Platelet Pool Research Articles

The lung is a megakaryocyte outpost that can defend against thrombocytopenic attack.

Lung megakaryocytes (Mks) are a unique subset of Mks that are distinct from their bone marrow counterparts. Recent evidence suggests that lung Mks favor an immune phenotype, but have unclear contributions to the total platelet mass. In this issue of the JCI, Livada et al. used an array of complementary in vivo labeling and tracing models in mice to investigate a longstanding question of where lung Mks are derived. By combining these models with stressed conditions, the authors assessed the contribution of lung Mks to total platelet counts in a homeostatic and thrombocytopenic state. Mks were minor contributors to the circulating pool of platelets during homeostasis but increased output during thrombocytopenia. These findings add critical understanding to the development of lung Mks and demonstrate the dynamic potential of these specialized cells to respond to thrombocytopenia.

Comparing quality parameters of apheresis and pooled platelet concentrates

Introduction. Harvesting of pooled platelet concentrates (PC) significantly expands the volume of transfusion support and could be used to create strategic platelet reserve.The aim of work. To compare the quality of pooled and apheresis platelet concentrates.Materials and methods. We studied platelet fractions (PF), isolated from the whole blood of cadre donors with Reveos system, platelet concentrates, obtained by pooling PF (Pooled PC), and platelet concentrates harvested by automatic apheresis. In all cases 2 days passed from the moment of blood donation to the platelet examination. In PC samples we studied cellular composition, morphofunctional parameters of platelets, pH and electrochemical parameters, hypotonic shock response.Results. The morphofunctional platelet rate in PF was significantly reduced, comparing to the normal levels. At the same time, the morphofunctional parameters of platelets did not significantly differ in PF and pooled PC. The pH values, the total content of platelets and leukocytes in the pooled PC corresponded to the requirements of The rules for procurement, storage, transportation and clinical use of donated blood and its components (2019). The total platelet count, the level of annexin-positive platelets, did not significantly differ in apheresis PC and pooled PC, hypotonic shock response was correct in apheresis PC and pooled PC. On the other hand, the level of platelets with granules in pooled PC was 1,8 times lower than in apheresis PC, other morphofunctional parameters of platelets in pooled PC were also reduced. Pooled PC were characterized by more positive level of open circuit potential and lower antioxidant capacity compared with apheresis PC on the same storage time.Conclusions. Platelet pooling with Reveos system is acceptable for obtaining PC doses that comply with Technical Regulations. Nevertheless, methods of pooled PC harvesting need further optimization.

In vitro regenerative effects of a pooled pathogen-reduced lyophilized human cord blood platelet lysate for wound healing applications.

Cord blood platelets, easily obtained from blood units not suitable for haematopoietic stem cell transplantation, represent an abundant source of growth factors for use in wound healing. Although several protocols have been described for platelet lysate production, no standard manufacturing protocol is available. The use of pooled cord blood platelets could thus facilitate standardization. In this study, the effect of varying concentrations (up to 20%) of a pooled pathogen-reduced lyophilized cord blood platelet lysate (PRL-CBPL) was investigated in different cell types involved in the wound healing process. The effect of heparin addition was also evaluated. In parallel, a comparison was performed with a single donor cord blood platelet lysate (SD-CBPL). The effect of PRL-CBPL on the viability and proliferation of different cell lines (L929 mouse fibroblasts and HaCaT keratinocytes) and human primary cells (fibroblasts-NHDF, coronary artery smooth muscle cells-HCASMC and coronary artery endothelial cells-HCAEC), on HaCaT migration and the chemotactic effect on human monocytes (THP-1) was evaluated. PRL-CBPL showed a lower PDGF-AB amount compared to SD-CBPL. Differing concentrations of both CBPL were necessary to influence cell viability and proliferation. 3% was the optimal concentration for L929 and HaCaT as well as for NHDF and HCASMC, while HCAEC required 10%. The effect of added heparin was more evident on SD-CBPL and in particular on NHDF and HCASMC proliferation. Keratinocyte scratch closure was obtained with 3 and 5% PRL-CBPL and SD-CBPL respectively. Both CBPLs caused an increase in the number of migrated THP-1 monocytes in a concentration-dependent manner up to 20% with a higher monocyte migration for SD-CBPL with respect to PRL-CBPL and in cells treated with heparin. The data obtained suggest that PRL-CBPL is an effective standardized alternative to SD-CBPL.

What It Takes To Be a Platelet: Evolving Concepts in Platelet Production.

Platelets are among the most abundant cells within the circulation. Given that the platelet lifespan is 7 to 10 days in humans, a constant production of around 100 billion platelets per day is required. Platelet production from precursor cells called megakaryocytes is one of the most enigmatic processes in human biology. Although it has been studied for over a century, there is still controversy about the exact mechanisms leading to platelet release into circulation. The formation of proplatelet extensions from megakaryocytes into bone marrow sinusoids is the best-described mechanism explaining the origin of blood platelets. However, using powerful imaging techniques, several emerging studies have recently raised challenging questions in the field, suggesting that small platelet-sized structures called buds might also contribute to the circulating platelet pool. How and whether these structures differ from microvesicles or membrane blebs, which have previously been described to be released from megakaryocytes, is still a matter of discussion. In this review, we will summarize what the past and present have revealed about platelet production and whether mature blood platelets might emerge via different mechanisms.

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.

Management of Concomitant Severe Thermal Injury and ST-Elevation Myocardial Infarction

Acute coronary thrombosis is a known, but rare, contributor to morbidity and mortality in patients with thermal and electrical injuries. The overall incidence of myocardial infarction among burn patients is 1%, with an in-hospital post-infarction mortality of approximately 67%, whereas the overall mortality rate of the general burn patient population is from 1.4% to 18%. As such, early detection and effective peri-operative management are essential to optimize patient outcomes. Here, we report the details of the management of an adult male patient with a 65% total body surface area severe thermal injury, who developed an ST-elevation myocardial infarction (STEMI) in the resuscitation period. The patient was found to have 100% occlusion of his left anterior descending coronary artery, for which prompt coronary artery stent placement with a drug-eluting stent (DES) was performed. Following stent placement, the patient required dual antiplatelet therapy. The ongoing dual antiplatelet therapy required the development of a detailed peri-operative protocol involving pooled platelets, packed red blood cells, desmopressin (DDAVP™) and intraoperative monitoring of the patient’s coagulation parameters with thromboelastography for three staged operative interventions to achieve complete debridement and skin grafting of his burn wounds.

Isohemagglutinin titration in pooled and apheresis platelets.

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. 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. 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%. 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.

Evaluation of platelets componentized with the Reveos Automated Blood Processing System and stored for 7 days at room temperature in a non-Di(2-ethylhexyl) phthalate (DEHP) platelet pooling set.

Platelet concentrates (PCs) used for transfusion can be produced by apheresis or derived from whole blood (WB). The Reveos device is the first US Food and Drug Administration-approved automated blood processing system that can produce PCs. In this work, we evaluated the quality and function of Reveos-collected PCs stored for 7 days at room temperature. WB was collected from healthy donors and componentized on the day of collection (Fresh) or after an overnight hold (Overnight). PCs were produced (n = 7 Fresh; n = 6 Overnight), stored at room temperature in plasma, and evaluated on days 1 and 7 for quality metrics, platelet activation, clot formation, and aggregation response. Platelet count was comparable between Fresh and Overnight PCs. A drop in pH was reported in Fresh day 7 PCs (p < .001, vs. day 1) but not in Overnight. Overnight units displayed the lowest levels of P-selectin expression (p = .0008, vs. day 7 Fresh). Reduced clot strength and increased lysis were observed in both Fresh and Overnight units on day 7 (vs. day 1). Overnight-hold PCs resulted in the highest clot strength on day 7 (p = .0084, vs. Fresh). No differences in aggregation were reported between groups. Reveos-processed PCs produced from overnight-hold WB performed better in hemostatic function assays and displayed reduced activation compared to fresh WB-derived PCs, although both PC groups maintained platelet quality throughout storage. Utilization of overnight WB for PC preparation with Reveos holds promise as an alternative method of producing platelets for transfusion purposes.

Assessment of the importance of platelet transfusion in patients with severe dengue: a systematic review

Introduction: Dengue is one of the most important vector-borne viral diseases in subtropical and tropical regions. The World Health Organization (WHO) 2009 classified dengue into three groups: dengue without alarm signs, dengue with alarm signs, and severe dengue. According to the type of dengue, various managements have been proposed. The authors suggest that a predictive factor to avoid a poor prognosis in this disease is to keep platelet levels stable in the patient to prevent a fatal outcome. The aim of this paper is to analyze the efficacy of transfusion of platelet agents in the outcome of patients with severe dengue and thrombocytopenia. Material and methods: A bibliographic search was carried out in the Medline, OVID, and Scielo databases from January 1, 2008, to April 31, 2023, using the MeSH terms. Results: 7 articles were included in the systematic review. A wide range in age was found (18 to 79 years). A minimal number of studies detail the comorbidities of patients in their enrollment. Regarding platelet transfusion, the average number of platelet units used in the transfusion case was 5 (2 - 14). Transfusions of ABO identical and compatible pooled platelets transfusion were found to be more successful in increasing platelets. Almost all of the patients included in the studies had platelets &lt;20,000 at enrollment. One study found a significant increase in platelets after transfusion, while the rest did not find it compelling. No mortality associated with platelet transfusion was recorded. Conclusions: Prophylactic platelet transfusion is not recommended as a routine measure in patients with severe dengue and thrombocytopenia. The uncertainty highlights need to reach a specific consensus establishing the appropriate indications for platelet transfusion and what type of patients with dengue virus would be beneficial.

Platelet RNA sequencing reveals profile of caffeic acid affecting hemostasis in mice

BackgroundCaffeic acid (CA) is a naturally occurring phenolic compound with diverse pharmacologic properties. CA plays a crucial role in hemostasis by increasing platelet count. However, the mechanism by which CA regulates platelets to promote hemostasis remains unclear. ObjectivesWe aim to identify the potential target pathways and genes by which CA regulates platelets to promote hemostasis. MethodsWe performed RNA sequencing (RNA-seq) analysis of mouse platelet pools in both the CA-gavaged group and phosphate-buffered saline–gavaged group. ResultsThe 12,934 expressed transcripts had been annotated after platelet RNA-seq. Compared with the phosphate-buffered saline group, 987 differentially expressed genes (DEGs) were identified, of which 466 were downregulated and 521 were upregulated in CA group. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Reactome gene set enrichment analysis demonstrated that upregulated DEGs were enriched in the pathways of hemostasis, platelet activation, signaling, aggregation, and degranulation. Moreover, Kyoto Encyclopedia of Genes and Genomes and Reactome gene set enrichment analysis revealed that 5 of the 25 cosignificantly upregulated DEGs were essential in CA-mediated platelet regulation to promote hemostasis. ConclusionOur findings of platelet RNA-seq analysis demonstrate that CA regulates the gene expression of hemostasis and platelet activation–related pathways to increase platelet count and promote hemostasis. It will also provide reference molecular resources for future research on the function and mechanism by which CA regulates platelets to promote hemostasis.

Deciphering cytopenias in internal medicine: a single-center observational study

Cytopenia is a common finding in patients admitted to internal medicine wards and the clinical workup may be long and time-consuming. In this single-center observational study, we analyzed a series of 151 inpatients who received hematologist referral due to cytopenia observed during hospital admission. Patients were mainly elderly (median 71 years, 15–96) and 87% had at least one comorbidity. Anemia was the most common cytopenia (91%), followed by thrombocytopenia (51%), and neutropenia (22%); 73 (48%) patients had a bicytopenia and 5 (3%) pancytopenia. Cytopenias were mainly severe, 66% of cases required RBC transfusions, and 21% platelet pools. During a median hospital stay of 15 days (1–166), 53 subjects (35%) received a hematologic discharge diagnosis, whilst the two-thirds had secondary cytopenia mainly due to associated comorbidities. Only about 34% of 2,728 diagnostic tests performed (including laboratory, imaging, and histology) clearly informed the discharge diagnosis in this heterogenous setting. Specifically, bone-marrow evaluation indicated in 46 (30%) patients, was diagnostic in 32 (69.6%). Eleven percent of patients died due to progression of the oncohematologic disease (29%), sepsis (24%), and solid tumor progression (24%). In conclusion, cytopenias in the internal medicine setting are mainly severe, more frequently secondary to associated comorbidities (2/3 of patients) and deserve proper workup before second/third-level tests (immune-hematological assays and CT scan or PET and bone-marrow evaluation, respectively).

Platelet versus fresh frozen plasma transfusion for coagulopathy in cardiac surgery patients.

Platelets (PLTS) and fresh frozen plasma (FFP) are often transfused in cardiac surgery patients for perioperative bleeding. Their relative effectiveness is unknown. We conducted an entropy-weighted retrospective cohort study using the Australian and New Zealand Society of Cardiac and Thoracic Surgeons National Cardiac Surgery Database. All adults undergoing cardiac surgery between 2005-2021 across 58 sites were included. The primary outcome was operative mortality. Of 174,796 eligible patients, 15,360 (8.79%) received PLTS in the absence of FFP and 6,189 (3.54%) patients received FFP in the absence of PLTS. The median cumulative dose was 1 unit of pooled platelets (IQR 1 to 3) and 2 units of FFP (IQR 0 to 4) respectively. After entropy weighting to achieve balanced cohorts, FFP was associated with increased perioperative (Risk Ratio [RR], 1.63; 95% Confidence Interval [CI], 1.40 to 1.91; P<0.001) and 1-year (RR, 1.50; 95% CI, 1.32 to 1.71; P<0.001) mortality. FFP was associated with increased rates of 4-hour chest drain tube output (Adjusted mean difference in ml, 28.37; 95% CI, 19.35 to 37.38; P<0.001), AKI (RR, 1.13; 95% CI, 1.01 to 1.27; P = 0.033) and readmission to ICU (RR, 1.24; 95% CI, 1.09 to 1.42; P = 0.001). In perioperative bleeding in cardiac surgery patient, platelets are associated with a relative mortality benefit over FFP. This information can be used by clinicians in their choice of procoagulant therapy in this setting.

Quality assessment of the preparation and storage of leukocyte-depleted pooled platelet concentrates

ABSTRACT Objective: To explore the feasibility of using a disposable platelet storage bag containing a leukocyte filter to prepare leukocyte-depleted pooled platelet concentrates with the buffy coat method. Methods: 150 bags of whole blood samples (400 mL/bag) were stored overnight at 22 ± 2°C, and buffy coats were separated on Day 2, then 5 units of ABO homotypic buffy coat and 1 unit of plasma were pooled into a disposable platelet storage bag containing a leukocyte filter to prepare leukocyte-depleted pooled platelet concentrates and stored in a Platelet Agitator. On Day 2, 4, 5 and 7 after the collection of whole blood, platelet content, pH value, pO2, pCO2, glucose (GLU), ATP, and other quality indicators were measured. Results: The quality indicators of leukocyte-depleted pooled platelet concentrates met the requirements for leukocyte-depleted aphaeresis platelets in the Chinese national standard Quality Requirements for Whole Blood and Blood Components (GB18469-2012). With the prolongation of storage time, MPV and PDW of platelets gradually increased, pH value, bicarbonate, and GLU gradually decreased, LA, LDH, and ATP gradually increased, pO2 slightly increased, pCO2 decreased, and HSR had no significant change. ESC decreased significantly on Day 7, CD62p decreased first and then increased, sP-selectin and GP V increased first and then decreased, but the results on Day 7 were higher than those on Day 2. Conclusion: The quality of leukocyte-depleted pooled platelet concentrates prepared by the buffy coat method using disposable platelet storage bags containing a leukocyte filter was comparable to that of leukocyte-depleted apheresis platelets, and could be used clinically.

Trend towards reduction of transfusion reactions using prestorage leukocyte-reduced and pooled whole blood-derived platelets and cost savings compared with poststorage whole blood-derived random platelets as evidenced by real-time hemovigilance.

Due to chemotherapy-induced neutropenia or hematologic malignancies, immunocompromised cancer patients may have higher incidence of febrile nonhemolytic transfusion reactions compared with the general population and frequently require platelet transfusions. This quality improvement project compared the safety of transfusion using prestorage leukocyte-reduced and pooled whole blood-derived platelets (Acrodose/WBD) with conventionally produced poststorage WBD platelets (RDP) using an active hemovigilance system. Every patient receiving a blood product at the hospital was virtually monitored in real time by trained nurses from a remote hemovigilance unit. These nurses monitor a digital dashboard, which populates a watch list of patients from the time blood product administration is initiated until 12 hours posttransfusion. Over the course of 6 months, 371 patients receiving 792 RDP transfusions and 423 patients receiving 780 Acrodose/WBD platelets transfusions were monitored for transfusion reactions. We identified 26 transfusion reactions in RDP but only 12 transfusion reactions in the Acrodose/WBD platelet group. Acrodose platelet transfusion was associated with fewer transfusion reactions, which resulted in significant cost savings.

Lung Megakaryocytes Arise from a Flt3 - Cell Lineage and Contribute to Recovery of Platelet Counts in Thrombocytopenic States

Platelet-producing megakaryocytes (Mks) are present in the lung, and we have described an immune modulatory phenotype of lung Mks. It is assumed that the extravascular lung Mks are ‘seeded’ from the bone marrow (BM). To investigate lung Mk origin and how origin may impact function, we used complementary models to lineage trace lung Mks and their platelets. FlkSwitch mTmG mice label all progeny from Flt3 + progenitors with GFP while all other cells are labeled with Tomato. We leveraged the FlkSwitch mouse model to compare BM and lung Mk origins. In 18 week old mice, the vast majority of BM and spleen Mks were GFP +, while lung Mks were largely Tomato + (Figure 1A). These data implied that in the adult mouse lung Mks arise from a Flt3 - pathway that is distinct from BM megakaryopoiesis in homeostatic conditions. We next used a lineage tracing model (MDS1-Cre-ERT2 Tomato) where tamoxifen (TAM) administration induces a Tomato reporter in hematopoietic stem cell (HSC)-derived cells. Mice were dosed with TAM at 18 weeks and sacrificed at 32 weeks. Lung and BM Mks were largely Tomato + (~75%) and matched Tomato levels of BM Lineage - Sca1 + c-Kit + (LSK). Next, we dosed mice with TAM at 8 weeks, sacrificed the mice at 14 weeks and found similarly high levels of Tomato in BM LSK, BM Mk, and lung Mk. In MDS1-Cre-ERT2 Tomato mice, both BM and lung Mks were Tomato +/HSC-dependent, suggesting that lung Mks arise from HSCs in adult mice. Next, we used CFSE dye given oropharyngeal (OP) to label only lung cells. We detected CFSE + lung Mks for up to 120 days post administration without any CFSE + Mks in BM or spleen. These data suggested there is a long-lived Mk population in the lung. Taken together, these complementary models imply that lung Mks arise from a Flt3 - lineage that is HSC-dependent, which yields Mks that can live in the lung for extended periods of time. The CFSE OP method labeled only lung cells, which enabled us to track lung Mk-derived platelets. We found that about 10% of the steady-state circulating platelet pool is lung derived. In a model of thrombocytopenia using diphtheria toxin mediated platelet depletion in PF4-Cre-iDTR mice, we observed a doubling of CFSE + lung-derived circulating platelets (about 20%). We also used Plasmodium yoelli murine malaria as a model of infection associated with more chronic thrombocytopenia. Using CFSE OP labeling of lung Mk-derived platelets, we saw a marked increase in CFSE + lung-derived platelets early post- P. Yoelli infection (Figure 1B). Our studies suggest that lung Mks arise from a Flt3 - HSC-dependent pathway and contribute relatively more platelets during acute and chronic thrombocytopenia. Ongoing work aims to describe the role of lung megakaryopoiesis via a Flt3 - HSC-dependent pathway in early development and in models of thrombocytopenia.

In vitro quality parameters of whole blood-derived platelets pooled using two different platelet pooling sets and stored up to 7 days are similar.

Increasing the number of collections of whole blood-derived platelets (WBDP) and lengthening the allowable storage time may alleviate platelet (PLT) shortages. There is a need for new PLT pooling sets that can provide acceptable quality on Day 7 of storage. This pool-and-split study compared WBDP prepared using the platelet-rich plasma method with the novel IMUGARD WB PLT pooling set and a control pooling set. After pooling and filtration, PLT products were tested on Days 1, 5, and 7. Large volume delayed sampling (LVDS) cultures were taken on Day 2. The median postfiltration residual white blood cell (rWBC) content was 0.18 million per product (maximum 1.26 million; n = 69) with mean PLT recovery of 88.5 ± 2.8% for the new set and median 0.23 million (maximum 1.83 million) rWBC with 87.5 ± 2.5% recovery for the control. Day 5 mean pH22°C were 7.18 ± 0.12 and 7.13 ± 0.10 for the new and control set, respectively. Day 5 in vitro quality parameters were within 20% between the two pooling sets. The new set Day 7 pH22°C was acceptable (7.07 ± 0.17, 100% ≥ 6.3), and most parameters were within 20% of Day 5 values. WBDP quality for the new pooling set is acceptable across a battery of in vitro tests when stored up to 7 days and meets FDA regulatory criteria. The quality parameters were similar between the new pooling set and the control set on Day 5. This new set is compatible with LVDS.

Changing Strategies for the Detection of Bacteria in Platelet Components in Ireland: From Primary and Secondary Culture (2010-2020) to Large Volume Delayed Sampling (2020-2023).

Bacterial contamination of platelet components (PC) poses the greatest microbial risk to recipients, as bacteria can multiply over the course of PC storage at room temperature. Between 2010 and 2020, the Irish Blood Transfusion Service (IBTS) screened over 170,000 buffy coat-derived pooled (BCDP) and single-donor apheresis platelets (SDAPs) with the BACT/ALERT 3D microbial detection system (Biomerieux, L'Etoile, France), using a two-step screening protocol which incorporated primary and secondary cultures. Although the protocol was successful in averting septic transfusion reactions (STRs), testing large sample volumes at later time points was reported to improve detection of bacterial contamination. A modified large-volume delayed sampling (LVDS)-type protocol was adopted in 2020, which in the case of SDAP was applied to collections rather than individual splits (2020-2023, 44,642 PC screened). Rates of bacterial contamination for BCDP were 0.125% on Day-2, 0.043% on Day-4 vs. 0.191% in the post-LVDS period. SDAP contamination rates in the pre-LVDS period were 0.065% on Day-1, 0.017% on Day-4 vs. 0.072% in the post-LVDS period. Confirmed STRs were absent, and the interdiction rate for possibly contaminated SDAP was over 70%. In the post-LVDS period, BCDPs had a higher total positivity rate than SDAPs, 0.191% (1:525) versus 0.072% (1:1385), respectively, (chi-squared 12.124, 1 df, p = 0.0005). The majority of organisms detected were skin-flora-type, low pathogenicity organisms, including coagulase-negative staphylococci and Cutibacterium acnes, with little change in the frequency of clinically significant organisms identified over time. Both protocols prevented the issue of potentially harmful components contaminated (rarely) with a range of pathogenic bacteria, including Escherichia coli, Serratia marcesens, Staphylococcus aureus, and streptococci. Culture positivity of outdates post-LVDS whereby 100% of expired platelets are retested provides a residual risk estimate of 0.06% (95% CI 0.016-0.150). However, bacterial contamination rates in expired platelets did not demonstrate a statistically significant difference between the pre-LVDS 0.100% (CI 0.033-0.234) and post-LVDS 0.059% (0.016-0.150) periods (chi-squared = 0.651, 1 df, p = 0.42).

The Bone Marrow Is the Primary Site of Thrombopoiesis

Megakaryocytes (MKs) are large cells predominantly residing within the bone marrow, generating thousands of platelets in an elaborate proplatelet formation process. Megakaryocytes, defined by their expression of CD41, have been observed in the bone marrow and distant organs such as the spleen and lung. However, whether these megakaryocytes genuinely contribute to the mature platelet pool remains controversial. Here, we aimed to delineate which organs participate in platelet production, which mechanisms affect platelet release, and how to define alternative tissue-dependent functions of megakaryocytes. Flow cytometry data revealed significantly higher numbers of hematopoietic stem and progenitor cells and megakaryocytes in the murine bone marrow compared to spleens or perfused lungs; megakaryocyte counts were also significantly higher in cryosections of the mouse bone marrow, and also significantly increased in the bone marrow compared to spleen and lungs when analyzed by both live cell bi photon in-vivo imaging and ex vivo 3D volumetric imaging (light-sheet). Moreover, megakaryocytes cultured and differentiated from the bone marrow ex vivo had a higher capacity for proplatelet formation than MKs derived from the spleen or lung. Our analysis of previously published murine and human scRNA-seq datasets supports our observations that only a marginal fraction of megakaryocyte-like cells can be found within the lung and spleen and most likely only marginally contribute to platelet production under a steady state.

The bone marrow is the primary site of thrombopoiesis

AbstractMegakaryocytes (MKs) generate thousands of platelets over their lifespan. The roles of platelets in infection and inflammation has guided an interest to the study of extramedullary thrombopoiesis and therefore MKs have been increasingly reported within the spleen and lung. However, the relative abundance of MKs in these organs compared to the bone marrow and the scale of their contribution to the platelet pool in a steady state remain controversial. We investigated the relative abundance of MKs in the adult murine bone marrow, spleen, and lung using whole-mount light-sheet and quantitative histological imaging, flow cytometry, intravital imaging, and an assessment of single-cell RNA sequencing (scRNA-seq) repositories. Flow cytometry revealed significantly higher numbers of hematopoietic stem and progenitor cells and MKs in the murine bone marrow than in spleens or perfused lungs. Two-photon intravital and light-sheet microscopy, as well as quantitative histological imaging, confirmed these findings. Moreover, ex vivo cultured MKs from the bone marrow subjected to static or microfluidic platelet production assays had a higher capacity for proplatelet formation than MKs from other organs. Analysis of previously published murine and human scRNA-seq data sets revealed that only a marginal fraction of MK-like cells can be found within the lung and most likely only marginally contribute to platelet production in the steady state.

Diversity of Megakaryocytes.

Megakaryocytes are commonly known as large, polyploid, bone marrow resident cells that contribute to hemostasis through the production of platelets. Soon after their discovery in the 19th century, megakaryocytes were described in tissue locations other than the bone marrow, specifically in the lungs and the blood circulation. However, the localization of megakaryocytes in the lungs and the contribution of lung megakaryocytes to the general platelet pool has only recently been appreciated. Moreover, the conception of megakaryocytes as uniform cells with the sole purpose of platelet production has been challenged. Here, we review the literature on megakaryocyte cell identity and location with a special focus on recent observations of megakaryocyte subpopulations identified by transcriptomic analyses.