Human platelets labeled at two discrete biotin densities are functional in vitro and are detected in vivo in the murine circulation: A promising approach to monitor platelet survival in vivo in clinical research.

BACKGROUND: The production of platelet concentrates (PCs) is evolving, and their survival capacity needs in vivo evaluation. This requires that the transfused platelets (PLTs) be distinguished from those of the recipient. Labeling at various biotin (Bio) densities allows one to concurrently trace multiple PLT populations, as reported for red blood cells. STUDY DESIGN AND METHODS: A method is described to label human PLTs at two densities of Bio for future clinical trials. Injectable-grade PLTs were prepared in a sterile environment, using injectable-grade buffers and good manufacturing practices (GMP)-grade Sulfo-NHS-Biotin. Sulfo-NHS-Biotin concentrations were chosen to maintain PLT integrity and avoid potential alloimmunization while enabling the detection of circulating BioPLTs. The impact of biotinylation on human PLT recirculation was evaluated in vivo in a severe immunodeficient mouse model using ex vivo flow cytometry. RESULTS: BioPLTs labeled with 1.2 or 10 µg/ml Sulfo-NHS-Biotin displayed normal ultrastructure and retained aggregation and secretion capacity and normal expression of the main surface glycoproteins. The procedure avoided detrimental PLT activation or apoptosis signals. Transfused human BioPLT populations could be distinguished from one another and from unlabeled circulating mouse PLTs, and their survival was comparable to that of unlabeled human PLTs in the mouse model. CONCLUSIONS: Provided low Sulfo-NHS-Biotin concentrations (<10 µg/ml) are used, injectable-grade BioPLTs comply with safety regulations, conserve PLT integrity, and permit accurate in vivo detection. This alternative to radioisotopes, which allows one to follow different PLT populations in the same recipient, should be valuable when assessing new PC preparations and monitoring PLT survival in clinical research.

Evaluation of survival and functionality in frozen platelets.

OBJECTIVE: Prolong platelet survival and functionality up to 28 days. METHODS: A sample of apheresis platelets was evaluated, distributed in 3 groups according to the cryopreservative solution used: DMSO5%+2%albumin; DMSO5%+NaCl0,9% and DMSO5%+Dextrose2%. They were then frozen at -80 °C and thawed at 7, 14 and 28 days. The in vitro survival and viability were assessed by the post-thaw platelet count and the CD41, CD61 and CD42a staining percentages by flow cytometry. The functionality was determined with the percentage of post-stimulation aggregation with 1Nm-thrombin using the Chromo-Log490 aggregometer. The control group (CG) consisted of fresh platelets under constant agitation at 22 °C. RESULTS: A total of 72 platelet aliquots was analyzed. The CG presented a platelet-count of 1934 ± 0.5 × 109/L and a 100% viability. The percentages of CD41, CD61 and CD42a labeling were 99, 98.5 and 96.5%, respectively. The percentage of aggregation was 99%. On day 7 of the post-freezing, the platelet count for groups 1, 2 and 3 was 1,844 ± 102, 1,856 ± 76 and 1,752 ± 226, with the viability of 98, 96 and 95%, respectively. On day 14, the counts were 1,722 ± 238, 1,649 ± 215 and 1,578 ± 223 with the viability of 96, 95 and 94% and, on day 28, they were 1,602 ± 374, 1,438.6 ± 429 and 1,406.6 ± 436, with the viability of 96, 94 and 93%, respectively. Group1 presented a higher expression of membrane antigens. Aggregation percentages were 90, 98 and 89% at day 7, 88%, 98 and 87% at day 14 and 84%, 95 and 82% at day of the 28 post-freezing, respectively, with group2 presenting the best results. CONCLUSION: The results support cryopreservation as a reasonable method to prolong platelet survival up to 28 days, maintaining its functionality and viability greater than 50%.

Biotinylated Platelets: A Promising Labeling Technique?

Labeling of platelets (PLTs) is essential for research purposes, in order to measure the recovery and survival of transfused PLTs in vivo. Biotinylation is a promising new alternative to the gold standard of radioactive labeling. This review highlights 4 key publications that provide significant insights into biotin-labeled PLTs (bioPLTs). Stohlawetz et al. established that transfusion of bioPLTs in human recipients is possible. De Bruin et al. developed a standardized, reproducible protocol for biotinylation of PLTs as a promising method to trace and isolate transfused PLTs in vivo, with reduced levels of PLT activation markers. Muret et al. developed a nonwashing biotin labeling method to implement in a blood bank environment. Finally, in a preclinical study, Ravanat et al. showed that different densities of biotin can be used to concurrently monitor multiple populations of human PLTs in the circulation of the same subject. These studies have made major contributions to the development of bioPLTs as a viable option for use in human research, and indicate that bioPLTs can be safely administered, preferably at a low density of biotin.

Evaluation of survival and functionality in frozen platelets

ABSTRACT Objective: Prolong platelet survival and functionality up to 28 days. Methods: A sample of apheresis platelets was evaluated, distributed in 3 groups according to the cryopreservative solution used: DMS05%+2%albumin; DMSO5%+NaC10,9% and DMS05%+Dextrose2%. They were then frozen at -80 °C and thawed at 7, 14 and 28 days. The in vitro survival and viability were assessed by the post-thaw platelet count and the CD41, CD61 and CD42a staining percentages by flow cytometry. The functionality was determined with the percentage of post-stimulation aggregation with INm-thrombin using the Chromo-Log490 aggregometer. The control group (CG) consisted of fresh platelets under constant agitation at 22 °C. Results: A total of 72 platelet aliquots was analyzed. The CG presented a platelet-count of 1934 ± 0.5 × 109/L and a 100% viability. The percentages of CD41, CD61 and CD42a labeling were 99, 98.5 and 96.5%, respectively. The percentage of aggregation was 99%. On day 7 of the post-freezing, the platelet count for groups 1, 2 and 3 was 1,844 ± 102, 1,856 ± 76 and 1,752 ± 226, with the viability of 98, 96 and 95%, respectively. On day 14, the counts were 1,722 ± 238, 1,649 ± 215 and 1,578 ± 223 with the viability of 96, 95 and 94% and, on day 28, they were 1,602 ± 374, 1,438.6 ± 429 and 1,406.6 ± 436, with the viability of 96, 94 and 93%, respectively. Groupl presented a higher expression of membrane antigens. Aggregation percentages were 90, 98 and 89% at day 7, 88%, 98 and 87% at day 14 and 84%, 95 and 82% at day of the 28 post-freezing, respectively, with group2 presenting the best results. Conclusion: The results support cryopreservation as a reasonable method to prolong platelet survival up to 28 days, maintaining its functionality and viability greater than 50%.

Potentials for prolonging shelf-life of platelets by near infrared low-level light.

Platelets are uniquely stored at room temperature, during which they gradually loss their quality owing to deteriorating functions of mitochondria over time. Given the well-documented beneficial effect of near infrared low-level light (LLL) on mitochondrial functions, we explored a potential for LLL to protect mitochondrial function and extend the shelf-life of platelets beyond the current 5 days. We found that exposure of a platelet-containing storage bag to 830 nm light-emitting diode (LED) light at 0.5 J/cm2 prior to storage could significantly retain a pH value and viability of the platelets stored for 8 days with improved quality compared to those stored similarly for 5 days in controls. The LLL inhibited reactive oxygen species (ROS) and lactate production, while sustaining ATP synthesis and mitochondrial membrane potential and morphology in the stored platelets. It also sustained aggregation capacity and in vivo survival of stored platelets, concomitant with no significant activation, as suggested by similar CD62p expression and enhanced agonist-induced aggregation and recovery following infusion in the presence compared to absence of LLL treatment. This simple, additive-free, cost-effective, noninvasive approach can be readily incorporated into the current platelet storage system to potentially improve quality of stored platelets.

Platelet Survival in Hematology Patients Assessed by the Corrected Count Increment and Other Formulas.

OBJECTIVES: To compare the performance of the corrected count increment (CCI) and three other formulas to assess 24-hour posttransfusion platelet survival in hematology patients. METHODS: Twenty-four-hour posttransfusion platelet counts were analyzed after apheresis platelet transfusion. Platelet increment (PI), percent platelet recovery (PPR), and percentage platelet increment (PPI) were compared with CCI by receiver operating characteristic analysis. Clinical factors that influence platelet survival were assessed by logistic regression. RESULTS: In total, 142 apheresis platelet transfusions in 85 hematology patients were studied. Mean (SD) CCI at 24 hours was 11,869 (10,125). Compared with CCI, the sensitivity of other formulas ranged from 89.4% to 95.7% and specificity from 94.7% to 100%. Cutoff values were 15.7 × 103/µL for PI, 11.4% for PPR, and 17% for PPI. For ABO-compatible vs incompatible transfusions, CCI was 14,070/µL vs 9,176/µL (P = .007). Negative factors for all formulas were sepsis, hypotension, and amphotericin B. CONCLUSIONS: PI, PPR, and PPI are comparable to CCI for assessing 24-hour platelet survival.