Effects of Obesity and Exercise on Bone Marrow Progenitor Cells after Radiation.

INTRODUCTION: The late effects of radiation therapy can have significant consequences for the health and quality of life of long-term cancer survivors. Radiation induces persistent alterations in hematopoietic stem and progenitor cells (HSPC) and the bone marrow environment; however, how relevant host factors such as obesity and exercise differentially regulate HSPC content and the bone marrow environment after radiation exposure remains unknown. The purpose of this investigation was to evaluate how the combination of obesity and exercise training modulates HSPC and their niche after sublethal radiation exposure in mice. METHODS: Mice fed either a control or a high-fat diet to induce obesity remained sedentary or underwent a progressive treadmill exercise program. At 13 wk of age, mice were irradiated (3 Gy) and continued their specific diets and exercise program for four more weeks. RESULTS: Exercise-trained mice had significantly higher quantities of several HSPC subpopulations and bone marrow stromal cell populations, whereas HSPC subpopulations were significantly lower in obese mice after radiation. Reactive oxygen species content was significantly decreased in HSPC with exercise training. Proteomics analysis of bone marrow supernatant revealed clustering of biologically relevant changes in exercise-trained mice. Functional evaluation of bone marrow supernatant revealed a significant increase in leukemia blast viability in obese mice but not in the exercise-trained mice (P < 0.05). CONCLUSION: Together, these data suggest that exercise training partially restores the negative effects of obesity on HSPC and their niche after radiation exposure. As such, exercise training should be considered to mitigate the late effects of radiation therapy on the hematopoietic system for cancer survivors with or without obesity who have undergone radiation therapy.

Granulocyte-Monocyte Progenitors and Monocyte-Dendritic Cell Progenitors Independently Produce Functionally Distinct Monocytes.

Granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) produce monocytes during homeostasis and in response to increased demand during infection. Both progenitor populations are thought to derive from common myeloid progenitors (CMPs), and a hierarchical relationship (CMP-GMP-MDP-monocyte) is presumed to underlie monocyte differentiation. Here, however, we demonstrate that mouse MDPs arose from CMPs independently of GMPs, and that GMPs and MDPs produced monocytes via similar but distinct monocyte-committed progenitors. GMPs and MDPs yielded classical (Ly6Chi) monocytes with gene expression signatures that were defined by their origins and impacted their function. GMPs produced a subset of "neutrophil-like" monocytes, whereas MDPs gave rise to a subset of monocytes that yielded monocyte-derived dendritic cells. GMPs and MDPs were also independently mobilized to produce specific combinations of myeloid cell types following the injection of microbial components. Thus, the balance of GMP and MDP differentiation shapes the myeloid cell repertoire during homeostasis and following infection.

An Effective Model of the Retinoic Acid Induced HL-60 Differentiation Program.

In this study, we present an effective model All-Trans Retinoic Acid (ATRA)-induced differentiation of HL-60 cells. The model describes reinforcing feedback between an ATRA-inducible signalsome complex involving many proteins including Vav1, a guanine nucleotide exchange factor, and the activation of the mitogen activated protein kinase (MAPK) cascade. We decomposed the effective model into three modules; a signal initiation module that sensed and transformed an ATRA signal into program activation signals; a signal integration module that controlled the expression of upstream transcription factors; and a phenotype module which encoded the expression of functional differentiation markers from the ATRA-inducible transcription factors. We identified an ensemble of effective model parameters using measurements taken from ATRA-induced HL-60 cells. Using these parameters, model analysis predicted that MAPK activation was bistable as a function of ATRA exposure. Conformational experiments supported ATRA-induced bistability. Additionally, the model captured intermediate and phenotypic gene expression data. Knockout analysis suggested Gfi-1 and PPARg were critical to the ATRAinduced differentiation program. These findings, combined with other literature evidence, suggested that reinforcing feedback is central to hyperactive signaling in a diversity of cell fate programs.

Reprint of: Heme oxygenase 1 affects granulopoiesis in mice through control of myelocyte proliferation.

Heme oxygenase-1 (HO-1) is stress-inducible, cytoprotective enzyme degrading heme to carbon monoxide (CO), biliverdin and Fe2+. We showed that HO-1 knock-out mice (HO-1-/-) have a twofold higher level of granulocytes than wild type (WT) mice, despite decreased concentration of granulocyte colony-stimulating factor (G-CSF) in the blood and reduced surface expression of G-CSF receptor on the hematopoietic precursors. This suggests the effect of HO-1 on granulopoiesis. Here we aimed to determine the stage of granulopoiesis regulated by HO-1. The earliest stages of hematopoiesis were not biased toward myeloid differentiation in HO-1-/- mice. Within committed granulocytic compartment, in WT mice, HO-1 was up-regulated starting from myelocyte stage. This was concomitant with up-regulation of miR-155, which targets Bach1, the HO-1 repressor. In HO-1-/- mice granulopoiesis was accelerated between myelocyte and metamyelocyte stage. There was a higher fraction of proliferating myelocytes, with increased nuclear expression of pro-proliferative C/EBPß (CCAAT/enhancer binding protein beta) protein, especially its active LAP (liver-enriched activator proteins) isoform. Also our mathematical model confirmed shortening the myelocyte cyclic-time and prolonged mitotic expansion in absence of HO-1. It seems that changes in C/EBPß expression and activity in HO-1-/- myelocytes can be associated with reduced level of its direct repressor miR-155 or with decreased concentration of CO, known to reduce nuclear translocation of C/EBPs. Mature HO-1-/- granulocytes were functionally competent as determined by oxidative burst capacity. In conclusion, HO-1 influences granulopoiesis through regulation of myelocyte proliferation. It is accompanied by changes in expression of transcriptionally active C/EBPß protein. As HO-1 expression vary in human and is up-regulated in response to chemotherapy, it can potentially influence chemotherapy-induced neutropenia.

The Granulocyte Progenitor Stage Is a Key Target of IRF8-Mediated Regulation of Myeloid-Derived Suppressor Cell Production.

Alterations in myelopoiesis are common across various tumor types, resulting in immature populations termed myeloid-derived suppressor cells (MDSCs). MDSC burden correlates with poorer clinical outcomes, credited to their ability to suppress antitumor immunity. MDSCs consist of two major subsets, monocytic and polymorphonuclear (PMN). Intriguingly, the latter subset predominates in many patients and tumor models, although the mechanisms favoring PMN-MDSC responses remain poorly understood. Ordinarily, lineage-restricted transcription factors regulate myelopoiesis that collectively dictate cell fate. One integral player is IFN regulatory factor (IRF)-8, which promotes monocyte/dendritic cell differentiation while limiting granulocyte development. We recently showed that IRF8 inversely controls MDSC burden in tumor models, particularly the PMN-MDSC subset. However, where IRF8 acts in the pathway of myeloid differentiation to influence PMN-MDSC production has remained unknown. In this study, we showed that: 1) tumor growth was associated with a selective expansion of newly defined IRF8lo granulocyte progenitors (GPs); 2) tumor-derived GPs had an increased ability to form PMN-MDSCs; 3) tumor-derived GPs shared gene expression patterns with IRF8-/- GPs, suggesting that IRF8 loss underlies GP expansion; and 4) enforced IRF8 overexpression in vivo selectively constrained tumor-induced GP expansion. These findings support the hypothesis that PMN-MDSCs result from selective expansion of IRF8lo GPs, and that strategies targeting IRF8 expression may limit their load to improve immunotherapy efficacy.

Multicenter Systems Analysis of Human Blood Reveals Immature Neutrophils in Males and During Pregnancy.

Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. In this study, we identified a gene signature of immature-like neutrophils, characterized by the overexpression of genes encoding for several granule-containing proteins, which was found at higher levels (up to 3-fold) in young (20-30 y old) but not older (60 to >89 y old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap formation at baseline and upon microbial or sterile autoimmune stimuli. The expression levels of the immature-like neutrophil signature increased linearly with pregnancy, an immune state of increased susceptibility to certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women during late pregnancy. Thus, our findings show novel sex differences in innate immunity and identify a common neutrophil signature in males and in pregnant women.

Serum levels of soluble adhesion molecules in newly diagnosed acute myeloid leukemia and in complete remission suggest endothelial cell activation by myeloblasts.

BACKGROUND AND AIMS: Despite high-dose multi-agent chemotherapy and allogeneic stem cell transplantation, the relapse rate of acute myeloid leukemia (AML) is high. Further, the disease is highly resistent to drugs. We speculated that deeper understanding of AML-endothelial cell interactions might provide new targets for selective modulation of the AML microenvironment and form the basis for novel treatment approaches. In this study, we evaluated levels of endothelium derived soluble adhesion molecules in active disease and in complete remission (CR) and their relationship with inflammatory cytokines. METHODS: Baseline serum levels of 25 cytokines and 5 soluble adhesion molecules were measured in 84 AML patients using biochip array technology. CR samples were evaluated in 44 patients of this cohort. The control group consisted of 15 healthy blood donors. RESULTS: All analytes were independent of age or disease origin. Some correlations were restricted to active AML, some were ubiquitous and some were found in remission. In active disease, E-selectin (E-SEL) and VCAM-1 correlated with leukocyte count, E-SEL correlated with P-selectin (P-SEL). Platelet count related to IL-7, EGF and VEGF but not to P-SEL. In CR, P-SEL correlated with platelet count and EGF but not with E-SEL. There was no relationship of P-SEL and E-SEL in the control group. CONCLUSIONS: Leukemic activity is associated with a different pattern of soluble adhesion molecule levels. Both E-SEL and P-SEL may be derived from endothelial cells. Their levels correlated in active disease. E-SEL correlated with leukocyte count. In CR, P-SEL physiologically correlated with platelet count. The correlation with E-SEL was insignificant and absent in the control group. Our data suggest activation of endothelial cells in the presence of myeloblasts.

Regulation of eosinophil development and survival.

PURPOSE OF REVIEW: Eosinophils are a subset of granulocytes generally associated with type 2 immune responses. They can contribute to protection against helminths but also mediate pro-inflammatory functions during allergic immune responses. Only recently, eosinophils were also found to exert many other functions such as regulation of glucose and fat metabolism, thermogenesis, survival of plasma cells, and antitumor activity. The mechanisms that control eosinophil development and survival are only partially understood. RECENT FINDINGS: Here we review new findings regarding the role of cell-extrinsic and cell-intrinsic factors for eosinophilopoiesis and eosinophil homeostasis. Several reports provide new insights in the regulation of eosinophil development by transcription factors, miRNAs and epigenetic modifications. Danger signals like lipopolysaccharide or alarmins can activate eosinophils but also prolong their lifespan. We further reflect on the observations that eosinophil development is tightly controlled by the unfolded protein stress response and formation of cytoplasmic granules. SUMMARY: Eosinophils emerge as important regulators of diverse biological processes. Their differentiation and survival is tightly regulated by factors that are still poorly understood. Newly identified pathways involved in eosinophilopoiesis and eosinophil homeostasis may lead to development of new therapeutic options for treatment of eosinophil-associated diseases.

Functionally identifiable apoptosis-insensitive subpopulations determine chemoresistance in acute myeloid leukemia.

Upfront resistance to chemotherapy and relapse following remission are critical problems in leukemia that are generally attributed to subpopulations of chemoresistant tumor cells. There are, however, limited means for prospectively identifying these subpopulations, which hinders an understanding of therapeutic resistance. BH3 profiling is a functional single-cell analysis using synthetic BCL-2 BH3 domain-like peptides that measures mitochondrial apoptotic sensitivity or "priming." Here, we observed that the extent of apoptotic priming is heterogeneous within multiple cancer cell lines and is not the result of experimental noise. Apoptotic priming was also heterogeneous in treatment-naive primary human acute myeloid leukemia (AML) myeloblasts, and this heterogeneity decreased in chemotherapy-treated AML patients. The priming of the most apoptosis-resistant tumor cells, rather than the median priming of the population, best predicted patient response to induction chemotherapy. For several patients, these poorly primed subpopulations of AML tumor cells were enriched for antiapoptotic proteins. Developing techniques to identify and understand these apoptosis-insensitive subpopulations of tumor cells may yield insights into clinical chemoresistance and potentially improve therapeutic outcomes in AML.

Comparison of newly diagnosed and relapsed patients with acute promyelocytic leukemia treated with arsenic trioxide: insight into mechanisms of resistance.

There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens.

Benzophenone-based photochemical micropatterning of biomolecules to create model substrates and instructive biomaterials.

The extracellular matrix (ECM) is a dynamic and heterogeneous environment that controls many aspects of cell behavior. Not surprisingly, many different approaches have focused on creating model substrates that recapitulate the biomolecular, topographical, and mechanical properties of the ECM for in vitro studies of cell behavior. This chapter details a general, versatile method for the spatially controlled deposition of multiple biomolecules onto both planar and topographically complex support structures with micrometer resolution. This approach is based upon the well-understood photochemical UV crosslinking of benzophenone (BP) to solution-phase biomolecules. This is a molecularly general strategy that can be utilized to immobilize biomolecules onto any surface prefunctionalized with BP. Examples described herein include modification of planar and corrugated glass substrates as well as collagen-glycosaminoglycan biomaterials configured either as highly porous scaffolds or nonporous membranes with a variety of biomolecular targets, including proteins, glycoproteins, and carbohydrates.

miRNA-130a regulates C/EBP-ε expression during granulopoiesis.

CCAAT/enhancer binding protein-ε (C/EBP-ε) is considered a master transcription factor regulating terminal neutrophil maturation. It is essential for expression of secondary granule proteins, but it also regulates proliferation, cell cycle, and maturation during granulopoiesis. Cebpe(-/-) mice have incomplete granulocytic differentiation and increased sensitivity toward bacterial infections. The amount of C/EBP-ε messenger RNA (mRNA) increases with maturation from myeloblasts with peak level in myelocytes (MC)/metamyelocytes (MM), when the cells stop proliferating followed by a decline in more mature cells. In contrast, C/EBP-ε protein is virtually detectable only in the MC/MM population, indicating that expression in more immature cells could be inhibited by microRNAs (miRNAs). We found that miRNA-130a (miR-130a) regulates C/EBP-ε protein expression in both murine and human granulocytic precursors. Overexpression of miR-130a in a murine cell line downregulated C/EBP-ε protein and lactoferrin (Ltf), cathelicidin antimicrobial protein (Camp), and lipocalin-2 (Lcn2) mRNA expression giving rise to cells with a more immature phenotype, as seen in the Cebpe(-/-) mouse. Introduction of a C/EBP-ε mRNA without target site for miR-130a restored both C/EBP-ε production, expression of Camp and Lcn2, and resulted in the cells having a more mature phenotype. We conclude that miR-130a is important for the regulation of the timed expression of C/EBP-ε during granulopoiesis.

Bone morphogenetic proteins regulate differentiation of human promyelocytic leukemia cells.

We investigated the role of bone morphogenetic proteins (BMPs) in suppression of all-trans retinoic acid (ATRA)-mediated differentiation of leukemic promyelocytes. In NB4 and HL60 cell lines, BMPs reduced the percentage of differentiated cells, and suppressed PU.1 and C/EBPε gene expression induced by ATRA. BMP and ATRA synergized in the induction of ID genes, causing suppression of differentiation. In primary acute promyelocytic leukemia bone-marrow samples, positive correlation of PML/RARα and negative of RARα with the expression of BMP-4, BMP-6 and ID genes were found. We concluded that BMPs may have oncogenic properties and mediate ATRA resistance by a mechanism that involves ID genes.

Increased promyelocytic-derived microparticles: a novel potential factor for coagulopathy in acute promyelocytic leukemia.

The frequent serious bleeding and thrombotic complications in acute promyelocytic leukemia (APL) are major causes of early mortality, but the complex mechanisms causing the bleeding have not been completely elucidated. Because microparticles (MPs) are known to be elevated in thromboembolic disorders, we hypothesized a role for MPs in the pathogenesis of coagulopathy in APL. MPs were isolated from 30 APL patients and 20 healthy subjects and from cultured NB4/APL cells. The morphology of the MPs was examined, and they were quantified and analyzed for their thrombin-generating potential. We confirmed the existence of promyelocytic-derived MPs by morphology using transmission electron microscopy and laser scanning confocal microscopy. Counts of MPs in APL were elevated and were typically from promyelocytic cells (CD33(+) TF(+) MPs). Importantly, the CD33(+) MPs strongly correlated with patient leukocyte count (R = 0.64, p = 0.002) and D-dimer (R = 0.51, p = 0.0038). Moreover, the MPs from patients with APL decreased the coagulation times and induced thrombin generation. APL MP-associated thrombin generation was reduced by 54 % when the extrinsic pathway was blocked using an anti-human tissue factor (TF) antibody. However, neither anti-factor XI nor anti-tissue factor pathway inhibitor had any significant inhibitory effect. Our results show that the procoagulant state in APL is partially due to the TF-dependent procoagulant properties of circulating promyelocytic-derived MPs. TF(+) MPs may be a novel potential risk factor for coagulopathy in APL.

Morphology and quantitative composition of hematopoietic cells in murine bone marrow and spleen of healthy subjects.

Laboratory mice play an outstanding role in modeling human development and disease. In contrast to human leukemia, the spleen is involved in almost all cases, and the bone marrow is only variably involved in murine models. Although mice have been used for medical research for over 100 years, there are only few reports with a small number of cases looking at morphology and quantitative composition of murine hematopoietic cells in the bone marrow of non-transplanted animals of most strains. To our knowledge, there is not even a single report describing the splenogram in C57BL/6J mice, one of the most commonly used strains for medical research. The present study illustrates the morphology of the hematopoietic cells in the bone marrow and spleen of non-treated C57BL/6J mice and establishes the murine myelogram from the largest healthy C57BL/6J cohort reported to date. Furthermore, we present the first murine splenogram described for C57BL/6J mice. Our study supports the acceptance of the presence of >5 % blast cells as providing clear evidence of abnormality in bone marrow like in humans. In addition, we are the first to show <1 % blast cells in the normal spleen. Interestingly, classical dysplastic changes were rare in normal healthy mice. Our study of the bone marrow and spleen of healthy non-transplanted animals provides reference ranges of each cell type and for the myeloid/erythroid ratio, which can be used to interpret preclinical gene therapy data, leukemogenesis, and hematopoiesis studies, and may improve the quality of such analyses.

A recombined protein (rSj16) derived from Schistosoma japonicum induces cell cycle arrest and apoptosis of murine myeloid leukemia cells.

rSj16, a recombined protein from Schistosoma japonicum, has been identified as an anti-inflammatory molecule. In this study, we demonstrated that rSj16 strongly suppressed the growth of murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. rSj16 induced apoptosis by increasing the proportion of sub-G1 apoptotic cells as well as causing cell cycle arrest at the G0/G1 phase. The expressions of cyclin D1, D2, D3, and E, and Cdk 2, 4, and 6 genes in WEHI-3B JCS cells were significantly down-regulated at 24 h as measured by real-time PCR. Furthermore, apoptosis induced by rSj16 was confirmed by 4,6-diamidino-2-phenylindole nuclear staining assay and annexin V/propidium iodide double staining. A reduction of the mitochondrial membrane potential indicated an active involvement of mitochondria in the apoptosis process. rSj16 treatment induced an increase in the activity of caspase 3, 6, and 9, and expression of pro-apoptotic Bax. Meanwhile, the decreased expression of anti-apoptotic Bcl-2 was observed after rSj16 treatment. Taken together, our results implied that rSj16 can inhibit proliferation by inducing G0/G1 cell cycle arrest and apoptosis of murine myeloid leukemia cells via activation of the caspase-mediated mechanism by regulating the expression of Bcl-2 family.

Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells.

The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

Analysis of human samples reveals impaired SHH-dependent cerebellar development in Joubert syndrome/Meckel syndrome.

Joubert syndrome (JS) and Meckel syndrome (MKS) are pleiotropic ciliopathies characterized by severe defects of the cerebellar vermis, ranging from hypoplasia to aplasia. Interestingly, ciliary conditional mutant mice have a hypoplastic cerebellum in which the proliferation of cerebellar granule cell progenitors (GCPs) in response to Sonic hedgehog (SHH) is severely reduced. This suggests that Shh signaling defects could contribute to the vermis hypoplasia observed in the human syndromes. As existing JS/MKS mutant mouse models suggest apparently contradictory hypotheses on JS/MKS etiology, we investigated Shh signaling directly on human fetal samples. First, in an examination of human cerebellar development, we linked the rates of GCP proliferation to the different levels and localizations of active Shh signaling and showed that the GCP possessed a primary cilium with CEP290 at its base. Second, we found that the proliferation of GCPs and their response to SHH were severely impaired in the cerebellum of subjects with JS/MKS and Jeune syndrome. Finally, we showed that the defect in GCP proliferation was similar in the cerebellar vermis and hemispheres in all patients with ciliopathy analyzed, suggesting that the specific cause of vermal hypo-/aplasia precedes this defect. Our results, obtained from the analysis of human samples, show that the hemispheres and the vermis are affected in JS/MKS and provide evidence of a defective cellular mechanism in these pathologic processes.

Altered neutrophil maturation patterns that limit identification of myelodysplastic syndromes.

BACKGROUND: Altered neutrophil maturation patterns have been reported useful for identification of myelodysplastic syndromes (MDS). METHODS: Neutrophil maturation patterns based on CD11b, CD13, and CD16 were visually and numerically evaluated in 19 control, 23 MDS, 37 nondiagnostic for MDS (NDM) specimens, and 19 also processed 1 and 2 days subsequently. RESULTS: In contrast to maturation patterns illustrated previously by others as "normal," 84% of controls displayed diminished acquisition of CD16, imparting a contracted appearance. Such divergence from published "normal" patterns was usually mild-moderate, considered nonspecific, and associated with delayed processing: longer intervals between collection and processing (median 20.5 vs. 5.2 h), and following 1 and 2 days delay. Findings restricted to nonspecific contraction were found in 56% MDS and 78% NDM specimens. Evaluation for aberrant patterns was still performed with mild-moderate contraction present, but concern for over interpretation led to use of an equivocal-aberrant category. Nine cases had aberrant or equivocal-aberrant patterns (seven MDS, two NDM) with distinct visual alterations that differed from nonspecific contraction and had numerical evidence for a left shift: myeloblasts increased (67%) and least mature neutrophils (CD11b-/low, CD16-/low) increased (78%). Although evidence for a left shift was associated with MDS, it was also seen in NDM specimens with a synchronous left shift. CONCLUSIONS: Neutrophil maturation patterns that diverge from previously illustrated "normal" patterns, not specific for MDS, may be common in some settings. Laboratories seeking to implement FC evaluation for MDS must determine which findings have sufficient specificity for MDS within their own practice and patient population.