Study on Leukapheresis of Hyperleukocyte Acute Myeloid Leukemia

Study on Leukapheresis of Hyperleukocyte Acute Myeloid Leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Study on Leukapheresis of Hyperleukocyte Acute Myeloid Leukemia Ruiyang Pan, Anjie Xu, Li Liu, Jinxian Wu, Xinqi Li, Guopeng Chen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4368848/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jul, 2024 Read the published version in BMC Cancer → Version 1 posted 10 You are reading this latest preprint version Abstract Respiratory failure, intracranial hemorrhage and infection were more common in hyperleukocyte acute myeloid leukemia patients than in non-hyperleukocyte leukemia patients. Compared with non-apheresis treatment, the white blood cells decreased significantly and the infection rate decreased after apheresis treatment. However, the treatment time of leukapheresis in patients with hyperleukocyte leukemia is very long, while the damage to cells is also large. In this study, a retrospective analysis was conducted on hyperleukocyte acute myeloid leukemia patients with hyperleukocytosis during induction. Centrifugation was performed at different rotational speeds and centrifugation times to observe whether there were changes in the number and morphology of peripheral blood cells in healthy people and patients with hyperleukocyte leukemia. The centrifugation of normal cells and hyperleukocyte was simulated in vitro, so as to explore optimal centrifugation parameters in the treatment of leukapheresis. The cells obtained by optimal centrifugation parameters were cryopreserved and animal models were established. Through the research, it is found that when the rotational speed is increased below 6000rpm, the damage to normal blood cells and blood cells in patients with hyperleukocyte leukemia is small. When the rotational speed is greater than 6000rpm, the platelets will be damaged. The cells obtained under optimal centrifugation parameters can be successfully cryopreserved and modeled in leukemia animals. Hyperleukocyte Acute Myeloid Leukemia Leukapheresis Centrifugation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Acute myeloid leukemia(AML) is a common hematopoietic malignant tumor with high heterogeneity and biological complexity. Of the tens of thousands of new cases in the United States each year, nearly one-third of leukemia diagnoses are acute myeloid leukemia, and the incidence increases with age( 1 – 3 ). Hyperleukocytic leukemia is a high-risk type of acute myeloid leukemia, which is characterized by an abnormally high number of peripheral white blood cells, exceeding 100×10 9 /L( 4 , 5 ). Hyperleukocytic leukemia accounts for 5% ~ 20% of adult acute myeloid leukemia( 6 ), among which M2 and M5 are the most common types of AML( 7 , 8 ). If not treated actively, the 1-week mortality can reach 40%( 8 – 10 ). Severe hyperleukocytosis, which causes leukocytostasis, is a medical emergency with a risk of organ damage and is a poor prognostic factor for early death in patients with hyperleukocytosis( 4 , 11 ). In clinical practice, leukocytosis syndrome most commonly affects the lungs, central nervous system and kidneys, and is associated with pulmonary congestion infection, intracranial hemorrhage or infarction, melanoma, hematuria and other complications, which makes patients progress quickly, and increases the risk of death( 9 , 12 ). However, leukocytosis is a poorly understood and life-threatening complication of acute leukemia. Therefore, it is necessary to rapidly reduce white blood cells during treatment. In addition, studies have confirmed that hyperleukocytosis is a poor prognostic factor for early death in hyperleukocyte patients, and the overall survival of patients with hyperleukocyte leukemia is low( 8 , 13 ). These studies also suggest that patients with hyperleukocytosis who are suitable for chemotherapy may benefit from leukapheresis to prevent complications such as leukocytostasis that occur before acute myeloid leukemia is diagnosed and chemotherapy is started. Leukapheresis is a type of physical therapy that reduces the number of white blood cells and blood viscosity in a patient's blood( 14 ), which is still the main treatment for patients with hyperleukocyte leukemia( 15 ). Leukapheresis is associated with improved prognosis and is generally safe. However, ,leukapheresis therapy does not achieve the ideal effect at present( 16 ), the reasons may be as follows: First, the time of leukapheresis is too long, which may delay the optimal treatment time of patients, and the leukemia cell bank may be quickly mobilized from the bone marrow, and the original cells in the blood circulation will increase, aggravating the disease( 17 ). Second, the intervention of leukapheresis may be too late to reverse the cascade of events that have already begun due to hyperleukocytosis. Third, leukapheresis may damage blood cells, and cell rupture releases a large number of harmful substances, aggravating the disease. Therefore, we should study the procedures of leukapheresis in more detail to improve the purity of blood cells in a short time. Our study asks whether it is possible to improve the separation efficiency by increasing the centrifugal speed while ensuring less cell damage, and to shorten the time of apheresis treatment and collect white blood cells more quickly by continuing to increase the centrifugal speed. Therefore, how to reduce the damage to cells, and what parameter range is safe to increase the rotational speed has become a major problem we have encountered. Therefore, in order to make up for the long time of apheresis, find the optimal rotational speed to improve the efficiency of apheresis treatment, gain time for further treatment, and further improve the survival rate of patients with hyperleukocyte acute myeloid leukemia. In this study, we explored the effect of mechanical force on the structure and function of peripheral blood cells in normal peripheral blood cells and patients with hyperleukocyte acute myeloid leukemia after in vitro centrifugation, hoping to find a suitable range of centrifugal rotation speed to improve the rotation speed without damaging normal cells, so as to achieve the purpose of improving the efficiency of apheresis. Materials and Method Patients Five healthy people donated blood. Inclusion criteria :1) Age: adult (≥ 18 years old); 2) Normal coagulation function; 3) No genetic history. Exclusion criteria :1) Drinking alcoholic beverages within 1 day before blood donation; 2) Take aspirin and other antiplatelet and anticoagulant drugs within 2 weeks before donating blood. Peripheral blood of patients with hyperleukocyte acute myeloid leukemia was extracted. It was extracted by the nurse and placed in purple, green and blue anticoagulant tubes. The donors for the sample study signed a written informed consent prior to having their blood drawn. The research program of blood donors has been approved by the local ethics committee, which is in line with China's blood donation guidelines and in line with ethics. Methods Whole blood cells were centrifuged at the same time and at different rotational speeds First of all, centrifuge at 0rpm (non-centrifuge), 1500rpm, 3000rpm, 4500rpm, 6000rpm, 7500rpm, 9000rpm, 10500rpm, 12000rpm for ten minutes in centrifuges imported from the regular company (Thermo company). Then centrifuge at room temperature at 3000rpm, 6000rpm, 9000rpm for 10min, 20min, 30min, 40min. The sample was kept consistent from the beginning of processing to the detection time, and the centrifuge temperature was 20℃. After treatment, a part of the blood samples in each group were obtained red blood cell layer, white blood cell platelet layer and plasma layer. After a part of the blood samples were separated, red blood cell lysate was added to the white blood cell platelet layer and supernatant was removed to obtain a relatively pure white blood cell platelet layer. The other part is mixed and tested after blood routine and other items. Cell number morphology, biochemical examination and electron microscope morphology A part of human whole blood was collected before and after centrifugation to detect blood routine, electrolyte, coagulation and other indexes and red blood cell morphology observation. Cell apoptosis was detected in the cell suspension obtained by centrifugation. The platelet activation rate was measured by flow cytometry after treatment with different centrifugal parameters. The morphology of red blood cell layer (pre-diluted), white blood cell layer and platelet layer were observed under transmission electron microscope. Chromosome breakage experiment and cell microstructure Peripheral blood samples after centrifugation were added to 3 identical peripheral blood media, and MMC was added to make the final concentrations of 0ng/ml, 50ng/ml and 100ng/ml, respectively. After a series of operations such as culture, harvest and preparation, chromosome breakage was observed under microscope. Homogeneous trace cells were recorded by taking photos, and the average cell size was measured, that is, the average cell radius was calculated to measure the changes in cell size, and the changes in cell center point, brightness, image contrast and other changes were calculated to understand the changes in cell surface. In addition, the red blood cell layer, white blood cell layer and platelet layer after centrifugation were separated and labeled, and the cell morphology was recorded by single cell imaging technology of photofluid time stretch microscope. The obtained cell images were processed and analyzed by computer. Cell cryopreservation was performed under optimal centrifugation parameters Peripheral blood samples of newly diagnosed patients with hyperleukocyte acute myeloid leukemia M5 requiring cryopreservation were collected. The experimental group used the optimal centrifugation parameters previously explored, and the control group was centrifuged at 3000rpm for 10min. After centrifugation, the mononuclear cell layer was absorbed and mixed with cleaning solution. The treated mononuclear cells were transferred to a sterile cryopreserved tube, and autologous plasma and DMSO (the ratio is 9:1) were added for cell cryopreserved. After 1 month, the frozen tube was taken out and placed in 37℃ water bath for cell resuscitation. The supernatant was removed by centrifugation and stained with pancreatic blue. The cell survival rate was calculated three times to take the average value. After 6 months of frozen storage, the frozen tube was taken out and placed in 37℃ water bath for cell resuscitation. After centrifugation, the uniformly obtained cell suspension was re-suspended with DMEN medium and cultured with CCK8 to determine the cell viability. Three times the volume of red cell lysate was added to the cell suspension obtained by centrifugation, and apoptosis was detected. Human acute myeloid leukemia mouse model was established with the cells obtained under optimal centrifugation parameters Experimental cells were taken from the above frozen cells and, again, divided into experimental and control groups according to whether or not centrifugation was performed with optimal parameters. The experimental animals were immunodeficient B-NSG mice (qualification number: SCXK(Beijing)2022-0004) purchased at 4–5 weeks. One week after purchase, each mouse was given 2.0Gy X-ray irradiation, and on the second day, acute myeloid leukemia cells (CD33CD117) were injected intravenously through the tail vein to establish a human acute myeloid leukemia mouse model. After the model was built, the weight changes of the mice were observed, and the mice were killed 3 weeks later, and the survival time of each group was recorded. The mouse bone marrow was collected and the erythrocytes were lysed after the bone marrow was rinsed. Then the mononuclear cells in the bone marrow were collected, and flow cytometry was performed to detect the change of the proportion of original cells in the bone marrow of mice in each group. The expression of CD117 in leukemia xenografts was detected by immunohistochemistry. Statistics The significance of differences between two groups was determined using unpaired two-tailed Student t tests. All results in bar graphs are mean value ± SEM. Overall survival curves were plotted according to the Kaplan-Meier method with the log-rank test applied for comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 Results 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. 1 A), HGB (Fig. 1 B), MCV (Fig. 1 D), HCT (Fig. 1 E), MCH (Fig. 1 G), RDW (Fig. 1 H), reticulocyte (Fig. 1 J), and MCHC (Fig. 1 K) under different centrifugal forces (P > 0.05). Red blood cells at 9000 and 12000rpm showed burr changes under erythroscope (Fig. 1 I, L).Normal red blood cell morphology at 6000rpm (Fig. 1 F). 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. 2 A), neutrophils (Fig. 2 B), lymphocytes (Fig. 2 C) and monocytes (Fig. 2 D) 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. 2 E). There was no significant difference in the number of white blood cells at 10min, 20min, 30min and 40min (Fig. 2 F). The morphology of white blood cells in a and b (6000rpm and 12000rpm) was normal under the peripheral blood smear microscope (Fig. 2 G, H). Meanwhile, difference in the morphology of leukocytes under electron microscopy was not significant (Fig. 2 I, 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. 3 A). The mean platelet volume increased at 12000rpm (P < 0.05), with significant differences (Fig. 3 B). 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. 3 C). At 6000rpm, platelet activation did not exceed 5%, which was within the normal range (Fig. 3 D). After centrifugation for 10, 20, 30, and 40min at 1500rpm, platelet activation rates gradually increased, but all were within the normal range (Fig. 3 E). Figure 3 G shows that platelet activation did not exceed the normal range at 10, 20, 30, and 40min centrifugation. Figure 3 F 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. 3 H, 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. 3 N). 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. 4 G, H, I, J), and APTT lengthens at 12000rpm (P < 0.05) (Fig. 4 E). 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. 4 K, M, ). It should be added that calcium ions did not decrease significantly under different centrifugal speeds (Fig. 4 K), 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. 5 A, 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. 5 D, 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. 5 J, 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. 6 A, 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. 6 E, 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. 6 G, 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. Discussion For different components of blood cells, in order to make different blood components better separated, in the specific separation operation, the parameter that is actually controllable and adjusted is the centrifuge speed. The main functions of centrifuge speed adjustment are as follows: 1) Quickly establish a good separation interface for different components of blood, use the shortest possible separation time, and improve the separation efficiency under the premise of preserving cell activity; 2) To ensure the separation effect of different components of blood, so that the separation of target demand components meets the corresponding standards, and meets the indicators of blood collection for medical use. A study found that rotational speed had the greatest effect on the separation efficiency of the platelet separation efficiency model, while the effect on the separation efficiency of red and white blood cells at high rotational speed was negligible( 18 ). Our study found that red blood cell morphology changed at and above 9000rpm, but the range of changes in hemoglobin, red blood cell number and hematocrit was too small, with no statistical difference, which was consistent with literature reports, and the loss of red blood cells at 6000rpm was almost zero. From this, we can see that at 9000rpm, the morphology of red blood cells is spiny, but the cell membrane is relatively intact, and the rate of red blood cell lysis is too small to be ignored. However, platelets have different degrees of damage at high rotational speed, so the best parameters for centrifugation should also refer to the damage of platelets. Our study found that the number of platelets did not decrease significantly below 6000rpm, while the number of platelets decreased at 7500 and 9000rpm.Platelet activation was within the normal range at 6000rpm, and beyond the normal range at 7500rpm and above. In addition, platelets had normal morphology under electron microscope with no foot process, fewer Alpha particles in cells, fewer mitochondrial vacuoles, and elongated morphology of foot process at 7500rpm when the rotational speed was 3000 and 6000rpm, respectively. At 9000rpm, the platelet foot mutation was varied and long, and at 12000rpm, the number of platelet Alpha particles was more and the platelets gathered together. When centrifuged at 12000rpm, the biggest changes in platelet morphology were alpha particles, dense particles and foot process. It has been documented that Alpha particles contain membrane binding proteins and soluble proteins that are involved in various processes, including cell adhesion, coagulation, inflammation, cell growth, and host defense( 19 ). After platelet activation, Alpha particles release fibrinogen and VWF, promoting platelet-platelet and platelet-endothelial cell interactions. In addition, components of the fibrinogen receptor Alpha ⅡBβ3, collagen receptor GPVI, and VWF receptor complex GPIb-IX-V found in Alpha particles are expressed on the surface of platelets and subsequently support platelet adhesion( 19 ). Alpha particles contain clotting mediators such as factor VIII and other clotting factors, which may affect the coagulation environment of the plasma, that is manifested as the reduction of some coagulation factors, the abnormal time of some thromboplastin, and the destruction of endogenous coagulation pathway. In this experiment, we tried for the first time to extract peripheral blood mononuclear cells with the optimal centrifugation parameters explored, and successfully frozen cells with autologous plasma. The survival rate of cells after resuscitation was above 95%, which laid a solid foundation for the establishment of blood cell sample bank in the future. We also successfully established a mouse model of acute myeloid leukemia with the optimum centrifugation parameters. We know that mouse models of acute myeloid leukemia are indispensable research tools. For example, extensive sequencing efforts have mapped the genomes of adults and children with acute myeloid leukemia, revealing many biological and prognostic drivers. In addition to identifying recurrent genetic aberrations, it is critical to adequately describe the complex mechanisms by which they contribute to the onset and evolution of disease, ultimately facilitating the development of targeted therapies. To achieve these goals, rapid advances in genetic engineering techniques over the past 20 years have greatly facilitated the use of mouse models to reflect specific genetic subtypes of human acute myeloid leukemia, define intracellular and external disease mechanisms, study interactions between co-occurring genetic lesions, and test new treatments. Therefore, it is of great significance to establish a mouse AML model. The cells obtained by using the best centrifugation parameters had less cell damage and improved the purity of mononuclear cells collected. The fast reading established a boundary layer to reduce the number and time of centrifugation. Compared with low-speed centrifugation, the cells could be separated faster and more accurately, and the survival rate after cell cryopreservation and recovery was similar to that obtained by low-speed centrifugation. The mouse model of AML was also established successfully. These results indicate that the centrifuged cells under optimal centrifugation parameters can be used for cryopreservation of clinical samples and establishment of mouse models of acute myeloid leukemia in laboratory. Conclusion At present, the clinical use of leukapheresis can not achieve the ideal effect, and it takes a long time and causes great damage to cells. It is an urgent problem to further improve leukapheresis, explore the best centrifugal parameters, realize fast and accurate leukocyte sorting, and minimize the possible adverse reactions in the process. In this study, by changing the centrifugal speed, it was observed that low-speed centrifugation was relatively safe for peripheral blood cells, while high-speed centrifugation would cause platelet activation and aggregation when it reached 12000rpm, and the best possible centrifugation parameter was found at 6000rpm, 10min. The cells obtained by centrifugation with the best parameters we explored could be frozen, and the cell viability was above 95%, and the mouse model of AML could be successfully established. The results of this experimental study are of great significance for the improvement of clinical leukocyte reduction therapy, which can improve the efficiency of leukapheresis therapy, reduce the treatment cost, guide the clinical prevention and treatment of adverse reactions in the process of leukapheresis therapy without increasing the risk related to treatment, and provide more scientific information for the evaluation of the collection effect of leukapheresis therapy and more scientific direction for the improvement of treatment instruments. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences (Approval No. 202020). The study scheme was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University. All patients signed informed consent forms and submitted them to Zhongnan Hospital for safekeeping. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interest All authors have read and approved the submission of the final manuscript. No conflicts of interest were declared. Authors’ Contributions Conceptualization: RYP, AJX, and FLZ; Methodology: RYP, AJX and LL; Validation: FLZ; Formal analysis: AJX and LL; Investigation: RYP, AJX, LL, JXW, XQL, GPC, WYY and RHL; Resources: AJX and DDL; Data Curation: RYP, AJX and LL; Writing - Original Draft: RYP and AJX; Writing - Review & Editing: FLZ; Visualization: RYP; Supervision: XYL and FLZ; Project administration: AJX, LL and FLZ; Funding acquisition: XYL, and FLZ. All authors reviewed and authorized the final manuscript. Acknowledgments and fundings We thank all of the patients who took part in this study, as well as their families. This study was funded by the Natural Science Foundation of China program [grant numbers 81900116, 82370176], and the Zhongnan Hospital of Wuhan University discipline construction platform project [grant numbers 202021, PDJH202217]. References Li Z, Philip M, Ferrell PB. Alterations of T-cell-mediated immunity in acute myeloid leukemia. Oncogene. 2020;39(18):3611–9. Marrero RJ, Lamba JK. Current Landscape of Genome-Wide Association Studies in Acute Myeloid Leukemia: A Review. Cancers (Basel). 2023;15(14). Pelcovits A, Niroula R. Acute Myeloid Leukemia: A Review. R I, Med J. (2013). 2020;103(3):38–40. Farid KMN, Sauer T, Schmitt M, Muller-Tidow C, Schmitt A. Symptomatic Patients with Hyperleukocytic FLT3-ITD Mutated Acute Myeloid Leukemia Might Benefit from Leukapheresis. Cancers (Basel). 2023;16(1). Marbello L, Ricci F, Nosari AM, Turrini M, Nador G, Nichelatti M, et al. 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Cite Share Download PDF Status: Published Journal Publication published 24 Jul, 2024 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 17 May, 2024 Reviews received at journal 16 May, 2024 Reviewers agreed at journal 08 May, 2024 Reviews received at journal 07 May, 2024 Reviewers agreed at journal 07 May, 2024 Reviewers invited by journal 07 May, 2024 Editor invited by journal 07 May, 2024 Submission checks completed at journal 07 May, 2024 Editor assigned by journal 07 May, 2024 First submitted to journal 04 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4368848","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":300304102,"identity":"8f7ae4a1-9fe6-4103-85af-1ad0c0684cef","order_by":0,"name":"Ruiyang Pan","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Ruiyang","middleName":"","lastName":"Pan","suffix":""},{"id":300304103,"identity":"68cd9896-b52b-45d2-bc4d-8ff277ed2518","order_by":1,"name":"Anjie Xu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Anjie","middleName":"","lastName":"Xu","suffix":""},{"id":300304104,"identity":"959bb72a-b687-4d28-a381-5aea8e3ad09b","order_by":2,"name":"Li Liu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Liu","suffix":""},{"id":300304105,"identity":"85d1820b-24a0-45df-8e9f-0a7743c988af","order_by":3,"name":"Jinxian Wu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Jinxian","middleName":"","lastName":"Wu","suffix":""},{"id":300304106,"identity":"9095badb-5ec7-432d-bc7c-cbceed7d356d","order_by":4,"name":"Xinqi Li","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xinqi","middleName":"","lastName":"Li","suffix":""},{"id":300304107,"identity":"db6f3f2f-1f96-4002-94d8-703e59548e16","order_by":5,"name":"Guopeng Chen","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Guopeng","middleName":"","lastName":"Chen","suffix":""},{"id":300304108,"identity":"8dc47b1f-ce48-443b-bed5-3e18341fa908","order_by":6,"name":"Ruihang Li","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Ruihang","middleName":"","lastName":"Li","suffix":""},{"id":300304109,"identity":"aaca2365-507c-499e-8830-87df9041c41e","order_by":7,"name":"Wanyue Yin","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Wanyue","middleName":"","lastName":"Yin","suffix":""},{"id":300304110,"identity":"80042ebd-ebef-43dd-85f7-e1f1d3361b0f","order_by":8,"name":"Dandan Liu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Liu","suffix":""},{"id":300304111,"identity":"639da2f3-be40-4254-8997-ccf0fd274ddc","order_by":9,"name":"Xiaoyan Liu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Liu","suffix":""},{"id":300304112,"identity":"09725c43-acc5-4769-a1fb-17b800aa2069","order_by":10,"name":"Fuling Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACCSCuYLDgYWBgPsAMFjlAjJYzDBJALWwJpGkBkjwGxGmRn918TOJgm4SMOf+ab9KFbQxyfDcSGD8X4NHCOOdYGkgLj+WMt9ukZ7YxGEveSGCWnoFHC7NEjpn0R6AWgxtnt93mbWNI3HAjgY2ZB48WNqAWsC0GN848A2mpJ6iFB67lfA8bSEuCASEtEhJpyRYHzoFsYTP/zXNOwnDmmYfN0vi0yM9IPnjjQJmNvcH5w4+Necps5PmOJx/8jE8Lkn0JYBKIGRuI0sDAwH+ASIWjYBSMglEw4gAAjeBIEO0DnsQAAAAASUVORK5CYII=","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Fuling","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-05-04 14:38:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4368848/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4368848/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-024-12644-5","type":"published","date":"2024-07-24T16:15:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56673622,"identity":"547de3d1-d954-41a5-b000-d1b9f52addec","added_by":"auto","created_at":"2024-05-17 15:28:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1164650,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The number of red blood cells at different centrifugal speeds (B) \u0026nbsp;The content of hemoglobin at different centrifugal speeds (C) Morphology of red blood cells under 3000rpm centrifugal force (D) mean corpuscular volume at different centrifugal speeds (E) Hematocrit at different centrifugal speeds (F) Morphology of red blood cells under 6000rpm centrifugal force (G) mean corpuscular hemoglobin at different centrifugal speeds (H) Red blood cell distribution width at different centrifugal speeds (I) Morphology of red blood cells under 9000rpm centrifugal force (J) The content of reticulocyte at different centrifugal speeds (K) mean corpuscular hemoglobin concentration at different centrifugal speeds (L) Morphology of red blood cells under 12000rpm centrifugal force\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/7f3c23be6cc5e8cea402e3ee.png"},{"id":56673621,"identity":"af0fcd24-72b9-4ec7-b7a1-b88d4122de49","added_by":"auto","created_at":"2024-05-17 15:28:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1779432,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The number of white blood cells at different centrifugal speeds (B) The number of neutrophils at different centrifugal speeds (C) The number of lymphocyte at different centrifugal speeds (D) The number of monocytes at different centrifugal speeds (E) Mononuclear cell activity at different centrifugal speeds (F) The number of white blood cells at different centrifugal times (G) Peripheral blood smear under 6000rpm centrifugal force (H) Peripheral blood smear under 12000rpm centrifugal force (I) Morphology of white blood cells under electron microscopy under 6000rpm centrifugal force (J) Morphology of white blood cells under electron microscopy under 12000rpm centrifugal force\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/98085ddfae2389790604ec57.png"},{"id":56673623,"identity":"32905dcf-c726-45b4-b3af-ff9d862e343b","added_by":"auto","created_at":"2024-05-17 15:28:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1861369,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The number of platelet at different centrifugal speeds (B) Average platelet volume at different centrifugal speeds (C) Platelet activation rate at different centrifugal speeds (D) Platelet activation rate at different centrifugal speeds (E) Platelet activation rate at different centrifugation times under 1500rpm centrifugal force (F) Peripheral blood smears under centrifugal force of 7500rpm and 12000rpm (G) Platelet activation rate at different centrifugation times (H~N) Platelet morphology under electron microscopy under different centrifugal forces\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/6e4dbc7888ddc685b229a0d0.png"},{"id":56673624,"identity":"f390ce18-3295-4ed6-bcf5-54ba7bd92032","added_by":"auto","created_at":"2024-05-17 15:28:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":249107,"visible":true,"origin":"","legend":"\u003cp\u003e(A) PT at different centrifugal speeds (B) TT at different centrifugal speeds (C) The content of FIB at different centrifugal speeds (D) \u0026nbsp;INR at different centrifugal speeds (E) APTT at different centrifugal speeds (F) PTTA at different centrifugal speeds (G~J) Coagulation factor content at different centrifugal speeds (K) Chromosome breakage rate at different centrifugal speeds (L) Calcium ion content at different centrifugal speeds (M) Chromosome structure and morphology under 9000rpm centrifugal force (N) Chromosome structure and morphology under 12000rpm centrifugal force\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/1e80e9936d41ece035364835.png"},{"id":56673625,"identity":"8e63a0ab-0d2f-4ec3-859e-d08804da8db3","added_by":"auto","created_at":"2024-05-17 15:28:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":264370,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The number of platelet at different centrifugal speeds (B) The number of red blood cells at different centrifugal speeds (C) The number of white blood cells at different centrifugal speeds (D~G) The number or content of white blood cells, red blood cells, platelets, and hemoglobin in peripheral blood of patients with hyperleukocyte acute myeloid leukemia patients (H~I) Platelet activation rate in peripheral blood of patients with hyperleukocyte acute myeloid leukemia patients (J) PLT cell radius at different centrifugal speeds (K) PLT image luminance at different centrifugal speeds (L) RBC cell radius at different centrifugal speeds (M) RBC image luminance at different centrifugal speeds (N) WBC cell radius at different centrifugal speeds (O) WBC image luminance at different centrifugal speeds\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/52268e21e705e57c94b02593.png"},{"id":56673626,"identity":"327405c0-1fd1-489b-b15f-6ff7ef8f8afb","added_by":"auto","created_at":"2024-05-17 15:28:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2495096,"visible":true,"origin":"","legend":"\u003cp\u003e(A~D) Morphology of white blood cells under electron microscopy at different centrifugal speeds (E~F) Platelet morphology under electron microscopy at different centrifugal speeds (G) Cell survival rate of control and treatment groups after CCK8 and trypan blue treatment (H) Flow cytometry of cell apoptosis in the control and treatment groups (I) Cell morphology under light microscopy in the control and treatment groups (J) Cell apoptosis of control and treatment groups\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/152ae618abd6e849d79a900e.png"},{"id":56674209,"identity":"a36bf342-17d2-4cd0-9aa2-a9f28a9733f0","added_by":"auto","created_at":"2024-05-17 15:36:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1959865,"visible":true,"origin":"","legend":"\u003cp\u003e(A) A flow diagram of leukemia mouse model (B) Survival curves of mice in the control and treatment groups (C) Changes in body weight of mice in the control and treatment groups (D) Changes in the number of white blood cells over time (E) Changes in hemoglobin content over time (F) Changes in the number of platelet over time (G) Changes in the number of red blood cells over time (H) Immunophenotype ratio of the control and treatment groups (I~L) The cell flow cytometry of immunophenotype ratio (M) Immunohistochemistry of the control and treatment groups (N) Staining smear of control and treatment groups\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/e82ffc0fbec47abbe7727d52.png"},{"id":61596130,"identity":"6d4f5cba-ff61-4e7d-b4a5-6e40e3780493","added_by":"auto","created_at":"2024-08-01 17:25:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12828518,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4368848/v1/d03a1bf8-6778-4650-b1bb-bd4c10982845.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on Leukapheresis of Hyperleukocyte Acute Myeloid Leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myeloid leukemia(AML) is a common hematopoietic malignant tumor with high heterogeneity and biological complexity. Of the tens of thousands of new cases in the United States each year, nearly one-third of leukemia diagnoses are acute myeloid leukemia, and the incidence increases with age(\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Hyperleukocytic leukemia is a high-risk type of acute myeloid leukemia, which is characterized by an abnormally high number of peripheral white blood cells, exceeding 100\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Hyperleukocytic leukemia accounts for 5% ~ 20% of adult acute myeloid leukemia(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), among which M2 and M5 are the most common types of AML(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). If not treated actively, the 1-week mortality can reach 40%(\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Severe hyperleukocytosis, which causes leukocytostasis, is a medical emergency with a risk of organ damage and is a poor prognostic factor for early death in patients with hyperleukocytosis(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In clinical practice, leukocytosis syndrome most commonly affects the lungs, central nervous system and kidneys, and is associated with pulmonary congestion infection, intracranial hemorrhage or infarction, melanoma, hematuria and other complications, which makes patients progress quickly, and increases the risk of death(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). However, leukocytosis is a poorly understood and life-threatening complication of acute leukemia. Therefore, it is necessary to rapidly reduce white blood cells during treatment. In addition, studies have confirmed that hyperleukocytosis is a poor prognostic factor for early death in hyperleukocyte patients, and the overall survival of patients with hyperleukocyte leukemia is low(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). These studies also suggest that patients with hyperleukocytosis who are suitable for chemotherapy may benefit from leukapheresis to prevent complications such as leukocytostasis that occur before acute myeloid leukemia is diagnosed and chemotherapy is started.\u003c/p\u003e \u003cp\u003eLeukapheresis is a type of physical therapy that reduces the number of white blood cells and blood viscosity in a patient's blood(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), which is still the main treatment for patients with hyperleukocyte leukemia(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Leukapheresis is associated with improved prognosis and is generally safe. However, ,leukapheresis therapy does not achieve the ideal effect at present(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), the reasons may be as follows: First, the time of leukapheresis is too long, which may delay the optimal treatment time of patients, and the leukemia cell bank may be quickly mobilized from the bone marrow, and the original cells in the blood circulation will increase, aggravating the disease(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Second, the intervention of leukapheresis may be too late to reverse the cascade of events that have already begun due to hyperleukocytosis. Third, leukapheresis may damage blood cells, and cell rupture releases a large number of harmful substances, aggravating the disease. Therefore, we should study the procedures of leukapheresis in more detail to improve the purity of blood cells in a short time.\u003c/p\u003e \u003cp\u003eOur study asks whether it is possible to improve the separation efficiency by increasing the centrifugal speed while ensuring less cell damage, and to shorten the time of apheresis treatment and collect white blood cells more quickly by continuing to increase the centrifugal speed. Therefore, how to reduce the damage to cells, and what parameter range is safe to increase the rotational speed has become a major problem we have encountered.\u003c/p\u003e \u003cp\u003eTherefore, in order to make up for the long time of apheresis, find the optimal rotational speed to improve the efficiency of apheresis treatment, gain time for further treatment, and further improve the survival rate of patients with hyperleukocyte acute myeloid leukemia. In this study, we explored the effect of mechanical force on the structure and function of peripheral blood cells in normal peripheral blood cells and patients with hyperleukocyte acute myeloid leukemia after in vitro centrifugation, hoping to find a suitable range of centrifugal rotation speed to improve the rotation speed without damaging normal cells, so as to achieve the purpose of improving the efficiency of apheresis.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cp\u003ePatients\u003c/p\u003e \u003cp\u003eFive healthy people donated blood. Inclusion criteria :1) Age: adult (\u0026ge;\u0026thinsp;18 years old); 2) Normal coagulation function; 3) No genetic history. Exclusion criteria :1) Drinking alcoholic beverages within 1 day before blood donation; 2) Take aspirin and other antiplatelet and anticoagulant drugs within 2 weeks before donating blood.\u003c/p\u003e \u003cp\u003ePeripheral blood of patients with hyperleukocyte acute myeloid leukemia was extracted. It was extracted by the nurse and placed in purple, green and blue anticoagulant tubes. The donors for the sample study signed a written informed consent prior to having their blood drawn. The research program of blood donors has been approved by the local ethics committee, which is in line with China's blood donation guidelines and in line with ethics.\u003c/p\u003e \u003cp\u003eMethods\u003c/p\u003e \u003cp\u003eWhole blood cells were centrifuged at the same time and at different rotational speeds\u003c/p\u003e \u003cp\u003eFirst of all, centrifuge at 0rpm (non-centrifuge), 1500rpm, 3000rpm, 4500rpm, 6000rpm, 7500rpm, 9000rpm, 10500rpm, 12000rpm for ten minutes in centrifuges imported from the regular company (Thermo company). Then centrifuge at room temperature at 3000rpm, 6000rpm, 9000rpm for 10min, 20min, 30min, 40min. The sample was kept consistent from the beginning of processing to the detection time, and the centrifuge temperature was 20℃.\u003c/p\u003e \u003cp\u003eAfter treatment, a part of the blood samples in each group were obtained red blood cell layer, white blood cell platelet layer and plasma layer. After a part of the blood samples were separated, red blood cell lysate was added to the white blood cell platelet layer and supernatant was removed to obtain a relatively pure white blood cell platelet layer. The other part is mixed and tested after blood routine and other items.\u003c/p\u003e \u003cp\u003eCell number morphology, biochemical examination and electron microscope morphology\u003c/p\u003e \u003cp\u003eA part of human whole blood was collected before and after centrifugation to detect blood routine, electrolyte, coagulation and other indexes and red blood cell morphology observation. Cell apoptosis was detected in the cell suspension obtained by centrifugation. The platelet activation rate was measured by flow cytometry after treatment with different centrifugal parameters. The morphology of red blood cell layer (pre-diluted), white blood cell layer and platelet layer were observed under transmission electron microscope.\u003c/p\u003e \u003cp\u003eChromosome breakage experiment and cell microstructure\u003c/p\u003e \u003cp\u003ePeripheral blood samples after centrifugation were added to 3 identical peripheral blood media, and MMC was added to make the final concentrations of 0ng/ml, 50ng/ml and 100ng/ml, respectively. After a series of operations such as culture, harvest and preparation, chromosome breakage was observed under microscope.\u003c/p\u003e \u003cp\u003eHomogeneous trace cells were recorded by taking photos, and the average cell size was measured, that is, the average cell radius was calculated to measure the changes in cell size, and the changes in cell center point, brightness, image contrast and other changes were calculated to understand the changes in cell surface. In addition, the red blood cell layer, white blood cell layer and platelet layer after centrifugation were separated and labeled, and the cell morphology was recorded by single cell imaging technology of photofluid time stretch microscope. The obtained cell images were processed and analyzed by computer.\u003c/p\u003e \u003cp\u003eCell cryopreservation was performed under optimal centrifugation parameters\u003c/p\u003e \u003cp\u003ePeripheral blood samples of newly diagnosed patients with hyperleukocyte acute myeloid leukemia M5 requiring cryopreservation were collected. The experimental group used the optimal centrifugation parameters previously explored, and the control group was centrifuged at 3000rpm for 10min. After centrifugation, the mononuclear cell layer was absorbed and mixed with cleaning solution. The treated mononuclear cells were transferred to a sterile cryopreserved tube, and autologous plasma and DMSO (the ratio is 9:1) were added for cell cryopreserved. After 1 month, the frozen tube was taken out and placed in 37℃ water bath for cell resuscitation. The supernatant was removed by centrifugation and stained with pancreatic blue. The cell survival rate was calculated three times to take the average value. After 6 months of frozen storage, the frozen tube was taken out and placed in 37℃ water bath for cell resuscitation. After centrifugation, the uniformly obtained cell suspension was re-suspended with DMEN medium and cultured with CCK8 to determine the cell viability. Three times the volume of red cell lysate was added to the cell suspension obtained by centrifugation, and apoptosis was detected.\u003c/p\u003e \u003cp\u003eHuman acute myeloid leukemia mouse model was established with the cells obtained under optimal centrifugation parameters\u003c/p\u003e \u003cp\u003eExperimental cells were taken from the above frozen cells and, again, divided into experimental and control groups according to whether or not centrifugation was performed with optimal parameters. The experimental animals were immunodeficient B-NSG mice (qualification number: SCXK(Beijing)2022-0004) purchased at 4\u0026ndash;5 weeks. One week after purchase, each mouse was given 2.0Gy X-ray irradiation, and on the second day, acute myeloid leukemia cells (CD33CD117) were injected intravenously through the tail vein to establish a human acute myeloid leukemia mouse model. After the model was built, the weight changes of the mice were observed, and the mice were killed 3 weeks later, and the survival time of each group was recorded. The mouse bone marrow was collected and the erythrocytes were lysed after the bone marrow was rinsed. Then the mononuclear cells in the bone marrow were collected, and flow cytometry was performed to detect the change of the proportion of original cells in the bone marrow of mice in each group. The expression of CD117 in leukemia xenografts was detected by immunohistochemistry.\u003c/p\u003e \u003cp\u003eStatistics\u003c/p\u003e \u003cp\u003eThe significance of differences between two groups was determined using unpaired two-tailed Student t tests. All results in bar graphs are mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Overall survival curves were plotted according to the Kaplan-Meier method with the log-rank test applied for comparisons. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe centrifugal speed affects the morphology of red blood cells, but does not affect the number of red blood cells\u003c/p\u003e \u003cp\u003eThere was no difference in the number of RBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), HGB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), MCV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), HCT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), MCH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), RDW (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), reticulocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), and MCHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK) under different centrifugal forces (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRed blood cells at 9000 and 12000rpm showed burr changes under erythroscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, L).Normal red blood cell morphology at 6000rpm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). 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.\u003c/p\u003e \u003cp\u003eThe centrifugal speed and time basically did not damage the white blood cells\u003c/p\u003e \u003cp\u003eThere were no significant differences in the number of leukocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and monocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) under different centrifugal forces (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). There was no significant difference in the number of white blood cells at 10min, 20min, 30min and 40min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The morphology of white blood cells in a and b (6000rpm and 12000rpm) was normal under the peripheral blood smear microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H). Meanwhile, difference in the morphology of leukocytes under electron microscopy was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Therefore, we know that the centrifugal speed and time have little effect on the damage of white blood cells.\u003c/p\u003e \u003cp\u003eWhen the centrifugal speed was 6000rpm, platelets could maintain low activation rate and normal morphology, but the activation rate increased with longer time\u003c/p\u003e \u003cp\u003eThe number of platelets decreased at 10500rpm (P\u0026thinsp;\u0026lt;\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The mean platelet volume increased at 12000rpm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At 6000rpm, platelet activation did not exceed 5%, which was within the normal range (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). After centrifugation for 10, 20, 30, and 40min at 1500rpm, platelet activation rates gradually increased, but all were within the normal range (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG shows that platelet activation did not exceed the normal range at 10, 20, 30, and 40min centrifugation. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF shows platelet aggregation in the 7500 and 12000rpm groups in peripheral blood smears.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I, J, K, L, M).\u003c/p\u003e \u003cp\u003eObservation 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN).\u003c/p\u003e \u003cp\u003eCoagulation factor decreased gradually with the increase of centrifugal speed\u003c/p\u003e \u003cp\u003eWith the increase of centrifugal speed, we can see a gradual decline in factors Ⅷ, Ⅸ, Ⅺ and Ⅻ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H, I, J), and APTT lengthens at 12000rpm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Therefore, we hypothesize that endogenous coagulation pathways are activated at centrifugal speeds above 9000rpm. Combined with Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, it is shown that different rotational speeds may change the structural changes within platelets, causing platelets to aggregate and activate, and affecting coagulation factors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe centrifugal speed did not affect the chromosome structure and morphology of blood cells\u003c/p\u003e \u003cp\u003eThe 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, M, ). It should be added that calcium ions did not decrease significantly under different centrifugal speeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), which may be due to the influence of EDTA and sodium tenuate in the blood collection tube, so the results may not be representative.\u003c/p\u003e \u003cp\u003eThe optimum centrifugal parameters were 6000rpm, 10min\u003c/p\u003e \u003cp\u003eFrom 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 9000rpm and above, the damage to platelets was greater, but the effect on red blood cells and white blood cells was less\u003c/p\u003e \u003cp\u003ePlatelets 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ, 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell cryopreservation and establishment of leukemia mouse model under optimal centrifugation parameters\u003c/p\u003e \u003cp\u003eFrom 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor different components of blood cells, in order to make different blood components better separated, in the specific separation operation, the parameter that is actually controllable and adjusted is the centrifuge speed. The main functions of centrifuge speed adjustment are as follows: 1) Quickly establish a good separation interface for different components of blood, use the shortest possible separation time, and improve the separation efficiency under the premise of preserving cell activity; 2) To ensure the separation effect of different components of blood, so that the separation of target demand components meets the corresponding standards, and meets the indicators of blood collection for medical use.\u003c/p\u003e \u003cp\u003eA study found that rotational speed had the greatest effect on the separation efficiency of the platelet separation efficiency model, while the effect on the separation efficiency of red and white blood cells at high rotational speed was negligible(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Our study found that red blood cell morphology changed at and above 9000rpm, but the range of changes in hemoglobin, red blood cell number and hematocrit was too small, with no statistical difference, which was consistent with literature reports, and the loss of red blood cells at 6000rpm was almost zero. From this, we can see that at 9000rpm, the morphology of red blood cells is spiny, but the cell membrane is relatively intact, and the rate of red blood cell lysis is too small to be ignored.\u003c/p\u003e \u003cp\u003eHowever, platelets have different degrees of damage at high rotational speed, so the best parameters for centrifugation should also refer to the damage of platelets. Our study found that the number of platelets did not decrease significantly below 6000rpm, while the number of platelets decreased at 7500 and 9000rpm.Platelet activation was within the normal range at 6000rpm, and beyond the normal range at 7500rpm and above. In addition, platelets had normal morphology under electron microscope with no foot process, fewer Alpha particles in cells, fewer mitochondrial vacuoles, and elongated morphology of foot process at 7500rpm when the rotational speed was 3000 and 6000rpm, respectively. At 9000rpm, the platelet foot mutation was varied and long, and at 12000rpm, the number of platelet Alpha particles was more and the platelets gathered together.\u003c/p\u003e \u003cp\u003eWhen centrifuged at 12000rpm, the biggest changes in platelet morphology were alpha particles, dense particles and foot process. It has been documented that Alpha particles contain membrane binding proteins and soluble proteins that are involved in various processes, including cell adhesion, coagulation, inflammation, cell growth, and host defense(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). After platelet activation, Alpha particles release fibrinogen and VWF, promoting platelet-platelet and platelet-endothelial cell interactions. In addition, components of the fibrinogen receptor Alpha ⅡBβ3, collagen receptor GPVI, and VWF receptor complex GPIb-IX-V found in Alpha particles are expressed on the surface of platelets and subsequently support platelet adhesion(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Alpha particles contain clotting mediators such as factor VIII and other clotting factors, which may affect the coagulation environment of the plasma, that is manifested as the reduction of some coagulation factors, the abnormal time of some thromboplastin, and the destruction of endogenous coagulation pathway.\u003c/p\u003e \u003cp\u003eIn this experiment, we tried for the first time to extract peripheral blood mononuclear cells with the optimal centrifugation parameters explored, and successfully frozen cells with autologous plasma. The survival rate of cells after resuscitation was above 95%, which laid a solid foundation for the establishment of blood cell sample bank in the future.\u003c/p\u003e \u003cp\u003eWe also successfully established a mouse model of acute myeloid leukemia with the optimum centrifugation parameters. We know that mouse models of acute myeloid leukemia are indispensable research tools. For example, extensive sequencing efforts have mapped the genomes of adults and children with acute myeloid leukemia, revealing many biological and prognostic drivers. In addition to identifying recurrent genetic aberrations, it is critical to adequately describe the complex mechanisms by which they contribute to the onset and evolution of disease, ultimately facilitating the development of targeted therapies. To achieve these goals, rapid advances in genetic engineering techniques over the past 20 years have greatly facilitated the use of mouse models to reflect specific genetic subtypes of human acute myeloid leukemia, define intracellular and external disease mechanisms, study interactions between co-occurring genetic lesions, and test new treatments. Therefore, it is of great significance to establish a mouse AML model. The cells obtained by using the best centrifugation parameters had less cell damage and improved the purity of mononuclear cells collected. The fast reading established a boundary layer to reduce the number and time of centrifugation. Compared with low-speed centrifugation, the cells could be separated faster and more accurately, and the survival rate after cell cryopreservation and recovery was similar to that obtained by low-speed centrifugation. The mouse model of AML was also established successfully. These results indicate that the centrifuged cells under optimal centrifugation parameters can be used for cryopreservation of clinical samples and establishment of mouse models of acute myeloid leukemia in laboratory.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAt present, the clinical use of leukapheresis can not achieve the ideal effect, and it takes a long time and causes great damage to cells. It is an urgent problem to further improve leukapheresis, explore the best centrifugal parameters, realize fast and accurate leukocyte sorting, and minimize the possible adverse reactions in the process. In this study, by changing the centrifugal speed, it was observed that low-speed centrifugation was relatively safe for peripheral blood cells, while high-speed centrifugation would cause platelet activation and aggregation when it reached 12000rpm, and the best possible centrifugation parameter was found at 6000rpm, 10min. The cells obtained by centrifugation with the best parameters we explored could be frozen, and the cell viability was above 95%, and the mouse model of AML could be successfully established.\u003c/p\u003e \u003cp\u003eThe results of this experimental study are of great significance for the improvement of clinical leukocyte reduction therapy, which can improve the efficiency of leukapheresis therapy, reduce the treatment cost, guide the clinical prevention and treatment of adverse reactions in the process of leukapheresis therapy without increasing the risk related to treatment, and provide more scientific information for the evaluation of the collection effect of leukapheresis therapy and more scientific direction for the improvement of treatment instruments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences (Approval No. 202020). The study scheme was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University. All patients signed informed consent forms and submitted them to Zhongnan Hospital for safekeeping.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the submission of the final manuscript. No conflicts of interest were declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: RYP, AJX, and FLZ; Methodology: RYP, AJX and LL; Validation: FLZ; Formal analysis: AJX and LL; Investigation: RYP, AJX, LL, JXW, XQL, GPC, WYY and RHL; Resources: AJX and DDL; Data Curation: RYP, AJX and LL; Writing - Original Draft: RYP and AJX; Writing - Review \u0026amp; Editing: FLZ; Visualization: RYP; Supervision: XYL and FLZ; Project administration: AJX, LL and FLZ; Funding acquisition: XYL, and FLZ. All authors reviewed and authorized the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments and fundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all of the patients who took part in this study, as well as their families. This study was funded by the Natural Science Foundation of China program [grant numbers 81900116, 82370176], and the Zhongnan Hospital of Wuhan University discipline construction platform project [grant numbers 202021, PDJH202217].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi Z, Philip M, Ferrell PB. Alterations of T-cell-mediated immunity in acute myeloid leukemia. Oncogene. 2020;39(18):3611\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarrero RJ, Lamba JK. Current Landscape of Genome-Wide Association Studies in Acute Myeloid Leukemia: A Review. Cancers (Basel). 2023;15(14).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePelcovits A, Niroula R. Acute Myeloid Leukemia: A Review. R I, Med J. (2013). 2020;103(3):38\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarid KMN, Sauer T, Schmitt M, Muller-Tidow C, Schmitt A. Symptomatic Patients with Hyperleukocytic FLT3-ITD Mutated Acute Myeloid Leukemia Might Benefit from Leukapheresis. Cancers (Basel). 2023;16(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarbello L, Ricci F, Nosari AM, Turrini M, Nador G, Nichelatti M, et al. Outcome of hyperleukocytic adult acute myeloid leukaemia: a single-center retrospective study and review of literature. Leuk Res. 2008;32(8):1221\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePorcu P, Cripe LD, Ng EW, Bhatia S, Danielson CM, Orazi A, et al. Hyperleukocytic leukemias and leukostasis: a review of pathophysiology, clinical presentation and management. Leuk Lymphoma. 2000;39(1\u0026ndash;2):1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStone RM, DeAngelo DJ, Janosova A, Galinsky I, Canning C, Ritz J, et al. Low dose interleukin-2 following intensification therapy with high dose cytarabine for acute myelogenous leukemia in first complete remission. Am J Hematol. 2008;83(10):771\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli AM, Mirrakhimov AE, Abboud CN, Cashen AF. Leukostasis in adult acute hyperleukocytic leukemia: a clinician's digest. Hematol Oncol. 2016;34(2):69\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng S, Zhou L, Zhang X, Tang B, Zhu X, Liu H, et al. Impact Of ELN Risk Stratification, Induction Chemotherapy Regimens And Hematopoietic Stem Cell Transplantation On Outcomes In Hyperleukocytic Acute Myeloid Leukemia With Initial White Blood Cell Count More Than 100 x 10(9)/L. Cancer Manag Res. 2019;11:9495\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, He H, He J, Gu X, Hu P, Zuo R, et al. Hyperleukocytic Acute Leukemia Circulating Exosomes Regulate HSCs and BM-MSCs. J Healthc Eng. 2021;2021:9457070.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerrano M, Chevret S, Raffoux E, Rabian F, Sebert M, Valade S, et al. Benefits of dexamethasone on early outcomes in patients with acute myeloid leukemia with hyperleukocytosis: a propensity score matched analysis. Ann Hematol. 2023;102(4):761\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShallis RM, Stahl M, Bewersdorf JP, Hendrickson JE, Zeidan AM. Leukocytapheresis for patients with acute myeloid leukemia presenting with hyperleukocytosis and leukostasis: a contemporary appraisal of outcomes and benefits. Expert Rev Hematol. 2020;13(5):489\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRollig C, Ehninger G. How I treat hyperleukocytosis in acute myeloid leukemia. Blood. 2015;125(21):3246\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIqbal M, Mukhamedshin A, Lezzar DL, Abhishek K, McLennan AL, Lam FW, et al. Recent advances in microfluidic cell separation to enable centrifugation-free, low extracorporeal volume leukapheresis in pediatric patients. Blood Transfus. 2023;21(6):494\u0026ndash;513.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBewersdorf JP, Zeidan AM. Hyperleukocytosis and Leukostasis in Acute Myeloid Leukemia: Can a Better Understanding of the Underlying Molecular Pathophysiology Lead to Novel Treatments? Cells. 2020;9(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovotny JR, Nuckel H, Duhrsen U. Correlation between expression of CD56/NCAM and severe leukostasis in hyperleukocytic acute myelomonocytic leukaemia. Eur J Haematol. 2006;76(4):299\u0026ndash;308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInaba H, Fan Y, Pounds S, Geiger TL, Rubnitz JE, Ribeiro RC, et al. Clinical and biologic features and treatment outcome of children with newly diagnosed acute myeloid leukemia and hyperleukocytosis. Cancer. 2008;113(3):522\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang MC, Chen TY, Tang JL, Lan YJ, Chao TY, Chiu CF, et al. Leukapheresis and cranial irradiation in patients with hyperleukocytic acute myeloid leukemia: no impact on early mortality and intracranial hemorrhage. Am J Hematol. 2007;82(11):976\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrower V, Adhesion, Molecules. Stem Cells, and the Microenvironment in Acute Myeloid Leukemia. J Natl Cancer Inst. 2016;108(4).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hyperleukocyte Acute Myeloid Leukemia, Leukapheresis, Centrifugation","lastPublishedDoi":"10.21203/rs.3.rs-4368848/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4368848/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRespiratory failure, intracranial hemorrhage and infection were more common in hyperleukocyte acute myeloid leukemia patients than in non-hyperleukocyte leukemia patients. Compared with non-apheresis treatment, the white blood cells decreased significantly and the infection rate decreased after apheresis treatment. However, the treatment time of leukapheresis in patients with hyperleukocyte leukemia is very long, while the damage to cells is also large. In this study, a retrospective analysis was conducted on hyperleukocyte acute myeloid leukemia patients with hyperleukocytosis during induction. Centrifugation was performed at different rotational speeds and centrifugation times to observe whether there were changes in the number and morphology of peripheral blood cells in healthy people and patients with hyperleukocyte leukemia. The centrifugation of normal cells and hyperleukocyte was simulated in vitro, so as to explore optimal centrifugation parameters in the treatment of leukapheresis. The cells obtained by optimal centrifugation parameters were cryopreserved and animal models were established. Through the research, it is found that when the rotational speed is increased below 6000rpm, the damage to normal blood cells and blood cells in patients with hyperleukocyte leukemia is small. When the rotational speed is greater than 6000rpm, the platelets will be damaged. The cells obtained under optimal centrifugation parameters can be successfully cryopreserved and modeled in leukemia animals.\u003c/p\u003e","manuscriptTitle":"Study on Leukapheresis of Hyperleukocyte Acute Myeloid Leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-17 15:28:13","doi":"10.21203/rs.3.rs-4368848/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-17T06:42:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-16T19:07:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266534647382339366727117023420817428019","date":"2024-05-08T17:02:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-07T22:18:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196620457193894718948405217593758924676","date":"2024-05-07T17:37:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-07T17:16:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-05-07T17:00:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-07T05:35:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-07T05:35:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2024-05-04T14:33:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b65c7bf7-9e40-46e3-ad35-a89cdcbce6c0","owner":[],"postedDate":"May 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:06:28+00:00","versionOfRecord":{"articleIdentity":"rs-4368848","link":"https://doi.org/10.1186/s12885-024-12644-5","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2024-07-24 16:15:34","publishedOnDateReadable":"July 24th, 2024"},"versionCreatedAt":"2024-05-17 15:28:13","video":"","vorDoi":"10.1186/s12885-024-12644-5","vorDoiUrl":"https://doi.org/10.1186/s12885-024-12644-5","workflowStages":[]},"version":"v1","identity":"rs-4368848","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4368848","identity":"rs-4368848","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}