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

Abstract

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.

Two novel platelet biotinylation methods and their impact on stored platelet concentrates in a blood bank environment

“…The response to collagen was also impacted in Bio‐Wash platelets in a storage time‐dependent manner. A similar decrease was observed when platelets were biotinylated at a high concentration 21 . Of importance, we were able to restore the ADP or collagen response with the use of epinephrine as a double‐agonist activation.…”

“…A similar decrease was observed when platelets were biotinylated at a high concentration. 21 Of importance, we were able to restore the ADP or collagen response with the use of epinephrine as a double-agonist activation. Co-aggregation might give a better understanding of the in vivo response by the fact that platelets naturally respond to more than one molecule, as multiple agonists are involved in injury sites for the aggregation of platelets, which is in agreement with previously published data.…”

“…Even though a decrease in the MFI was detected in both conditions over time, this decrease should not be a limiting factor on the detection for further in vivo analysis since the biotinylated population was still clearly segregated from the native one at the end of the storage (SI Figure S1). Based on the literature, we can assume that biotinylated platelets should be detected for up to 1 week in the human 20 or 5 days in canine 23,30 and up to 48 h in mice 21 blood.…”

“…As an alternative, the use of chemical tags has provided new opportunities. [18][19][20][21][22] To label platelets, biotin, a watersoluble vitamin naturally present in the human body from food intake, has been widely selected.…”

“…23 Biotin, in our case, a Sulfo-NHS-SS-Biotin, develops strong covalent bonds with the free amino groups at the surface of proteins and is easily detectable with streptavidin probes. Until now, platelet preparations required at least a washing step to isolate them from plasma prior to biotinylation, [19][20][21] as plasma proteins also react with biotin, which reduces the labeling efficiency on platelets. On the other hand, platelets are very sensitive to manipulation and washing steps, which significantly increase platelet activation (e.g., increase of CD62-P expression 19 ) and consequently change their phenotype.…”