# Three Milliliters of Peripheral Blood Is Sufficient for Preparing Liquid Platelet-Rich Fibrin (PRF): An In Vitro Study

## TL;DR

This study shows that only 3 mL of blood is enough to make platelet-rich fibrin, which supports tissue regeneration and could be useful in research and pediatric surgery.

## Contribution

The study demonstrates that 3 mL of blood can produce effective liquid PRF comparable to the standard 10 mL protocol.

## Key findings

- 3 mL of blood produces PRF with similar platelet and leukocyte concentrations as 10 mL.
- Growth factor release from PRF made with 3 mL blood is comparable to that from 10 mL blood.
- PRF-conditioned media from 3 mL blood supports cell proliferation similar to 20% FBS-conditioned media.

## Abstract

Platelet-rich fibrin (PRF) has assumed an important role in supporting tissue regeneration in different fields. To date, the standard protocol for liquid PRF requires at least 10 mL of peripheral blood. The present study aimed to analyze the composition, growth factor release, and effects on the cell proliferation of PRF samples produced using 3 mL vs. 10 mL of peripheral blood in vitro. Peripheral venous blood from six healthy donors was used to prepare liquid PRF using either 3 mL or 10 mL tubes. Three different centrifugation protocols were used according to the low-speed centrifugation concept. The cellular distribution was evaluated using immunohistology and automated cell count. ELISA was used to determine the release of different growth factors (EGF, TGF-β1, and PDGF) and interleukin 8 at different time points. Primary human osteoblasts (pOBs) were cultivated for 7 days using PRF-conditioned media acquired from either 3 mL or 10 mL of peripheral blood. The results showed that 3 mL of peripheral blood is sufficient to produce a liquid PRF concentrate similar to that acquired when using 10 mL blood. The concentrations of platelets and leukocytes were comparable regardless of the initial blood volume (3 mL vs. 10 mL). Similarly, the release of growth factors (EGF, TGF-β1, and PDGF) and interleukin 8 was often comparable in both groups over 7 days. The cultivation of pOBs using PRF-conditioned media showed a similar proliferation rate regardless of the initial blood volume. This proliferation rate was also similar to that of pOBs treated with 20% FBS-conditioned media. These findings validated the use of 3 mL of peripheral blood to generate liquid PRF matrices according to the low-speed centrifugation concept, which may open new application fields for research purposes such as in vivo experiments and clinical applications such as pediatric surgery.