The centrifugal speed affects the morphology of red blood cells, but does not affect the number of red blood cells
There was no difference in the number of RBC (Fig. 1A), HGB (Fig. 1B), MCV (Fig. 1D), HCT (Fig. 1E), MCH (Fig. 1G), RDW (Fig. 1H), reticulocyte (Fig. 1J), and MCHC (Fig. 1K) under different centrifugal forces (P > 0.05).
Red blood cells at 9000 and 12000rpm showed burr changes under erythroscope (Fig. 1I, L).Normal red blood cell morphology at 6000rpm (Fig. 1F). Therefore, we know that the red blood cell morphology is normal at 6000rpm and the cell membrane is intact, and the red blood cell morphology is wrinkled and spiny at 9000rpm and 12000rpm. Therefore, we speculate that the centrifugal speed has some effect on the morphology of red blood cells, but does not change the number of red blood cells.
The centrifugal speed and time basically did not damage the white blood cells
There were no significant differences in the number of leukocytes (Fig. 2A), neutrophils (Fig. 2B), lymphocytes (Fig. 2C) and monocytes (Fig. 2D) under different centrifugal forces (P > 0.05).
We can not observe the significant difference in the activity of peripheral blood mononuclear cells obtained at different rotational speeds, and the activity was about 90%. The activity of peripheral blood mononuclear cells obtained by centrifugation at 6000rpm had little difference with that of centrifugation at 1500rpm and 3000rpm (Fig. 2E). There was no significant difference in the number of white blood cells at 10min, 20min, 30min and 40min (Fig. 2F). The morphology of white blood cells in a and b (6000rpm and 12000rpm) was normal under the peripheral blood smear microscope (Fig. 2G, H). Meanwhile, difference in the morphology of leukocytes under electron microscopy was not significant (Fig. 2I, J). Therefore, we know that the centrifugal speed and time have little effect on the damage of white blood cells.
When the centrifugal speed was 6000rpm, platelets could maintain low activation rate and normal morphology, but the activation rate increased with longer time
The number of platelets decreased at 10500rpm (P < 0.05), while the number of platelets at 7500 and 9000rpm also decreased, but there was no significant difference. At 1500, 3000, 4500, and 6000rpm, platelet levels did not decrease significantly (Fig. 3A). The mean platelet volume increased at 12000rpm (P < 0.05), with significant differences (Fig. 3B). Platelet volume also increased at 10500rpm, but there was no significant difference. The platelet activation rate was gradually increased by flow cytometry (CD61 was a platelet-specific marker, CD62p was a platelet-specific marker). Platelet activation in the normal body cannot exceed 5%, so 7500rpm, 9000rpm, 10500rpm, and 12000rpm do not meet the requirements (Fig. 3C). At 6000rpm, platelet activation did not exceed 5%, which was within the normal range (Fig. 3D). After centrifugation for 10, 20, 30, and 40min at 1500rpm, platelet activation rates gradually increased, but all were within the normal range (Fig. 3E). Figure 3G shows that platelet activation did not exceed the normal range at 10, 20, 30, and 40min centrifugation. Figure 3F shows platelet aggregation in the 7500 and 12000rpm groups in peripheral blood smears.
When the rotational speed was 3000rpm and 6000rpm, the platelets had normal morphology under electron microscope, no foot process, fewer intracellular Alpha particles and fewer mitochondrial vacuoles, and the platelets had elongated foot process morphology at 7500rpm. At 9000rpm, the platelet foot mutations varied and long, and at 12000rpm, the number of platelet Alpha particles increased, the platelets clustered together, the intracellular vacuoles became larger and more numerous, and the platelets fused into sheets. Multiple ruptures of platelet membrane; The particles were significantly reduced, mitochondria were swollen and vacuolated (Fig. 3H, I, J, K, L, M).
Observation under scanning electron microscopy showed that the fibrin intermediate layer after 12000rpm centrifugation contained a large number of clustered white blood cells, and the fibrin matrix was interleaved in a grid pattern and wrapped white blood cells and platelets (Fig. 3N).
Coagulation factor decreased gradually with the increase of centrifugal speed
With the increase of centrifugal speed, we can see a gradual decline in factors Ⅷ, Ⅸ, Ⅺ and Ⅻ (Fig. 4G, H, I, J), and APTT lengthens at 12000rpm (P < 0.05) (Fig. 4E). Therefore, we hypothesize that endogenous coagulation pathways are activated at centrifugal speeds above 9000rpm. Combined with Figs. 3 and 4, it is shown that different rotational speeds may change the structural changes within platelets, causing platelets to aggregate and activate, and affecting coagulation factors.
The centrifugal speed did not affect the chromosome structure and morphology of blood cells
The incidence of chromosome breakage did not change much when the rotational speed was 3000rpm, 6000rpm, 9000rpm and 12000rpm, and it could be considered that the different rotational speed did not affect the structural and morphological changes of chromosomes in the nucleus (Fig. 4K, M, ). It should be added that calcium ions did not decrease significantly under different centrifugal speeds (Fig. 4K), which may be due to the influence of EDTA and sodium tenuate in the blood collection tube, so the results may not be representative.
The optimum centrifugal parameters were 6000rpm, 10min
From our above experimental results, we can see that under different rotational speeds and different centrifugation times, the number loss and cell damage of red and white blood cells are less. For platelets, the centrifugal loss of platelets at 3000 and 6000rpm is relatively small, and the centrifugal loss of platelets at 6000rpm has little change compared with that at 3000rpm (Fig. 5A, B, C). However, when the centrifugal speed increases to 7500rpm, the platelet activation rate exceeds 5%. The number of platelet decreased significantly at 10500rpm. From these results, we can see that 6000rpm centrifugation has little damage to cells, and the platelet activation rate increases with the increase of centrifugation time. At this time, we found that the optimal centrifugal parameter was 6000rpm,10min.
At 9000rpm and above, the damage to platelets was greater, but the effect on red blood cells and white blood cells was less
Platelets also decreased significantly when the centrifugal speed was changed to 9000rpm. However, different centrifugal speed had little effect on red blood cells and white blood cells in hyperleukocyte acute myeloid leukemia patients. At 3000 and 6000rpm, the platelet activation rate was in the normal range, while at 12000rpm, platelets showed a large amount of activation and decreased in number (Fig. 5D, E, F, G, H, I). After the rotational speed was 3000rpm, 6000rpm and 12000rpm, there was no statistical difference in the morphological changes of red blood cells and white blood cells in the peripheral blood of hyperleukocyte acute myeloid leukemia patients, such as average cell radius, average image brightness and average cell image energy gradient. After centrifugation at 12000rpm, platelets clustered a lot, resulting in increased mean cell image brightness, larger mean image energy gradient, and larger mean cell radius (Fig. 5J, K, L, M, N, O). The morphology of white blood cells, such as neutrophils, lymphocytes and monocytes, did not change significantly under electron microscopy whether centrifugation was performed at a low rotational speed below 6000rpm or at a high rotational speed below 12000rpm (Fig. 6A, B, C, D). Under electron microscope, the number of Alpha particles in platelets was higher when the rotational speed was more than 6000rpm. At 12000rpm, there were more platelet foot processes and more platelet aggregation. This indicates that platelets are activated more thoroughly, which is more conducive to platelet aggregation and adhesion and other functions (Fig. 6E, F).
Cell cryopreservation and establishment of leukemia mouse model under optimal centrifugation parameters
From the above results, we obtained the optimal centrifugation parameters (6000rpm, 10min) with minimal damage to cells. The leukemia cells centrifuged with the optimal centrifugation parameters were frozen, and their resuscitation activity reached more than 95% (Fig. 6G, H, I, J). Resuscitated cells (experimental group) and uncentrifuged fresh leukemia cells (control group) were injected into NSG mice by tail vein respectively, and the incidence cycle, infiltration and distribution of leukemia cells in mice were observed and compared. The results (Fig. 7) showed that there was no significant difference in weight change and survival curve between the experimental group and the control group, and all mice successfully developed acute myeloid leukemia and died successively. CD45 positivity was seen by cytometry and immunohistochemistry, as well as infiltration of leukemia cells in HE staining of the spleen and bone marrow, indicating successful modeling of AML mice and showing the aggressiveness of acute myeloid leukemia.