CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

PlateletSeq: A novel method for discovery of blood-based biomarkers.

Platelets are small circulating fragments of cells that play important roles in thrombosis, haemostasis, immune response, inflammation and cancer growth. Although anucleate, they contain a rich RNA repertoire which offers an opportunity to characterise changes in platelet gene expression in health and disease. Whilst this can be achieved with conventional RNA sequencing, a large input of high-quality RNA, and hence blood volume, is required (unless a pre-amplification step is added), along with specialist bioinformatic skills for data analysis and interpretation. We have developed a transcriptomics next-generation sequencing-based approach that overcomes these limitations. Termed PlateletSeq, this method requires very low levels of RNA input and does not require specialist bioinformatic analytical skills. Here we describe the methodology, from sample collection to processing and data analysis. Specifically, blood samples can be stored for up to 8 days at 4 °C prior to analysis. Platelets are isolated using multi-step centrifugation and a purity of ≤ 1 leucocyte per 0.26x106 platelets is optimal for gene expression analysis. We have applied PlateletSeq to normal adult blood samples and show there are no age-associated variations and only minor gender-associated differences. In contrast, platelets from patients with myeloproliferative neoplasms show differences in platelet transcript profiles from normal and between disease subtypes. This illustrates the potential applicability of PlateletSeq for biomarker discovery and studying platelet biology in patient samples. It also opens avenues for assessing platelet quality in other fields such as transfusion research.

Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Osteohematology: To be or Notch to be.

Osteohematology is an emerging research field that studies the crosstalk between hematopoietic and bone stromal cells, to elucidate the mechanisms of hematological and skeletal malignancies and diseases. The Notch is an evolutionary conserved developmental signaling pathway, with critical roles in embryonic development by controlling cell proliferation and differentiation. However, the Notch pathway is also critically involved in cancer initiation and progression, such as osteosarcoma, leukemia, and multiple myeloma. The Notch-mediated malignant cells dysregulate bone and bone marrow cells in the tumour microenvironment, resulting in disorders ranging from osteoporosis to bone marrow dysfunction. To date, the complex interplay of Notch signaling molecules in hematopoietic and bone stromal cells is still poorly understood. In this mini-review, we summarize the crosstalk between cells in bone and bone marrow and their influence under the Notch signaling pathway in physiological conditions and in tumour microenvironment.

Hospital-Acquired Infection, Length of Stay, and Readmission in Elective Surgery Patients Transfused 1 Unit of Red Blood Cells: A Retrospective Cohort Study.

BACKGROUND: Most patients transfused red blood cells in elective surgery receive small volumes of blood, which is likely to be discretionary and avoidable. We investigated the outcomes of patients who received a single unit of packed red blood cells during their hospital admission for an elective surgical procedure when compared to those not transfused. METHODS: This retrospective cohort study included elective surgical admissions to 4 hospitals in Western Australia over a 6-year period. Participants were included if they were at least 18 years of age and were admitted for elective surgery between July 2014 and June 2020. We compared outcomes of patients who had received 1 unit of red blood cells to patients who had not been transfused. To balance differences in patient characteristics, we weighted our multivariable regression models using the inverse probability of treatment. In addition to propensity score weighting, our multivariable regression models adjusted for hemoglobin level, surgical procedure, patient age, gender, comorbidities, and the transfusion of fresh-frozen plasma or platelets. Outcomes studied were hospital-acquired infection, hospital length of stay, and all-cause emergency readmissions within 28 days. RESULTS: Overall, 767 (3.2%) patients received a transfusion of 1 unit of red blood cells throughout their admission. In the propensity score weighted analysis, the transfusion of a single unit of red blood cells was associated with higher odds of hospital-acquired infection (odds ratio, 3.94; 95% confidence interval [CI], 2.99-5.20; P < .001). Patients who received 1 unit of red blood cells throughout their admission were more likely to have a longer hospital stay (rate ratio, 1.57; 95% CI, 1.51-1.63; P < .001) and had 1.42 (95% CI, 1.20-1.69; P < .001) times higher odds of 28-day readmission. CONCLUSIONS: These results suggest that avoidance of even small volumes of packed red blood cells may prevent adverse clinical outcomes. This may encourage hospital administrators to implement strategies to avoid the transfusion of even small volumes of red blood cells by applying patient blood management practices.

A novel translocation t(10;17)(p13;q11.2) harboring two cryptic deletions identified by array-CGH and characterized by SUZ12 overexpression in a patient with chronic thrombocytosis.

No specific translocation is associated with myeloproliferative neoplasms (MPNs). However, an interstitial deletion involving subband 17q11.2 which includes the NF1 gene, although rare, is a recurrent aberration in several myeloid disorders including MPNs. For the first time, we report an acquired novel translocation involving 10p13 and 17q11.2 in a 62-year-old Caucasian female which was referred for investigation of chronic and persistent unexplained thrombocytosis. The patient had no history of hematological sequelae and genomic testing for JAK2, CALR, and MPL mutations were negative. She was subsequently diagnosed with a triple negative essential thrombocythemia. Array-CGH analysis noted that the translocation harbored two cryptic deletions, one of which involved 17q11.2 encompassing the NF1 gene. One of the junction breakpoints involved the SUZ12 gene. Immunohistochemical assessment of the marrow trephine showed increased megakaryocytic expression of the SUZ12 protein, as well as EZH2 and Ki67; biochemical abnormalities suggestive of excess megakaryocytic hyperplasia. This novel translocation may affect the expression of SUZ12 and its downstream targets, and may represent a unique pathogenomic etiology which drives chronic thrombocytosis in essential thrombocythemia.

Platelets in myeloproliferative neoplasms have a distinct transcript signature in the presence of marrow fibrosis.

Marrow fibrosis is a significant complication of myeloproliferative neoplasms (MPN) that affects up to 20% of patients and is associated with a poor prognosis. The pathological processes that lead to fibrotic progression are not well understood, but megakaryocytes have been implicated in the process. The aim of this study was to determine whether platelets, derived from megakaryocytes, have transcriptomic alterations associated with fibrosis. Platelets from MPN patients with and without fibrosis and non-malignant control individuals were assessed using next generation sequencing. Results from the initial training cohort showed discrete changes in platelet transcripts in the presence of marrow fibrosis. We identified more than 1000 differentially expressed transcripts from which a putative 3-gene fibrotic platelet signature (CCND1, H2AX [previously termed H2AFX] and CEP55) could be identified. This fibrosis-associated signature was assessed blinded on platelets from an independent test MPN patient cohort. The 3-gene signature was able to discriminate between patients with and without marrow fibrosis with a positive predictive value of 71% (93% specificity, 71% sensitivity). This demonstrates that assessment of dysregulated transcripts in platelets may be a useful monitoring tool in MPN to identify progression to marrow fibrosis. Further, sequential monitoring could have clinical applications for early prediction of progression to fibrosis.

"Immuno-flowFISH" for the Assessment of Cytogenetic Abnormalities in Chronic Lymphocytic Leukemia.

Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high-resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high-throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called "Immuno-flowFISH". The aim of this study was to demonstrate the application of immuno-flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus-specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH-positive CLL cells and the ratio of FISH spot counts for CD5/CD19-positive CLL cells to CD3/CD5-positive T cells (FISH "mean spot ratio"). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5-22.8% and the FISH "mean spot ratio" 0.86-0.96. Immuno-flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno-flowFISH could detect del(17p) in phenotypically identified CD5/CD19-positive B-cells. The 100-fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno-flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry.

Imaging flow cytometry to assess chromosomal abnormalities in chronic lymphocytic leukaemia.

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in the Western world, has a characteristic phenotype and prognosis largely defined by the presence of cytogenetic aberrations. The gold standard for detecting these cytogenetic abnormalities is interphase fluorescence in situ hybridisation (FISH) performed on cell smears or tissue sections on glass slides. Fluorescently labelled DNA probes bind to specific chromosomal regions and the signal detected by fluorescent microscopy. Generally only 200 cells are assessed and the limit of sensitivity is 3% positive cells. Here we report the development and use of imaging flow cytometry to assess chromosomes by FISH in phenotyped CLL cells in suspension. Thousands of CLL cells, identified by their phenotype, are assessed for specific FISH probe signals using an automated, high throughput imaging flow cytometer. The "extended depth of field" capability of the imaging flow cytometer enables FISH probe signals ("spots") to be resolved and localised within the (stained) nucleus of the immunophenotyped cells. We report the development of the automated "immuno-flowFISH" on normal blood using the Amnis ImageStreamX mark II platform and illustrate the clinical application of the method for the assessment of chromosome 12 in CLL. It is a powerful new method which has potential to be applied at diagnosis for disease stratification, and following treatment to assess residual disease. These applications will assist clinicians in optimising therapeutic decision making and thereby improve patient outcome.

Megakaryocytes in Myeloproliferative Neoplasms Have Unique Somatic Mutations.

Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2V617F, CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Ten of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss.

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.

Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.

Automated imaging flow cytometry integrates flow cytometry with digital microscopy to produce high-resolution digital imaging with quantitative analysis. This enables cell identification based on morphology (cell size, shape), antigen expression, quantification of fluorescence signal intensity and localisation of detected signals (i.e. surface, cytoplasm, nuclear). We describe applications of imaging flow cytometry for the diagnostic assessment of acute leukaemia. These bone marrow malignancies are traditionally diagnosed and classified by cell morphology, phenotype and cytogenetic abnormalities. Traditionally morphology is assessed by light microscopy, phenotyping by conventional flow cytometry and genetics by karyotype and fluorescence in situ hybridisation (FISH) on interphase nuclei/metaphase spreads of cells on slides. Imaging flow cytometry adds a new dimension to the diagnostic assessment of these neoplasms. We describe three specific applications: From this we conclude that imaging flow cytometry offers benefits over conventional diagnostic methods. Specifically the ability to visualise the cells of interest, the pattern and localisation of expressed antigens and assess cytogenetic abnormalities in one integrated automated high-throughput test. Imaging flow cytometry presents a new paradigm for the diagnostic assessment of leukaemia.

Imaging flow cytometry in the assessment of leukocyte-platelet aggregates.

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an 'internal' mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.

Disruption of IKAROS activity in primitive chronic-phase CML cells mimics myeloid disease progression.

Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.

Development of a robust immuno-S-FISH protocol using imaging flow cytometry.

Fluorescence in situ hybridization (FISH) is a microscopy technique which uses a fluorescent probe to detect DNA sequences and is generally performed on metaphase spreads or interphase nuclei of intact cells on a slide. In a diagnostic laboratory, cells are hybridized with fluorescent probes and up to 200 cells counted for the number of cells with probe "spots." Recent modifications to standard FISH include immuno-FISH, where chromosomal abnormalities are detected only in cells by their phenotype, and S-FISH where probe hybridization is performed on whole cells in suspension. Here we describe the development of an immuno-S-FISH method that combines immunophenotyping and FISH analysis of cells in suspension followed by analysis on an imaging flow cytometer. This single platform technique couples microscopy with flow cytometry and "spot" detection of bound FISH probe. Automated immuno-S-FISH enables large numbers of analyzed cells to be identified by phenotype and assessed for specific chromosomal determinants by FISH. This novel robust method enables quantitative cell population analysis and "spot" counting for large numbers of cells. We report method optimization of this imaging immuno-S-FISH flow cytometry protocol which has capability for many clinical applications. © 2016 International Society for Advancement of Cytometry.

Gray platelet syndrome: proinflammatory megakaryocytes and α-granule loss cause myelofibrosis and confer metastasis resistance in mice.

NBEAL2 encodes a multidomain scaffolding protein with a putative role in granule ontogeny in human platelets. Mutations in NBEAL2 underlie gray platelet syndrome (GPS), a rare inherited bleeding disorder characterized by a lack of α-granules within blood platelets and progressive bone marrow fibrosis. We present here a novel Nbeal2(-/-) murine model of GPS and demonstrate that the lack of α-granules is due to their loss from platelets/mature megakaryocytes (MKs), and not by initial impaired formation. We show that the lack of Nbeal2 confers a proinflammatory phenotype to the bone marrow MKs, which in combination with the loss of proteins from α-granules drives the development of bone marrow fibrosis. In addition, we demonstrate that α-granule deficiency impairs platelet function beyond their purely hemostatic role and that Nbeal2 deficiency has a protective effect against cancer metastasis.

An active, collaborative approach to learning skills in flow cytometry.

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula.

Measurement of monocyte-platelet aggregates by imaging flow cytometry.

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets express P-Selectin (CD62P) on the cell membrane and bind to P-Selectin glycoprotein ligand 1 expressing monocytes, influencing them toward a pro-adhesive and inflammatory phenotype. It is well established that elevated circulating monocyte-platelet aggregates (MPAs) are linked to atherothrombosis in high risk patients. However, whole blood flow cytometry (FCM) has recently shown that circulating MPAs may also occur in the absence of platelet activation, particularly in healthy children. A potential limitation of conventional FCM is the potential for coincident events to resemble monocyte platelet aggregates. Here we report a novel imaging cytometry approach to further characterize monocyte-platelet aggregate formation by P-Selectin dependent and P-Selectin independent mechanisms and distinguish circulating MPAs from coincidental events. Monocytes were identified by expression of the lipopolysachharide receptor (CD14 BV421), while platelets were identified by expression of the glycoprotein Ib (CD42b APC). Differentiation of P-Selectin dependent and P-Selectin independent binding was achieved with AF488 labeled CD62P. Overall analysis of circulating and in vitro generated MPAs by conventional and imaging cytometry methods showed very strong correlation (r(2) = >0.99, P < 0.01). The Bland-Altman bias of -1.72 was not significantly different to zero. However, when measuring only P-Selectin negative MPAs, a lack of correlation (r(2) = 0.27, P = n.s.) likely reflects better discrimination of coincidence events using imaging cytometry. Our data demonstrate that IFC is more accurate in enumerating MPAs than conventional FCM, which over-estimates the number of MPAs due to the presence of coincident events.