Flow cytometric analysis of membrane CD11b, CD11c and CD14 expression in acute myeloid leukaemia: relationships with monocytic subtypes and the concept of relative antigen expression.

Blast cells from 26 cases of acute myeloid leukaemia (AML) were examined, by single and "two-colour" flow cytometry, for relationships between membrane CD11b (monoclonal antibody OKM1), CD11c (KB90) and CD14 (Leu-M3). Increased expression of all three determinants was associated with myelomonocytic leukaemias, with their relative diagnostic value in discriminating monocytic (M4 and M5) from non-monocytic (M1, M2 and M3) subtypes being CD14 greater than CD11c greater than CD11b. However, the results also indicated, because of the heterogenous expression of CD11c in particular, and to a lesser extent CD11b, that the patterns or histograms of fluorescent staining were potentially more informative than an empirical subdivision of blasts into positive and negative subpopulations. In addition, analysis of phenotypic correlations by simultaneous two-colour fluorescence showed that the expression of CD11b and CD11c determinants by leukaemic myeloid blasts was highly correlated, in contrast to the expression of CD14 and CD11c which were relatively independent. Consequently, CD11c+ myeloid blasts almost always coexpressed CD11b whereas CD14+ cases of AML often comprised CD14+ CD11c+ and CD14+ CD11c- subpopulations. It is concluded from these observations that CD11c immunophenotyping is a useful supplementary investigation, particularly in CD14- cases of myelomonocytic leukaemia. However, it is also apparent that the presence of membrane CD11c per se is not lineage-specific and that the level of expression is perhaps a more discriminatory factor.     

Evaluation of CD11b expression on peripheral blood neutrophils for early detection of neonatal sepsis

Neonatal sepsis is a disease of infants who are less than 1 month of age. These infants are clinically ill, and their blood culture are positive for bacteria. The reported incidence of neonatal sepsis for all infants is 1 to 10 per 1000 live births. The mortality rate is 4.2-26%. The clinical signs are not specific and diagnosis of neonatal sepsis is one of the most difficult tasks in clinical medicine. The aim of this work was determination of CD11b sensitivity and specificity for early detection of neonatal sepsis. We studied 65 neonates with gestational age of 27 to 38 weeks who were suspected for sepsis within the 28 days of life. Whole blood was obtained from neonates to determine CD11b expression on peripheral blood neutrophils by flow cytometry. C-Reactive protein (CRP) was measured qualitatively. Neonates were divided into two groups. Classification was based on the result of the blood culture. In the sepsis group all of the neonates (n=8) showed positive blood culture and clinical symptoms. In the suspected group (n=57) the neonates showed clinical signs but blood cultures were negative. Sensitivity and specificity of CD11b were 75%, 100% respectively. Also positive and negative predictive values of CD11b were 100% and 86% respectively. Results of present study and previous studies showed that measurement of neutrophil surface markers can be useful for diagnosis of infection in the early phases. Also, the quantitative measurement of CRP in addition to CD11b further enhances the ability to diagnose infections and improves sensitivity and negative predictive value by 100%.     

急性髓系白血病细胞表面抗原CD11b的表达与其预后的关系

目的:探讨急性髓系白血病(AML)细胞表面抗原表达与预后的关系.方法:回顾性分析90例初治AML患者,流式细胞仪检测骨髓白血病细胞表面抗原的表达情况,按染色体危险分层分为好、中、差3组,x2分析骨髓白血病细胞表面抗原阳性率的差异;Kaplan- Meier生存分析和COX回归分析骨髓白血病细胞表面抗原的阳性率与患者预后...     

Surface expression of CD 11b/CD18 of pseudo-Pelger granulocytes in chronic myeloid leukaemia

The aim of the present study was to evaluate the surface membrane glycoproteins of pseudo-Pelger granulocytes in six patients suffering from chronic myeloid leukaemia (CML). We studied the functional and immunochemical activities of five monoclonal antibodies (MoAb) minimally reactive with integrin familial antigens of pseudo-Pelger granulocytes. The study conducted with cytofluorimetric and immunological alkaline phosphatase anti-alkaline phosphatase (APAAP) analysis showed a decreased expression of CD11b/CD18 detected by antibodies OKM1, 60.1 and 60.3 (P less than 0.001). Lymphocyte function associated antigen (LFA-1) was expressed in normal amounts in pseudo-Pelger granulocytes. There was decreased expression of CD11b/CD18 in pseudo-Pelger granulocytes with respect to controls (P less than 0.001) after stimulation with formyl-met-leu-phe (FMLP). We conclude that acquired pseudo-Pelger granulocyte dysfunction may be correlated to decrease of surface glycoprotein expression of CD11b/CD18.     

冠心病患者中性粒细胞和单核细胞CD11b/CD18表达的研究

目的 :探讨不同类型冠心病患者中性粒细胞和单核细胞膜 CD11b/CD18表达的变化。方法 :选择经冠状动脉造影确诊的 49例心绞痛患者 ,30例急性心肌梗死患者和 2 0例正常人 ,用流式细胞仪直接免疫荧光法检测中性粒细胞和单核细胞膜 CD11b/CD18表达。结果 :冠心病患者中性粒细胞和单核细胞膜 CD11b/CD18表达较正常对照组均显著增加 （P<0 .0 1） ;不稳定性心绞痛和急性心肌梗死患者中性粒细胞和单核细胞膜 CD11b/CD18表达显著高于稳定性心绞痛患者 （P<0 .0 1）。与正常对照组比较 ,心绞痛患者组中性粒细胞和单核细胞计数无变化 （P>0 .0 5 ） ,而急性心肌梗死组明显增加 （P<0 .0 1）。急性心肌梗死患者中性粒细胞和单核细胞膜 CD11b/CD18表达与梗死范围无关。结论 :冠心病患者中性粒细胞和单核细胞膜 CD11b/CD18表达明显增加 ,其增加程度与心肌缺血的类型有关。     

Correlation of CD11b and CD56 expression in adult acute myeloid leukemia with cytogenetic risk groups and prognosis

Among other phenotypic markers, CD11b expression has been considered as an unfavorable prognostic factor, both in terms of overall survival (OS), disease-free survival (DFS), and attainment and duration of complete remissions (CRs) in adult patients with acute myeloid leukemia (AML). Recently, some groups have restricted its prognostic impact to poor prognostic karyotypic risk groups. The aim of this study was to retrospectively analyze the prevalence of CD11b and of CD56 expression in blast cells of 158 AML patients [excluding those with t(15;17)] stratified according to their cytogenetic risk and to correlate these phenotypic characteristics with OS, DFS, and CR. CD11b was more frequently expressed in intermediate and unfavorable cytogenetic prognostic groups (38.9 and 35.5 %, respectively) than in the favorable group (9.5 %). No differences were observed in CD56 expression according to the cytogenetic risk groups. When OS, DFS, and CR were analyzed according to these two markers, no statistical differences were recorded in any cytogenetic risk group. In conclusion, although CD11b was more frequently expressed in blast cells of patients with intermediate and unfavorable cytogenetic risk groups, this feature did not translate into different clinical outcome. Similarly, CD56 positivity did not have any influence on the prognosis of these patients.     

Differential expression of CD11b/CD18 (Mo1) and myeloperoxidase genes during myeloid differentiation

During the course of differentiation of early human myeloid cells toward monocytes and granulocytes, cell surface expression of the cell adhesion molecule, CD11b/CD18 (Mo1) increases dramatically and expression of myeloperoxidase (MPO), a bacteriocidal enzyme, decreases markedly. Using the inducible promyelocytic cell line HL-60 as a model, we studied the mRNA expression of these genes. Differentiation of these cells along both a monocytic and a granulocytic pathway demonstrated that the mRNA levels of the two subunits of CD11b/CD18 increased in a pattern temporally and quantitatively similar to the increase in cell surface expression of this heterodimer. In contrast, the expression of MPO mRNA decreased in a temporal and quantitative pattern similar to the known decrease in MPO protein during differentiation, suggesting that regulation of these myeloid-specific proteins may occur at the level of mRNA expression. These findings have important implications with regard to the nature of the block in differentiation in acute nonlymphocytic leukemia and the regulation of myeloid gene expression.     

Continuous activation and deactivation of integrin CD11b/CD18 during de novo expression enables rolling neutrophils to immobilize on platelets

In an in vitro flow model, unstimulated neutrophils rolled steadily over a surface coated with platelets, until superfusion of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) caused a dose-dependent (10(-11) to 10(-7) mol/L) transition from rolling to stationary attachment in seconds, followed more slowly by neutrophil shape change and spreading on the surface, However, at low concentrations of Ca2+ and Mg2+ (0.1 mmol/L and 0.05 mmol/L, respectively, rather than physiologic 1 mmol/L and 0.5 mmol/L), neutrophils first halted but then started to roll again and to detach from the surface over 5 to 10 minutes. At the low cation concentration, stopping was largely inhibited by antibodies to the neutrophil integrins CD18 or CD11b, but not CD11a. When neutrophils were pretreated with antibodies to CD11b or CD18 in 1 mmol/L Ca2+ 0.5 mmol/L Mg2+, stopping was not prevented but delayed. However, if antibodies were also included with the superfused fMLP, stopping was inhibited, and detachment followed. This indicates that CD11b/CD18 was newly expressed during shape change and mediated the second phase of neutrophil immobilization and spreading in a cation-dependent manner. Prestimulated neutrophils also bound to platelets and spread, but immobilization was blocked if they were perfused with antibody to CD18 or CD11b or with low Ca2+ and Mg2+. Examining the cation-dependence further, it was evident that the presence of Mg2+ was essential for integrin-mediated adhesion and that the Mg2+ concentration determined whether immobilization could be maintained or was transient. Continuous superfusion of fMLP was also essential for maintenance of stable adhesion and spreading. Thus, activation of constitutive CD11b/CD18 rapidly and reversibly converted rolling to stationary attachment, whereas maintenance of adhesion and neutrophil spreading required continual expression of additional CD11b/CD18 that was only functional at physiologic Mg2+. Continual activation and deactivation of CD11b/CD18 during de novo expression could mediate immobilization and onward migration of neutrophils in vivo, and activated platelets appear capable of supporting this process as well as endothelial cells.     

Hypoxia-induced upregulation of CD11b expression in granulocytes

The aim of this study is to evaluate leukocyte-endothelial adhesion during ischemia/reperfusion injury. Polymorphonuclear neutrophils (PMN) were isolated from healthy volunteers and incubated in the presence of 2% O₂ (hypoxia) or 21% O₂ (normoxia) at 37°C for two hours. In some experiments, whole blood was subjected to hypoxia or normoxia prior to PMN isolation. Flowcytometric analysis and adhesion assays were carried out using these PMN. Moreover, adhesion assay was carried out using PMN, prepared from the patients who underwent infrarenal aortic aneurysmectomy with aortic clamping at various time points as an in vivo model of ischemial reperfusion injury. Flowcytometric analysis revealed an increased expression of CD11b in PMN subjected to hypoxia compared with those subjected to normoxia prior to isolation. Interestingly, these phenomena were not observed with PMN isolated prior to being subjected with hypoxia. Enzyme-linked immunosorbent assay (ELISA) using serum prepared from whole blood subjected to hypoxia revealed elevated levels of tumor necrosis factor (TNF)- compared with those subjected to normoxia. PMN obtained during aneurysmectomy exhibited an increased adhesion to activated human umbilical vein endothelial cells (HUVEC), compared with those taken from the same patients prior to the operation. In contrast, PMN prepared after aortic clamp, exhibited a significant reduced adhesion to activated HUVEC. Hypoxic condition, induced CD11b expression on PMN in vitro. PMN subjected to hypoxic condition in vivo exhibited an increased adhesion to activated endothelium. Elevated level of serum TNF- may be involved in this phenomenon.     

Subcellular distribution and mobilization of MAC-1 (CD11b/CD18) in neonatal neutrophils

The CD11b/CD18 (Mac-1) heterodimeric surface glycoprotein contributes to a broad range of adherence-dependent neutrophil inflammatory functions. Previous investigations have indicated that diminished expression or regulation of Mac-1 may underlie abnormalities of stimulated adhesion and chemotaxis of neonatal neutrophils in vitro and inflammatory deficits in human neonates. To define the pathogenic mechanisms contributing to these findings, we compared the distribution and translocation of Mac-1 in subcellular fractions of neonatal and adult neutrophils before and after chemotactic stimulation. The total cell content of Mac-1 and the proportions of Mac-1 in beta fractions (vitamin B12 binding protein-rich granules), pre-gamma fractions (gelatinase-rich granules), or gamma fractions (plasma membrane) of neonatal neutrophils were comparable with those of adult neutrophils. However, after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10 nmol/L, 37 degrees C, 15 minutes), neonatal neutrophils demonstrated (1) diminished translocation of Mac-1 from pre-gamma fractions (P less than .05), and (2) diminished surface expression of Mac-1 (P less than .05), as compared with healthy adult neutrophils. As shown in enzymatic and immunochemical assays, neonatal cells contained significantly (P less than .01) diminished levels of neutrophil gelatinase. In response to FMLP (0.1 to 10 nmol/L, 37 degrees C, 15 minutes), neonatal suspensions also released significantly (P less than .001) less gelatinase, as compared with adult neutrophil suspensions. These observations demonstrate that diminished mobilization of Mac-1 from gelatinase-rich granular pools in neonatal neutrophils is associated with abnormal surface expression of this glycoprotein after chemotactic stimulation. This abnormality may contribute, in part, to abnormal migratory properties of neonatal neutrophils in response to inflammatory stimuli.     

Neutrophil surface expression of CD11b and CD62L in diabetic microangiopathy

The aims of the study are (1) assessment of cell surface expression of adhesion molecules CD11b and CD62L on peripheral blood neutrophils in patients with type 2 diabetes and microangiopathy; (2) analysis of serum levels of soluble adhesion molecules: E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and von Willebrand factor (vWF) and; (3) evaluation of systemic inflammatory markers like interleukin-6 (IL-6), soluble interleukin-6 receptor (IL-6Rs), high sensitivity C-reactive protein (hsCRP) and fibrinogen. Thirty patients with type 2 diabetes and microangiopathy were enrolled in the study. The study group was compared to 22 patients with type 2 diabetes without microangiopathic compliations. The control group included 20 healthy volunteers. Flow cytometry was used to analyse surface expression of adhesion molecules. Both inflammatory markers and soluble adhesion molecules were determined by immunoenzymatic assay. A significant increase in neutrophil surface CD11b expression (P < 0.01) as well as decrease in surface CD62L expression (P < 0.01) were observed in the group with diabetic microangiopathy in comparison with diabetic group without microangiopathic complications and healthy controls. Moreover, significantly higher concentrations of sICAM-1 (P < 0.05), sVCAM-1 (P < 0.05), sE-selectin (P < 0.05), vWF (P < 0.01), hsCRP (P < 0.01), IL-6 (P < 0.01) and fibrinogen (P < 0.001) were also found in patients with microangiopathy in comparison with the control group. IL-6Rs concentrations did not significantly vary between groups. We concluded (1) diabetic microangiopathy is accompanied by increase in CD11b expression and decrease in CD62L expression on peripheral blood neutrophils; (2) in diabetic microangiopathy rise in CD11b expression indicates neutrophil activation and intensified adhesion; (3) the development of diabetic microangiopathy is accompanied by an increase in soluble adhesion molecules and inflammatory markers concentrations in the blood.     

Effect of surgically-induced weight loss on inflammatory mediators and peripheral blood monocyte CD11b expression in morbid obesity

Obesity is characterized by a state of chronic mild inflammation, with raised circulating levels of inflammatory markers. Expression and release of inflammation-related adipokines, generally, rise as adipose tissue expands. In the present study we evaluated the level of serum mediators concerned in inflammation and monocyte activation (TNF-alpha, hs-CRP, MCP-1) together with percentage of CD11-b expression on monocytes in a group of morbidly obese individuals (n = 20) before and (3-6 months) after restrictive surgery, and in 15 healthy normal weight individuals. Serum MCP-1, TNF-alpha and hs-CRP were assayed by enzymatic immunoassay, while the percentage of CD11b expression on monocytes was assayed by flow cytometry. The total lipid profile and random blood glucose levels were also assessed. Morbidly obese individuals ( before surgical weight loss) had significantly increased levels of MCP-1, TNF-alpha, hs-CRP, CD11b expression on monocytes as compared to controls (P < 0.01). Levels of MCP-1, TNF-alpha, hs-CRP were significantly decreased 3 to 6 months after restrictive surgery than before the operation (P < 0.01). hs-CRP, MCP-1 and TNF-alpha were positively correlated versus each other. TNF-alpha and hs-CRP also showed positive correlation with the body mass index. Our data suggested that the studied serum and monocyte parameters may link obesity with systemic inflammation and metabolic disorders. The interactions of MCP-1, CD11b and other inflammatory parameters might provide the basis for development of new therapies for this syndrome.     

Granulocyte-macrophage colony-stimulating factor induces the expression of the CD11b surface adhesion molecule on human granulocytes in vivo

The CD11b (Mol) molecule is a member of a family of surface glycoproteins that are essential for adhesion-dependent granulocyte functions. Brief exposure of granulocytes to human granulocyte- macrophage colony-stimulating factor (GM-CSF) in vitro increases the surface expression of CD11b and increases granulocyte adhesiveness. To assess the possible in vivo significance of these observations we studied the effect of GM-CSF on CD11b, CD11a (LFA-1), and CD11c (gp 150, 95) expression on granulocytes from nine adult patients with sarcoma who were receiving GM-CSF as part of a phase I trial. GM-CSF was administered as a continuous infusion at a dose of 32 or 64 micrograms/kg/d. Granulocyte CD11b, CD11a, and CD11c expression was determined by indirect immunofluorescence staining of whole blood, thereby minimizing in vitro manipulation. A transient leukopenia developed within 15 minutes of initiation of GM-CSF treatment that was associated with a marked increase in the surface antigen density of CD11b. A mean 1.7-fold increase (P = .001) in the percentage of CD11b- positive granulocytes and a mean 2.1-fold increase (P = .002) in CD11b surface antigen density was noted after 12 hours of treatment. No change in CD11a or CD11c expression was observed over the first 12 hours. The level of CD11b expression was followed in six patients for up to 5 days of treatment with GM-CSF. Compared with the 12-hour value, three of six patients showed a subsequent decrease in CD11b expression, two remained constant, and one showed a continued increase in CD11b surface density. Fluorescence-activated cell sorting of granulocytes into high- and low-density CD11b-positive groups revealed a preponderance of immature myeloid forms in the low-density CD11b fraction, which suggests that the late decrease in CD11b expression in some patients may be related to a greater proportion of circulating immature myeloid forms in the peripheral blood. This study suggests that GM-CSF administered as a continuous infusion rapidly upregulates the expression of granulocyte CD11b in vivo. The influence of this phenomenon on in vivo granulocyte aggregation may be clinically relevant with regard to the toxicity of GM-CSF and deserves further investigation.     

Erythromycin inhibits beta2-integrins (CD11b/CD18) expression, interleukin-8 release and intracellular oxidative metabolism in neutrophils

Macrolides have therapeutic benefits on chronic inflammatory airway diseases. Thus, macrolides are supposed to have variable biological effects apart from antimicrobial activity. Neutrophil adherence and influx with oxidants and cytokines production implicates involvement in airway inflammation. To investigate whether erythromycin (EM) affects neutrophil activity in vitro, lipopolysaccharide (LPS)-treated neutrophils were continuously incubated for 4 h in the absence or presence of increasing doses of EM from 1 microg ml(-1) to 100 microg ml(-1) in the last 2 h. Leukocyte adhesion molecules Mac-1 and intracellular H2O2(DCFH) were determined by flowcytometric assay. IL-8 and TNFalpha in supernatant was measured by ELISA method. The expression of Mac-1 and mean intracellular DCF fluorescence intensity (DCFH) of neutrophils significantly increased after stimulation with LPS. Pretreatment with EM significantly decreased LPS induced Mac-1 expression on neutrophils compared with LPS stimulation only. EM alone (100 microg ml(-1)) also decreased Mac-1 expression on neutrophils. EM significantly reduced the LPS-increased DCFH. EM alone (100 microg ml(-1)) also caused a decrease in DCFH. Increasing doses of EM also significantly decreased the IL-8 released by LPS-stimulated neutrophils. In conclusion, EM exerts a direct effect on the neutrophils by downregulating the expression of beta2-integrin on neutrophils, thus leading to a decrease in the intracellular H2O2, as well as the production of IL-8. Our conclusion provides an explanation for the clinical efficacy of erythromycin in neutrophil-mediated airway inflammation.     

同型半胱氨酸与白细胞黏附分子CD11b、CD18关系的体内外研究

目的 研究探讨同型半胱氨酸(HCY)与炎性指标白细胞黏附分子CD11b/CD18之间的关系.方法 体外研究:用免疫萤光流式细胞仪检测体外HCY和健康人血白细胞表面黏附分子CD11b、CD18的表达.体内研究:选择75例急性冠脉综合征(ACS)患者为ACS组,35例稳定性心绞痛(NACS)患者为NACS组,35名健康人为对照组.采用酶联免疫法测定血浆HCY水平,通过流式细胞仪采用直接免疫荧光法测定白细胞黏附分子CD11b/CD18的表达,同时对血浆C-反应蛋白(CRP)含量和白细胞计数等进行检测和分析.结果 ①HCY在体外明显增加CD11b和CD18在各种白细胞的表达,这种作用随HCY浓度升高而增强.②ACS组血浆HCY水平、平均白细胞表面黏附分子CD11b和CD18表达、CRP含量和白细胞计数显著高于NACS组和对照组(P<0.05).③与对照组相比,稳定型心绞痛组仅HCY水平轻度升高,余差异无统计学意义.结论 ①同型半胱氨酸导致体外白细胞黏附分子的表达;ACS患者同型半胱氨酸、CD11b/CD18,C-反应蛋白含量和白细胞计数明显升高.②炎症可能是ACS发生的重要原因之一,同型半胱氨酸、白细胞、及其CD11b/CD18表达和CRP等共同参与了ACS的发生.     

Adipose tissue metabolism and CD11b expression on monocytes in obese hypertensives

At a given degree of adiposity, metabolic and cardiovascular risk varies markedly between individuals. Animal studies suggest that differentially expressed systemic activation of monocytes contributes to the obesity-associated risk variability. We tested the hypothesis that systemic monocyte activation is associated with changes in adipose tissue and skeletal muscle metabolism. In 17 obese hypertensive patients, we assessed CD11b expression on circulating monocytes, gene expression in adipose tissue biopsies, and obtained blood samples and adipose tissue and skeletal muscle microdialysis samples in the fasted state and during a glucose load. Patients were stratified into groups with higher and lower CD11b expression on monocytes. Expression of the macrophage marker CD68 was increased markedly in adipose tissue of subjects with higher CD11b expression. Although no differences in systemic insulin sensitivity were found between both groups, patients with higher peripheral CD11b expression showed a markedly augmented increase in dialysate glucose in adipose tissue during oral glucose tolerance testing and increased adipose tissue lipolysis as well. Our data demonstrate that human monocyte activation is associated with tissue-specific changes in glucose and lipid metabolism. These findings may be explained in part by monocyte/macrophage infiltration of adipose tissue, which appears to interfere with insulin responsiveness.     

Deficient total cell content of CR3 (CD11b) in neonatal neutrophils

Neonatal neutrophils (PMN) show a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b), a critical adhesion molecule, on chemoattractant-activated PMN. After activation of PMN with additional stimuli including calcium ionophores, we also found deficient surface CR3 (but normal CR1) expression on neonatal PMN suggesting that abnormal signaling mechanisms are not likely to explain the deficient CR3 expression on activated neonatal PMN. Therefore, we hypothesized that deficient surface expression of CR3 on stimulated neonatal neutrophils is caused by a deficiency in total cell content of CR3. We tested this hypothesis using three different methods to compare the total quantity of CR3 in neonatal versus adult PMN. Western blotting of serial twofold dilutions of PMN lysates from five adult and neonatal pairs, using a monoclonal antibody (MoAb) against CR3 (21PM19C), consistently showed diminished CR3 content in neonatal PMN. A sandwich enzyme-linked immunosorbent assay, in which the CR3 heterodimers in PMN lysates were captured by MoAb to the beta-chain, CD18 (R15.7), then detected with a biotinylated MoAb to the alpha-chain, CD11b (anti-Mac-1), showed that neonatal PMN lysates contain about 66% of adult PMN levels of CR3 (P < 0.03; n = 6). PMN fixed with paraformaldehyde and permeabilized with saponin were studied by immunofluorescence flow cytometry to determine total (surface plus intracellular) CR3 content using phycoerythrin-conjugated MoAb to CR3 (anti-Leu15). Mean total cell CR3 content (in relative fluorescence units) was 58 +/- 14 for adult PMN and 27 +/- 6 for neonatal PMN (n = 5; P = 0.013). In each method, total cell content of CR1 was equivalent for neonatal versus adult PMN. We conclude that neonatal PMN are markedly deficient in total cell CR3 content compared with adult PMN. This result provides a primary explanation for deficient CR3 surface expression on activated neonatal PMN that, in turn, may be important in the chemotactic defect of these cells.     

Flow cytometric identification of myeloid disorders by asynchronous expression of the CD14 and CD66 antigens

Abstract: Using a multiparameter flow cytometry assay enumerating cells positive for CD13, CD14 and CD66 antigens, we determined the asynchronous CD14/CD66 co-expression in unselected bone marrow and peripheral blood samples with suspected malignant blood disorders. CD14/CD66 co-expression >5% were found in 131/691 bone marrow samples. Only 55 of these exhibited an identifiable population in 2-parameter flow cytometry histograms. Of the 55 samples 43 (78%) came from patients with myeloid disorders; e.g. 11 with myelodysplastic syndromes, 15 with chronic myeloproliferative disorders and 17 with acute myeloid leukaemia. Only one of these 17 cases was a de novo case, while 8 were secondary to another malignant haematological disease and 8 were from the period after cytoreductive therapy. Notably, CD14/CD66 co-expression patterns were related to disease categories; e.g. in chronic myelomonocytic leukaemia and acute myeloid leukaemia following a dysplastic phase the co-expression displayed two subsets in peripheral blood, low-avidity CD14 and low-avidity CD66, respectively. The latter disease category also exhibited these 2 subsets in bone marrow. In all other cases, the CD14/CD66 co-expression in bone marrow was heterogeneous. In conclusion, abnormal CD14/CD66 co-expression might be a valuable parameter in defining asynchronous myelopoiesis in malignant myeloid disorders, especially myeloproliferative disorders and secondary acute myeloid leukemias.