Exploration of Optimizing Density Gradient Centrifugation in Semen Processing to Improve Fertilization Outcomes / 中山大学学报(医学科学版)

ObjectiveThis study aimed to improve the existing semen processing methods in the field of reproductive male medicine， particularly focusing on the 300 ×g 20 min treatment condition in the double-layer density gradient method， to enhance fertilization outcome. MethodsSemen specimens from 1 623 patients undergoing assisted reproductive techniques at the Reproductive Medicine Center of the Sixth Affiliated Hospital of Sun Yat-sen University from July and September 2020 and March and May 2022 were collected for preliminary experiments. Four different double-layer density gradient methods （200 ×g 10 min， 200 ×g 20 min， 300 ×g 10 min， and 300 ×g 20 min） were compared for sperm DNA fragmentation rates and recovery rates after processing. Subsequently， the optimal method was selected as the new approach and compared with the current method in use （300 ×g 20 min double-layer gradient method） to assess any statistical differences in fertilization rates. Further optimization to a single-layer density gradient method was performed based on the new method and compared with the double-layer density gradient method to determine any statistical differences. Experimental conditions were strictly controlled for temperature， centrifugation speed， and duration， with the quantity and processing conditions of each sample recorded. ResultsAmong the four double-layer density gradient methods， the sperm DNA fragmentation rate was lower with the 300 ×g 10 min treatment compared to 300 ×g 20 min while ensuring sufficient sperm recovery rates. Consequently， the 300 ×g 10 min method was selected as the new approach for experimentation. Results indicated that the total fertilization rate and 2 pronuclei （2PN） fertilization rate with the new 300 ×g 10 min method were higher than with the 300 ×g 20 min method， the difference was statistically significant （P < 0.05）. Although the cleavage rate with 300 ×g 10 min was slightly higher than 300 ×g 20 min， the difference was not statistically significant （P > 0.05）. The total fertilization rate and 2PN fertilization rate were slightly higher with the single-layer density gradient method compared to the double-layer density gradient method， but the difference was not statistically significant （P > 0.05）. The cleavage rate with the single-layer density gradient method was higher than the double-layer density gradient method， and the blastocyst formation rate is lower than that of the double-layer density gradient method， and the differences are statistically significant （P < 0.05）. ConclusionThe 300 ×g 10 min double-layer density gradient method successfully improved total fertilization rates， 2PN fertilization rates， and cleavage rates compared to the existing 300 ×g 20 min method， while reducing the time required for semen optimization processing. Although the single-layer density gradient method improves the cleavage rate， and saves reagent costs and operation time， its blastocyst formation rate has decreased. These findings provide valuable guidance and insights for semen processing methods in the field of reproductive andrology.

Timing of semen cryopreservation: before or after processing?

Abstract Objective: Seminal cryopreservation causes significant damage to the sperm; therefore, different methods of cryopreservation have been studied. The aim of the study was to compare the effects of density gradient processing and washing/centrifugation with seminal plasma removal for cryopreservation in semen parameters. Methods: Seminal samples of 26 normozoospermic patients were divided into 3 parts: with seminal plasma; after washing/centrifugation; and after selection through density gradient. The samples were cryopreserved for at least two weeks. Motility, sperm count, morphology and viability were evaluated before cryopreservation and after thawing. Results: Density gradient processing selected motile and viable sperm with normal morphology in fresh samples (p<0.05). Cryopreservation negatively affected all sperm parameters regardless of the processing performed, and even if the sperm recovery was lower in the density gradient after the thawing, progressive motility, total motility, viability and morphology remained higher (p<0.05). Conclusion: Cryopreservation significantly compromises sperm parameters (motility, morphology, viability). In normozoospermic patients, the density gradients select better quality spermatozoa compared to other processing methods; this benefit was kept after thawing.

Antibacterial effect of low-temperature plasma on Enterococcus faecalis in dentinal tubules in vitro / 北京大学学报(医学版)

OBJECTIVE@#To construct a model of Enterococcus faecalis (E. faecalis) infection in dentinal tubules by gradient centrifugation and to evaluate the antibacterial effect of low-temperature plasma on E. faecalis in dentinal tubules.@*METHODS@#Standard dentin blocks of 4 mm×4 mm×2 mm size were prepared from single root canal isolated teeth without caries, placed in the E. faecalis bacterial solution, centrifuged in gradient and incubated for 24 h to establish the model of dentinal tubule infection with E. faecalis. The twenty dentin blocks of were divided into five groups, low-temperature plasma jet treatment for 0, 5 and 10 min, calcium hydroxide paste sealing for 7 d and 2% chlorhexidine gel sealing for 7 d. Scanning electron microscopy and confocal laser scanning microscope were used to assess the infection in the dentinal tubules and the antibacterial effect of low-temperature plasma.@*RESULTS@#The results of scanning electron microscopy and confocal laser scanning microscopy showed that after 24 h of incubation by gradient centrifugation, E. faecalis could fully enter the dentinal tubules to a depth of more than 600μm indicating that this method was time-saving and efficient and could successfully construct a model of E. faecalis infection in dentinal tubules. Low-temperature plasma could enter the dentinal tubules and play a role, the structure of E. faecalis was still intact after 5 min of low-temperature plasma treatment, with no obvious damage, and after 10 min of low-temperature plasma treatment, the surface morphology of E. faecalis was crumpled and deformed, the cell wall was seriously collapsed, and the normal physiological morphology was damaged indicating that the majority of E. faecalis was killed in the dentinal tubules. The antibacterial effect of low-temperature plasma treatment for 10 min exceeded that of the calcium hydroxide paste sealing for 7 d and the 2% chlorhexidine gel sealing for 7 d. These two chemicals had difficulty entering deep into the dentinal tubules, and therefore only had a few of antibacterial effect on the bacterial biofilm on the root canal wall, and there was also no significant damage to the E. faecalis bacterial structure.@*CONCLUSION@#Gradient centrifugation could establish the model of E. faecalis dentin infection successfully. Low-temperature plasma treatment for 10 min could kill E. faecalis in dentinal tubules effectively, which is superior to the calcium hydroxide paste sealing for 7 d and the 2% chlorhexidine gel sealing for 7 d.

Evaluation for optimization effects of sperm floating plate / 临床检验杂志

Objective To compare the difference between sperm floating plate and density gradient centrifugation combined with swim-up in human sperm preparation.Methods The semen samples were obtained from 50 infertile men in the clinic of Reproductive Medi-cine of Northwest Women's and Children's Hospital excluding azoospermia,severe oligoasthenozoospermia and semen volume less than 2 mL.After semen liquefaction,the differences of sperm concentration,total progressively motile sperm count(TMSC),percentages of progressively motile sperm and normal morphology sperm,recovery rate and DNA fragmentation index(DFI)were measured by both the methods of sperm floating plate and density gradient centrifugation combined with swim-up,and the results were compared.Results Compared with the pre-sorting samples,sperm concentrations[(16.08±13.39)x 106/mL,(8.88±8.06)x 106/mL vs(60.05± 27.21)×106/mL],TMSC[(7.41±6.14)×106,(3.98±3.57)×106vs(22.24±13.74)×106]and DFI[(2.20±3.44)％,(5.20± 10.79)％vs(26.38±13.92)％]in the sorting groups by sperm floating plate and density gradient centrifugation combined with swim-up were decreased significantly,and the percentages of progressive motile sperm[(91.67±4.75)％,(87.86±7.90)％vs(40.21± 16.83)％]and normal morphology sperm[(9.58±5.08)％,(7.72±4.01)％vs(3.58±2.06)％]were increased significantly.Com-pared with the density gradient centrifugation combined with swim-up,the results of sperm floating plate were higher in sperm concen-tration,percentages of progressively motile sperm and normal morphology sperm,TMSC and sperm recovery rate[(30.74±13.70)％vs(17.09±9.20)％],but DFI was lower,time-consuming was shorter[(32.38±1.01)min vs(60.08±2.06)min],and the difference between the two groups was statistically significant(P＜0.05).Conclusion The sperm floating plate may have certain clinical applica-tion prospects in the future due to better parameters of sperm preparation than those of density gradient centrifugation combined with swim-up in simple operation and shorter time-consuming.

Research progress on platelet-rich fibrin preparation and preservation technology / 国际外科学杂志

Platelet-rich fibrin(PRF), a second-generation of platelet concentrate with stereo fibrin structure, leukocyte and platelets, can release various cytokines and growth factors to promote wound healing. PRF has been widely used in the treatment of various acute and chronic wounds in traumatic surgery, plastic surgery and stomatology. Chronic wounds are common in traumatic surgery, but conventional treatment is often ineffective. Accumulating evidences have confirmed that PRF has excellent therapeutic effect on chronic wounds. According to various methods such as different specimen, centrifugation, centrifuge tube material, and freeze-drying, the researchers tried to improve the efficiency of PRF. The progress of platelet-rich fibrin preparation technology and preservation technology are reviewed in this paper.

Blood transfusion compatibility in patients with delayed serologic transfusion reaction: a retrospective analysis of 6 cases / 中国输血杂志

【Objective】 To retrospectively analyze the characteristics of blood transfusion compatibility detection in patients with delayed serologic transfusion reaction ( DSTR), in order to provide reference for safe and effective blood transfusion in clinical practice. 【Methods】 From April 2020 to July 2021, 6 samples of patients who applied for blood type identification, unexpected antibody screening and transfusion from the Third People′s Hospital of Chengdu or People′s Hospital of Sichuan Province were collected. Microcolumn method was used for identification of ABO and RhD blood type of patients; unexpected antibody screening, blood cross-match, antibody identification and direct anti-human globulin tests were also conducted. The sensitizing antibodies on the surface of red blood cells were identified by acid release solution, and the antigen-antibody reaction was enhanced by polyethylene glycol. The patients′ own red blood cells and input red blood cells were separated by capillary high-speed centrifugation, and the surface antigens of red blood cells were detected by serological method. Meanwhile,the characteristics of patients before and after transfusing antigen-positive red blood cells were summarized. 【Results】 Anti-E was detected in the plasma of patients 1 and 2, and anti-c,-E were detected in the red blood cell release solution, while anti-C, anti-E, anti-JKa and anti-Fyb were detected in the plasma and red blood cell release solution of patients 3, 4, 5 and 6, respectively. After capillary high-speed centrifugation, antigen-positive red blood cells were detected in the distal end of the blood samples of 6 patients. 【Conclusion】 For patients with multiple blood transfusions and a recent history of blood transfusion, when newly emerging erythrocyte antibodies with clinically significance, direct anti-human globulin test(+) or erythrocyte antibody screening(+) are detected, and the patient has no clinical symptoms of hemolysis, it should be suspected as DSTR occurrence, and the transfusion reaction investigation procedure should be initiated in time.

Potential effect of platelet rich fibrin prepared under differen centrifugation protocols on stem cells from the apical papila / O potencial efeito da fibrina rica em plaquetas preparada sob diferentes protocolos de centrifugação nas células tronco da papila apical

Objetivo: Este estudo objetivou avaliar o potencial proliferativo e de diferenciação das células tronco da papila cultivadas conjuntamente com fibrina rica em plaquetas (PRF) preparados sob dois protocolos de centrifugação distintos. Material e Métodos: Protocolos padrão e avançado de PRF foram utilizados. As células foram divididas em 4 grupos: controle negativo, controle positivo, padrão (L-PRF) e avançado (A-PRF). A contagem de células e ensaio de viabilidade foram realizados para verificar a capacidade proliferativa. Coloração vermelho de alizarina S, atividade de fosfatase alcalina e imunofluorescência para o receptor ativador do fator nuclear kappa-B (RANKL) foram utilizados para avaliar o potencial osteogênico e de diferenciação celular. Resultados: Ambos os tipos de PRF aumentaram o número de células, viabilidade celular sem toxicidade o que refletiu no aumento da proliferação e diferenciação de acordo com os testes realizados. Conclusão: O grupo A-PRF aumentou significativamente a proliferação e diferenciação comparado com o grupo L-PRF.(AU)

Objectives: The present work was designed to evaluate the proliferation and differentiation potential of stem cells from the apical papilla (SCAP) seeded along with platelet rich fibrin (PRF) scaffolds prepared under two different centrifugation protocols. Materials and Methods: Standard and advanced PRF protocols were used. Cells were divided into 4 groups: negative control, positive control, standard (L-PRF) and advanced (A-PRF) groups. Cell count and cell viability assays were carried out to assess the proliferation capacity. Alizarin red S (ARS) stain, Alkaline phosphatase (ALP) activity and Receptor activator of nuclear factor-kappa B ligand (RANKL) immunofluorescence staining were used to evaluate the osteogenic potential in the differentiated cells. Results:Both types of platelet rich fibrin increased the cell count, cell viability with no cytotoxicity that was reflected on increased proliferation and differentiation in terms of the performed tests. Conclusion: A-PRF group showed significant increase in proliferation and differentiation potentials compared to L-PRF group

Guia prático de uso da centrífuga IntraSpin® para a obtenção de fibrina rica em plaquetas / Practical guide for using the IntraSpin® centrifuge to obtain platelet-rich fibrin

Obter corretamente a Fibrina Rica em Plaquetas (PRF) depende da execução de cada etapa de maneira protocolada. Nesse sentido, no Laboratório Associado de Pesquisa Clínica (LPCO), da Faculdade de Odontologia da Universidade Federal Fluminense (FO-UFF) é utilizado o sistema IntraSpin® de centrifugação do sangue coletado, que caracteriza o segundo passo para preparo deste material autólogo. O objetivo do presente trabalho é realizar uma descrição prática do correto manuseio da Centrífuga IntraSpin para garantir a obtenção do PRF e sua efetiva aplicabilidade clínica.

Obtaining Platelet Rich Fibrin (PRF) correctly depends on the execution of each step in a protocol manner. In this sense, at the Associated Clinical Research Laboratory (LPCO), of the Faculty of Dentistry in the Federal Fluminense University (FO-UFF) the IntraSpin® centrifugation system is used in the collected blood, that characterizes the second step to prepare this autologous material. The objective of this present work is to make a practical description of the correct handling of the IntraSpin Centrifuge to guarantee the PRF's biocompatibility and its effective clinical applicability.

Evaluation of Effectiveness of Different Regimens of Centrifugation for a Single-spin Method of Pure Platelet-containing Plasma Preparation

Aims: The aim of the study is to determine the optimal parameters of single centrifugation for the preparation of platelet-containing plasma (PCP) with maximum reduction of other cellular elements of the blood.Materials and Methods:30 conditionally healthy persons aged 18 to 60 years (36.9±11.2 years) were included in the study. A total of 12 centrifugation modes were studied: 110g×5min, 110g×10min, 110g×15min, 140g×5min, 140g×10min, 140g×15min, 160g×5min, 160g×10min, 160g×15min, 190g×5min, 190g×10min, 190g×15min. To evaluate the effectiveness of different centrifugation modes, in addition to the number of basic cellular elements, such indicators as platelet capture efficiency, platelet enrichment factor, erythrocyte-reducing efficiencies and leukocyte-reducing efficiencies were studied.Results:When examining the volumes of the obtained plasma containing platelets, it was found that almost all centrifugation modes allow obtaining significantly different volumes of the investigated blood fraction from the others (p<0.001). For clarity, the regimens were sorted according to the volume of platelet-containing plasma obtained, from the smallest to the largest. There was a progressive decrease in the numerical values of the concentration of platelets, erythrocytes and leukocytes in plasma samples. Also there was a progressive decrease in the numerical values of the coefficient of platelet enrichment.Conclusions:With a singlecentrifugation for the preparation of plasma containing platelets, the most effective mode is 160g × 10 min, which allows achieving a platelet enrichment factor of about 1.71 at a platelet concentration of 483.6 ± 45.4 × 109/l, a platelet capture efficiency of 85, 7 ± 0.1% and reductions of erythrocytes and leukocytes 98.76 ± 0.09% and 98.46 ± 0.14%, respectively.

Harvesting of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium using different techniques / Colheita de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio utilizando diferentes técnicas

ABSTRACT: The harvesting process is a current challenge for the commercial production of microalgae because the biomass is diluted in the culture medium. Several methods have been proposed to harvest microalgae cells, but there is not a consensus about the optimum method for such application. Herein, the methods based on sedimentation, flocculation, and centrifugation were evaluated on the recovery of Chlorella sorokiniana BR001 cultivated in a low-nitrogen medium. C. sorokiniana BR001 was cultivated using a low-nitrogen medium to trigger the accumulation of neutral lipids and neutral carbohydrates. The biomass of C. sorokiniana BR001 cultivated in a low-nitrogen medium showed a total lipid content of 1.9 times higher (23.8 ± 4.5%) when compared to the biomass produced in a high-nitrogen medium (12.3 ± 1.2%). In addition, the biomass of the BR001 strain cultivated in a low-nitrogen medium showed a high content of neutral carbohydrates (52.1 ± 1.5%). The natural sedimentation-based process was evaluated using a sedimentation column, and it was concluded that C. sorokiniana BR001 is a non-flocculent strain. Therefore, it was evaluated the effect of different concentrations of ferric sulfate (0.005 to 1 g L-1) or aluminum sulfate (0.025 to 0.83 g L-1) on the flocculation process of C. sorokiniana BR001, but high doses of flocculant agents were required for an efficient harvest of biomass. It was evaluated the centrifugation at low speed (300 to 3,000 g) as well, and it was possible to conclude that this process was the most adequate to harvest the non-flocculent strain C. sorokiniana BR001.

RESUMO: O processo de colheita é um desafio atual para a produção comercial de microalgas porque a biomassa é diluída no meio de cultivo. Diversos métodos têm sido propostos para coletar células de microalgas, porém não existe um consenso sobre um método ótimo para tal aplicação. Neste estudo, métodos baseados em sedimentação, floculação e centrifugação foram avaliados na recuperação de Chlorella sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio. C. sorokiniana BR001 foi cultivada em um meio com baixo teor de nitrogênio para induzir ao acúmulo de lipídeos e carboidratos neutros. A biomassa de C. sorokiniana BR001 cultivada em um meio com baixo teor de nitrogênio apresentou um teor de lipídeos 1,9 vezes superior (23,8 ± 4,5%), quando comparada à biomassa produzida em um meio com alto teor de nitrogênio (12,3 ± 1,2%). Adicionalmente, a biomassa da linhagem BR001 cultivada em um meio com baixo teor de nitrogênio apresentou alto teor de carboidratos neutros (52,1 ± 1,5%). O processo baseado em sedimentação natural foi avaliado utilizando uma coluna de sedimentação e concluiu-se que C. sorokiniana BR001 é uma linhagem não floculante. Portanto, o efeito de diferentes concentrações de sulfato férrico (0,005 a 1 g L-1) ou sulfato de alumínio (0,025 a 0,83 g L-1) foram avaliados no processo de floculação de C. sorokiniana BR001, mas altas doses de floculantes foram necessárias para uma colheita de biomassa eficiente. Também foi avaliada a centrifugação em baixa velocidade (300 a 3.000 g), e foi possível concluir que este processo constituiu o mais adequado para a colheita da linhagem não floculante C. sorokiniana BR001.

Progress in research on the determination of entrapment efficiency of liposomes / 中国药科大学学报

@#Liposome, a new dosage form, has become important in improving in vivo behavior of drugs or realizing targeted drug delivery. Study and control of its critical processes and quality attributes are the main challenges in the current research on liposomes. The degree of encapsulation can determine drug''s effect in vivo directly, thus entrapment efficiency (EE) has turned into one of the critical quality attributes of liposome.In this paper some methods commonly used for the determination of EE and their characteristics are summarized and analyzed, and the main factors to be considered for the determination are discussed.

Preparation and in vitro evaluation of forsythoside A-loaded exosomes / 中国中药杂志

A drug delivery system of forsythoside A-loaded exosomes(FTA-Exos) with high biocompatibility and low immunogenicity was established to investigate its impact on the migration of human lung epithelial adenocarcinoma A549 cells. The exosomes from A549 cells were extracted and purified by ultra-high speed centrifugation and ultrafiltration. FTA-Exos were prepared by ultrasonic incubation, and characterized by particle size analysis, transmission electron microscopy, and Western blot assay. The uptake of FTA-Exos by A549 cells was observed under the laser confocal microscope, and the impact of FTA-Exos on the migration of A549 cells was investigated by cell scratch assay. The results showed that the average particle size of the prepared FTA-Exos was(138.90±2.37) nm, which increased slightly after drug loading. The PDI was 0.291±0.013, and the average potential was(-10.1±0.66) mV. The FTA-Exos were spheroidal in appearance as observed by transmission electron microscope, with an obvious saucer-like double-layer membrane. Western blot assay indicated that the specific proteins CD63 and Alix were both expressed in exosomes. The laser confocal microscopy suggested that FTA-Exos were taken up by A549 cells and stably maintained in the cell for 4-8 h, and the fluorescence was significantly enhanced at 4 h. The scratch assay showed that the inhibitory effect of FTA-Exos on the migration of A549 cells was significantly stronger than that of forsythoside A(P < 0.05). In conclusion, the drug delivery system of FTA-Exos established in this study had good stability, reliable preparation process, and potent inhibitory effect on the migration of A549 cells in vitro, which can provide an important reference for subsequent in-depth research and application.

Preparation of triptolide-loaded dissolving microneedles and its transdermal penetration / 中国中药杂志

Triptolide(TP), the main active and toxic component of Tripterygium wilfordii, has the limitations of low bioavailability, poor absorption, low concentration in plasma, and small lethal dose. Microneedle(MN), the hybrid of hypodermic needle and transdermal patch, is a physical penetration-enhancing system. Dissolving microneedles(DMNs) can be tailored to specific needs of degradation rate. In this study, the TP-loaded DMNs(DMNs-TP) were prepared with the two-step centrifugation method. The optimal ratio of PVA to PVP K30, water content in matrix solution, demoulding method, and plasticizer for preparing DMNs were investigated with the indexes of formability and mechanical strength. The drug loading capacity was determined by HPLC and morphological characteristics were observed under an optical microscope. The mechanical properties were investigated by H&E staining and Franz diffusion cell was used to detect the in vitro skin permeation characteristics. Through the experiment, we confirmed that the optimal backing material should be PVA and PVP K30(3∶1) and the optimal ratio of matrix material to water should be 3∶4. The prepared DMNs-TP were pyramidal with smooth surface and length of approximately 550 μm. Each patch(2.75 cm~2) had the drug loading capacity of(153.41±2.29) μg, and TP was located in the upper part of the needle. The results of in vitro skin permeation assay demonstrated that the cumulative penetration of TP in DMNs-TP reached 80% in 24 h, while little TP solution penetrated the skin, which proved that DMNs promoted the transdermal delivery of TP.

Correlation between adhesion molecules on the surface of red blood cell membrane and red blood cell lifespan / 中国输血杂志

【Objective】 To explore the correlation between red blood cell lifespan and adhesion molecules on the surface of red blood cell membrane, in order to establish a method to detect the duration of red blood cell storage. 【Methods】 10 samples(10 mL each) of fresh red blood cell, collectedf rom 10 healthy voluntary blood donors, were divided into 5 age groups (layers) by Percoll density gradient centrifugation. The expression of CD47, CD44 and CD147 on the surface of red blood cell membrane in each layer was detected using flow cytometry. The variance of protein expression in each layer of red blood cells was analyzed by SPSS statistical software. 【Results】 The expression levels (%) of 3 adhesion molecules on the surface of red blood cell membranes from young to old were CD47: 14.44±2.61, 9.30±1.75, 7.84±1.49, 6.54±1.32 and 5.53±1.12 (P<0.01); CD44: 25.01±1.94, 19.22±1.52, 17.10±1.28, 15.18±1.11 and 13.56±1.08 (P<0.01); CD147: 33.46±1.99, 28.31±2.95, 23.83±1.59, 20.40±1.56 and 18.03±1.65 (P<0.01). 【Conclusion】 The expression levels of CD47, CD44 and CD147 on the surface of red blood cell membranes have showed a downward trend as the storage extended. These three protein adhesion molecules have showed a correlation with red blood cells lifespan, and could be used as detection markers of cell age.

How to improve one-time yield in blood plasma separation / 中国输血杂志

【Objective】 To analyze the factors affecting "one-time yield" in blood plasma separation (BPS) and explore the methods to improve the rate of it. 【Methods】 The rate of "one-time yield" in BPS of 2018 was calculated and the influencing factors were analyzed. Such influencing factors as air release after leukodepletion, poor performance of leukodepletion filter, selection of blood bag barrel, cup filling method, standing time after centrifugation, separation method were improved in 2019, and the difference of the "one-time plasma yield" rate of fresh frozen plasma/frozen plasma and the centrifugal damage before and after the improvement were compared. 【Results】 After the improvement, the rate of "one-time yield" of fresh frozen and frozen plasma increased to 89% and 80%, respectively, which was significantly increased as compared with that before(P<0.05); the centrifugal damage rate decreased to 0.01%, showed no statistical significance compared with that before. 【Conclusion】 With accurate analysis of the factors affecting "one-time plasma yield", the improved method proved to be effective, the rate of "one-time yield" has been improved significantly, and the blood quality has been guaranteed.

Determination of Encapsulation Efficiency of Novel Solid Lipid Nanoparticles Containing Oleic Acid-CAT3 Conjugates by Mini-Column Centrifugation Method / 中国药学杂志

OBJECTIVE: To establish a mini-column centrifugation method to determine the encapsulation efficiency (EE) of novel solid lipid nanoparticles containing oleic acid-CAT3 conjugates (OA-CAT3-SLN) and normal CAT3 SLN (CAT3-SLN). METHODS: Sephadex G-50 mini-columns were used to separate the encapsulated drug and free drug in the solid lipid nanoparticles (SLN) with the help of centrifugation. The boundary point of the elution between the encapsulated drug and free drug was established by the elution curve. The encapsulated drug in the SLN was eluted with 1 mL water for three times. Then 2 mL of ethanol was used to elute the separated free drug for three times. The eluted CAT3 was quantified by the verified HPLC-UV method and used to calculate the EE. The method was verified with the recovery and repeatability test. Finally, the EE of three batches of OA-CAT3-SLN and CAT3-SLN were determined. RESULTS: The mini-column centrifugation method could effectively separate the free drug from the encapsulated drug in SLN. The column recovery was (99.64±1.97)% (n=9), and the result of EE repeatability test of OA-CAT3-SLN was (83.71±0.70)% (n=5). The EE of OA-CAT3-SLN and CAT3-SLN were (86.26±2.65)% and (72.22±4.52)%, respectively (n=3). CONCLUSION: The established separation method is simple and reliable, with high recovery and good repeatability, and can distinguish different preparation processes.

Preparation of neurotoxin loaded dissolving microneedles and its transdermal penetration / 中草药

Objective: To prepare Naja atra neurotoxin (NT) loaded dissolving microneedles (DMNs-NT), and investigate the physicochemical properties and in vitro transdermal properties. Methods: DMNs-NT was prepared by a two-step centrifugation method. The ratio of CS and PVP K30, the water content of the matrix solution, and the backing layer material were optimized by the indexes of formability and mechanical strength of the microneedles and flexibility of the backing layer. The drug loading content was determined by HPLC, and the morphological characteristics were observed under an optical microscope, and the stability was also examined. Franz diffusion cell was used to investigate its in vitro skin permeation characteristics. Results: Through the single-factor exploration, we confirmed that the optimal prescription technique for DMNs-NT preparation was a 1:1 ratio of CS and PVP k30, a 5:4 ratio of matrix material and water, with CMC as the backing layer material. The DMNs-NT had a pyramidal shape with a smooth surface and a length of approximately 500 μm. The drug loading content of per tablet was (15.4 ± 0.5) μg. The drug was located in the upper part of the needle. DMNs-NT had good stability within 3 months. The results of in vitro skin permeation assay showed that the cumulative penetration of NT in DMNs-NT could reach 95.8% in 4 h, while NT solution barely penetrated the skin, which proved that it had a good promoting effect on NT transdermal delivery. Conclusion: In this study, DMNs-NT had good mechanical properties and good skin penetration, which realized the transdermal drug delivery of macromolecular drugs.

Comparison of the clinical effect of intra-articular injection of platelet-rich plasma and methylprednisolone in primary osteoarthritis of knee: a randomized controlled trial

Background: Osteoarthritis of knee is one of the commonest musculoskeletal disorder causing mobility impairment affecting 3.3% in urban areas and 5.5% in rural areas. Intra-articular injection of Platelet-Rich Plasma (PRP) delivers activated platelets that may reduce inflammation, provide pain relief, improve function and stimulate possible cartilage regeneration at the site of worn cartilage area of the knee.Methods: Eighty patients with primary osteoarthritis of the knee fulfilling inclusion and exclusion criteria were recruited in the study conducted in the Department of Physical Medicine and Rehabilitation, RIMS, Imphal from October 2014 to September 2017. Six ml of PRP prepared by conventional bench top centrifugation system was injected intra-articularly, two weeks apart in the PRP group. Steroid group received 80mg of methylprednisolone, two weeks apart by the same technique. The outcome variables (VAS and WOMAC score) were measured before starting intervention (baseline) and at 8 and 24-weeks post-intervention follow up.Results: Significant improvement seen in VAS, WOMAC-pain, stiffness and physical function and total scores in both the groups at 8- and 24-weeks follow-ups (p˂0.001). Steroid group showed better result than the PRP group in VAS (2.78±0.76 vs 3.58±1.03) and WOMAC-total (30.42±6.85 vs 36.25±10.87) scores at 8 weeks respectively (p˂0.001). But at 24 weeks follow-up, PRP showed significantly more effective than the steroid group in reducing pain (2.0±.0.87 vs 2.45±0.78) and disability (22.95±3.78 vs 25.25±6.67) respectively (p˂0.001).Conclusions: Intra-articular injection of methylprednisolone was found to be more effective in reducing pain and disability in primary knee osteoarthritis of KL grade 2 and 3 at the end of 8 weeks whereas 2 doses of PRP intra-articular injection 2 weeks apart was significantly more effective than methylprednisolone at the end of 24 weeks. However, the long-term benefit of PRP is to be determined by studies with a larger sample size and longer duration of follow-up.

Influência do tempo de centrifugação em testes de coagulação: tempo de protrombina e tempo de tromboplastina parcial / Influence of the centrifuge time in coagulation tests: prothrombin time and partial thromboplastin time

Objetivo: Analisar os testes de coagulação: tempo de protrombina (TP) e tempo de tromboplastina parcial (TTP) em diferentes tempos de centrifugação da amostra da biológica, com relação ao protocolo padrão do Clinical Laboratory Standards Institute (CLSI). Métodos: As amostras foram divididas em cinco alíquotas de 1 mL. Foi realizada a centrifugação em 15, 10, 5, 2 e 1 minuto, com velocidade de 1500 g. O TP e TTP foram imediatamente analisados em aparelho automatizado. Os plasmas foram analisados para presença de elementos residuais: eritrócitos, leucócitos e plaquetas. Resultados: Observou-se alteração dos valores do TP nos tempos de centrifugação 10, 5, 2 e 1 minuto e do TTP em 5, 2 e 1 minuto, com relação ao protocolo padrão. Na interpretação de Bland Altman, observou-se um viés significativo do limite clínico aceitável para o TP e para o TTP em todos os tempos de centrifugação, com relação ao protocolo padrão. Apenas no tempo de centrifugação de 15 minutos não foram encontradas células residuais nas amostras analisadas. Conclusão: O tempo de centrifugação de 15 minutos é o ideal para remoção completa das células sanguíneas residuais e para garantia da confiabilidade dos resultados dos testes de coagulação TP e TTP.

Objective: To analyze the coagulation tests: prothrombin test (PT) and partial thromboplastin time (PTT) in different centrifugation times of the sample, in relation to the standard protocol of the Clinical Laboratory Standards Institute (CLSI). Methods: The selected samples were splitted up into five aliquots of 1 mL. Centrifugation of these aliquots was carried out at 15, 10, 5, 2 and 1 minute at 1500 g. The PT and PTT were analyzed in an automated apparatus. The plasmas were analyzed for presence of residual elements: erythrocytes, leukocytes and platelets. Results: The results showed a change in the values of PT at the 10, 5, 2 and 1 minute centrifugation times and the PTT at 5, 2 and 1 minutes, relative to the standard protocol. In the interpretation of Bland Altman, a significant bias of the acceptable clinical limit for TP and TTP at all centrifugation times was observed, relative to the standard protocol. Only in the 15 minute centrifugation time no residual cells were found in the analyzed samples. Conclusion: The present study demonstrated that the 15-minute centrifugation time is ideal for complete removal of residual blood cells and to ensure the reliability of the results of the PT and PTT coagulation

Investigation of Physicochemical Properties and Determination Methods of Liposomal Entrapment Efficiency of Dichroa Alkali Hydrochloride / 中国实验方剂学杂志

Objective: To study on the physical and chemical properties of dichroa alkali hydrochloride and to establish a method for the determination of entrapment efficiency of dichroa alkali hydrochloride liposomes. Method: HPLC was used to determine the content of dichroa alkali hydrochloride with mobile phase of acetonitrile-water-triethylamine-glacial acetic acid(9:91:0.35:0.75) and detection wavelength at 265 nm.The oil-water partition coefficient of this compound in different pH range was measured by shake flask method.The stability of the dichroa alkali hydrochloride in phosphate buffer solution with different pH after sterilization at 125℃ for 30 min was investigated.Ammonium sulfate gradient method was used to prepare dichroa alkali hydrochloride liposomes.The microcolumn was prepared by dextran gel and cation exchange resin,respectively.Then the free drug and liposome were separated by centrifugation,the drug content was measured,and the encapsulation efficiency was calculated.The t-test was performed using SPSS 20.0 software,the differences between these two methods were compared. Result: In the pH 6-9,the oil-water partition coefficient of dichroa alkali hydrochloride increased with increasing of pH,which was between 0.016 and 1.44;the recovery rate of dichroa alkali hydrochloride after sterilization was 37.16%-57.91%.Between the dextran gel microcolumn centrifugation and the cation exchange resin microcolumn centrifugation,there was no significant difference in the entrapment efficiency of the liposomes. Conclusion: Dichroa alkali hydrochloride is suitable for preparation of liposomes.However,its stability is not ideal,so the experimental temperature should be strictly controlled in the preparation process.Dextran gel microcolumn centrifugation and cation exchange resin microcolumn centrifugation can be used to determine the entrapment efficiency of dichroa alkali hydrochloride liposomes,and the cation exchange resin microcolumn centrifugation is suggested after comparison.