Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin.
BACKGROUND
To avoid radioisotopic labeling and permit comparison of the survival of two platelet populations concurrently in one animal, we compared simultaneous recoveries and survival times of homologous rabbit platelets labeled in vitro with the lipophilic dyes PKH26 (red fluorescing) and PKH67 (green fluorescing) and with two levels of biotin (low, 1 microg/ml; high, 10 microg/ml).


METHODS
Blood samples were drawn up to 96 h postinfusion and analyzed by flow cytometry. Biotin-labeled samples were incubated with phycoerythrin-streptavidin before analysis.


RESULTS
Recovery of PKH26-labeled platelets at 1 h was lower (37.5%) than that of PKH67-labeled platelets (47.3%; P < 0.001). Platelet survival times were 62.4 and 61.9 h. Recoveries at 1 h of platelets labeled with two levels of biotin were similar (86.6% and 84.6%) and greater than those of PKH-labeled platelets (P < 0.001). Survival of platelets labeled with biotin did not differ (low, 83.3 h; high, 85.2 h) and was longer than for PKH-labeled platelets (P < 0.01). Labeling methods did not activate platelets (measured by P-selectin expression), nor did they affect platelet responses to adenosine diphosphate (ADP), collagen, or thrombin.


CONCLUSIONS
Labeling with two levels of biotin is superior to labeling with PKH dyes, and is useful for measuring concurrently the survival of two differing platelet populations.
Rand
M L
ML
Division of Hematology/Oncology, The Hospital for Sick Children, Toronto, Ontario, Canada. margaret.rand@sickkids.ca
Wang
H
H
Mody
M
M
Chu
I
I
Treutiger
I
I
Nguyen
A
A
Packham
M A
MA
Freedman
J
J
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001710:Biotin / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002470:Cell Survival; D003094:Collagen / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology*; D066298:In Vitro Techniques; D019041:L-Selectin / Q000032:analysis; D009930:Organic Chemicals; D010974:Platelet Aggregation / Q000187:drug effects; D011817:Rabbits; D012701:Serotonin / Q000378:metabolism; D013194:Staining and Labeling; D013917:Thrombin / Q000494:pharmacology
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C070080:PKH 26; C451241:PKH67; D019041:L-Selectin; D012701:Serotonin; D000244:Adenosine Diphosphate; D001710:Biotin; D003094:Collagen; D013917:Thrombin