In vivo fluorescence flow cytometry reveals that the nanoparticle tumor vaccine OVA@HA-PEI effectively clears circulating tumor cells

Tumor vaccine therapy offers significant advantages over conventional treatments,including reduced toxic side effects.However,it currently functions primarily as an adjuvant treatment modality in clinical oncology due to limitations in tumor antigen selection and delivery methods.Tumor vaccines often fail to elicit a sufficiently robust immune response against progressive tumors,thereby limiting their clinical efficacy.In this study,we developed a nanoparticle-based tumor vaccine,OVA@HA-PEI,utilizing ovalbumin(OVA)as the presenting antigen and hyaluronic acid(HA)and polyethyleneimine(PEI)as adjuvants and carriers.This formulation significantly enhanced the proliferation of immune cells and cytokines,such as CD3,CD8,interferon-,and tumor necrosis factor-,in vivo,effectively activating an immune response against B16–F10 tumors.In vivofluorescenceflow cytometry(IVFC)has already become an effective method for monitoring circulating tumor cells(CTCs)due to its direct,noninvasive,and long-term detection capabilities.Our study utilized a laboratory-constructed IVFC system to monitor the immune processes induced by the OVA@HA-PEI tumor vaccine and an anti-programmed death-1(PD-1)antibody.The results demonstrated that the combined treatment of OVA@HA-PEI and anti-PD-1 antibody significantly improved the survival time of mice compared to anti-PD-1 antibody treatment alone.Additionally,this combination therapy substantially reduced the number of CTCs in vivo,increased the clearance rate of CTCs by the immune system,and slowed tumor progression.Thesefindings greatly enhance the clinical application prospects of IVFC and tumor vaccines.

In Vivo Studies and Flow Cytometric Investigation on Anticancer Potential of Selenium Nanoparticles Synthesized via Aqueous Extract of Clerodendron phlomidis

Nowadays,doctors and nutritionists recommend individuals incorporate selenium-rich foods such as nuts,cereals,and mushrooms into their regular diet to maintain fitness and overall health.Selenium nanoparticles(SeNPs)exhibit strong chemopreventive capabilities.The anticipations for SeNPs with enhanced and tunable bioactive activities have led to a keen interest in phytofabrication.In this study,the aqueous extract of Clerodendron phlomidis plant leaves was utilized for the synthesis of SeNPs.In traditional Indian medicine,this plant extract is recognized as a significant anti-diabetic agent.The flavonoids tetrahydroxylflavone,7-hydroxyflavanone,and 6,4’-dimethyl-7-acetoxy-scutellarein present in this plant leaf extract demonstrate excellent anticancer activity.These secondary metabolites exhibit the ability to reduce sodium selenite into SeNPs.At a concentration of 13μg/mL,the synthesized SeNPs effectively inhibited the proliferation of the HepG2 cell line.The results suggest that the SeNPs possess promising anti-cancer potential against liver cancer and can be considered as a therapeutic agent for liver cancer treatment.Additionally,the cell cycle arrest induced by SeNPs was further confirmed by the fluorescence-activated cell sorting(FACS)method,indicating that SeNPs could efficiently differentiate cancer cells from normal cells.Notably,it showed a significant improvement in diethylnitrosamine(DEN)-induced Swiss Wistar rat groups.This scientific investigation highlights the high anti-cancer potential of SeNPs,positioning them as a promising therapeutic agent for liver cancer treatment.

Detection of cells by flow cytometry:Counting,imaging,and cell classification

The study of circulating cells in the blood stream is critical,as it covers many felds of biomed-icine,including immunology,cell biology,oncology,and reproductive medicine.In-viuo flowcytometry(IVFC)is a new tool to monitor and count cells in real time for long durations in theirnative biological environment.This review describes two main categories of IVFC,ie.,labeledand label-free IVFC.It focuses on label-free IVFC and introduces its technological developmentand related biological applications.Because cell recognition is the basis of flow cytometrycounting,this review also describes various methods for the classification of unlabeled cells,including the latest machine learning-based technologies.

抑癌基因Kmt2c杂合缺失对小鼠造血系统的影响

目的:探究组蛋白甲基转移酶Kmt2c基因杂合缺失对小鼠血液系统的影响。方法:使用CRISPR/Cas9技术构建Kmt2c基因杂合缺失的模型小鼠,血常规连续监测小鼠全血细胞计数的变化;通过体外集落形成实验探究骨髓细胞的克隆性扩增能力;流式细胞术分析突变小鼠体内原始造血细胞群(长期造血干细胞、短期造血干细胞、多能祖细胞)的比例变化。结果:成功构建Kmt2c基因杂合缺失小鼠(Kmt2c^(+/-))模型,其Kmt2c mRNA表达水平是C57BL/6J小鼠的28%。Kmt2c^(+/-)小鼠骨髓细胞体外集落形成能力随着传代次数的增加而增强,并在第四代时集落数显著高于对照组(P<0.05)。Kmt2c^(+/-)小鼠原始造血细胞群中的长期造血干细胞和短期造血干细胞比例分别为19.6%±3.3%及28.9%±4.9%较对照组的16.9%±2.6%及18.9%±2.5%有增高趋势,但差异无统计学意义(P>0.05)。Kmt2c^(+/-)小鼠的白细胞计数在监测的第12周后逐渐上升在第14周时为(9.8±1.0)×10^(9)/L,显著高于对照组的(7.3±1.4)×10^(9)/L(P<0.05)。结论:Kmt2c^(+/-)小鼠的骨髓细胞具有克隆性扩增的潜能。

A multichannel thermal bubble-actuated impedance flow cytometer with on-chip TIA based on CMOS-MEMS

Electrochemical impedance spectroscopy(EIS)flow cytometry offers the advantages of speed,affordability,and portability in cell analysis and cytometry applications.However,the integration challenges of microfluidic and EIS read-out circuits hinder the downsizing of cytometry devices.To address this,we developed a thermal-bubble-driven impedance flow cytometric application-specific integrated circuit(ASIC).The thermal-bubble micropump avoids external piping and equipment,enabling high-throughput designs.With a total of 36 cell counting channels,each measuring 884×220μm^(2),the chip significantly enhances the throughput of flow cytometers.Each cell counting channel incorporates a differential trans-impedance amplifier(TIA)to amplify weak biosensing signals.By eliminating the parasitic parameters created at the complementary metal-oxidesemiconductor transistor(CMOS)-micro-electromechanical systems(MEMS)interface,the counting accuracy can be increased.The on-chip TIA can adjust feedback resistance from 5 to 60 kΩto accommodate solutions with different impedances.The chip effectively classifies particles of varying sizes,demonstrated by the average peak voltages of 0.0529 and 0.4510 mV for 7 and 14μm polystyrene beads,respectively.Moreover,the counting accuracies of the chip for polystyrene beads and MSTO-211H cells are both greater than 97.6%.The chip exhibits potential for impedance flow cytometer at low cost,high-throughput,and miniaturization for the application of point-of-care diagnostics.

Performance Evaluation of Combined Detection of Serum CEA,CYFRA21-1,CA125,and NSE in Patients with Lung Cancer by Fluorescence Flow Cytometry

Objective:To investigate the effect of combined detection of serum carcinoembryonic antigen(CEA),cytokeratin 19 fragment(CYFRA21-1),cancer antigen 125(CA125),and neuron-specific enolase(NSE)in patients with lung cancer by fluorescence flow cytometry.Methods:From August 2019 to July 2022,200 patients with lung cancer diagnosed by pathology in our hospital were retrospectively analyzed.2 mL venous blood was collected in a fasting state and centrifuged to separate the serum(containing human chorionic gonadotropin antibody[anti-hCG antibody],hepatitis B surface antibody[anti-HBs antibody],and CEA).Results:The sensitivities of CEA and CYFRA21-1 detected via enzyme-linked immunosorbent assay(ELISA)were 100%,and the detection limits were 0.5 ng/mL and 0.1 ng/mL,respectively;the sensitivities of CA125 and NSE detected via flow cytometry were 100%,and the detection limits were 10 U/mL and 2 ng/mL,respectively.Compared with ELISA,the sensitivities of CA125 and NSE detected via flow cytometry were higher.When the concentration of CEA was 10-40 ng/mL,the sensitivities of the three markers CYFRA21-1,CA125,and NSE showed no significant changes(P>0.05);when the concentration of CEA was 40-80 ng/mL,the sensitivity of CEA significantly decreased(P<0.01),but the sensitivities of the three markers CYFRA21-1,CA125,and NSE showed no significant changes(P>0.05);when the concentration of CEA was 80-200 ng/mL,the sensitivities of all four markers showed no significant changes(P>0.05).Conclusion:Compared with the double-antibody sandwich ELISA,fluorescence flow cytometry has certain advantages,including high sensitivity,good precision,short detection time,low sample usage,and low medical cost;thus,it is worthy of clinical promotion.

The Clinical Value of Detecting the Level of Exfoliated Cells in Pleural Effusion by Flow Cytometry in the Differential Diagnosis of Non-

Objective:To explore the value of flow cytometry(FCM)in detecting the level of exfoliated cells in pleural effusion in the differential diagnosis of non-small cell lung cancer and benign lung diseases.Methods:Clinical data of patients with non-small cell lung cancer who were hospitalized in Hebei hospital from June 2019 to March 2022 were collected.A total of 98 patients were included,and 63 patients with alveolar lung disease were screened during the same period,and the two groups of patients were analyzed.Results:Compared with alveolar lung disease group,FCM detection and analysis showed that the level of exfoliated cells in the pleural effusion of non-small cell lung cancer(NSCLC)patients was 99(3-969)/100,000,and patients with alveolar lung disease was 4(0~19)/100,000.Additionally,compared with the alveolar lung disease group,the level of exfoliated cells in the pleural effusion of patients with non-small cell lung cancer(NSCLC)was significantly increased(P<0.001).The diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in non-small cell lung cancer was assessed using ROC curves and using 95%CI(-11.1,-13.2)with a sensitivity of 0.75 and specificity of 0.94,and the diagnostic efficacy of FCM for detecting pleural fluid exfoliated cells in alveolar lung disease was assessed using 95%CI(-11.1,-13.2)with a sensitivity of 0.71 and specificity of 0.87.Conclusion:Flow cytometry has a wider range of clinical applications,simple operation,low cost,and high sensitivity,which makes it of great significance in disease diagnosis.

mT/mG;Wnt1-Cre小鼠肺组织神经嵴细胞的分离培养与特征鉴定

目的体外分离培养小鼠肺组织中的神经嵴细胞(NCCs),并鉴定该细胞的特性,为研究肺损伤及修复提供新的细胞模型。方法将mT/mG双荧光报告小鼠和Wnt1-Cre转基因小鼠杂交,筛选获得增强型绿色荧光蛋白(EGFP)永久标记NCCs的mT/mG;Wnt1-Cre转基因小鼠。用酶解法制备小鼠肺组织单细胞悬液,用流式细胞仪分选获得EGFP+的细胞(即为NCCs)。用经实验室优化的DMEM/F12培养基进行原代培养,用细胞免疫荧光染色法对NCCs进行特征鉴定,使用小鼠骨髓间充质干细胞成骨诱导分化培养基定向诱导NCCs分化。结果成功通过杂交筛选得到EGFP永久标记NCCs的mT/mG;Wnt1-Cre转基因小鼠,并从肺组织中分离获得高纯度的NCCs,其能够在体外进行培养,形态为梭形,类似成纤维样贴壁增殖,NCCs表达神经嵴干细胞标志物Sox10,能被定向诱导分化为成骨细胞。结论从mT/mG;Wnt1-Cre转基因小鼠肺组织中分离、培养的NCCs增殖稳定,具有神经嵴干细胞特征,可为肺组织损伤和修复研究提供新的细胞模型。

Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts

Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.

A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle

To set up a three-parameter method for cell cycle analysis by two-laser flowcy-tometer, which can detect two types of cyclin plus DNA content in one measurement, and thatanalyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysisof two types of cyclins and DNA content simultaneously in individual cells by two-laser flowcytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. ATriton-X100 permeabilization procedure was optimized for detection of two types of cyclins. Onecyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other,indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochromeDAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method.Results: Permeabilization with 0.5% Triton-XlOO in PBS containing 1% BSA for 5 min on ice providedoptimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It wasfound that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured withno compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growingMOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously,allowing for analysis of cell cycle phase specific perturbations without the necessity of cellsynchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine andcyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleledexpected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only beapplied to measure unscheduled vs. expected cyclin expression but may also be used to estimate thefraction of cycling cells in up to 6 cell populations.

Radio-protective effect of vitamin E on spermatogenesis in mice exposed to γ-irradiation: a flow cytometric study

Aim: To investigate the effect of vitamin E on the radioprotection of spermatogenesis and chromatin condensation of spermatozoa during passage through the epididymis in mice exposed to irradiation. Methods: Adult outbred male ICR mice were orally administered natural vitamin E (VE, D-α-tocopheryl acetate) at 400 IU/kg for 7 days before exposure to 1 Gy of γ-irradiation. The animals were sacrificed at day 1, 7, 14, 21, 28, 35 and 70 post-irradiation (IR) and the percentage of testicular germ cells and epididymal sperm chromatin condensation was analyzed using flow cytometry. Results: Serum D-α-tocopheryl acetate levels were 47.4 ± 3.2 μg/dL in the treated group, yet it could not be detected in the control group. The testicular weight of irradiated mice pretreated with VE+IR was significantly (P<0.05) higher than that of those without VE treatment (IR) at day 14 and 21 post-irradiation. The percentage of primary spermatocytes (4C) in the VE+IR group was comparable to the controls but significantly (P<0.05) higher than those in the IR group from day 7 to 35 post-irradiation. The percentage of round spermatids (1C) in the VE+IR group was also significantly (P<0.05) higher than those in the IR group at day 28 post-irradiation. The primary spermatocytes : spermatogonia ratio in the IR group was significantly (P<0.05) declined at day 7 to 35 post-irradiation when compared to the VE+IR and control groups. The round spermatid : spermatogonia ratio in the VE+IR group was significantly (P<0.05) higher than that of the IR group at day 14 and 28 post-irradiation. The chromatin condensation of epididymal spermatozoa measured by propidium iodide uptake was not affected by 1 Gy of γ-irradiation. Conclusion: The administration of VE prior to irradiation protects spermatogenic cells from radiation.

Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

Summary: The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52 %. There were 40 cases of LPI (64.5 %) including 15 negative cases and 22 cases of HPI (35 5 %) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.

Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques （Macaca leonina）

Pig-tailed macaques(Macaca nemistrina group) have been extensively used as non-human primate animal models for various human diseases in recent years, notably for AIDS research due to their sensitivity to HIV-1. Northern pig-tailed macaques(M. leonina) are distributed in China and other surrounding Southeast Asia countries. Although northern pig-tailed macaques have been bred on a large scale as experimental animals since 2012, the reference value of normal levels of leukocytes is not available. To obtain such information, 62 blood samples from male and female healthy northern pig-tailed macaques at different ages were collected. The normal range of major leukocyte subpopulations, such as T lymphocytes, B lymphocytes, natural killer(NK) cells, monocytes, and the expression levels of activation or differentiation related molecules(CD38, HLA-DR, CCR5, CD21, IgD, CD80 and CD86) on lymphocytes were analyzed by flow cytometry. The counts of B cells decreased with age, but those of CD8+ T cells and NK cells and the frequency of CD38+HLA-DR+CD4+ T cells were positively correlated with age. The counts of leukocyte subpopulations were higher in males than those in females except for CD4+ T cells. Males also showed higher expression levels of Ig D and CD21 within B cells. This study provides basic data about the leukocyte subpopulations of northern pig-tailed macaques and compares this species with commonly used Chinese rhesus macaques(M. mulatta), which is meaningful for the biomedical application of northern pig-tailed macaques.

Application of CD45/SSC Gating Multiparameter Flow Cytometry in the Classification of Acute Leukemia——An Analysis of 139 Cases

In order to study the significance of flow cytometry immunophenotyping in the diagnosis of acute leukemia, CD45/SSC gating multiparameter flow cytometry (FCM) was utilized to analyze the immunophenotypes of 139 cases of acute leukemia. 139 cases of acute leukemia were enrolled in our hospital from April 1998 to April 2000. Morphological analysis and FCM immunophenotypic tests were conducted on all cases. Our results showed that CD45/SSC gating multiparameter flow cytometry immunophenotyping could reflect the origin of leukemic cells specifically. It is one of the important methods for the diagnosis of ALL, AML, and HAL. CD45/SSC gating multiparameter FCM analysis is a good technique for immunophenotyping. FCM immunophenotypic analysis can help improve the diagnosis and classification of acute leukemia, and extend the use of FCM in clinical practice.

Flow cytometric analysis of DNA content for four commercially important crabs in China

The genome size（C-value） of an organism is referring to the DNA content of its non-replicated haploid chromosome complement,generally deduced from measuring somatic diploid nuclei.We presented genome size（C-value） data obtained by flow cytometry for four commercially important crabs（Portunus trituberculatus,Charybdis japonica,Scylla paramamosain,and Eriocheir sinensis） common in the coast of China.Gallus domesticus（2C=2.5 pg） was used as the internal standard.The results showed that the C-value for P.trituberculatus,C.japonica,S.paramamosain,and E.sinensis were（2.31±0.01） pg,（2.33±0.03） pg,（1.64±0.02） pg,and（2.29±0.03） pg,respectively.The C-value of P.trituberculatus,C.japonica and S.paramamosain were reported for the first time.The data represented by the four species indicated that they had lower DNA contents than average DNA values in crustaceans（（4.99±0.48） pg）,and three of the four values were very similar if not identical.The results provide useful data for future studies in the fields of biodiversity,species conservation,and phylogeny of these commercial crabs.They will also be helpful in instructing the hybridization breeding program and estimating the cost of the whole genome sequencing project.

Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （El）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and El detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

Flow cytometry for the assessment of animal sperm ntegrity and functionality： state of the art

Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of ＇bench-top＇ flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

Recent advances in fuorescence-based in vivo flow cytometry

The fuorescence-based in vivo flow cytometry(IVFC)is an emerging tool to monitor eirculating cells in vivo.As a noninvasive and real-timne diagnostic technology,the fluorescence based IVFC allows long-term monitoring of circulating cells without changing their native biological environment.It has been applied for various biological applications(eg,monitoring circulating tumor cells).In this work,we will review our recent works on fluorescence-based IVFC.The operation principle and typical biological applications will be introduced.In addition,the recent advances in IVFC flow cytometry based on photoacoustic effects and other label free detection methods such as imaging based methods,difuse-light methods,hybrid multimodality methods and multispectral methods are also summarized.

DETECTION OF PLATELET-DERIVED MICROPARTICLES USING FLOW CYTOMETRY AND ITS CLINICAL APPLICATION

Objective.To establish a flow cytometric internal standard method for counting platelet-derived microparti-cles(PMPs)and to study its clinical significance. Methods. PMPs suspension(platelet poor plasma,PPP) was extracted by gradual centrifugation. According to the size of PMPs,3 μm and 0.8μm latex beads were used as internal standards for the quantitation. PMPs were counted by adjusting flow cytometric discrimination and voltage of forward scatter and side scatter. Results. In 30 healthy donors,the average concentration of resting PMPs was(1.2×105±5.7×104 )/ml and that of activated PMPs was(1.6×106±9.1×105)/ml. Compared with healthy donors,PMPs mean value was significantly higher(P< 0.001)in 18 patients with coronary artery disease,12 with acute cerebral infraction and 23 with chronic renal failure[the average PMPs concentration,( 6.1×105±2.5×105 )/ml, ( 6.8×105±3.4×105)/ml and(5.9×105±3.1×105)/ml respectively]. However,no significant difference in PMPs concentration was observed in 25 patients with acute leukemia and severe thrombocytopenia during the aplastic phase after chemotherapy [1.3×105±6.1×104)/ml,(P >0.05)] .Conclusions. PMPs is a useful indicator in monitoring platelet activation,and plays an important role in thrombotic disease. By flow cytometric internal standard method,PMPs can be counted rapidly and accurately,which may be very helpful in interlaboratory comparative studies.