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.

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.

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.

A new method for separation of human pancreatic stellate cells / 中华肝胆外科杂志

Objective To explore a new method for the separation of human pancreatic stellate cells.Methods Single-cell suspension of normal pancreatic tissue and pancreatic cancer tissue was prepared by gentle MACSTM tissue processor-constant temperature shaking digestion.Human pancreatic stellate cells of quiescent and activated state were isolated by density gradient centrifugation.Results A new type of isolation method could obtain about (2.6 ± 0.7) × 106 quiescent pancreatic stellate cells in 1 g of human normal pancreatic tissue,with a viability of about 90.0％.The morphology of the cells were conformed to the representative for the quiescent state characteristics and transient blue-green autofluorescence was observed at the 328 nm excitation wavelength;1 g of human pancreatic cancer was able to obtain approximately (4.1 ± 1.1) × 106 activated PSCs with a viability of 92.0％,and all of the activated cells expressed α-SMA vimentin,FSP-1 and other characteristic markers.Conclusions The new separation method of this experiment is suitable for both human resting and activated human pancreatic stellate cells.At the same time,the purity is high and the separation time is greatly shortened,which is worth promoting.

Sperm DNA fragmentation and sex chromosome aneuploidy after swim-up versus density gradient centrifugation / 대한생식의학회지

OBJECTIVE: The aim of this study was to compare the efficacy of swim-up and density gradient centrifugation (DGC) for reducing the amount of sperm with fragmented DNA, sex chromosome aneuploidy, and abnormal chromatin structure. METHODS: Semen samples were obtained from 18 healthy male partners who attended infertility clinics for infertility investigations and were processed with swim-up and DGC. The percentages of sperm cells with fragmented DNA measured by the sperm chromatin dispersion test, normal sex chromosomes assessed by fluorescence in situ hybridization, and abnormal chromatin structure identified by toluidine blue staining were examined. RESULTS: The percentage of sperm cells with fragmented DNA was significantly lower in the swim-up fraction (9.7%, p=0.001) than in the unprocessed fraction (27.0%), but not in the DGC fraction (27.8%, p=0.098). The percentage of sperm cells with normal X or Y chromosomes was comparable in the three fractions. The percentage of sperm cells with abnormal chromatin structure significantly decreased after DGC (from 15.7% to 10.3%, p=0.002). The swim-up method also tended to reduce the percentage of sperm cells with abnormal chromatin structure, but the difference was not significant (from 15.7% to 11.6%, p=0.316). CONCLUSION: The swim-up method is superior for enriching genetically competent sperm.

The effect of semen optimization on the rate of sperm DNA fragmentation and its effect on pregnancy out-come of IVF-ET / 实用医学杂志

Objective To investigate the method of effectively density gradient centrifugation combined with swim-up improves sperm nuclear integrity and determine whether the sperm chromatin dispersion test of sperm DNA fragmentation in raw or DGC-swim-up treated semen can influence the outcome of IVF. Method The DNA integrity of spermatozoa from 120 patients underwent IVF were analyzed by SCD before and after DGC and swim-up. The predictive value of the SDFI for IVF outcomes were assessed in a cohort of 100 patients who were underwent new embryo transfer. Result In male infertility group,DGC combined with swim-up decreased the SDFI from 22.75(14.44,30.25)to 11.50(5.60,22.79),while the control group decreased the SDFI from 20.86(15.00,26.81) to 7.50(3.63,15.44),respectively(P<0.05);SDFI after optimization in clinical pregnancy group was significantly lower than that of non-pregnant group. The area under the receiver operating characteristic curve was 0.667. The patients with low sperm DFI had a higher implantation rate and pregnancy rate compared with patients with high sperm DFI. Conclusions DGC and swim-up treated Sperm DNA fragmentation can predict the outcome of IVF. The effect of semen optimization on the rate of sperm DNA fragmentation is limited,once exceed,pregnancy rate and birth rate are decreased although fertilization is normal.

The effect of semen optimization on the rate of sperm DNA fragmentation and its effect on pregnancy out-come of IVF-ET / 实用医学杂志

Objective To investigate the method of effectively density gradient centrifugation combined with swim-up improves sperm nuclear integrity and determine whether the sperm chromatin dispersion test of sperm DNA fragmentation in raw or DGC-swim-up treated semen can influence the outcome of IVF. Method The DNA integrity of spermatozoa from 120 patients underwent IVF were analyzed by SCD before and after DGC and swim-up. The predictive value of the SDFI for IVF outcomes were assessed in a cohort of 100 patients who were underwent new embryo transfer. Result In male infertility group,DGC combined with swim-up decreased the SDFI from 22.75(14.44,30.25)to 11.50(5.60,22.79),while the control group decreased the SDFI from 20.86(15.00,26.81) to 7.50(3.63,15.44),respectively(P<0.05);SDFI after optimization in clinical pregnancy group was significantly lower than that of non-pregnant group. The area under the receiver operating characteristic curve was 0.667. The patients with low sperm DFI had a higher implantation rate and pregnancy rate compared with patients with high sperm DFI. Conclusions DGC and swim-up treated Sperm DNA fragmentation can predict the outcome of IVF. The effect of semen optimization on the rate of sperm DNA fragmentation is limited,once exceed,pregnancy rate and birth rate are decreased although fertilization is normal.

A comparative study of tumor-associated macrophages isolation in colorectal carcinoma by Ficoll-Hy-paque density gradient centrifugation and Percoll density gradient centrifugation / 实用医学杂志

Objective Percoll density gradient centrifugation and Ficoll-Hypaque density gradient cen-trifugation, which are frequently-used methods for separation of tumor-associated macrophages (TAMs) from solid carcinoma were compared, in order to find an effective way to separate TAMs from colorectal carcinoma (CRC). Furthermore, we studied the best adherence time of separating macrophage among mononuclear cells. Methods specimens were collected from CRC patients , after digesting into single cells , TAMs were separated from the same specimen by 100% Ficoll, 35% percoll and 25% combined with 65% percoll respectively. After these pre-liminary separation, the collected cells were purified a second time by adherence separation. The purity of TAMs were detected by immunofluorescence. Results TAMs purity from Ficoll-Hypaque density gradient centrifugation was 80.18%, statistically higher than that from Percoll density gradient centrifugations (54.33% and 10.93% re-spectively). Conclusion Compared to Percoll density gradient centrifugation, Ficoll-Hypaque density gradient centrifugation is a more effective and simple way to isolate TAMs from colorectal carcinoma , suggesting it can be wildly used in clinical and basic medical research. 2-4 hours is the best adherence time for isolating macrophage.

Progress in the isolation and purification methods of adipose-derived stem cells / 中国比较医学杂志

Adipose-derived stem cells ( ASCs ) as potential seeded cells have been widely used in tissue engineering.Thus to obtain enough, high activity, high purity adipose-derived stem cells is the particular important premise of the application in tissue engineering.In this paper, the isolation and purification methods of ASCs were reviewed and the merit and demerit of different methods were compared in order to provide theoretical basis for safe and high-effective isolation and purification of ASCs.

An optimized extraction protocol of prefrontal cortical and striatal synaptosomes from SHR rat / 中国比较医学杂志

Objective To introduce an improved extraction method of prefrontal cortical and striatal synaptosomes from SHR rat. Methods Synaptosomes were prepared from SHR rat brain tissue by Percoll density gradient centrifugation.Transmission electron microscopy was used to assess the morphology and structural integrity of the synaptosomes.Results The obtained synaptosomes showed oval structures surrounded by an intact membrane.Presynaptic components contained one or more mitochondria and a large number of synaptic vesicles.The synaptic clefts were clearly visible, and prominent part of the characteristic compact structure was clear, complete and with higher electron-density. The synaptosome presynaptic membrane, synaptic cleft, and postsynaptic membrane were well preserved, and the synaptosomes were densely distributed, showing typical morphological characteristics of synaptosomes.Conclusions The results of our study improved the traditional preparation method and provide a less time-consuming, highly productive protocol for preparation of structurally typical and intact synaptosomes, suitable for further research on neuroscience and neurological diseases.

Comparison of the effect of different media on the clinical outcomes of the density-gradient centrifugation/swim-up and swim-up methods / 대한생식의학회지

OBJECTIVE: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. METHODS: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). RESULTS: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. CONCLUSION: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.

Detection and clinical significance of circulating tumor cells in pa-tients with gastric cancer / 中国肿瘤临床

Objective: To detect circulating tumor cells (CTCs) in patients with gastric cancer and evaluate the relationship among CTCs, clinico-pathological characteristics, and prognosis of gastric cancer. Methods: Peripheral blood samples (10 mL in EDTA) were obtained from 45 patients with gastric cancer. CTCs were detected using density-gradient centrifugation and immunofluo-rescence staining. The clinical significance of the two methods were also compared and investigated. Results:CTC-positive case was defined by the presence of at least one CK19 (+)-CTC per 10 mL of the sample. CTCs were found in 27 of the 45 patients with gastric cancer. The presence of CTCs was significantly correlated with lymph node metastasis, distant metastasis, and recurrence (P=0.007, 0.035, 0.035, respectively). However, CTCs were not significantly correlated with sex, age, tumor location, TNM staging, and tumor differentiation (P>0.05). Conclusion:CTCs were associated with poor prognosis of gastric cancer.

Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production / Avaliação do swim-up e do gradiente descontinuo de densidade na pré-seleção do sexo de espermatozoides na produção de embriões bovinos

The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y- specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05) in deviation of sex ratio when comparing the control group (45.2% females) with the other spermatozoa selection procedures (60.6% females) (P<0.05). The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively) and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.

O objetivo do presente trabalho foi associar o método de swim-up modificado à centrifugação em gradiente de densidade para a separação de espermatozoides portadores do cromossomo X. A viabilidade e a integridade espermática foram avaliadas pelo método de coloração Azul de Tripan e Giemsa. O controle de qualidade dos espermatozoides centrifugados foi realizado por meio da produção in vitro de embriões bovinos. Os resultados foram validados pela técnica de PCR para verificar a proporção sexual dos embriões produzidos in vitro, com o uso de sequências Y especificas presente no DNA genômico de machos bovinos. Após determinar o sexo genético dos embriões produzidos in vitro, os resultados não mostraram diferença (P<0,05) no desvio da proporção do sexo quando comparou o grupo controle (45,2% de fêmeas) com os outros processos de seleção de espermatozoides (60,6% de fêmeas) (P<0,05). Os métodos de seleção de espermatozoides são capazes de selecionar espermatozoides portadores do cromossomo X sem comprometer a fertilidade, medida pelas taxas de clivagem e blastocisto de 70% e 26%, respectivamente, e foram considerados métodos de relevância para serem introduzidos nos programas de produção in vitro de embriões bovinos.

Subpopulation of Rabbit Articular Chondrocytes Separated by Percoll Density Gradient / 대한정형외과연구학회지

Articular chondrocytes have been known to have heterogeneity in articular cartilage. Four layers are generally recognized from the articular surface to the subchondral bone. We have used Percoll density gradients to separate chondrocytes from articular cartilage into distinct subpopulations. Non-fibrillated articular cartilage was obtained from rabbit knee. The cells were carefully layered on the top of the preformed gradient and spun. After centrifugation, we obtained four fractions: Fraction A referred boundary between 0% and 10%, fraction B from between 10% and 20%, fraction C from between 20% and 30%, and fraction D from between 40% and 50%. In the A fraction, cells are relatively larger and round in shape, while their nuclei are relatively smaller. In the cytoplasm many lipid droplets were found and rough endoplasmic reticulum were disrupted. In the D fraction, chondrocyte is small, with large nucleus which surrounded by well-developed rough endoplasmic reticulum. The type II collagen proteins were expressed strongly and more proteoglycans synthesized in fractions A and B. And chondrocytes from the fraction D divided more slowly than those from the fractions A, B, and C. We have succeeded in separating chondrocytes from articular cartilage into distinct subpopulations by Percoll density gradients, as well as characterized growth rate, histological appearances and phenotypic expression. This study is the first report about the Percoll density gradients to separate articular chondrocytes.

Improvement of Amplification Method for Cryptosporidium parvum Oocysts from Mice / 中国寄生虫学与寄生虫病杂志

Objective To establish a simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from infected C57BL/6N mice. Methods All mice in the experimental groups were immunosuppressed by given different concentrations of dexamethasone phosphate added in drinking water throughout the experiment. The recovery and purity of the oocysts obtained using different purification methods was compared. The infectivity of the oocysts obtained from the same origin but different animals and different purification methods in a bovine fallopian tube epithelial cell culture system was studied. Results 4.16?10 9 oocysts were obtained in 30 mice in the 3rd group with dexamethasone of 20 ?g/ml in drinking water. No significant difference in the oocyst recovery, purity and infectivity was found between methods using saturated saline floatation and sucrose density gradient centrifugation. The infectivity of the oocysts obtained from the same origin but different animals was similar. Conclusion A simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from the infected mice was established.

Purification of treponema pallidum from rabbit testicular tissue / 대한피부과학회지

There are several different methods of purifying Treponema pallidum(TP) from rabbit testicular tissue. Among them, we compared the use of differential centrifugation, which has been most widely used, to Percoll density gradient centrifugatian, a newly applied method, in purifying TP from rabbit testicular tissue by checking the protein concentration of the TP suspension, hemagglutination assay using sheep erythrocytes sensitized by TP, IgM-TP-enzyme-linked immunosorhent, assay(IgM-TP-ELISA) and eJect,ron microscopic observation. The protein concent,ration af TP antigen suspension (2x10(8)TP/ml) purified by Percoll density gradient centrifugation (lower band) was the lowest (129.0pg/ml) when compared to those purified by differential centrifugation (324.0pg/ml) and Percoll density gradient centrifugatian (upper band) (560.2pg/ml). Sheep erythrocytes sensitized by TP purified by Percoll density gradient centrifugation(lower band) showed the same resiilts as those using a commercii1 TPHA kit when tested with positive and negative control sera. The sensitivity and specificity of the IgM-TP-ELISA were 88.5%(23/26') and 86.4%(19/2Z) respectively using TP as an antigen purified by differential centrifugation. The rates were 96.29% (25/26) and 95.5%(2l/22) using TP purified by Percoll density gradient centrifugation. As shown by the electron microscopy, T. pizllida purified by clifferential centritugation and Percoll density gradient centrifugatiori were structurally unaltered, and Percoll-purified TP contained much less tissue debris than TP prepared by differ ential centrifugation. Therefor e, Percoll density gradient centrifugation is considered to be a better method of purifying TP from rabbit testicular tissue when compared to differential centrifugatian, as a matter of fact, Perrol1 density gradient centrifugation has been applieci successfully in the study of the physiology, recombinant DNA techniques, and antigenic structure of TP and to the preparation of the antigen for the FTA-ARS and TP-ELISA

IsoIation and 2-DE analysis of mitochondria of cultured hepatoma cells / 重庆医科大学学报

Objective:To make the isolation and 2-DE analysis of mitochondria,in order to prepare the following proteomic study on mitochondria of hepatoma-cell. Methods:Mitochondria was separated from cell homogenate by means of density gradient centrifugation,and 2-DE was conducted to examine the protein profile of mitochondrial preparations.Results:The increase in purity of mitochondria was found to be 12 times by density gradient centrifugation.Mitochondrial proteins were displayed well in the 2-DE pattern after purification.Conclusion:The isolation procedure is practicable,which provides a basis for the following proteomic study on mitochondria.

Immunological Assay for Duck Hepatitis B Virus and Its Uneven Geographic Distribution / 中国免疫学杂志

The duck hepatitis B surface antigen(DHBsAg)particles were purified from the s-era of ducks by sucrose rate zonal and KBr isopycnic centrifugation.Analysis of DH-BsAg by SDS-PAGE revealed the major polypeptide to be 18Kd.The anti-DHBsAg-serum was made by immunizing cavy or rabbit and the anti-DHBsA-IgG was purifiedby DEAE-Sephadex A50.A simple,specific and sensitive Reverse Passive Hemgglutin-ation Assay for DHBsAg have been established with the method of Glutaraldehyde T-wo-step Procedure.The sera of ducks from three districts determined by RPHA andelectromicroscopy showed that the geographic distribution of DHBV is uneven and thearea of higher incidence of hepatitis B is also that of duck DHBV infection.So.therewould be other environmental facts influencing the uneven distribution of DHBV,andprobably that of HBV.

The phenotypic analysis of Human fetal thymocytes / 中国免疫学杂志

The phenotypes of human fetal thymocytes has been analysed with fflowcytometry.The findings suggest that the composition of thymocyte subsets shows no obvious changes from the 17th week of gestation to birth.In the pieriod of human fetus,features of thymocyte phenotypes are showed:1.Expression density of CD3/TCR?? shows a continuous distribution from low to high.According to the density of D3/TCR??,tyhmocytes can be divided into three subsets,that is,L—CD3/TCR??(low—density),M—CD3/TCR?? medium—density)and H—CD3/TCR??(high— density).The proportion of three subsets is different in different fetuses.2.Single positive thymocytes express CD1 antigens.H—CD3/TCR?? thymocytes also express CD1 antigens.But,Part of those thymocytes express low—density of CD1 antigens,other part of the hymocytes express high—density of CD1 antigens.3.HLA class Ⅰ antigen density varies with thymocytes.Most thymocytes express low density of HLA class Ⅰ antitgens,minor thymocytes express high density of HLA class Ⅰantigens.HLA class Ⅰ antigen density correlates with maturity of thymocytes.4.Thymocytes can be divided into two subsets:Thy-L and Thy-H on the basis of thymocyte special density by means of density gradient centrifugation.Thy-L subset includes more mature and early thymocytes than Thy-H subset,while thy-H subset includes much more common thymocytes than Thy-L subset.