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. 2015 Oct 20;50(6):729-38.
doi: 10.1016/j.rboe.2015.10.008. eCollection 2015 Nov-Dec.

Quantification of platelets obtained by different centrifugation protocols in SHR rats

João Alberto Yazigi Junior  1 , João Baptista Gomes Dos Santos  1 , Bruno Rodrigues Xavier  1 , Marcela Fernandes  1 , Sandra Gomes Valente  1 , Vilnei Mattiolli Leite  1
Affiliations

Quantification of platelets obtained by different centrifugation protocols in SHR rats

João Alberto Yazigi Junior et al. Rev Bras Ortop. .

Abstract
in English, Portuguese

Objective: To quantify the platelet concentration in the blood of SHR rats, by means of different centrifugation protocols, and to evaluate what the most effective method for obtaining platelets is.

Methods: We used 40 male rats of the isogenic SHR lineage. The animals were divided into three groups: control, using whole blood without centrifugation; single centrifugation, using whole blood subjected to a single centrifugation at 200 × g and 400 × g; and double centrifugation, using whole blood subjected one centrifugation at different rotations, followed by collection of whole plasma subjected to another centrifugation at different rotations: 200 × g + 200 × g; 200 × g + 400 × g; 200 × g + 800 × g; 400 × g + 400 × g; 400 × g + 800 × g. Samples of 3 ml of blood were drawn from each animal by means of cardiac puncture. The blood was stored in Vacutainer collection tubes containing 3.2% sodium citrate. The blood from the control group animals was analyzed without being subjected to centrifugation. After the blood from the other groups of animals had been subjected to centrifugation, the whole plasma was collected and subjected to platelet counting in the lower third of the sample.

Results: We obtained greatest platelet enrichment in the subgroup with two centrifugations comprising 400 × g for 10 min + 400 × g for 10 min, in which the mean platelet concentration was 11.30 times higher than that of the control group.

Conclusion: It was possible to obtain a high platelet concentration using viable simple techniques, by means of centrifugation of whole blood and use of commonly used materials. The most effective method for obtaining platelet concentrate was found in samples subjected to two centrifugations.

Objetivo: Quantificar a concentração de plaquetas do sangue de ratos SHR, por meio de diferentes protocolos de centrifugação, e avaliar qual o método mais eficaz de obtenção de plaquetas.

Métodos: Usamos 40 ratos machos da linhagem isogênica SHR. Os animais foram divididos em três grupos: Controle (GCT) - sangue total sem centrifugação; Única Centrifugação (GUC) - sangue total submetido a uma única centrifugação: 200 g e 400 g; Dupla Centrifugação (GDC) - sangue total submetido a uma centrifugação, seguido de coleta do plasma total, e realizado uma centrifugação, em diferentes rotações: 200 g + 200 g; 200 g + 400 g; 200 g + 800 g; 400 g + 400 g; 400 g + 800 g. Foram retirados 3 ml de sangue de cada animal por meio de punção cardíaca. O sangue foi acondicionado em tubo de coleta vacutainer com citrato de sódio 3,2%. O sangue dos animais do grupo controle não foi submetido à centrifugação e foi analisado. Após a centrifugação do sangue dos animais, submetido à centrifugação, o plasma total foi coletado e submetido à contagem de plaquetas no terço inferior da amostra.

Resultados: Obtivemos maior enriquecimento de plaquetas no subgrupo de duas centrifugações (400 g por 10 minutos + 400 g por 10 minutos), no qual ocorreu uma concentração média de plaquetas 11,30 vezes superior em relação ao grupo controle.

Conclusão: Foi possível obter uma alta concentração plaquetária, com técnica simples e viável, por meio de centrifugação do sangue total e uso de materiais de uso corriqueiro; e método mais eficaz de obtenção de concentrado de plaquetas ocorreu nas amostras submetidas a duas centrifugações.

Keywords: Centrifugation; Platelet count; Platelet-rich plasma.

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Figures

Fig. 1
Fig. 1
Plasma collection in the 1.5 mL Eppendorf tube.
Fig. 2
Fig. 2
Platelet count in the hematology analyzer.
Fig. 3
Fig. 3
Median leukocyte values according to subgroups.
Fig. 4
Fig. 4
Median erythrocyte values according to subgroups.
Fig. 5
Fig. 5
Median platelet values according to subgroups.
Fig. 6
Fig. 6
Median plasma volume values.
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