Storage properties of platelet concentrates from umbilical cord blood prepared using three different methods.

BACKGROUND: Thrombocytopenia, common in preterm newborns, may increase bleeding risk and is often treated with transfusions. Recent studies reveal that transfusing platelets at a high threshold worsens outcomes, possibly due to a "developmental mismatch" between adult-derived platelets and neonatal hemostatic system. Cord blood-derived platelet concentrates (CBPCs) could be an alternative for newborns. Our study aims to produce and evaluate the quality parameters of CBPCs during storage. STUDY DESIGN AND METHODS: Cord blood was collected from placentas after near-term and full-term pregnancies. Several production methods were attempted to obtain CBPCs, varying centrifugation settings, filtration, and dilution procedures. Adult-derived platelet concentrates (PCs) processed with the same methods, and standard PCs from five buffy-coats were used as controls. Storage tests were performed on days 2, 4-5, 7 from the collection. RESULTS: CBPCs parameters were compared with adult-derived PCs, and no significant differences were found for mean platelet volume (MPV), swirling, morphology, glucose, lactate, pCO2, and pO2. pH and bicarbonate were lower in CBPCs. Some significant differences between methods in CD62P expression and JC-1 ratio were observed. Compared with standard PCs, CBPCs showed lower platelet concentration, pH, and JC-1. Additionally, both in CBPCs as well as in control PCs, the apoptosis marker phosphatidylserine was elevated. DISCUSSION: CBPCs were of comparable quality to control PCs during storage. However, apoptosis markers in both groups were elevated, suggesting processing and storage of low volumes of PCs require further optimization. Also, filtration of low volumes leads to significant platelet loss, an issue that requires remedy.

A modern overview of the process of platelet formation (thrombocytopoiesis) and its dependence on several factors.

Structural and functional alterations in platelets are an actual problem that requires more attention. The treatment of these illnesses proves challenging, inefficient and heavily relies on platelet donations. A difficult task confronting science is producing platelets in vitro, which calls for meticulous examination of factors affecting platelet generation. It is known that megakaryocytes produce platelets in vitro and in vivo differently: in the laboratory we can get a smaller number of platelets compared to the human body. This review primarily examines the stages of megakaryocyte maturation and the processes involved in platelet formation. The article reflects the results of both fundamental research on the problem and the new results obtained over the past decade. Currently, most scientists accept the pro-platelets theory of platelet formation. This review aims to explore in detail each stage of pro-platelet formation and the platelet formation process. It explains on the processes of polyploidization, endomitosis, and apoptosis, as well as the functions of structural cell components (microtubules, mitochondria, T- and α-granules) and pro-platelet migration. The microenvironment influence is acknowledged for the osteoblastic and vascular niches that affect thrombocytopoiesis. The additional aspect is the contribution of specific proteins to thrombocytopoiesis such as RhoA, ß1-tubulin, cytokines IL-6, IL-8, Toll-like receptors, etc.

Integrated microfluidic multiple electrode aggregometry for point-of-care platelet function analysis.

Point-of-care (POC) platelet function analysis can enable timely and precise management of bleeding and clotting in emergency rooms, operation rooms and intensive care units. However, POC platelet testing is currently not commonly performed, due to the complexity of sample preparation and limitations of existing technologies. Here, we report the development of an integrated microfluidic multiple electrode aggregometry (µMEA) sensor which uses multi-frequency impedance measurement of an embedded microelectrode array to perform platelet aggregometry directly from whole blood, sensing and measuring platelet activation in a label-free manner and without requiring any additional sample preparation. Additionally, the sensor incorporates blood flow during the assay to account for physiological flow and shear conditions. We show that the impedance signal from the sensor can be used to accurately detect and quantify platelet aggregation in a label-free manner, which was further validated by simultaneous fluorometric measurement and visualization of platelet aggregation. Further, we optimized the sensitivity and repeatability of the sensor using its frequency response and demonstrated that the sensor could be used to characterize drug dose-response in antiplatelet therapy with a frequency-tunable dynamic range. We also demonstrate that the sensor provides high sensitivity to perform platelet aggregometry under thrombocytopenic or low platelet count conditions. The µMEA sensor could thus enable POC platelet function analysis across several clinical applications.

Plateletcrit and absolute immature platelet count are not impacted by platelet transfusions: a single-centre prospective study.

BACKGROUND: This is the first study in which the impact of platelet transfusions on seven platelet indices was evaluated in platelet transfusion-dependent patients admitted in the ICU. STUDY DESIGN AND METHODS: Among a cohort of 21 ICU patients prospectively studied over eleven months, a total of 19 ICU patients were enrolled. Seven platelet indices were measured before and then, within 18-24 h, after platelet transfusions using the Sysmex XN-10 analyser and statistically investigated as follows: i) apheresis vs. pooled platelet transfusions; ii) pre- vs. post-platelet transfusions; and iii) platelet count (PC) increment vs. PC decrement group. RESULTS: A 79.2% of platelet transfusion episodes in ICU patients showed an increase in PC increment within 18-24 h, of which 73.7% had a peak percentage immature platelet fraction (%-IPF) above 10.0% during their stay. No difference was observed in the measurements of platelet indices between the apheresis and pooled platelet transfusion doses (all p > 0.05). Of the seven platelet indices investigated, plateletcrit (PCT) and absolute immature platelet count (A-IPF) were not influenced by platelet transfusions and thus proven to be stable (0.06 vs. 0.07%, p = 0.0901 and 4.6 vs. 4.9 × 109/L, p = 0.4559, respectively), despite their close proximity to platelet transfusion. But the overall effectiveness of these indices in detecting changes over time was not hindered. CONCLUSION: A-IPF and PCT are stable after platelet transfusions, regardless of whether patient's respond to or do not respond to platelet transfusion doses. PCT and A-IPF may thus prove useful in monitoring patient transfusion support and guiding management in thrombocytopenic patients.

Differential composition and yield of leukocytes isolated from various blood component leukoreduction filters.

In Flanders, an estimated 300,000 leukoreduction filters are discarded as biological waste in the blood establishment each year. These filters are a possible source of fresh donor leukocytes for downstream purposes including research. We investigated leukocyte isolation from two types of filters either used for the preparation of platelet concentrates (PC-LRF) or erythrocyte concentrates (EC-LRF). Outcome parameters were leukocyte yield, differential count, turnaround time and effect of storage conditions. Leukocytes were harvested by reverse flow of a buffer solution. Control was the gold standard density gradient centrifugation of buffy coats. Total leukocyte number isolated from PC-LRF (1049 (± 40) x 106) was almost double that of control (632 (± 66) x 106) but the differential count was comparable. Total leukocyte number isolated from EC-LRF (78 (± 9) x 106) was significantly lower than control, but the sample was specifically enriched in granulocytes (81 ± 4%) compared to control (30 ± 1%). Isolation of leukocytes from either PC- or EC-LRF takes 20 min compared to 240 min for control density gradient centrifugation. Leukocyte viability is optimal when harvested on day 1 post donation (95 ± 0.9%) compared to day 3 (76.4 ± 2.4%). In conclusion, our study demonstrates that leukoreduction filters from specific blood component processing are easy to use and present a valuable source for viable leukocytes of all types.

The effect of flow-derived mechanical cues on the growth and morphology of platelet aggregates under low, medium, and high shear rates.

Platelet aggregation is a dynamic process that can obstruct blood flow, leading to cardiovascular diseases. While many studies have demonstrated clear connections between shear rate and platelet aggregation, the impact of flow-derived mechanical signals on this process is not fully understood. The objective of this work is to investigate the role of flow conditions on platelet aggregation dynamics, including effects on growth, shape, density composition, and their potential correlation with binding processes that are characterised by longer (e.g., via αIIbß3 integrin) and shorter (e.g., via VWF) initial binding times. In vitro blood perfusion experiments were conducted at wall shear rates of 800, 1600 and 4000 s-1. Detailed analysis of two modalities of experimental images was performed to offer insights into the morphology of platelet aggregates. A consistent structural pattern was observed across all samples: a high-density core enveloped by a low-density outer shell. An image-based 3D computational blood flow model was subsequently employed to study the local flow conditions, including binding availability time and flow-derived mechanical signals via shear rate and rate of elongation. The results show substantial dependence of the aggregation dynamics on these flow parameters. We found that the different binding mechanisms that prefer different flow regimes do not have a monotonic cross-over in efficiency as the flow increases. There is a significant dip in the cumulative aggregation potential in-between the preferred regimes. The results suggest that treatments targeting the biomechanical pathways could benefit from creating conditions that exploit these low-efficiency zones of aggregation.

Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Develop Tandem Tension Sensor to Gauge Integrin-Transmitted Molecular Forces.

DNA-based tension sensors have innovated the imaging and calibration of mechanosensitive receptor-transmitted molecular forces, such as integrin tensions. However, these sensors mainly serve as binary reporters, only indicating if molecular forces exceed one predefined threshold. Here, we have developed tandem tension sensor (TTS), which comprises two consecutive force-sensing units, each with unique force detection thresholds and distinct fluorescence spectra, thereby enabling the quantification of molecular forces with dual reference levels. With TTS, we revealed that vinculin is not required for transmitting integrin tensions at approximately 10 pN (piconewtons) but is essential for elevating integrin tensions beyond 20 pN in focal adhesions (FAs). Such high tensions have emerged during the early stage of FA formation. TTS also successfully detected changes in integrin tensions in response to disrupted actin formation, inhibited myosin activity, and tuned substrate elasticity. We also applied TTS to examine integrin tensions in platelets and revealed two force regimes, with integrin tensions surpassing 20 pN at cell central regions and 13-20 pN integrin tensions at the cell edge. Overall, TTS, especially the construct consisting of a hairpin DNA (13 pN opening force) and a shearing DNA (20 pN opening force), stands as a valuable tool for the quantification of receptor-transmitted molecular forces within living cells.

Light microscopic morphology of blood cells of non-descript indigenous Zoar chicken of Mizoram, India.

Identifying and analysing distinct blood cells is crucial for the diagnosis and treatment of diseases in the field of biomedicine. The present study was undertaken to study the cytomorphological and cytochemical characteristics of the blood cells of Zoar, a non-descript indigenous breed of chicken extensively reared under backyard poultry farming in Mizoram, India. For this study, 2 mL of blood samples were aseptically collected from the wings veins of 12 chickens and were processed for light microscopic study under standard protocols. The matured erythrocytes were elliptical, while the immature erythrocytes appeared oval. The heterophils were positive for SBB (SBB), Periodic Acid Schiff (PAS), acid phosphatase, alkaline phosphatase and Arylsulphatase while the eosinophils were positive for SBB, PAS, alkaline phosphatase, cytochrome oxidase and peroxidase. The basophils of were positive for toluidine blue while the thrombocytes were positive for PAS. These cytochemical and cytoenzymatic staining properties plays a very important role in diagnosis, differentiation, and classification of leukaemias.

Gelsolin regulates receptor-mediated and fluid-phase endocytosis in platelets.

BACKGROUND: Endocytosis is the process by which platelets incorporate extracellular molecules into their secretory granules. Endocytosis is mediated by the actin cytoskeleton in nucleated cells; however, the endocytic mechanisms in platelets are undefined. OBJECTIVES: To better understand platelet endocytosis, we studied gelsolin (Gsn), an actin-severing protein that promotes actin assembly. METHODS: Mouse platelets from Gsn-null (Gsn-/-) and wild-type (WT) controls were used. The uptake of fluorescent cargo molecules was compared as a measure of their endocytic efficiency. Receptor-mediated endocytosis was measured by the uptake of fibrinogen and transferrin; fluid-phase endocytosis was monitored by the uptake of fluorescent dextrans. RESULTS: Adenosine diphosphate (ADP)-stimulated WT platelets readily internalized both receptor-mediated and fluid-phase cargoes. In contrast, Gsn-/- platelets showed a severe defect in the endocytosis of both types of cargo. The treatment of WT platelets with the actin-disrupting drugs cytochalasin D and jasplakinolide also reduced endocytosis. Notably, the individual and combined effects of Gsn deletion and drug treatment were similar for both receptor-mediated and fluid-phase endocytosis, indicating that Gsn mediates endocytosis via its action on the actin cytoskeleton. CONCLUSION: Our study demonstrates that Gsn plays a key role in the uptake of bioactive mediators by platelets.

Evaluation of a method to fluorescently label platelets for in-human recovery and survival studies.

BACKGROUND AND OBJECTIVES: Platelets for transfusion are evaluated for in vivo quality using recovery and survival measurements in healthy human subjects. Radiolabelling is the standard for tracing platelets post-transfusion but imposes logistical and technical limitations. This study investigates the in vitro feasibility of labelling platelets with the calcein family of fluorescent dyes as an alternative to radioisotopes or biotin. MATERIALS AND METHODS: Protocols for radiolabelling were adapted for use with calcein acetoxymethyl ester (CAM) and biotin. Labelled platelets were analysed by flow cytometry and evaluated for activation and function. We tested feasibility for labelling without manipulation of platelets and for multiplexing of samples. RESULTS: Labelling at 2 µg CAM/1010 platelets resulted in >99% of CAM+ platelets. There was no significant difference in activation or aggregation between CAM-labelled or biotinylated platelets and vehicle controls although %CD62P+ was significantly lower in platelets that were not processed for labelling. Addition of CAM to the platelet storage bag labelled >95% of platelets. Platelet populations labelled with different dyes could be distinguished by flow cytometry. CONCLUSION: These data provide a rationale for further development of CAM and other fluorescent dyes as tools for measuring post-transfusion kinetics of platelets.

Temporal dynamics of in vitro hemostatic function in platelets cryopreserved using a novel approach for rapid issuance.

BACKGROUND: Current procedures for thawing and issuing of cryopreserved platelets (CPPs) are laborious and have remained challenging in emergency settings such as blood banks and military operations. In this prospective study, a novel processing method designed to facilitate the rapid issuance of CPPs with no postthaw handling required was developed and functionally characterized in parallel with standard CPPs manufactured. STUDY DESIGN AND METHODS: Double-dose plateletpheresis units (n = 42) were cryopreserved at -80°C in 5%-6% dimethyl sulfoxide to produce matched pairs thawed successively over a 27-month period for comparison between two processing arms. In contrast to the standard CPPs manufactured as standalone units, platelets were frozen in tandem with resuspending plasma in a distinct partition as a single unit in the novel method, herein referred to as tandem CPPs. Postthaw (PT) CPPs from both arms were assessed at PT0-, 12-, and 24-h to measure platelet recovery, R-time (time to clot initiation; min), and maximum amplitude (MA; clot strength; mm) using thromboelastography. RESULTS: In the overall dataset, mean platelet recovery was higher (p < .0005) for tandem CPPs (83.9%) compared with standard CPPs (73.3%) at PT0; mean R-times were faster (p < .0005) for tandem CPPs (2.5-3.6 min) compared with standard CPPs (3.0-3.8 min); mean MA was higher for tandem CPPs (57.8-59.5 mm) compared with standard CPPs (52.1-55.8 mm) at each postthaw time point (p < .05). CONCLUSION: Robust temporal dynamics of superior hemostatic functionality were established for tandem CPPs over extended cryopreservation up to 27 months and 24 h of postthaw storage.

Isohemagglutinin titration in pooled and apheresis platelets.

BACKGROUND: Platelet inventory constraints necessitate ABO-incompatible platelet transfusion. Many minimize the hemolytic impact by confirming low titre (LT) donor isohemagglutinins. This process is costly. Pathogen-reduced platelets (PRP) in platelet additive solutions (PAS) will dilute plasma and decrease high-titre isohemagglutinins (HT). We determined the proportion of HT platelets and incompatible transfusions for units suspended in plasma to reassess the need for titres following introduction of PRP/PAS. STUDY DESIGN AND METHODS: Our titre method is manual tube (1:50) dilution of platelet supernatant from apheresis or whole blood derived buffy coat pools suspended in plasma, tested with A1/B red cells. Testing included 49,058 pooled and 11,738 apheresis platelets over 4 years. The HT proportion, rate of out-of-group transfusions, and hemolytic reactions were determined. The impact of PAS dilution was estimated. RESULTS: Totally 60,796 platelet units were tested. Group O pooled and group B apheresis platelets had HT in 6.6% and 5.7%, respectively. Group A pooled and apheresis platelets included 2% with HT. Approximately 25% of platelets transfused were ABO-incompatible and no hemolytic reactions were reported. Based on the proportions of PAS-E and plasma for PRP platelets, plasma from each donor comprises 11 mL (6% of total volume) vs 20-257 mL in untreated pools. PAS-E will replace and dilute residual plasma by at least 50%. DISCUSSION: Rare platelet pools may demonstrate HT. PRP platelets with PAS will reduce titres and may abrogate the need for titration. A strategy of group specific transfusion or transfusion of group A PRP platelet transfusions may be a safe alternative.

The effect of near-infrared low-level light on the in vitro quality of platelets during storage.

BACKGROUND AND OBJECTIVES: Near-infrared (NIR) light has been successfully applied to improve the quality of mouse platelets during storage. Because it is suspected that the mitochondria contain the primary photon acceptor, we hypothesized that human platelets for transfusion may be affected similarly and could benefit from NIR light treatment. MATERIALS AND METHODS: The optimal light dose was determined using portions of platelet concentrates (PCs) in PAS-E. A pool-and-split design was used to prepare PCs in PAS-E or plasma (n = 6). On day 1, one unit of both pairs was illuminated with 830 nm light (light-emitting diodes, 15 J/cm2). PCs were stored at 22°C and sampled regularly for analysis. Data were compared with their corresponding controls with a paired two-sided t-test. RESULTS: Illuminated platelets in PAS-E were less activated with significantly lower CD62P expression (day 8: 10.8 ± 1.8 vs. 12.2 ± 2.6, p < 0.05) and lower Annexin A5 binding (day 8: 11.8 ± 1.9 vs. 13.1 ± 2.4, ns). They produced significantly less lactate resulting in a higher pH (days 6-10). ATP content and mitochondrial membrane potential were not affected. Although these trends were also observed for PCs in plasma, the differences did not reach statistical significance as compared with the control group. CONCLUSION: Our study demonstrates that the glycolysis rate of human platelets can be modulated through the use of NIR, possibly through mitochondrial aerobic metabolism, but this requires confirmation. If NIR illumination can be further optimized, it may potentially become a useful tool in situations in which glycolysis and platelet activation are exacerbated.

Activation of AMPK in platelets promotes the production of offspring.

Platelets are terminally differentiated anucleated cells, but they still have cell-like functions and can even produce progeny platelets. However, the mechanism of platelet sprouting has not been elucidated so far. Here, we show that when platelet-rich plasma(PRP) was cultured at 37°C, platelets showed a spore phenomenon. The number of platelets increased when given a specific shear force. It is found that AMP-related signaling pathways, such as PKA and AMPK are activated in platelets in the spore state. Meanwhile, the mRNA expression levels of genes, such as CNN3, CAPZB, DBNL, KRT19, and ESPN related to PLS1 skeleton proteins also changed. Moreover, when we use the AMPK activator AICAR(AI) to treat washed platelets, cultured platelets can still appear spore phenomenon. We further demonstrate that washed platelets treated with Forskolin, an activator of PKA, not only platelet sprouting after culture but also the AMPK is activated. Taken together, these data demonstrate that AMPK plays a key role in the process of platelet budding and proliferation, suggesting a novel strategy to solve the problem of clinical platelet shortage.

What is new? In this study, we showed that when platelet-rich plasma(PRP) was cultured at 37°C, platelets showed spore phenomenon and increased.It was found that AMP-related signaling pathways, such as PKA and AMPK were activated in platelets in the spore state.In addition, we found that PKA acts as an upstream kinase of AMPK.In the process of platelet sprouting and proliferation, the mRNA expression levels of skeleton protein PLS1 and its related genes, such as CNN3, CAPZB, DBNL, KRT19, andESPN also changed.What is the impact? Our study proposes a new strategy to solve the problem of clinical platelet shortage.

Why might cord blood be a better source of platelets for transfusion to neonates?

Thrombocytopenia (defined as a platelet count <150×109/L) is a common condition in preterm neonates and may occur in 18-35% of all infants admitted to the Neonatal Intensive Care Unit (NICU). Neonatal platelet functionality in terms of reactivity is often described as reduced compared to adults, even in healthy, term neonates. However, this platelet "hyporeactivity" does not correspond to a global functional impairment of the normal delicately balanced neonatal hemostatic system. The extent to which neonatal thrombocytopenia and platelet hyporeactivity contribute to the bleeding risk in preterm neonates remains unknown. Prophylactic platelet transfusions are often administered to them to reduce the risk of bleeding. However, recent literature indicates that adopting a higher platelet transfusion threshold than a lower one results in significantly higher death rates or major bleeding and can be harmful. Although the mechanism by which this occurs is not entirely clear, a mismatch between adult transfused platelets and the neonatal hemostatic system, as well as volume overload, are speculated to be potentially involved. Therefore, future research should consider novel transfusion products that may be more suitable for premature neonates. Blood products derived from umbilical cord blood (UCB) are promising, as they might perfectly match neonatal blood features. Here, we discuss the current knowledge about UCB-derived products, focusing on UCB-derived platelet concentrates and their potential for future clinical application. We will discuss how they may overcome the potential risks of transfusing adult-derived platelets to premature infants while maintaining efficacy.

Integration of an MC-80 Digital Image Analyzer With an Automated BC-6800Plus Hematology Analyzer Enables Accurate Platelet Counting in Samples With EDTA-Induced Pseudothrombocytopenia.

Background: EDTA-induced pseudothrombocytopenia (PTCP) during whole blood collection requires significant laboratory resources to obtain accurate results. We evaluated platelet-deaggregation function in EDTA-induced PTCP and platelet-clump flagging by the BC-6800Plus hematology analyzer using integrated digital image analysis. Methods: We prospectively collected 132 whole blood samples suspected of platelet clumping (102 in EDTA and 30 in sodium citrate) from 88 individuals. We compared platelet counts determined using the platelet count by impedance (PLT-I) function of the DxH 900 hematology analyzer and the PLT-I or optical platelet count (PLT-O) function of the BC-6800Plus. Platelet clumping was verified through manual inspection and the MC-80 digital image analyzer. Results: Among the 132 whole blood samples, 43 EDTA samples showed platelet clumping. The DxH 900 PLT-I and BC-6800Plus PLT-I results demonstrated a strong correlation (r=0.711) for the EDTA samples but only a moderate correlation with the BC-6800Plus PLT-O results (r=0.506 and 0.545, respectively). The BC-6800Plus PLT-O results were consistent with the sodium citrate platelet counts, with a median dissociation rate of 102.5% (range, 74.9%-123.1%). The DxH 900 and BC-6800Plus analyzers had sensitivity values of 0.79 and 0.72, respectively, for platelet-clump flagging. When integrating the MC-80 digital image analysis results, the sensitivity of BC-6800Plus improved to 0.89 (standard mode) or 1.0 (PLT-Pro mode). Conclusions: BC-6800Plus PLT-O measurement results are close to the actual values obtained by platelet deaggregation with PTCP samples. Integrating the BC-6800Plus with a digital imaging analyzer effectively improved the diagnosis of PTCP and reduced the requirement for additional laboratory procedures.

Reliability of the DI-60 Digital Image Analyzer for Detecting Platelet Clumping and Obtaining Accurate Platelet Counts.

Pseudothrombocytopenia caused by platelet clumping (PC) can lead to unnecessary platelet transfusions or underdiagnosis of hematologic neoplasms. To overcome these limitations, we assessed the capacity of the Sysmex DI-60 digital morphology analyzer (Sysmex, Kobe, Japan) for detecting PC and determining an accurate platelet count in the presence of PC. For this purpose, 135 samples with or without PC (groups Y and N, respectively) were processed by an examiner (a hematologic specialist) using both the Sysmex XN-9000 and DI-60 analyzers. Although the platelet aggregate (PA) and giant platelet (GP) counts reported by the DI-60 and the examiner exhibited strong correlations, they proved inadequate as effective indicators for screening samples containing PC. Between the PA and GP counts and four platelet indices (the platelet distribution width [PDW], mean platelet volume [MPV], platelet large cell ratio [P_LCR], and plateletcrit [PCT]) reported by the XN-9000, we observed statistically significant correlations (both overall and with group Y), but they were relatively weak. The platelet counts determined using the DI-60 and light microscopy in group Y showed substantial variations. Although the performance of the DI-60 was reliable for detecting PA and GP in smear images, such fixed areas are not representative of whole samples. Further, in the presence of PC, the resulting platelet counts determined using the DI-60 were not sufficiently accurate to be accepted as the final count.