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Platelet-rich plasma/prp, tissue regeneration, platelet activation, glucose proliferative therapy, platelets, proliferative therapy
Cite this article as: Harrison TE, Bowler J, Reeves K, et al. (May 17, 2022) The effect of glucose on platelet count and volume: implications for regenerative medicine. Cure 14(5): e25081. doi:10.7759/cureus.25081
Platelet-rich plasma (PRP) and hypertonic glucose solutions are commonly used for injection in regenerative medicine, sometimes together. The effect of hypertonic glucose on platelet lysis and activation has not been previously reported. We tested the effect of elevated glucose concentrations on platelet and erythrocyte counts, as well as cell volumes in PRP and whole blood (WB). A rapid partial reduction in platelet count occurred with all glucose mixtures mixed with PRP or whole blood, consistent with partial lysis. After the first minute, platelet counts remained stable, suggesting a rapid accommodation of residual platelets to extreme (>2000 mOsm) hypertonicity. After the first minute, platelet counts remained stable, suggesting a rapid accommodation of residual platelets to extreme (>2000 mOsm) hypertonicity. После первой минуты количество тромбоцитов оставалось стабильным, что указывает на быструю аккомодацию остаточных тромбоцитов до экстремального (>2000 мОсм) гипертонуса. After the first minute, the platelet count remained stable, indicating a rapid accommodation of the residual platelets to extreme (>2000 mOsm) hypertonicity.第一分钟后，血小板计数保持稳定，表明残余血小板迅速适应极端（> 2000 mOsm）高渗状态。 2000 mOsm）高渗状态。 После первой минуты количество тромбоцитов оставалось стабильным, что указывает на быструю адаптацию остаточных тромбоцитов к экстремальному (>2000 мОсм) гиперосмолярному состоянию. After the first minute, the platelet count remained stable, indicating a rapid adaptation of the residual platelets to the extreme (>2000 mOsm) hyperosmolar state. Glucose concentrations of 25% and above resulted in a significant increase in mean platelet volume (MPV), indicating an early stage of platelet activation. Further studies are needed to determine whether platelet lysis or activation occurs and whether hypertonic glucose injection alone or in combination with PRP may provide additional clinical benefit.
In the 1950s, American surgeon George Hackett discovered that he could permanently relieve joint and back pain in many patients by injecting a proliferative solution into tendons and ligaments. His experiments on rabbits showed that the treatment, which he called proliferative therapy, caused the tendons to enlarge and strengthen. Histological studies have confirmed that new collagen is produced during this process [1].
During the first few decades, many different distribution solutions were tried. By the 1990s, most practitioners considered high concentrations of glucose to be the safest and most effective method. However, the mechanism of action remains unclear.
Few clinical studies were conducted in the 20th century following Hackett’s work. However, in the 2000s there was renewed interest and several successful clinical trials of proliferative therapy were completed for the treatment of low back pain [2], osteoarthritis of the knee [3], and lateral epicondylitis [4].
Tissue regeneration requires the participation of stem cells. Therefore, high concentrations of glucose must somehow induce migration, replication, and differentiation of stem cells. We hypothesize that platelets may act as messengers and that high glucose concentrations may cause platelets to release cytokines and growth factors, thereby promoting regenerative processes, especially stem cell migration to areas of high glucose concentrations.
Platelet activation always precedes an increase in intracellular calcium [5]. Liu et al. in 2008 showed that high glucose levels increase the activity of transient receptor potential canonical type 6 (TRPC6) channels in the plasma membrane, which leads to an influx of calcium ions into platelets [6]. Another study showed that exposure of the microtubule marginal zone to calcium ions causes relaxation, expansion, and deformation of the marginal zone, which in turn causes a change in shape from disc to spherical, resulting in mean platelet volume (MPV) [7].
Our hypothesis in this study is that exposure of platelets to high concentrations of glucose affects the microtubule marginal zone and intracellular environment, leading to an increase in MPV.
All participants signed an informed consent form after the details of the study were explained and prior to receiving the samples. In this study, only PRP samples with a hematocrit greater than 2% were used so that erythrocyte (erythrocyte) count and mean corpuscular volume of red blood cells (MCV) could be included for comparison.
The study was conducted in four phases, the first phase was PRP and the remaining phases were whole blood (Table 1). As described previously [8], all relative centrifugal forces (RCF, g-force) were calculated from the midpoint (Rmid, in cm) of the blood column in the centrifugal syringe. We chose to use MPV as a marker of platelet sensitization and platelet count as an indicator of potential platelet lysis, both of which can be easily measured on standard hematology analyzers.
In the first phase, 47 volunteers donated blood samples—one tube of ethylenediaminetetraacetic acid (EDTA) and one PRP whole blood sample (anticoagulated with sodium citrate (NaCl, 3%)) (Table 1). Place the rocker in the tube immediately. Complete blood count (CBC) was performed on EDTA samples in triplicate, and NaCl samples were analyzed in triplicate for CBC analysis, and then PRP was prepared by various methods described above [8]. All PRP samples were prepared by centrifugation at 900–1000 g. Mix each PRP sample on a vortex mixer for 5–10 seconds, then divide five 0.5 ml aliquots into tubes.
To evaluate the effect of platelet exposure on elevated glucose concentrations, equal amounts (0.5 ml) of 0%, 5%, 12.5%, 25%, and 50% glucose in water were mixed with platelet samples to obtain 0%, 2.5 % 6.25%, 12.5% ​​and 25% concentrations of the glucose mixture and mix the tubes on a test tube shaker for 15 minutes. The TAC of each mixture was analyzed in triplicate after 15 min. Platelet count (PLT), RBC count, MCV, and MPV were averaged for each tube, and mean platelet count, RBC count, MCV, and MPV were calculated for all PRP samples.
After the first phase of data collection was completed, we noticed a significant increase in platelet volume in PRP platelets after the addition of D50W. PRP platelets do not necessarily represent all platelets in the blood, and PRP medium differs from WB medium. Therefore, we decided to conduct a second phase trial of the effect of adding D50W to whole blood.
For the second round, we chose a sample size of 30 based on the results from the first series, as described in the Analysis section. In this series, 20 volunteers donated blood samples (Table 1). Whole blood (1.8 ml) was drawn into a 3 ml syringe and anticoagulated with 0.2 ml 40% NaCl. The whole blood syringe was mixed for five seconds with a vortex mixer and the CBC was analyzed in triplicate. After analysis, anticoagulated blood was added to 2 ml of 50% glucose in a 5 ml syringe (final glucose concentration was approximately 25% (D25) and placed in a shake tube for 30 minutes. After 30 minutes, D25/CBC in WB syringes were analyzed in triplicate. Platelet count, RBC count, MCV, and MPV per syringe were averaged, and mean PLT, RBC count, MCV, and MPV were calculated for each sample before and after adding glucose.
Because platelets in whole blood are commonly exposed to hypertonic glucose during proliferative glucose therapy due to minimally invasive injection, and it is not common to combine PRP with hypertonic glucose just prior to injection, we decided to study hypertonic glucose in combination with WB in Section 1. Step Three and four. At each stage, 20 volunteers donated 7-8 ml of ACD-A (acid containing trisodium citrate (22.0 g/l), citric acid (8.0 g/l) and glucose (24.5 g/l), solution dextrose citrate) for blood anticoagulants (Table 1). Only mixtures of glucose greater than 12.5% ​​were used to determine the threshold percentage associated with an increase in MPV. At the third stage, 1 ml of blood is placed in a test tube. Then mix the blood on a vortex mixer for 10 seconds by adding 1 ml of 30% glucose, 40% glucose, or 50% glucose to the tube to obtain a final glucose concentration of 15%, 20%, and 25%, respectively. Glucose blood samples were analyzed for CBC immediately after mixing and repeated every two minutes for 30 minutes.
During initial mixing, the addition of 1:1 hypertonic glucose and WB or PRP exposes platelets to concentrations above 25% for several seconds. In the fourth step, to evaluate the effect of hypertonic glucose with minimal initial peak concentrations and test the upper limit of the effect of glucose, we added only a small amount of blood to D25W or D50W. Place 1 ml of D25W or D50W in a tube and add 0.2 ml of WB while vortexing the sample for 10 seconds. In these cases, the blood was exposed to glucose at a concentration approximately 20% above the final concentration, rather than 50% above the final concentration as in Phase 3, resulting in final glucose concentrations of 20.8% and 41.6%. Mixed samples were analyzed at the same time interval as in step 3.
In the first step of each glucose dilution series, 30 samples were taken as this was the appropriate sample size for the pilot study [9]. At the end of each phase (including the first phase), evaluate the adequacy of the sample size using the formula used to determine the sample size needed to estimate the mean of the continuous outcome variable in one population. Formula n = Z2 x SD2 /E2. In this equation, Z is the Z-score, SD is the standard deviation, and E is the desired error [10]. Our alpha is 0.05, which corresponds to a Z value of 1.96, and we expect an error of 5 (in percent). Hence we solve for n = (1.962 x SD2)/52. The results showed that the sample size required for each stage was smaller than the actual number collected.
During periods 1, 3 and 4 using more than one glucose concentration, the effect of different glucose concentrations was analyzed by comparing the fractional change between time 0 and each subsequent time (phase 1 at 15 minutes, period 3 at 15 minutes). and four at 15 seconds, then every two minutes.) Change rates for each time period were compared using the Mann-Whitney U-test because the data did not follow a normal distribution as determined by the Shapiro-Wilk normality test. Since a 1-to-1 analysis of several groups (five) was performed in the first, third and fourth steps (five in total), a Bonferroni correction was performed to adjust the desired alpha value to ≤0.01 but not ≤0.05.
Reduction of platelet count with all concentrations of hypertonic dextrose and an increase in MPV in PRP platelets at >12.5% dextrose concentration: PRP platelet counts rose from one to five times concentration compared to baseline whole blood, varying by the method (not depicted). Reduction of platelet count with all concentrations of hypertonic dextrose and an increase in MPV in PRP platelets at >12.5% ​​dextrose concentration: PRP platelet counts rose from one to five times concentration compared to baseline whole blood, varying by the method (not depicted). Уменьшение количества тромбоцитов при всех концентрациях гипертонической декстрозы и увеличение MPV в тромбоцитах PRP при концентрации декстрозы > 12,5%: количество тромбоцитов PRP увеличилось в 1-5 раз по сравнению с исходной цельной кровью, в зависимости от метода (не показано). Decreased platelet count at all hypertonic dextrose concentrations and increased MPV in PRP platelets at >12.5% ​​dextrose concentration: PRP platelet count increased 1-5 times compared to baseline whole blood, depending on method (not shown). ).在> 12.5% 的葡萄糖浓度下，所有浓度的高渗葡萄糖降低血小板计数，PRP 血小板中MPV 增加：与基线全血相比，PRP 血小板计数从浓度的1 倍上升到5 倍，因方法而异（未描述）。 At >12.5% ​​glucose concentration, the high concentration of glucose reduces the blood count, PRP blood MPV increases: compared to 与基线全血, the PRP blood count increases from 1 to 5 times that of the concentration (not described). При концентрациях глюкозы >12,5% все концентрации гипертонической глюкозы снижали количество тромбоцитов, а MPV повышали в тромбоцитах PRP: количество тромбоцитов PRP увеличивалось от 1- до 5-кратных концентраций по сравнению с исходными концентрациями цельной крови, в зависимости от метода (не описано ). At glucose concentrations >12.5%, all hypertensive glucose concentrations decreased platelet counts and increased MPV in PRP platelets: PRP platelet counts increased 1- to 5-fold compared to baseline whole blood concentrations, depending on the method (as described ). Figure 1 shows that the number of platelets decreased by almost 75% after dilution in water and by 20-30% after 15 minutes of dilution with different concentrations of glucose compared to baseline PRP and a 1:1 dilution adjusted for volume (1- k1 with volume correction). k -1 breeding).1 breeding).
The number of cells in each dilution is expressed as a fraction of the original number before dilution.
MPV decreased minimally during PRP production, without further change in dilution concentrations to 12.5% ​​in water or glucose (including 25% PRP glucose mixtures) and increased by more than 20% after dilution in 50% glucose solution (Fig. .2). ). In contrast, erythrocytes showed no significant change in volume at any dilution other than H2O.
The average volume of cells in each dilution is expressed as a percentage of the original volume before dilution.
A similar but less pronounced reduction in platelet count and increase in CVR were observed in BC exposed to 50% glucose (to formulate with 25% glucose). Table 2 compares cell numbers and cell volumes in whole blood diluted in 50% dextrose with phase 1 PRP data diluted in 50% dextrose. Changes in RBC count and RBC MCV were not obvious and were not the focus of our attention.
SD = standard deviation, MD = mean difference between groups, SE = standard deviation of mean difference, RBC = erythrocytes, PLT = platelets, PRP = platelet rich plasma, WB = whole blood
After adding D50W to WB, the percentage dilution-adjusted platelet loss was 7.7% (310±73 vs. 286±96) compared to 17.8% for PRP dilution in D50W (664±348 vs. 544±277). MPV WB increased by 16.8% (from 10.1 ± 0.5 to 11.8 ± 0.6), while MPV PRP increased by 26% (9.2 ± 0.8 vs. 11.6 ± 0. 7). Although the mean differences in both platelet count reduction and MPV increase were significantly more with PRP, the changes in platelet count reduction within WB were nearly significant (310 ± 73 to 286 ± 96 (-7.7%); p = .06) and the increase in MPV was significant (10.1 ± 0.5 to 11.8 ± 0.6 (+16.8) p < .001). Although the mean differences in both platelet count reduction and MPV increase were significantly more with PRP, the changes in platelet count reduction within WB were nearly significant (310 ± 73 to 286 ± 96 (-7.7%); p = .06) and the increase in MPV was significant (10.1 ± 0.5 to 11.8 ± 0.6 (+16.8) p < .001). Although the mean differences in both platelet count reduction and CVR increase were significantly greater with PRP, changes in platelet count decline within WB were almost significant (310 ± 73 to 286 ± 96 (-7.7%); p = 0.06). увеличение MPV было значительным (от 10,1 ± 0,5 до 11,8 ± 0,6 (+16,8) p < 0,001). the increase in MPV was significant (from 10.1 ± 0.5 to 11.8 ± 0.6 (+16.8) p < 0.001).尽管PRP 在血小板计数减少和MPV 增加方面的平均差异显着更大，但WB 内血小板计数减少的变化几乎是显着的（310 ± 73 至286 ± 96 (-7.7%)；p = .06）和MPV 的增加是显着的（10.1 ± 0.5 到11.8 ± 0.6 (+16.8) p < .001）。尽管 PRP 在 血小板 计数 和 和 增加 方面 的 平均 差异 显着 大 ， 但 但 内血小板 计数 减少 的 几乎 是 显着 的 （（（310 ± 73 至 286 ± 96 (-7.7%) ； p = .06）和MPV 的增加是显着的（10.1 ± 0.5 到11.8 ± 0.6 (+16.8) p < .001）。 The change in platelet count reduction within the WB was nearly significant (from 310 ± 73 to 286 ± 96 (-7.7%); p = 0.06), although PRP had significantly larger mean differences in platelet count decline and MPV increase. and the increase in MPV was significant. (от 10,1 ± 0,5 до 11,8 ± 0,6 (+16,8) р < 0,001). (from 10.1 ± 0.5 to 11.8 ± 0.6 (+16.8) p < 0.001).
A final concentration of 20% glucose was required to see a significant change in MPV, but the change in MPV was more pronounced at the final concentration of 25%. Platelet loss stabilized after the initial decline. We noted an initial sharp decrease in CVR, however, CVR was rapidly restored at the 25% final glucose concentration, which was significantly higher than the CVR levels observed at the final glucose concentrations of 20% and 15% (Fig. 3 and to the left of Table 3; shaded boxes). indicate p-values ​​≤ alpha with a Bonferroni correction of 0.01). There was also an initial sharp drop in the number of PLT, observed in the initial phase of 0-15 s, and then remained stable (from 15 s to 30 min; left of table 4).
The addition of various concentrations of glucose to whole blood resulted in an initial rapid decrease in MPV followed by a concentration-dependent recovery of more than 20%. The legend shows the concentration of glucose after dilution. D15, D20 and D25 were carried out in a 1:1 dilution. D21 and D41 were carried out at a 1:5 dilution.
Table 4 shows the change in platelet count when diluted in hypertonic glucose. We observed a dose-dependent relationship between the immediate drop in PLT numbers at the 1:1 dilution and at the 1:5 dilution. Comparing the 1:1 dilutions as a single group with the 1:5 dilutions, the 1:1 group had an immediate decrease in platelet count less than the 1:5 group 66±48,000 (23%) versus 99±69,000 (37%). , p = 0.014) in the 1:5 group. After an initial drop at the first measurement point, the platelet count as a percentage of glucose stabilized (Fig. 4).
When whole blood is added to glucose in a 1:1 ratio, the platelet count is reduced by about 25%. However, when whole blood was added at a ratio of 1:5, the reduction was much greater – about 50%.
41% glucose increased MPV faster and more dramatically than 25% or 21%. MPV results are shown in Figure 3. At all other dilutions, no immediate initial decrease in MPV was observed after addition of 50% glucose. When using 25% glucose (glucose concentration 20.8% at the final dilution), the change in MPV was comparable to the change in 20% glucose at a 1:1 dilution (Fig. 3). Although changes in MPV were initially greater at the 41% mixed concentration than at 25%, the difference in MPV between 41% and 25% after 16 minutes was no longer significant (Table 3, right). It is also interesting that 25% glucose increased MPV more effectively than 20.8%.
This in vitro study partially confirmed our hypothesis. It showed potential partial platelet lysis by dextrose admixture, a rapid accommodation of platelets to extreme hypertonicity, and a significant rise in MPV in response to > 25% concentrations of hypertonic dextrose. It showed potential partial platelet lysis by dextrose admixture, a rapid accommodation of platelets to extreme hypertonicity, and a significant rise in MPV in response to > 25% concentrations of hypertonic dextrose. Он показал потенциальный частичный лизис тромбоцитов примесью декстрозы, быструю аккомодацию тромбоцитов до экстремального гипертонуса и значительное повышение MPV в ответ на гипертоническую концентрацию декстрозы > 25%. It showed potential partial platelet lysis with dextrose, rapid platelet accommodation to extreme hypertonicity, and a significant increase in MPV in response to hypertonic dextrose levels >25%.它显示出通过葡萄糖混合物潜在的部分血小板溶解，血小板快速适应极端高渗，以及响应> 25% 浓度的高渗葡萄糖时MPV 显着上升。它 显示 出 通过 葡萄糖 潜在 的 部分 血小板 溶解 血小板 快速 适应 极端 高渗 ， 以及 响应> 25% 浓度 高渗 葡萄糖 时 时 mpv 显着。。。。。 Он показывает потенциальный частичный лизис тромбоцитов смесями с глюкозой, быструю адаптацию тромбоцитов к экстремальному гипертонусу и значительное увеличение MPV в ответ на концентрацию гипертонической глюкозы > 25%. It shows potential partial platelet lysis by glucose mixtures, rapid platelet adaptation to extreme hypertonicity, and a significant increase in MPV in response to hypertonic glucose >25%. The initial increase was maximal at 41.6% glucose exposure, but the increase in MPV approached 25% glucose exposure approximately 20 minutes after exposure.
The concentration of platelets is affected by glucose. We noticed that the amount of PLT decreased at all dilutions of glucose. A sharp drop in the number of platelets in H2O (0%) dilutions of the PRP series may be associated with osmotic lysis. Alternatively, this could be an artifact caused by platelet clumping, but this is in contrast to the lack of MPV change at this dilution. This finding means that some platelets are very sensitive to hypoosmolarity.
In all 1:1 dilutions of glucose, the amount of PLT decreased by 20-30%, even by D5W (hypotonic at 252 mOsm), which may indicate a specific non-osmotic effect of glucose, since both PLT and MPV remained unchanged at a three-fold increase in concentration. glucose. from D5W to D25W. In fact, PLT concentrations tended to increase slightly with increasing osmolarity.
The decrease in PLT between 1:1 and 1:5 dilutions means that the dissolution effect depends on the initial and final glucose concentration. If it depended only on the initial concentration, then one would expect to see a difference in PLT reduction between 1:1 concentrations. But we don’t. If the lysis effect depends only on the final glucose concentration, then we do not expect much difference between a 20% 1:1 dilution and a 20.8% 1:5 dilution. And yet we did it.
If platelet loss occurs due to platelet lysis, a partial lysate is formed, after which cytokines and growth factors are released into the extracellular environment. Several studies have shown that platelet lysate is almost as effective as PRP as a proliferation solution [11]. PRP itself has been shown to be an effective solution for the treatment of proliferation [12-14].
Inactive platelets circulate in the form of a disk reinforced with several internal structures. During activation, they take on a more spherical or amoeba shape, resulting in an increase in volume. The increase in volume requires an increase in surface area, which is the result of the extrusion of the open tubule system (OCS) and the addition of exocytic granules to the membrane. It remains to be determined whether the increase in MPV induced by hypertonic glucose involves one or both of these mechanisms, but if the latter, then an increase in MPV would indicate degranulation.
This study showed that exposure to high concentrations of glucose on PRP or whole blood platelets resulted in an increase in MPV within 15 minutes with a glucose concentration of 25% and 41.6%, respectively.
The increase in platelet MPV may be due to dilatation of the surrounding microtubule tangles in response to calcium influx. Liu et al. Glucose has been shown to mediate calcium influx through the platelet TRPC6 channel [6]. Our hypothesis is that glucose induces relaxation of microtubule tangles, leading to an increase in MPV and platelet sensitization and/or activation. However, judging by our results, this is only part of the story. In our tests, no concentration below D25W resulted in an increase in MPV. Given that we have not tested exposure to glucose concentrations between 12.5% ​​and 25%, our phase 1 results suggest that there may be a threshold in this range of glucose concentrations that leads to an increase in MPV. Further testing in stages 3 and 4 showed that 20-25% glucose appears to be the threshold for this, but it remains unclear why.
We also observed a ~9% decrease in MPV after centrifugation. It is not clear whether this decrease in MPV is due to larger and denser platelets trapped in the RBC layer of the centrifuge. This observation may be important to clinicians as it may imply that PRP platelets are a smaller and less dense subset of WB platelets.
In a previous study, we showed that PRP preparation by manual methods is inexpensive [8]. If glucose sensitizes tissue platelets or PRP, making them more susceptible to activation, or if PRP is produced with partial lysate properties, this may enhance regeneration and reduce the need for therapy. Therefore, the combination of PRP and highly concentrated glucose may be more cost-effective than PRP or glucose alone.
Our study has several shortcomings. First, we use PRP obtained from several different methods. This can lead to conflicting results. Second, we were unable to perform a biochemical analysis of any of our samples to more accurately determine if platelet activation had occurred. We would like to measure P-selectin, platelet factor 4, monocytic platelet aggregates, or other markers of platelet activation to better understand the degree or presence of alpha granule degranulation, but this is beyond the scope of this study. Third, we were unable to confirm by electron microscopy or other methods that the increase in MPV in glucose-exposed platelets was due to the effect on microtubule tangles.
Mixtures of WB or PRP with 25% glucose increased MPV, signaling the onset of platelet activation, although this study did not demonstrate progression of aggregation or degranulation. The hypertonic glucose mixture resulted in platelet loss, possibly representing a lytic effect. Partial activation or lysis of platelets can cause tissue regeneration after platelet injection. It is not clear what clinical consequences these changes may lead to. Further studies have demonstrated more accurate measurements of activation or lysis and have evaluated the different clinical effects of hypertonic glucose mixtures with WB or PRP.
Glucose proliferative therapy is a simple and inexpensive regenerative therapy that is rapidly expanding and supporting clinical research. This study suggests a physiological mechanism that, if confirmed, could help us understand part of the regenerative mechanism of proliferative therapy.
Biomedical and Health Informatics at the University of Missouri, Kansas City School of Medicine, Kansas City, USA
Human Subjects: All participants in this study gave or did not give consent. The International Society for Cellular Medicine has issued ICMS-2017-003 approval. The following protocol has been approved for further use by the Institutional Review Board of the International Society for Cellular Medicine: Title: Calculation of platelet-rich plasma drug yield based on baseline CBC platelet count. Animal Subjects: All authors confirmed that no animals or tissues were involved in this study. Conflicts of Interest: In accordance with the ICMJE Uniform Disclosure Form, all authors declare the following: Payment/service information: All authors declare that they did not receive financial support from any organization for the submitted work. Financial Relationships: All authors declare that they do not currently or within the past three years have financial relationships with any organization that may be interested in the submitted work. Other Relationships: All authors declare that there are no other relationships or activities that may affect the submitted work.
Harrison TE, Bowler J, Reeves K et al. (May 17, 2022) The effect of glucose on platelet count and volume: implications for regenerative medicine. Cure 14(5): e25081. doi:10.7759/cureus.25081
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