Quantification of platelets obtained by different centrifugation protocols in SHR rats

Yazigi Junior, João Alberto; Santos, João Baptista Gomes dos; Xavier, Bruno Rodrigues; Fernandes, Marcela; Valente, Sandra Gomes; Leite, Vilnei Mattiolli

doi:10.1016/j.rboe.2015.10.008

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ORIGINAL ARTICLE • Rev. bras. ortop. 50 (6) • Nov-Dec 2015 • https://doi.org/10.1016/j.rboe.2015.10.008 copy

Quantification of platelets obtained by different centrifugation protocols in SHR rats ^☆ ☆ Study carried out at the Laboratory of Microsurgery of the Discipline of Hand and Upper Limb Surgery, Department of Orthopedics and Traumatology, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brazil.

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

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 × gand 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 × gfor 10 min + 400 ×gfor 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.

Keywords:
Platelet count; Centrifugation; Platelet-rich plasma

Variable	Group	Mean	SD	Median	Minimum	Maximum	N	p
Leukocytes	CTRL	3060	219.1	3200	2700	3200	5	0.016
	200 × g-10 min	420	465.8	200	100	1200	5
	400 × g-10 min	420	549.5	200	100	1400	5
	200 × g-10 min + 200 × g-10 min	1160	2260.1	100	100	5200	5
	200 × g-10 min + 400 × g-10 min	1860	1105.9	1400	500	3100	5
	200 × g-10 min + 800 × g-10 min	2.700	3755.7	700	300	9200	5
	400 × g-10 min + 400 × g-10 min	3.020	2983.6	1500	400	7800	5
	400 × g-10 min + 800 × g-10 min	3160	3943.7	500	200	8700	5
Erythrocytes	CTRL	7,120,000	358,956.8	7,020,000	6.640.000	7,530,000	5	<0.001
	200 × g-10 min	22,000	16,431.7	20,000	10,000	50,000	5
	400 × g-10 min	14,000	11,401.8	10,000	0	30,000	5
	200 × g-10 min + 200 × g-10 min	80,000	134,721.9	20,000	10,000	320,000	5
	200 × g-10 min + 400 × g-10 min	262,000	895,545	240,000	150,000	380,000	5
	200 × g-10 min + 800 × g-10 min	170,000	101,242.3	150,000	60,000	330,000	5
	400 × g-10 min + 400 × g-10 min	120,000	86,313.4	150,000	20,000	220,000	5
	400 × g-10 min + 800 × g-10 min	188,000	199,549.5	70,000	10,000	420,000	5
Platelets	CTRL	648,800	30,094.9	648,000	614,000	695,000	5	0.013
	200 × g-10 min	1,632,400	580,962.4	1650.000	682,000	2,122,000	5
	400 × g-10 min	939,600	687,417.1	863,000	110,000	1,764,000	5
	200 × g-10 min + 200 × g-10 min	3,826,000	4920.808.4	456,000	29,000	970.0000	5
	200 × g-10 min + 400 × g-10 min	4,760,000	2254.550.953	530.0000	130.0000	720.0000	5
	200 × g-10 min + 800 × g-10 min	4,080,000	1685.823,241	310.0000	270.0000	6500.000	5
	400 × g-10 min + 400 × g-10 min	7,320,000	2819.929,077	7,300,000	2,800,000	9,700,000	5
	400 × g-10 min + 800 × g-10 min	5,500,000	1813.835,715	5200.000	3400.000	7600.000	5
Plasma volume (mL)	CTRL	0	0	0	0	0	5	<0.001
	200 × g-10 min	0.72	0.11	0.8	0.6	0.8	5
	400 × g-10 min	1.10	0.10	1.1	1	1.2	5
	200 × g-10 min + 200 × g-10 min	1.46	0.11	1.5	1.3	1.6	5
	200 × g-10 min + 400 × g-10 min	1.60	0.10	1.6	1.5	1.7	5
	200 × g-10 min + 800 × g-10 min	1.62	0.04	1.6	1.6	1.7	5
	400 × g-10 min + 400 × g-10 min	1.62	0.08	1.6	1.5	1.7	5
	400 × g-10 min + 800 × g-10 min	1.72	0.08	1.7	1.6	1.8	5

Leukocytes
Comparison		Z value	p
CTRL VS	200 × g-10 min	2.84	0.004
CTRL VS	400 × g-10 min	2.84	0.004
CTRL VS	200 × g-10 min + 200 × g-10 min	2.70	0.007
CTRL VS	200 × g-10 min + 400 × g-10 min	0.90	0.367
CTRL VS	200 × g-10 min + 800 × g-10 min	1.04	0.298
CTRL VS	400 × g-10 min + 400 × g-10 min	0.54	0.589
CTRL VS	400 × g-10 min + 800 × g-10 min	1.11	0.267
200 × g-10 min VS	400 × g-10 min	0.00	>0.999
200 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-0.14	0.890
200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-1.94	0.052
200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.80	0.071
200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-2.30	0.021
200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.73	0.083
400 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-0.14	0.890
400 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-1.94	0.052
400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.80	0.071
400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-2.30	0.021
400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.73	0.083
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-1.80	0.071
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.66	0.096
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-2.16	0.031
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.59	0.111
200 × g-10 min + 400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	0.14	0.890
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-0.36	0.718
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	0.21	0.835
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-0.50	0.618
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 800 × g-10 min	0.07	0.945
400 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	0.57	0.570

Erythrocytes
Comparison		Z value	p
CTRL VS	200 × g-10 min	3.90	<0.001
CTRL VS	400 × g-10 min	4.27	<0.001
CTRL VS	200 × g-10 min + 200 × g-10 min	3.41	0.001
CTRL VS	200 × g-10 min + 400 × g-10 min	1.30	0.192
CTRL VS	200 × g-10 min + 800 × g-10 min	1.91	0.056
CTRL VS	400 × g-10 min + 400 × g-10 min	2.44	0.015
CTRL VS	400 × g-10 min + 800 × g-10 min	2.18	0.029
200 × g-10 min VS	400 × g-10 min	0.37	0.708
200 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-0.49	0.627
200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-2.59	0.010
200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.98	0.047
200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-1.46	0.145
200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.72	0.086
400 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-0.86	0.390
400 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-2.97	0.003
400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-2.36	0.018
400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-1.83	0.067
400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-2.09	0.036
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-2.11	0.035
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.50	0.134
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-0.97	0.332
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.23	0.217
200 × g-10 min + 400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	0.61	0.542
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	1.14	0.255
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	0.87	0.382
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 400 × g-10 min	0.53	0.598
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 800 × g-10 min	0.26	0.792
400 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-0.26	0.792

Platelets
Comparison		Z value	p
CTRL VS	200 × g-10 min	-1.03	0.305
CTRL VS	400 × g-10 min	-0.25	0.803
CTRL VS	200 × g-10 min + 200 × g-10 min	-1.19	0.233
CTRL VS	200 × g-10 min + 400 × g-10 min	-2.50	0.013
CTRL VS	200 × g-10 min + 800 × g-10 min	-2.29	0.022
CTRL VS	400 × g-10 min + 400 × g-10 min	-3.48	0.001
CTRL VS	400 × g-10 min + 800 × g-10 min	-2.91	0.004
200 × g-10 min VS	400 × g-10 min	0.78	0.437
200 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-0.17	0.868
200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-1.47	0.142
200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.26	0.207
200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-2.45	0.014
200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.89	0.059
400 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-0.94	0.346
400 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-2.25	0.025
400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-2.04	0.041
400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-3.23	0.001
400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-2.66	0.008
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-1.30	0.192
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.10	0.273
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-2.29	0.022
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.72	0.086
200 × g-10 min + 400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	0.21	0.835
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-0.98	0.325
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-0.42	0.677
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-1.19	0.233
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-0.62	0.533
400 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	0.57	0.570

Plasma volume
Comparison		Z value	p
CTRL VS	200 × g-10 min	-0.69	0.488
CTRL VS	400 × g-10 min	-1.39	0.166
CTRL VS	200 × g-10 min + 200 × g-10 min	-2.36	0.018
CTRL VS	200 × g-10 min + 400 × g-10 min	-3.38	0.001
CTRL VS	200 × g-10 min + 800 × g-10 min	-3.55	<0.001
CTRL VS	400 × g-10 min + 400 × g-10 min	-3.58	<0.001
CTRL VS	400 × g-10 min + 800 × g-10 min	-4.47	<0.001
200 × g-10 min VS	400 × g-10 min	-0.69	0.488
200 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-1.66	0.096
200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-2.69	0.007
200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-2.86	0.004
200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-2.88	0.004
200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-3.77	<0.001
400 × g-10 min VS	200 × g-10 min + 200 × g-10 min	-0.97	0.332
400 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-2.00	0.046
400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-2.16	0.031
400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-2.19	0.028
400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-3.08	0.002
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 400 × g-10 min	-1.03	0.305
200 × g-10 min + 200 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-1.19	0.233
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-1.22	0.222
200 × g-10 min + 200 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-2.11	0.035
200 × g-10 min + 400 × g-10 min VS	200 × g-10 min + 800 × g-10 min	-0.17	0.868
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-0.19	0.846
200 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-1.08	0.279
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 400 × g-10 min	-0.03	0.978
200 × g-10 min + 800 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-0.92	0.360
400 × g-10 min + 400 × g-10 min VS	400 × g-10 min + 800 × g-10 min	-0.89	0.375

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[1] * Corresponding author. E-mail:junioryazigi73@yahoo.com.br(J.A. Yazigi Junior).