationship was observed only for NMC, which also failed to stain after formalin fixation. Metachromatic cells in colonies were similar to formalin-sensitive NMC and to peripheral blood basophils in their sensitivity to different fixatives. Nasal symptoms correlated inversely with the number of basophil CFU and directly with the number of either formalin-sensitive NMC or peripheral blood basophils. These findings confirm and extend evidence for increased nasal metachromatic cells, basophilia, and alterations in basophilopoiesis in atopy.

The immunologic surface marker profile of human mast cells (MCs) was established using a combined toluidine/immunofluorescence staining procedure [49 monoclonal antibodies (MoAbs) tested]. Ascites (n = 9) MCs as well as enzymatically dispersed MCs from all organs tested (lung n = 11, skin n = 7, intestinum n = 10) exhibited an identical marker profile. MCs were recognized by MoAbs clustered as CD9 (anti-gp24), CD33 (anti-gp67), and CD45 (anti-gp220) as well as by MoAbs directed against membrane-bound IgE. MoAB YB5B8 (anti-gp145) selectively recognized MCs. Most significantly, however, MCs were stained by MoAbs MAX1 (anti-gp65), MAX3 (anti-gp68), MAX11 (anti-gp65), and MAX24 (anti-gp65). These antibodies bind to surface membrane antigens associated with a late stage of monocyte/macrophage differentiation. Thus, our results provide definite evidence that MCs share surface membrane markers with mononuclear phagocytes. In contrast, MCs are stained neither by lymphatic markers (CD1-8, 10, 19-24) nor by myelomonocytic markers (CD11-17). MCs also lack the interleukin-2 (IL-2) receptor (CD25), the T10 antigen (CD38), and most of the myelocytic markers expressed on peripheral blood (PB) basophils. Thus, MCs displayed a unique phenotype within the hematopoietic system. This new approach enabled us to enrich human lung MCs to a purity greater than 95% by means of negative selection with complement-mediated cell lysis. Purified MCs were subsequently stained with MoAbs and analyzed by flow cytometry, which confirmed the results obtained from the double-staining experiments. We next examined cultured metachromatic cells derived from bone marrow (BM) and peripheral blood colony-forming units (CFU). These metachromatic cells previously could not be classified by morphologic criteria alone and have therefore been termed basophil-like/MC-like cells. In this study, toluidine blue-positive cells obtained from either pooled multipotential colonies (day 14-CFU-GEM) or pooled myelocytic colonies (day 16/17-CFU-GM/G/M) were recognized by MoAbs MY7 (CD13), VIM12 (CD11b), and VIM2, as well as by an anti-IgE MoAb, after preincubation with IgE. In contrast, CFU-derived metachromatic cells were not stained by MoAb YB5B8. This marker profile corresponds to the immunologic phenotype of blood basophils and excluded a detectable formation of mature MCs in colonies derived from cultured hematopoietic stem cells.

The effect of recombinant human (rh) cytokines, interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), granulocyte/macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte/macrophage colony stimulating factor (M-CSF), interferon-alpha (IF-alpha), interferon-gamma (IF-gamma), and the tumor necrosis factor-alpha (TNF-alpha) on differentiation and function of metachromatic cells (MCS) was studied. Among all cytokines tested, rh interleukin-3 (rhIL-3) selectively induced a significant formation of MCS (IL-3: 1.1 +/- 0.6 x 10(5) v control: 0.02 +/- 0.15 x 10(5) MCS/mL suspension) and dose dependent increase in formation of intracellular histamine (IL-3, 100 U/mL: 95 +/- 23 ng/mL v control: 1.8 +/- 0.8 ng/mL) in a bone marrow suspension culture system (analyzed on day 14 of culture). Besides MCS, formation of eosinophils was observed in this culture system in the continuous presence of rhIL-3, whereas IL-3 pulse-stimulation for three hours and subsequent exposure to control medium induced growth of MCS but not of eosinophils. By combined immunofluorescence/toluidine blue staining, MCS were found to express a cell surface marker profile that corresponds to the immunological phenotype of peripheral blood basophils (MY-7(CD13)+, VIM12(CD11b)+, VIM2+, MAX1-, MAX24- and YB5B8-). Furthermore, cultured MCS expressed surface membrane receptors for IgE and could be triggered for nontoxic histamine release by a monoclonal anti-IgE antibody. To evaluate a possible influence of IL-3 on basophil function, studies were extended to freshly obtained blood basophils (healthy volunteers, n = 3). However, like all other cytokines tested, rhIL-3 failed to induce basophil histamine release. Taken together, our studies demonstrate that IL-3 is a differentiation factor for human basophils.

It has been reported that blood basophils comprise two populations of different densities. In this study, we compared the responses of two populations of basophils (band-1 and 2 basophils) to several secretagogues and anti-allergic agents. Twelve patients with bronchial asthma, 3 patients with Japanese cedar pollinosis and 6 healthy subjects were included in this study. Band-2 basophils collected from the interface between Percoll of density 1.078 and 1.068 g/ml released histamine better in response to anti-IgE than band-1 basophils taken from the interface between plasma and Percoll 1.068 (band 1 vs. band 2, 13.2 +/- 2.7 vs. 21.4 +/- 3.6% at 1:10(5), p less than 0.01; 47.1 +/- 5.0 vs. 57.7 +/- 6.1% at 1:10(4), p less than 0.01). Band-2 basophils also responded better to formyl-methionyl-leucyl-phenylalanine than band-1 basophils (34.4 +/- 4.9 vs. 43.2 +/- 5.6% at 10(-6) M, p less than 0.01; 35.6 +/- 5.0 vs. 45.2 +/- 5.7% at 10(-5) M, p less than 0.01). In contrast, band-1 basophils were much more sensitive to the calcium ionophore A23187 stimulation than band-2 basophils (13.0 +/- 3.7 vs. 6.3 +/- 1.8% at 0.05 microgram/ml, p less than 0.05; 52.5 +/- 4.3 vs. 33.8 +/- 3.9% at 0.1 microgram/ml, p less than 0.01; 71.3 +/- 3.3 vs. 51.5 +/- 4.2% at 0.2 microgram/ml, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

We designed experiments in this study to test the hypothesis suggested by recent purification data that blood basophils comprise two populations of different density, which circulate in numbers characteristic for each human subject. Basophils were separated into two density bands by single step centrifugation on a discontinuous Percoll gradient. Band 1 cells were at the interface between plasma and Percoll of density 1.070 gm/ml. Band 2 cells were at the Percoll 1.070 to 1.080 interface. When the number of band 1 basophils was expressed as a percentage of the total in bands 1 and 2, this relative amount generally remained in a narrow range for blood obtained from the same donor on 3 successive days but differed markedly in different individuals. In a series of leukapheresis experiments, we demonstrated that the percentage of band 1 basophils in postleukapheresis venous blood was strikingly similar to the preleukapheresis value. If basophils that repopulated the leukapheresis-depleted circulation came from the bone marrow, we can conclude that blood levels of basophils in bands 1 and 2 are under physiologic control and that the two types of basophils are released in amounts characteristic for each human subject. Additional evidence for two distinct blood basophil populations was provided by histamine measurements. The histamine content per basophil was consistently higher in cells from band 1 than from band 2, the mean difference between pairs of values for 30 subjects being 0.3 +/- 0.04 pg or about 27% of the band 1 basophil histamine content of 1.1 pg.