Simple and Rapid Quantification of Thrombocytes in Zebrafish Larvae

Abstract

Platelets are a critical component of hemostasis, with disorders of number or function resulting in coagulation disturbances. Insights into these processes have primarily been realized through studies using mammalian models or tissues. Increasingly, zebrafish embryos and larvae have been used to study the protein and cellular components of hemostasis and thrombosis, including the thrombocyte, a nucleated platelet analog. However, investigations of thrombocytes have been somewhat limited due to lack of a robust and simple methodology for quantitation, an important component of platelet studies in mammals. Using video capture, we have devised an assay that produces a rapid, reproducible, and precise measurement of thrombocyte number in zebrafish larvae by counting fluorescently tagged cells. Averaging 1000 frames, we were able to subtract background fluorescence, thus limiting assessment to circulating thrombocytes. This method facilitated rapid assessment of relative thrombocyte counts in a population of 372 zebrafish larvae by a single operator in less than 3 days. This technique requires basic microscopy equipment and rudimentary programming, lends itself to high throughput analysis, and will enhance future studies of thrombopoiesis in the zebrafish.

Quantitative methods for studying hemostasis in zebrafish larvae

“…The video processing techniques applied in this method account for and eliminate background autofluorescence as well as these stationary cells, resulting in only quantification of circulating thrombocytes. This was modified from our previous description (Huarng & Shavit, 2015) so that video processing now uses freely available nonproprietary software.…”

“…Fluorescent thrombocytes are first visible at 4 dpf, and the number increases over days 5 and 6 (Huarng & Shavit, 2015). Thus, we find that the optimal time for analysis is at 6–7 dpf.…”

“…Once captured, a simple algorithm using freely available software is utilized to eliminate background and count thrombocytes. The setup and video capture described is a modification to a method we have previously published (Huarng & Shavit, 2015), with the added description of an updated analysis algorithm using nonproprietary software.…”

“…Video Processing: automated counting of thrombocytes is performed as previously described (Huarng & Shavit, 2015) with slight modifications. Videos are processed using the freeware program VirtualDub version 1.10.4 (www.virtualdub.org) and freely available Channel Mixer v1.4 filter from Emiliano Ferrari (emiliano.deepabyss.org).…”

Analysis of factor V in zebrafish demonstrates minimal levels needed for hemostasis and risk stratifies human variants

“…New developments in genome editing technology have facilitated quick and robust systems for targeted genetic modification [19][20][21] . We and others have demonstrated the benefits of this system for modeling hemostasis and thrombosis [22][23][24][25][26][27][28][29][30][31][32][33][34] . Knockout models have demonstrated longer than expected survival in comparison to their mammalian counterparts 25,35,36 , enabling additional characterization of the individual phenotypes.…”