MDS and AML show elevated fractions of CD34-positive blast cell populations with a high anti-apoptotic versus proliferation ratio.

This study investigates the intertwined processes of (anti-)apoptosis and cell proliferation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Utilizing antibodies to Bcl-2 and Ki-67, the CD34-positive blast cell compartments in bone marrow aspirates from 50 non-malignant cases, 25 MDS patients, and 25 AML patients were analyzed for their anti-apoptotic and proliferative cell fractions through ten-color flow cytometry. MDS patients exhibited a significantly increased anti-apoptotic (p=0.0014) and reduced proliferative cell fraction (p=0.0030) in their blast cell population as compared to non-malignant cases. AML patients showed an even more exacerbated trend than MDS patients. The resulting Bcl-2:Ki-67 cell fraction ratios in MDS and AML were significantly increased as compared to the non-malignant cases (p=0.0004 and p<0.0001, respectively). AML patients displayed, however, a high degree of variability in their anti-apoptotic and proliferation index, attributed to heterogeneity in maturation stage and severity of the disease at diagnosis. Using double-labeling for Bcl-2 and Ki-67 it could be shown that besides blast cells with a mutually exclusive Ki-67 and Bcl-2 expression, also blast cells concurrently exhibiting anti-apoptotic and proliferative marker expression were found. Integrating these two dynamic markers into MDS and AML diagnostic workups may enable informed conclusions about their biological behavior, facilitating individualized therapy decisions for patients.

Impact of the gating strategy for Ki-67 and Bcl-2 on the determination of proliferation and anti-apoptosis data by flow cytometry in non-malignant bone marrow aspirates and aspirates from patients with myeloid malignancies.

This Data in Brief article displays a flow cytometric assay that was used for the acquisition and analyses of proliferative and anti-apoptotic activity in hematopoietic cells. This dataset includes analyses of the Ki-67 positive fraction (Ki-67 proliferation index) and Bcl-2 positive fraction (Bcl-2 anti-apoptotic index) of the different myeloid bone marrow (BM) cell populations in non-malignant BM, and in BM disorders, i.e. myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The present dataset comprises 1) the percentage of the CD34 positive blast cells, erythroid cells, myeloid cells and monocytic cells, and 2) the determined Ki-67 positive fraction and Bcl-2 positive fraction of these cell populations in tabular form. This allows the comparison and reproduction of the data when these analyses are repeated in a different setting. Because gating the Ki-67 positive and Bcl-2 positive cells is a critical step in this assay, different gating approaches were compared to determine the most sensitive and specific approach. BM cells from aspirates of 50 non-malignant, 25 MDS and 27 AML cases were stained with 7 different antibody panels and subjected to flow cytometry for determination of the Ki-67 positive cells and Bcl-2 positive cells of the different myeloid cell populations. The Ki-67 or Bcl-2 positive cells were then divided by the total number of cells of the respective cell population to generate the Ki-67 positive fraction (Ki-67 proliferation index) or the Bcl-2 positive fraction (Bcl-2 anti-apoptotic index). The presented data may facilitate the establishment and standardization of flow cytometric analyses of the Ki-67 proliferation index and Bcl-2 anti-apoptotic index of the different myeloid cell populations in non-malignant BM as well as MDS and AML patients in other laboratories. Directions for proper gating of the Ki-67 positive and Bcl-2 positive fraction are crucial for achieving standardization among different laboratories. In addition, the data and the presented assay allows application of Ki-67 and Bcl-2 in a research and clinical setting and this approach can serve as the basis for optimization of the gating strategy and subsequent investigation of other cell biological processes besides proliferation and anti-apoptosis. These data can also promote future research into the role of these parameters in diagnosis of myeloid malignancies, prognosis of myeloid malignancies and therapeutic resistance against anti-cancer therapies in these malignancies. As specific populations were identified based on cell biological characteristics, these data can be useful for evaluating gating algorithms in flow cytometry in general by confirming the outcome (e.g. MDS or AML diagnosis) with the respective proliferation and anti-apoptotic profile of these malignancies. The Ki-67 proliferation index and Bcl-2 anti-apoptotic index may potentially be used for classification of MDS and AML based on supervised machine learning algorithms, while unsupervised machine learning can be deployed at the level of single cells to potentially distinguish non-malignant from malignant cells in the identification of minimal residual disease. Therefore, the present dataset may be of interest for internist-hematologists, immunologists with affinity for hemato-oncology, clinical chemists with sub-specialization of hematology and researchers in the field of hemato-oncology.

Optimized gating strategy and supporting flow cytometry data for the determination of the Ki-67 proliferation index in the diagnosis of myelodysplastic syndrome.

This Data in Brief article presents a novel flow cytometric assay used to acquire and process the data presented and discussed in the research paper by Mestrum et al., co-submitted to Leukemia Research, entitled: "Integration of the Ki-67 proliferation index into the Ogata score improves its diagnostic sensitivity for low-grade myelodysplastic syndromes." [1]. The dataset includes the gated fractions of the different myeloid populations in bone marrow (BM) aspirates (total BM cells, CD34 positive blast cells, erythroid cells, granulocytes and monocytes. The raw data is hosted in FlowRepository, while the analyzed data of 1) the fractions of the different myeloid cell populations and 2) the Ki-67 proliferation indices of these myeloid cell populations are provided in tabular form to allow comparison and reproduction of the data when such analyses are performed in a different setting. BM cells from aspirates of 50 myelodysplastic syndrome (MDS) patients and 20 non-clonal cytopenic controls were stained using specific antibody panels and proper fixation and permeabilization to determine the Ki-67 proliferation indices of the different myeloid cell populations. Data was acquired with the three laser, 10-color Navios™ Flow cytometer (Beckman Coulter, Marseille, France) with a blue diode Argon laser (488 nm, 22 mW), red diode Helium/Neon laser (638 nm, 25 mW) and violet air-cooled solid-state diode laser laser (405 nm, 50 mW). A minimum of 100,000 relevant events were acquired per sample, while we aimed at acquiring 500,000 events per sample. Gating was performed with the Infinicyt v2.0 software package (Cytognos SL, Salamanca, Spain). These data may guide the development and standardization of the flow cytometric analysis of the Ki-67 proliferation index (and other markers for cell behavior) for differentiation between non-clonal cytopenic patients and MDS patients. In addition, this assay may be used in myeloid malignancies for research and clinical purposes in other laboratories. This data can be used to encourage future research regarding stem-/progenitor cell resistance against anti-cancer therapies for myeloid malignancies, diagnostics of myeloid malignancies and prognosis of myeloid malignancies. Therefore, these data are of relevance to internist-hematologists, clinical chemists with sub-specialization of hematology and hemato-oncology oriented researchers.

Integration of the Ki-67 proliferation index into the Ogata score improves its diagnostic sensitivity for low-grade myelodysplastic syndromes.

BACKGROUND: Although flow cytometric detection of myelodysplastic syndrome (MDS) with the Ogata score has a high specificity, its sensitivity for low-grade MDS is low. Additional markers are needed to improve its diagnostic reliability. Therefore, we investigated the diagnostic performance of the Ki-67 proliferation index in bone marrow (BM) cell populations for detection of MDS. METHODS: BM aspirates from 50 MDS patients and 20 non-clonal cytopenic controls were analyzed with flow cytometry to determine the Ogata score and the Ki-67 proliferation indices in different cell populations. RESULTS: Ki-67 proliferation indices alone could be used to detect MDS with a sensitivity of up to 80 % and specificity of up to 70 %. Combining the Ogata score with the Ki-67 proliferation index of erythroid cells significantly improved its sensitivity for detection of MDS from 66 % to 90 %, while maintaining a specificity of 100 %. Particularly, the sensitivity for detection of low-grade MDS improved from 56 % to 91 %. CONCLUSIONS: This is the first study using Ki-67 proliferation indices to detect MDS and shows their particularly high diagnostic sensitivity for detection of low-grade MDS. Integration of the Ki-67 proliferation index of erythroid cells into the Ogata score significantly improved its sensitivity without loss of the high specificity.

Proliferative activity is disturbed in myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), and MDS/MPN diseases. Differences between MDS and MDS/MPN.

The proliferation marker Ki-67 is widely used within the field of diagnostic histopathology as a prognostic marker for solid cancers. However, Ki-67 is hardly used for prognostic and diagnostic purposes in flow cytometric analyses of hematologic neoplasms. In the present study, we investigated to what extent the proliferative activity, as determined by Ki-67 expression, is disturbed in myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), and MDS/MPN diseases. Bone marrow aspirates from 74 patients suffering from MPN, MDS, or MDS/MPN, and aspirates from 50 non-malignant cases were analyzed by flow cytometry for Ki-67 expression in the erythro-, myelo-, and monopoiesis. Ki-67 expression was used to investigate the proliferative activity during the various maturation steps within these hematopoietic cell lineages. In the MPN patient cohort, the proliferative activity of all cell lineages is significantly higher during almost all maturation stages compared to those of the benign control cohort. In the MDS and MDS/MPN cohort, a significantly lower proliferative activity is observed in the early maturation stages. In the MDS/MPN patient cohort, increased proliferative activity is seen in the later stages of the maturation. MDS and MDS/MPN display a distinct pattern in the proliferating fraction of maturing hematopoietic cells. This could become of added value in order to classify these malignancies based on their biological background and behavior, as well as in gaining a better understanding into the pathobiology of these malignancies.

Multicenter study to evaluate a new enumeration method for hematopoietic stem cell collection management.

BACKGROUND: CD34 flow cytometry is the gold standard for stem cell enumeration in peripheral blood at the mobilization stage and in the final apheresis product. The new stem cell mode of the Sysmex XN Series analyzer enumerates an immature cell population in the white progenitor and pathological cell (WPC) channel, based on the cell size, internal cellular complexity, and fluorescence intensity. STUDY DESIGN AND METHODS: In this multicenter study we analyzed 147 peripheral blood samples, 22 samples during collection of stem cells, and 45 samples from the apheresis product of 18 healthy allogeneic donors and 84 autologous patients. RESULTS: In this multicenter study we demonstrate that the XN stem cell enumeration method correlates well with viable CD34+ cells determined by flow cytometry during the stem cell mobilization phase to determine apheresis start time, during apheresis for real-time monitoring and adjustment, and for quality control of the final stem cell harvest. CONCLUSION: Our data show that there is an improvement in the correlation of XN stem cells and CD34+ cells in the peripheral blood during stem cell mobilization as well as in stem cell harvests compared to SE or XE Series analyzers. The XN stem cell enumeration method has a number of advantages compared to CD34 flow cytometry: it is fast, simple, reproducible, and less expensive. CE marking for the European market has been obtained, making the stem cell count on the XN analyzer a reportable clinical variable.

Within-day biological variation and hour-to-hour reference change values for hematological parameters.

BACKGROUND: Middle- and long-term biological variation data for hematological parameters have been reported in the literature. Within-day 24-h variability profiles for hematological parameters are currently lacking. However, comprehensive hour-to-hour variability data are critical to detect diurnal cyclical rhythms, and to take into account the 'time of sample collection' as a possible determinant of natural fluctuation. In this study, we assessed 24-h variation profiles for 20 hematological parameters. METHODS: Blood samples were collected under standardized conditions from 24 subjects every hour for 24 h. At each measurement, 20 hematological parameters were determined in duplicate. Analytical variation (CVA), within-subject biological variation (CVI), between-subject biological variation (CVG), index of individuality (II), and reference change values (RCVs) were calculated. For the parameters with a diurnal rhythm, hour-to-hour RCVs were determined. RESULTS: All parameters showed higher CVG than CVI. Highest CVG was found for eosinophils (46.6%; 95% CI, 34.9%-70.1%) and the lowest value was mean corpuscular hemoglobin concentration (MCHC) (3.2%; 95% CI, 2.4%-4.8%). CVI varied from 0.4% (95% CI, 0.32%-0.42%) to 20.9% (95% CI, 19.4%-22.6%) for red cell distribution width (RDW) and eosinophils, respectively. Six hematological parameters showed a diurnal rhythm. CONCLUSIONS: We present complete 24-h variability profiles for 20 hematological parameters. Hour-to-hour reference changes values may help to better discriminate between random fluctuations and true changes in parameters with rhythmic diurnal oscillations.

Hemolysis during cardiac surgery is associated with increased intravascular nitric oxide consumption and perioperative kidney and intestinal tissue damage.

INTRODUCTION: Acute kidney injury (AKI) and intestinal injury negatively impact patient outcome after cardiac surgery. Enhanced nitric oxide (NO) consumption due to intraoperative intravascular hemolysis, may play an important role in this setting. This study investigated the impact of hemolysis on plasma NO consumption, AKI, and intestinal tissue damage, after cardiac surgery. METHODS: Hemolysis (by plasma extracellular (free) hemoglobin; fHb), plasma NO-consumption, plasma fHb-binding capacity by haptoglobin (Hp), renal tubular injury (using urinary N-Acetyl-ß-D-glucosaminidase; NAG), intestinal mucosal injury (through plasma intestinal fatty acid binding protein; IFABP), and AKI were studied in patients undergoing off-pump cardiac surgery (OPCAB, N = 7), on-pump coronary artery bypass grafting (CABG, N = 30), or combined CABG and valve surgery (CABG+Valve, N = 30). RESULTS: FHb plasma levels and NO-consumption significantly increased, while plasma Hp concentrations significantly decreased in CABG and CABG+Valve patients (p < 0.0001) during surgery. The extent of hemolysis and NO-consumption correlated significantly (r (2) = 0.75, p < 0.0001). Also, NAG and IFABP increased in both groups (p < 0.0001, and p < 0.001, respectively), and both were significantly associated with hemolysis (R s = 0.70, p < 0.0001, and R s = 0.26, p = 0.04, respectively) and NO-consumption (Rs = 0.55, p = 0.002, and R s = 0.41, p = 0.03, respectively), also after multivariable logistic regression analysis. OPCAB patients did not show increased fHb, NO-consumption, NAG, or IFABP levels. Patients suffering from AKI (N = 9, 13.4%) displayed significantly higher fHb and NAG levels already during surgery compared to non-AKI patients. CONCLUSIONS: Hemolysis appears to be an important contributor to postoperative kidney injury and intestinal mucosal damage, potentially by limiting NO-bioavailability. This observation offers a novel diagnostic and therapeutic target to improve patient outcome after cardiothoracic surgery.

Immature platelet fraction measured on the Sysmex XN hemocytometer predicts thrombopoietic recovery after autologous stem cell transplantation.

OBJECTIVES: A period of thrombocytopenia is common after stem cell transplantation (SCT). To prevent serious bleeding complications, prophylactic platelet transfusions are administered. Previous studies have shown that a rise in immature platelets precedes recovery of platelet count. Our aim was to define a cutoff value for immature platelets predicting thrombopoietic recovery within 2 d. METHODS: Hematological parameters were measured on the Sysmex XN hemocytometer. We calculated reference change values (RCV) for platelets in eight healthy individuals as marker for platelet recovery. To define a cutoff value, we performed ROC analysis using data from 16 autologous SCT patients. RESULTS: RCV for platelet concentration was 14.1%. Platelet recovery was observed 13 (median; range 9-31) days after SCT. Increase in immature platelet fraction (IPF) before platelet recovery was seen in all autologous SCT patients. Optimal cutoff IPF was found to be 5.3% for platelet recovery within 2 d (specificity 0.98, sensitivity 0.47, positive predictive value 0.93). CONCLUSIONS: We identified an optimal cutoff value for IPF 5.3% to predict platelet recovery after autologous SCT within 2 d. Implementing this cutoff value in transfusion strategy may reduce the number of prophylactic platelet transfusions.

Blood transfusions increase circulating plasma free hemoglobin levels and plasma nitric oxide consumption: a prospective observational pilot study.

INTRODUCTION: The increasing number of reports on the relation between transfusion of stored red blood cells (RBCs) and adverse patient outcome has sparked an intense debate on the benefits and risks of blood transfusions. Meanwhile, the pathophysiological mechanisms underlying this postulated relation remain unclear. The development of hemolysis during storage might contribute to this mechanism by release of free hemoglobin (fHb), a potent nitric oxide (NO) scavenger, which may impair vasodilation and microcirculatory perfusion after transfusion. The objective of this prospective observational pilot study was to establish whether RBC transfusion results in increased circulating fHb levels and plasma NO consumption. In addition, the relation between increased fHb values and circulating haptoglobin, its natural scavenger, was studied. METHODS: Thirty patients electively received 1 stored packed RBC unit (n = 8) or 2 stored packed RBC units (n = 22). Blood samples were drawn to analyze plasma levels of fHb, haptoglobin, and NO consumption prior to transfusion, and 15, 30, 60 and 120 minutes and 24 hours after transfusion. Differences were compared using Pearson's chi-square test or Fisher's exact test for dichotomous variables, or an independent-sample t test or Mann-Whitney U test for continuous data. Continuous, multiple-timepoint data were analyzed using repeated one-way analysis of variance or the Kruskall-Wallis test. Correlations were analyzed using Spearman or Pearson correlation. RESULTS: Storage duration correlated significantly with fHb concentrations and NO consumption within the storage medium (r = 0.51, P < 0.001 and r = 0.62, P = 0.002). fHb also significantly correlated with NO consumption directly (r = 0.61, P = 0.002). Transfusion of 2 RBC units significantly increased circulating fHb and NO consumption in the recipient (P < 0.001 and P < 0.05, respectively), in contrast to transfusion of 1 stored RBC unit. Storage duration of the blood products did not correlate with changes in fHb and NO consumption in the recipient. In contrast, pre-transfusion recipient plasma haptoglobin levels inversely influenced post-transfusion fHb concentrations. CONCLUSION: These data suggest that RBC transfusion can significantly increase post-transfusion plasma fHb levels and plasma NO consumption in the recipient. This finding may contribute to the potential pathophysiological mechanism underlying the much-discussed adverse relation between blood transfusions and patient outcome. This observation may be of particular importance for patients with substantial transfusion requirements.

The effect of oxytocin and an oxytocin antagonist on the human myometrial proteome.

OBJECTIVE: The immediate effects of oxytocin on myometrial signal transduction have been described. However, the longer term effects (up to an hour) on the myometrial proteome have not. We combined in vitro contractility with proteomic analysis to determine the protein changes associated with oxytocin-induced myometrial activity. STUDY DESIGN: Human myometrial biopsies were taken at elective caesarean section prior to administration of oxytocics. Strips were mounted in an organ bath and exposed to oxytocin (10(-8) mol/L), the oxytocin antagonist L372,662 (10(-7) mol/L), or vehicle (n = 5 for each) for 60 minutes. Contractility was determined and expressed as a percentage of pretreatment for each strip. At the end of the contractility experiment, proteins were extracted and separated by two-dimensional (2D) gel electrophoresis. Two-dimensional gels were analyzed by PDQuest and proteins of interest identified by mass spectrometry. RESULTS: Identified proteins that demonstrated differences as a result of treatment with oxytocin or the oxytocin receptor antagonist L372,662 were Annexin-A3, Osteoglycin, HSP70-protein 2, 2 isoforms of Cytokeratin 19, Desmin, EHD2, Protein disulfide isomerase A3, BIGH3, transgelin, thioredoxin reductase, triose phosphate isomerase, pyruvate kinase, elongation factor 1gamma, and alpha-Actin-3. These proteins can be grouped into 5 classes of protein, those associated with cytoskeletal function, contractile/oxidative stress, protein synthesis, extracellular matrix proteins, and energy metabolism. CONCLUSION: This study demonstrates that oxytocin has longer term (1 hour) effects on the myometrial proteome.

Markers of infection in inpatients and outpatients with acute Q-fever.

BACKGROUND: Query-fever (Q-fever) is a zoonotic infection caused by the intracellular Gram-negative coccobacillus Coxiella burnetii. A large ongoing outbreak of Q-fever has been reported in the Netherlands. We studied various markers of infection in inpatients (hospitalised) and outpatients (treated by a general physician) with acute Q-fever in relation to disease severity. METHODS: Leukocyte counts, C-reactive protein (CRP) and procalcitonin (PCT) concentrations were measured in 25 inpatients and 40 outpatients upon presentation with acute Q-fever. Chest X-rays, if available, were analysed and confusion, urea, respiratory rate, blood pressure-age 65 (CURB-65) scores, indicating severity of pneumonia, were calculated. RESULTS: CRP was the only marker that significantly differentiated between inpatients and outpatients. It was increased in all patients from both groups. Leukocyte counts and PCT concentrations did not differ between inpatients and outpatients. Overall, only 13/65 patients had an increased leukocyte count and only 11/65 patients presented with PCT concentrations indicative of possible bacterial respiratory tract infection. Infiltrative changes on the chest X-ray were observed in the majority of patients. CURB-65 score was 0+/-1 (mean+/-SD). CONCLUSIONS: Acute Q-fever, a relatively mild pneumonia with low CURB-65 scores, specifically induces a response in CRP, while PCT concentrations and leukocytes are within the normal range or increased only marginally.

Anti-inflammatory and relaxatory effects of prostaglandin E2 in myometrial smooth muscle.

The onset of human labour is complex and involves multiple mediators, prostaglandins, cytokines and chemokines. However, whilst prostaglandins are routinely used for labour induction and inhibitors of prostaglandin synthesis are used to prevent pre-term labour, these practices are not invariably successful, and the rationale for their use is equivocal. As COX-2 and prostaglandin E(2) (PGE(2)) production is increased towards term, we have investigated the effect of PGE(2) and other cAMP-elevating agents on events associated with labour induction. Time-dependent increases in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8) release were observed following treatment of primary human myometrial smooth muscle (HMSM) cells with IL-1beta, via mechanisms that required de novo transcription and translation. Prior treatment with PGE(2) (1 microM) produced 86 and 80% decreases in GM-CSF and IL-8 release, respectively. Similarly, the cAMP analogue, 8-bromo-cAMP (8Br-cAMP) and the phosphodiesterase-4 (PDE(4)) inhibitor, rolipram, also repressed GM-CSF and IL-8 release. In addition, PGE(2), 8Br-cAMP, rolipram and salbutamol all had a dose-dependent inhibitory effect on spontaneous myometrial contractions in vitro. In this study, PGE(2) reduced the release of factors associated with cervical ripening and attenuated force development in myometrial smooth muscle, raising the possibility that in myometrium, PGE(2) may act to down-regulate some of the processes that contribute to the onset of human labour and may be beneficial in helping to maintain pregnancy towards term.

Paracrine oxytocin and estradiol demonstrate a spatial increase in human intrauterine tissues with labor.

In this study we investigated the spatial and temporal relationship among oxytocin (OT), oxytocin receptor (OTR), and estradiol (E2) at term, with (LAB) and without labor (NIL), in human amnion (AM), chorio-decidua (CD), fundal (FU), and lower segment (LS) myometrium. RT-PCR and RIA demonstrated a labor-associated increase in OT mRNA and peptide in CD, AM, and FU, but not LS. HPLC purification and mass spectrometry analysis confirmed that immunoreactive OT corresponded to alpha-amidated OT. Immunohistochemistry localized OT to chorionic trophoblast, decidual stroma, and glandular epithelium. RT-PCR analysis of OTR mRNA demonstrated a significant difference between FU and LS samples, which remained unchanged with labor in all tissues. Immunohistochemistry localized OTR to amniotic epithelium, decidual stroma, and myometrium. Tissue E2 concentrations, as determined by ELISA, demonstrated a significant increase with labor in all tissues. E2 was highest in CD, followed by FU, AM, and LS, respectively. E2 correlated with OT in samples of FU and CD taken from NIL women and in FU, CD, and AM taken from LAB women. We conclude that a significant increase in both OT and E2 occurs at the myometrial decidual interface with labor, and this increase is reflected in both the fundal and lower segments of the uterus. In contrast to OT and E2 the OTR is spatially regulated, with significantly greater expression in the fundal region of the uterus. Paracrine OT production stimulated by E2 may be important in activating the uterus at term.