Monocyte subsets to differentiate chronic myelomonocytic leukemia from reactive monocytosis

BackgroundChronic myelomonocytic leukemia (CMML) is characterized by persistent monocytosis and dysplastic features of blood cells. No specific genetic abnormalities are present in CMML, and reactive monocytosis should be excluded. An increase in classical monocytes (MO1) has been suggested as a screening tool for CMML.MethodsWe evaluated monocyte subsets in the peripheral blood of patients with CMML (n = 16), patients with reactive monocytosis (n = 19), and normal controls (n = 15) with flow cytometry using antibodies against CD14, CD16, CD56, CD24, CD45, and CD2. The cutoff of MO1 ≥94% was validated, and the optimal cutoff was analyzed with receiver operating curve analysis.ResultsThe sensitivity of monocyte subset testing for screening for CMML was 0.938 (0.717‐0.997), and the specificity was 0.882 (0.734 ‐ 0.953) using the cutoff of MO1 ≥94%. Serial samples from patients who responded to hypomethylating therapy showed an MO1 < 94%. However, few patients with reactive monocytosis, including patients with nonhematologic malignancies and acute myeloid leukemia, showed an increase in the MO1 ≥ 94%. Monocyte subset results were correlated with the response to hypomethylating therapy in follow‐up samples.ConclusionMonocyte subset analysis is useful in screening for and monitoring CMML. Harmonization of the protocols for monocyte subset analysis is required.     

THE CYTOKINETICS OF MONOCYTOSIS IN ACUTE SALMONELLA INFECTION IN THE RAT

The mechanisms responsible for monocytosis occurring in acute Salmonella infection were studied by means of isotopic labeling and autoradiography. Male (Lewis x BN)F₁ hybrid rats (160–180 g) were pulse-labeled with [³H]TdR at varying intervals with respect to the time of i.v. injection of about 10⁶ living Salmonella enteritidis. The half time for monocytes in the blood was estimated from the exponential decline in the percentage of labeled monocytes. The average generation time for dividing monocyte precursors in bone marrow was estimated by fitting a regression line to the decline in median grain counts (halving-time = T_G). After an initial fall, the absolute number of blood monocytes rose to a plateau about 2.5 x normal on day 5, suggesting the reimposition of steady state conditions. The half time of monocytes in the blood of infected rats was shortened to 25 h throughout the infection, compared with 61 h estimated in uninfected rats. T_G was reduced to 15 h (days 1–3) but later reverted to the preinfection level of 34 h (days 4–8). Another early response to infection was the release of immature monocytes into the blood. These cells, however, were too few to offset the initial monocytopenia. Under these conditions, with little or no division of blood monocytes, the sustained monocytosis (days 4–8) must have been due to enlargement of the dividing precursor pool. Excessive loss of monocytes from the blood thus appears to activate a feedback mechanism. However, a more direct stimulating effect on monocyte production by endotoxin could have contributed substantially to the monocytosis.     

细胞免疫表型检测在慢性粒单核细胞白血病诊断和治疗监测中的应用

目的评估流式细胞仪细胞免疫表型检测(FCI)在慢性粒单核细胞白血病(CMML)诊断和监测治疗反应中的临床价值和意义。方法采用多色FCI分析法和形态学观察法对109例CMML患者和39例随访患者的骨髓标本的免疫表型和形态学进行检测和评价,并对结果进行统计学分析。结果采用FCI测得骨髓单核细胞百分比为15%。与形态学分出的单核细胞百分比相关(r=0.35,P=0.000 3)。96%患者单核细胞FCI发生改变;FCI观察到的CMML粒细胞生成障碍为91.7%明显高于形态学方法测得的82.6%(χ~2=4.10,P0.05)。CMML患者FCI CD34+细胞测定结果为中位数0.7%(范围0.15%~11.60%),但其免疫表型改变非常显著改变个数的中位数为7(范围1~11个)。91%患者出现1阶段原始血细胞缺失或明显减少。39例随访患者中35例使用低甲基化剂(HMA)治疗和4例等待治疗患者的骨髓样品CD34+原始细胞均被检测到持续的免疫表型改变,对HMA治疗有应答的13例患者单核细胞CD14表达均正常;而40.9%(9/22)HMA治疗无应答者单核细胞CD14表达发生改变(P=0.030),4例接受造血干细胞移植治疗的患者免疫表型于移植后均正常。结论 CMML患者的CD34+细胞,单核细胞和粒细胞表现出多种FCI异常,FCI能够将恶性增生从反应性单核细胞增多中区分出来,同时也可用于监测CMML患者治疗反应。     

Usefulness of thresholds for smear review of neutropenic samples analyzed with a Sysmex XN-10 analyzer

Neutropenia is one of the main criteria for a blood smear review. The objective of this study was to compare the thresholds proposed by the international consensus group for hematology review (1.0 10⁹/L) and the French speaking Group for Cellular Haematology (1.5 10⁹/L) in terms of the number of useless smears. We collected 112,097 analyzed samples from four laboratories equipped with XN instruments (Sysmex, Kobe, Japan) during early 2016. The only exclusion criterion was a leucocyte count below 0.5 10⁹/L. In the absence of abnormal cells and/or morphology suggesting haematological disease, samples were classified as ‘negative for morphology’ and the differential from the XN-10 was reported. These smear procedures were considered as uninformative. Some 2202 samples met the criterion for neutropenia (<1.5 10⁹/L) for slide review representing 1.96% of the total. These included 1031 with neutropenia alone and 1171 neutropenia plus other abnormalities. Of the 1031 with neutropenia alone, 886 had a neutrophil count between 1.0 10⁹/L and 1.5 10⁹/L. The smear was uninformative for all of these samples. In conclusion, microscopic examination of a blood smear provided very limited information in cases of neutropenia without other abnormalities.     

Elastase of U-937 Monocytelike Cells: COMPARISONS WITH ELASTASES DERIVED FROM HUMAN MONOCYTES AND NEUTROPHILS AND MURINE MACROPHAGELIKE CELLS

As an approach to facilitating the understanding of proteinases associated with monocytes we have studied U-937 monocytelike cells. Elastase activity was identified in U-937 cell extracts and compared to monocyte elastase activity, neutrophil elastase, and the elastase activity from a continuous line of murine macrophagelike cells (P388D1). Serine proteinase activity which solubilized ¹⁴C-labeled elastin accounted for >90% of the neutral proteinase activity of both U-937 cells and monocyte extracts. U-937 cell and monocyte elastase activities were similar catalytically, resembling neutrophil elastase. U-937 cells and monocytes showed other similarities: (a) both had activities reacting with [³H]diisopropylfluorophosphate that migrated in sodium dodecyl sulfate (SDS) polyacrylamide gels at ~30,000 and 60,000 daltons and (b) both contained material that cross-reacted with antiserum raised to neutrophil elastase. Preliminary characterization of U-937 cell elastase activity by affinity chromatography and ion-exchange chromatography suggested the presence of at least two distinct elastases. Minimal elastase activity was found in U-937 cell-conditioned medium, indicating that the activity is not spontaneously released by the cells. In contrast to the elastase activity associated with U-937 cells and monocytes, the elastase activity associated with P388D1 cells was a metalloproteinase and was found principally in the culture medium. These results indicate (a) U-937 cells will be useful for further investigation of proteinases associated with normal monocytes; (b) monocytes and U-937 cells contain material with catalytic and immunologic similarities to neutrophil elastase; (c) monocyte elastase activity differs from elastase activity secreted by murine macrophages and murine macrophagelike cells of the P388D1 line.     

The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions

Healing of myocardial infarction (MI) requires monocytes/macrophages. These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling. The mechanisms that orchestrate such divergent functions remain unknown. In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI. We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing. Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6C^hi and -6C^lo monocytes via CCR2 and CX₃CR1, respectively. Ly-6C^hi monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions. Ly-6C^lo monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular–endothelial growth factor. Consequently, Ly-6C^hi monocytes digest damaged tissue, whereas Ly-6C^lo monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen. MI in atherosclerotic mice with chronic Ly-6C^hi monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response. These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI.     

Technique and Practical Problems: Comparative Studies of the Value of Two Cyanide-Xitroprusside Methods ix the Diagnosis of Cystixuria: Cystinuria in Sweden,Part ix

We have tested a Quantitative Buffy Coat (QBC) instrument (Becton Dickinson, USA), and compared results obtained with this instrument to results obtained with standard methods in a haematology clinic.

The basic principle of the method is a classical haematocrit centrifuge. The analysis provides a haematocrit value, platelet count, a total white blood count, and separates the white blood cells in granulocytes and mononuclear cells (lymphocytes plus monocytes).

The instrument is easy to use but requires a trained observer. All results are available in 15 min. We have found the accuracy of the method good for all parameters. The precision of the instrument is good but for estimation of granulocytes and lymphocytes plus monocytes we did not find the high sensitivity claimed by the manufacturer (manufacturer's lower limit 0.02x 10⁹/1; standard deviation for low levels of granulocytes: 0.300×10⁹71 and lymphocytes plus monocytes: 0.235×10⁹/1).

A large fraction of samples (leukocytes 27%, platelets 40%) from a haematological clinic falls beyond the limits for reliable results set by the manufacturer, which reduces the utility of the instrument in such patients. Furthermore, in 21% and 10% of samples within the recommended range for leukocytes and platelets, respectively, QBC results could not be read owing to ill-defined boundaries. For granulocytes and lymphocytes plus monocytes 25% and 34% of the samples, respectively, could not be read owing to ill-defined boundaries. The instrument is not constructed to protect against blood contamination of the centrifuge and, therefore, the safety of the instrument is not satisfactory.     

ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice

Leukocytosis is associated with increased cardiovascular disease risk in humans and develops in hypercholesterolemic atherosclerotic animal models. Leukocytosis is associated with the proliferation of hematopoietic stem and multipotential progenitor cells (HSPCs) in mice with deficiencies of the cholesterol efflux–promoting ABC transporters ABCA1 and ABCG1 in BM cells. Here, we have determined the role of endogenous apolipoprotein-mediated cholesterol efflux pathways in these processes. In Apoe^–/– mice fed a chow or Western-type diet, monocytosis and neutrophilia developed in association with the proliferation and expansion of HSPCs in the BM. In contrast, Apoa1^–/– mice showed no monocytosis compared with controls. ApoE was found on the surface of HSPCs, in a proteoglycan-bound pool, where it acted in an ABCA1- and ABCG1-dependent fashion to decrease cell proliferation. Accordingly, competitive BM transplantation experiments showed that ApoE acted cell autonomously to control HSPC proliferation, monocytosis, neutrophilia, and monocyte accumulation in atherosclerotic lesions. Infusion of reconstituted HDL and LXR activator treatment each reduced HSPC proliferation and monocytosis in Apoe^–/– mice. These studies suggest a specific role for proteoglycan-bound ApoE at the surface of HSPCs to promote cholesterol efflux via ABCA1/ABCG1 and decrease cell proliferation, monocytosis, and atherosclerosis. Although endogenous apoA-I was ineffective, pharmacologic approaches to increasing cholesterol efflux suppressed stem cell proliferative responses.     

Lenograstim with or without dexamethasone for neutrophil mobilization in healthy donors: short-term kinetics of white blood cells and effects of granulocyte apheresis

Objectives : To determine the optimal time schedule for neutrophil collection after single mobilization with glycosylated recombinant granulocyte colony‐stimulating factor (G‐CSF, lenograstim) with or without dexamethasone (DXM). Donors and Methods : In this prospective randomized trial, 26 healthy volunteers were randomly assigned to a single subcutaneous dose of lenograstim 6 μg/kg plus 8‐mg DXM (G‐CSF/DXM, n = 13) or placebo (G‐CSF/placebo, n = 13). Hematological and biochemical parameters were analyzed before and 12, 15, 18, 21, 24, 27, 29, 36, 48, 60, 72, and 84 h and 7 and 30 days after mobilization. Six G‐CSF/DXM subjects underwent standard neutrophil apheresis (NA) 12 and 36 h after mobilization. Results : Polymorphonuclear neutrophil (PMN) counts 12 and 21 h after mobilization were 22.7 (16.6?32.8) × 10⁹/L and 22.4 (18.6?30.6) × 10⁹/L for G‐CSF/placebo versus 33.1 (24.2–44.9) × 10⁹/L and 32.5 (17.4–39.6) × 10⁹/L for G‐CSF/DXM. This mobilization plateau was followed by slow normalization at 72–84 h. The six NA subjects had median PMN yields of 62 (47–101) × 10⁹ and 39 (23–42) × 10⁹ per therapeutic unit. After the first apheresis, PMN counts sharply decreased to 21.1 (14.8–26.3) × 10⁹/L and then temporarily recovered to 25.9 (18.9–36.5) × 10⁹/L (P ≤ 0.001) over the next 8 h. Conclusions : Single doses of lenograstim with or without DXM induced a PMN plateau that lasted 9 h (12–21 h after mobilization), with PMN counts suitable for neutrophil collection. Lenograstim plus DXM made it possible to perform NA twice, 12 and 36 h after mobilization. © 2011 Wiley Periodicals, Inc.     

Cryopreservation of peripheral blood stem cell: the influence of cell concentration on cellular and hematopoietic recovery

BACKGROUND: The optimal cryopreservation cell concentration during the peripheral blood stem cell (PBSC) collection is a controversial topic. We evaluated the influence of cryopreservation concentration on the recovery of hematopoietic progenitor cells and the kinetics of hematopoietic recovery of autologous stem cell transplant patients. STUDY DESIGN AND METHODS: In this retrospective study, we compared two different cryopreservation protocols: 1 × 10⁸ cells/mL (Protocol A) and 2 × 10⁸ cells/mL (Protocol B). A total of 419 PBSCs were analyzed with regard to the number of viable cells and colony‐forming units–granulocytes‐monocytes (CFU‐GM) progenitors. The hematopoietic recovery of 275 patients who received PBSCs cryopreserved at a dose of 1 × 10⁸ cells/mL (Group A) and 2 × 10⁸ cells/mL (Group B) were compared. RESULTS: There were no significant differences in recovery of viable cells between Protocol A and Protocol B. The median of recovery of CFU‐GM progenitors was significantly higher in Protocol B (41.2 vs. 57.3, p < 0.01). The median times to neutrophil recovery (≥500 × 10⁶/L) and platelet (PLT) recovery (≥20 × 10⁹/L) in Groups A and B were 11 days versus 11 days and 12 days versus 12 days, respectively. However, by Kaplan and Meier analyses, Group B recovered neutrophils with a little delay (p = 0.01). No difference was observed with regard to time to PLT recovery. On multivariate analysis, we found that the number of CD34+ cells and CFU‐GM progenitors had a significant influence on hematopoietic recovery. CONCLUSION: Cryopreservation of PBSCs at a dose of 2 × 10⁸ cells/mL did not affect the recovery rate of viable cells or the hematopoietic recovery of autologous stem cell transplant patients.     

Increased sperm count may account for high population frequency of factor V Leiden

Summary. Background: Factor V Leiden (FVL) increases the risk of venous thrombosis and pregnancy loss in carriers. Nevertheless, this relatively old mutation is prevalent in Caucasion populations, which could be explained by positive selection pressure. Men with FVL have previously been found to have higher fecundity (the time between marriage and first pregnancy). Whether this is caused by increased sperm counts in men with FVL is unknown. Objectives: To assess whether men with factor V Leiden have increased sperm counts. Patients/methods: We performed a prospective cohort study among 1139 consecutively included male partners of subfertile couples presenting at our university hospital for fertility workup between January 2000 and July 2007. All potential candidates who gave informed consent were included, irrespective of their fertility workup. In this retrospective analysis, we excluded participants with known causes of spermatogenic function or azoospermia. Subsequently, we genotyped all participants and compared sperm counts between FVL carriers and non‐carriers. Results: We identified 37 FVL carriers and 921 non‐carriers. FVL carriers had higher total sperm counts and total motile sperm counts than non‐carriers: 236 × 10⁶ (95% CI 158–292 × 10⁶) vs. 163 × 10⁶ (95% CI 147–178 × 10⁶) and 81 × 10⁶ (95% CI 54–105 × 10⁶) vs. 52 × 10⁶ (95% CI 48–57 × 10⁶), respectively. Conclusions: To our knowledge, this is the first study that indicates that an increased incidence of a genotype may be controlled by increased sperm counts. However, the finding that men with FVL had higher total (motile) sperm counts was not statistically significant and needs confirmation in other studies.     

Monocytes/Macrophages Control Resolution of Transient Inflammatory Pain

Insights into mechanisms governing resolution of inflammatory pain are of great importance for many chronic pain–associated diseases. Here we investigate the role of macrophages/monocytes and the anti-inflammatory cytokine interleukin-10 (IL-10) in the resolution of transient inflammatory pain. Depletion of mice from peripheral monocytes/macrophages delayed resolution of intraplantar IL-1β- and carrageenan-induced inflammatory hyperalgesia from 1 to 3 days to >1 week. Intrathecal administration of a neutralizing IL-10 antibody also markedly delayed resolution of IL-1β- and carrageenan-induced inflammatory hyperalgesia. Recently, we showed that IL-1β- and carrageenan-induced hyperalgesia is significantly prolonged in LysM-GRK2^+/− mice, which have reduced levels of G-protein-coupled receptor kinase 2 (GRK2) in LysM⁺ myeloid cells. Here we show that adoptive transfer of wild-type, but not of GRK2^+/−, bone marrow-derived monocytes normalizes the resolution of IL-1β-induced hyperalgesia in LysM-GRK2^+/− mice. Adoptive transfer of IL-10^−/− bone marrow-derived monocytes failed to normalize the duration of IL-1β-induced hyperalgesia in LysM-GRK2^+/− mice. Mechanistically, we show that GRK2^+/− macrophages produce less IL-10 in vitro. In addition, intrathecal IL-10 administration attenuated IL-1β-induced hyperalgesia in LysM-GRK2^+/− mice, whereas it had no effect in wild-type mice. Our data uncover a key role for monocytes/macrophages in promoting resolution of inflammatory hyperalgesia via a mechanism dependent on IL-10 signaling in dorsal root ganglia.PerspectiveWe show that IL-10-producing monocytes/macrophages promote resolution of transient inflammatory hyperalgesia. Additionally, we show that reduced monocyte/macrophage GRK2 impairs resolution of hyperalgesia and reduces IL-10 production. We propose that low GRK2 expression and/or impaired IL-10 production by monocytes/macrophages represent peripheral biomarkers for the risk of developing chronic pain after inflammation.     

Leukapheresis in non–cytokine‐stimulated donors with a new apheresis system: first‐time collection results and evaluation of subsequent cryopreservation

BACKGROUND: Adoptive cell therapy based on mononuclear cells (MNCs) became an important modality of cancer immunotherapy. Data about collection results and donor response of leukapheresis with the Spectra Optia v.5.0 (Terumo BCT) in nonmobilized donors are required. STUDY DESIGN AND METHODS: Twelve MNC collections were performed using the Spectra Optia v.5.0 in non–cytokine‐stimulated donors. Leukapheresis products and peripheral blood samples from donors were assayed for CD45+, CD34+, CD3+, and CD14+ cells by flow cytometry. Prefreeze and postthaw cell counts, cell viability, and numbers of colony‐forming units were assessed in cryobags and compared to data from cryovials. RESULTS: Leukapheresis yielded a mean of 5.26 × 10⁹ ± 2.2 × 10⁹ CD45+ cells, 1.5 × 10⁹ ± 0.77 × 10⁹ CD14+ monocytes, and 2.28 × 10⁹ ± 1.2 × 10⁹ CD3+ T cells by processing 6690 ± 930 mL of whole blood. A significant positive correlation between yield of CD3+ T cells and residual platelets (PLTs) and red blood cells (RBCs) was observed. This did not apply for CD34+ and CD14+ white blood cell subsets. Mean collection efficiencies for CD14+ monocytes and CD3+ T cells were 61.8 ± 17 and 37.2 ± 18%, respectively. Recovery of CD14+ cells after cryopreservation was 75.2 ± 8.2%, which was significantly lower than recovery of CD45+ cells (81.4 ± 5.5%; p = 0.01). CONCLUSION: This study of a small cohort demonstrates that the Spectra Optia v.5.0 is capable of collecting low product volumes with satisfactory MNC yields and low residual RBCs and PLTs in non–cytokine‐mobilized apheresis. Our data suggest that cryovials can serve as a representative surrogate for the primary product cryobag.     

Identification of Human Neutrophil-derived Cathepsin G and Azurocidin/CAP37 as Chemoattractants for Mononuclear Cells and Neutrophils

Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated with a protein of 30 kD, according to SDS-PAGE analysis. NH₂-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human cathepsin G was dose dependent with optimal concentration at 0.5–1 μg/ml. Cathepsin G is chemotactic rather than chemokinetic for monocytes, as demonstrated by checkerboard analysis. Cathepsin G–induced monocyte chemotaxis is partially pertussis toxin sensitive implying the involvement of a G protein–coupled receptor. Enzymatic activity of cathepsin G is associated with its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic activity of cathepsin G can also be completely blocked by α₁ antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 μg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.     

Factors influencing haematological recovery after allogeneic bone marrow transplantation in leukaemia patients treated with methotrexate-containing GVHD prophylaxis

 In the present single institution study of 66 leukaemia patients (28 AML, 23 ALL, 15 CML), the factors influencing haematological recovery after allogeneic bone marrow transplantation (alloBMT) were analysed retrospectively to identify the optimal conditions required for rapid haematological recovery after alloBMT. All patients received GVHD prophylaxis with cyclosporine A plus methotrexate. The mean number of days required to achieve a neutrophil count ≥0.5×10⁹/l after alloBMT was 17 (range 9–27), 19 patients (28.8%) had rapid neutrophil recovery within 15 days after alloBMT. Haematological recovery was more rapid in the 38 patients without GVHD or with only grade I GVHD. Also, 50% and 40% of patients receiving 10 (n=18) or 5 (n=20) μg/kg G-CSF per day, respectively, had rapid neutrophil recovery within 15 days after alloBMT, as against only 7.1% of patients not receiving G-CSF after the transplant (n=28);P<0.001. The neutrophil recovery was similar in patients receiving either fresh or cryopreserved allografts and either a TBI-containing or a busulfan-containing conditioning regimen. A significant correlation was found between the neutrophil recovery and either the MNCs or CFU-GM contents of the allografts. The mean number of days required for neutrophil recovery was only 16 (range 9–24) in patients receiving allografts containing >1×10⁵ CFU-GM/kg (n=28), as against 19 (range 13–27) in patients receiving allografts containing ≤1×10⁵ CFU-GM/kg (n=35). Three patients receiving allografts containing <0.5×10⁵ CFU-GM/kg had primary neutrophil engraftment failure. The mean number of days required to achieve a platelet count ≥20×10⁹/l was 21 (range 11–50), and 30 patients (46.9%) had platelet recovery within 20 days after alloBMT. The platelet recovery after alloBMT was not affected by the type of leukaemia, conditioning regimen, or G-CSF administration. The mean number of days required for platelet recovery after alloBMT was 20 in patients receiving allografts containing >1.0×10⁵ BFU-E/kg (n=35), as against 23 days in patients receiving allografts containing ≤1.0×10⁵ BFU-E/kg (n=24). Seven patients receiving allografts containing <0.5×10⁵ BFU-E/kg had primary platelet engraftment failure. The present study has identified the high number of progenitor cells in the allografts infused and the daily administration of G-CSF posttransplant as the optimal combination for rapid neutrophil recovery after alloBMT. More significantly, the number of BFU-E in allografts was the most significant determining factor in platelet recovery after alloBMT. The development of GVHD of grade II or more during the first weeks after alloBMT was associated with slower haematological recovery, a longer period of fever during neutropenia and longer hospitalization.     

Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells

The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-alpha). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-alpha, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-alpha-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.     

Pre-emptive plerixafor injection increases blood neutrophil, lymphocyte and monocyte counts in addition to CD34+ counts in patients with non-Hodgkin lymphoma mobilizing poorly with chemotherapy plus G-CSF: potential implications for apheresis and graft composition

BackgroundCXCR4 receptor antagonist plerixafor is used to mobilize hematopoietic stem cells. No detailed data regarding the effects of plerixafor on other blood cell components have been published but may be of importance in regard to graft composition collected after plerixafor injection.Patients and methodsThe study included thirty-nine patients with non-Hodgkin lymphoma (NHL) mobilized with chemotherapy plus G-CSF. Plerixafor was given pre-emptively in twenty patients due to poor mobilization or low collection yield. Nineteen NHL patients served as controls. We evaluated CD34⁺ counts and WBC counts and differential from the morning of the first plerixafor injection and 8 h after the plerixafor injection. From the control patients the corresponding values were evaluated on the morning of the first apheresis and 24 h before it.ResultsThe first plerixafor dose increased CD34⁺ counts and number of leukocytes, neutrophils, lymphocytes, eosinophils and monocytes. Leukocyte, neutrophil, lymphocyte, monocyte and eosinophil counts were higher after plerixafor injection compared to the control group at the time of the first apheresis. Minimal graft (?2 × 10⁶/kg CD34⁺ cells) was collected in 85% of plerixafor treated patients, with a single apheresis in 45% of the patients.DiscussionPlerixafor significantly increases B-CD34⁺ cell counts on the next morning making effective blood stem cell collection possible in the majority of the patients mobilizing poorly. It also influences other blood cell components but impact of this observation in regard to graft content and post-transplant course needs to be assessed in further studies.     

Criteria for reflex peripheral smear review in infants

Abstract

Criteria for peripheral smear review are designed to include those samples with results outside the reference interval and can be more extreme based on what is considered to have clinical utility. However, we are unaware of previous studies that reported the distributions of various complete blood cell count (CBC) parameters in infants. In the following study we reviewed screening CBC results of 692 infants aged 9–15 months in order to determine the proportion of peripheral smear reviews recommended according to consensus criteria and that after adjusting for the observed distributions of the various parameters. According to consensus criteria the recommended reflex peripheral smear review rate was 39.7% (95% CI 36.1–43.4) whereas after adjustment for the observed distributions, the rate fell to 5.6% (95% CI 3.9–7.3) (p < 0.001). The major reasons for the difference in rates were the high proportion of infants with an absolute lymphocyte count > 7 × 10⁹/L (17.5%), the presence of a plus one blast flag (4.3%), and a large unstained cell count of ≥ 5% (26.2%) (equivalent to + 1 atypical flag). We found that international consensus criteria for reflex peripheral smear review results in a very high peripheral smear review rate in well infants, and might be inappropriate.     

Outcomes of Immunocompetent Children Presenting with Fever and Neutropenia

Background

Neutropenia may alarm clinicians and prompt extensive evaluation in children with fever, even in immunocompetent patients.

Granulomonocytapheresis as a cell-dependent treatment option for patients with inflammatory bowel disease: Concepts and clinical features for better therapeutic outcomes

Ulcerative colitis (UC) and Crohn's disease (CD) are major phenotypes of the chronic inflammatory bowel disease (IBD), which afflicts millions of individuals throughout the world with debilitating symptoms. The chronic nature of IBD means that patients require life-long medications, and this may lead to drug dependency, loss of response together with adverse side effects as additional morbidity factors. The efficacy of antitumour necrosis factor (TNF)-α biologics has validated the role of inflammatory cytokines notably TNF-α in the exacerbation and perpetuation of IBD. However, cytokines are released by myeloid lineage leucocytes like the CD14⁺CD16⁺ monocyte phenotype. Additionally in IBD, myeloid leucocytes are elevated with activation behavior, while lymphocytes are compromised. Therefore, patients' leucocytes appear logical targets of therapy. Adsorptive granulomonocytapheresis (GMA) with an Adacolumn uses carriers, which interact with the Fcγ receptor expressing leucocytes and deplete the elevated myeloid leucocytes, while the neutrophils, which re-enter the circulation via the Adacolumn outflow (≥40%) are phagocytosed by CD19 B-cells to become interleukin (IL)-10 producing Bregs or CD19^highCD1D^high B-cells. IL-10 is an anti-inflammatory cytokine. GMA has been applied to treat patients with IBD. The efficacy outcomes have been impressive as well as disappointing, the clinical response to GMA defines the patients' disease course and severity at entry. Efficacy outcomes in patients with deep ulcers together with extensive loss of the mucosal tissue are not encouraging, while patients without these features respond well and attain a favorable long-term disease course. Accordingly, for responder patients, GMA fulfills a desire to be treated without drugs.