Double Centrifugation Research Articles

Characterization of a protein S assay measuring the APC cofactor activity / Charakterisierung eines APC-Kofaktor basierten Protein S-Aktivitätstests

Abstract The Protein S Ac activity assay is based on the role of protein S as a cofactor to activated protein C (APC). Tests performed on three coagulation analyzers were studied to determine assay performance characteristics. The overall mean imprecision was 5.5%, 5.0% and 7.5% for within-run coefficient of variation (CV), run-to-run CV and total CV, respectively. A total of 239 sample results yielded a correlation to the STA Protein S Clotting assay of r=0.92 (slope=0.96). A group of patients with APC resistance and additional underlying conditions, such as oral contraception, oral anticoagulation, pregnancy and recurrent thrombosis showed pathologically low protein S values with activity assays as well as with free antigen assays. To differentiate between the effects of APC resistance and other influencing factors, samples from healthy blood donors with APC resistance were measured with both assay types. Based on comparison of antigen and activity results, it can be concluded that APC resistance lowers protein S activity values by approximately 15% (rel.). No interference was observed from physiologically relevant concentration of factor V, factor VIII, triglycerides, hemoglobin, bilirubin and heparin (low molecular weight heparin and unfractionated heparin). When samples are to be frozen, double centrifugation is the best procedure to reduce the platelet count. The stability of the sample was at least 4 h at room temperature, regardless of whether fresh or frozen samples were tested. Freezing had no observable effect on samples stored for up to 12 months at −70°C.

Adenovirus E1A proteins are closely associated with chromatin in productively infected and transformed cells

The adenovirus E1A 243R oncoprotein encodes a potent transcription-repression function within the N-terminal 80 amino acids. Our proposed model of E1A repression predicts that E1A interacts with important cellular proteins on chromatin. Consistent with this idea, we report here that E1A proteins from in vivo formaldehyde cross-linked 293 cells are closely associated with chromatin even after several stringent purification steps including double isopycnic CsCl density gradient centrifugation and size exclusion chromatography. Likewise, E1A proteins expressed from virus during productive infection of HeLa cells are closely associated with chromatin starting at early times after infection. No other adenoviral proteins are necessary for E1A 243R protein to associate with chromatin. Analyses of chromatin from HeLa cells infected with adenovirus vectors expressing E1A 243R protein with deletions in different E1A functional domains indicate that sequences within the E1A N-terminal repression domain are needed for the majority of E1A's interactions with chromatin.

Flow cytometry for the rapid detection of bacteria in cell culture production medium

The rapid and sensitive detection of microbial contaminants is critical for timely contamination investigations and monitoring of process control for in vitro protein production systems. A flow cytometric method was developed to monitor two types of cell culture media used in large scale protein production. The process used a DNA dye, thiazole orange, which binds to nucleic acids of viable and nonviable organisms. To ensure a representative sample was tested and to enhance the detection limit, a concentration step before staining and analysis included a double centrifugation. In addition, a washing step was included to eliminate background fluorescence caused by material in the media formulations. The staining and analysis of concentrated and washed samples takes approximately 0.5 h and provides objective results. The feasibility of the method was demonstrated by spiking sterile media with six bacterial species that represent the most commonly encountered bacterial contaminants. In addition to the bacterial spiking study, 164 lots of large scale production medium were tested by the flow cytometric method in parallel with conventional culture using Trypticase Soy Broth (TSB). In the bacterial spiking study, the concentration method increased the microbial titer by 2 logs. The detection limit of organisms within the medium was determined to be 1 CFU/ml. There was 100% correlation between the flow cytometric method and the standard aerobic plate count method using Trypticase Soy Agar. In the parallel study on 164 lots of large scale production media, the flow cytometric method was compared against culture in TSB. The sensitivity of the method was determined to be 100%, the specificity was 99.4%, the positive predictive value was 83%, and the negative predictive value was 100%. To reduce reporting of false positives, an initial positive result should be confirmed by an additional sample. The analytic sensitivity, specificity, and objectivity of this flow cytometric method indicate this method is valuable in the monitoring of production media for microbial contamination.

Assessment of sperm motility, viability and apoptosis in human spermatozoa after hydrogen peroxide exposure

ObjectiveMale infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2).DesignProspective study.Materials and methodsSemen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence.ResultsTableComparing different sperm parameters after exposure to H2O2Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. Open table in a new tab ConclusionsExposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage. ObjectiveMale infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2). Male infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2). DesignProspective study. Prospective study. Materials and methodsSemen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence. Semen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence. ResultsTableComparing different sperm parameters after exposure to H2O2Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. Open table in a new tab Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. ConclusionsExposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage. Exposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage.

Evaluation of poly (ADP-ribose) polymerase cleavage (cleaved-PARP) in sperm fractions after sperm apoptosis induction

Sperm cell apoptosis is an energy and protein dependent process that results in sperm DNA damage. PARP cleavage (cPARP) is stimulated by DNA breaks for help in repair process, so it is considered as an early marker for apoptosis. Staurosporine (STS) is a PARP inducer while 3-aminobenzamide (3ABA), is a PARP inhibitor. Sperm DNA damage due to oxidative stress is considered as one of the causes of male infertility. Conflicting reports exist regarding the presence of whole or cPARP in human ejaculated spermatozoa. We evaluated: 1) the presence of cPARP in mature ejaculated spermatozoa and 2) if we can control its level in sperm fraction using apoptosis inducers with or without PARP inhibitor. Prospective-controlled study. Semen samples were collected from 10 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation. Mature and immature sperm fractions were resuspended into HTF media and divided in 5 aliquots (control, H2O2 treated, H2O2 + 3ABA, STS treated, and STS + 3ABA) incubated at 37°C with 5 % CO2 for 1 hour. The final concentrations were 2.5 μM for STS, 30 μM for H2O2, and 0.3 mM for 3ABA. After incubation, the mature and immature aliquots were evaluated for the early and late apoptosis by 1) annexin V, activated caspase-3, 2) sperm DNA damage by TUNEL and 3) cPARP by FACS. cPARP was detected in both neat and mature fractions. There were no significant differences between mature versus immature fractions regarding most of molecular apoptosis markers for treated and untreated sperm fractions for cPARP modulation.Table 1Correlation between the measured parameters in different treatment groupsUntreated sperm fractionsTreatmentActivated Caspase-3 (a C-3)TUNEL + sperm%Cleaved PARP + %Early apoptotic sperm %Late apoptotic sperm %rPrPrPrPrPSTS treated sperm0.960.120.30.50.720.90.420.130.60.12STS + 3ABA treated sperm0.940.0040.60.570.80.170.240.040.16<0.001H2O2 treated sperm0.940.110.80.20.80.26-0.190.002-0.13<0.001H2O2 + 3ABA treated sperm0.970.0020.510.560.81-0.7-0.20.003-0.06<0.001Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Open table in a new tab Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Cleaved PARP is present in neat as well as in washed ejaculated human spermatozoa. Its level is related to the extent of sperm DNA damage. PARP inhibitors can not prevent/repair oxidative or chemical-induced sperm DNA damage.

Calidad del plasma rico en plaquetas: estudio de la activación plaquetaria

Objective. Platelet Rich Plasma (PRP) is an autologous preparation currently used in oral and maxillofacial reconstructive surgery. Blood collection and preparation of platelet concentrates may lead to platelet activation and the premature loss of their granular load. In this study, we have analyzed the quality of the PRP obtained from a small volume of whole blood through a double centrifugation technique, so called tube method. Design. We obtained 50 ml of whole blood from 45 patients and centrifuged at 200 g for 10 minutes. The plasma and buffy-coat were collected and we then centrifuged at 700g for 15 minutes. The pellet was resuspended after discarding 2/3 of the plasma. The platelet concentration, platelet activation and the functional response to thrombin were analyzed in these samples. Results. By using this method of PRP preparation, we obtained a 364 ± 177% increase in platelet concentration in comparison with whole blood levels. Platelet activation, measured by flow cytometry analysis of CD62 expression, was of 2.7% in unprocessed blood and 3.6% in fresh PRP. This figure increased to 16% in PRP samples after overnight storage at room temperature. A percentage of 96% of platelets showed activation in PRP samples after thrombin stimulation. Conclusion. Our results show that platelets contained in PRP concentrates obtained by this method are not significantly activated. A good functional platelet reserve is preserved through the procedure, since platelets maintained a satisfactory response to thrombin after PRP preparation.

Increased Plasma Concentrations of Soluble CD40 Ligand in Acute Coronary Syndrome Depend on in Vitro Platelet Activation

Soluble CD40 ligand (sCD40L) was suggested as a novel biomarker of cardiovascular risk. We examined the effect of preanalytical variation on the measurement of sCD40L concentration. From healthy control individuals (n = 20) and patients with acute coronary syndrome (ACS) (n = 20) or sepsis (n = 20), we obtained blood drawn into 5 tubes containing citrate or a mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD). The tubes were incubated for 30 min at room temperature or 0 degrees C before a single or double centrifugation (15 min, 2500 g) at room temperature or 4 degrees C, respectively. sCD40L, beta-thromboglobulin (betaTG), and platelet factor 4 (PF4) concentrations were measured using immunoassays. Concentrations of sCD40L were very low in all CTAD and citrated samples maintained at 0 degrees C (median < or = 0.076 microg/L). Although increased betaTG and PF4 confirmed disease-related in vivo platelet activation, sCD40L was not higher in patients than in controls. In contrast, if the samples were processed at room temperature, sCD40L was significantly higher in ACS patients than in controls (P <0.02 in CTAD and citrated plasma at room temperature). Moreover, the betaTG:PF4 ratio decreased in patient but not control CTAD samples, suggesting a greater susceptibility of patient platelets to in vitro activation. Increased sCD40L concentrations resulted from in vitro platelet activation during sample preparation. Disease-related in vivo activation did not contribute to sCD40L concentrations in plasma. Therefore, published studies of sCD40L demand cautious interpretation, because their preanalytical conditions were not standardized.

Relationship between sperm apoptosis signalling and oocyte penetration capacity

Human sperm have been documented to display apoptosis-like features such as externalization of phosphatidylserine (EPS), disruption of the transmembrane mitochondrial potential (MMP) and activation of caspases. Our aim was to evaluate possible association between activation of the apoptosis cascade in human sperm and its oocyte penetration capacity using the zona free hamster oocyte penetration assay (SPA). Semen specimens from 76 unselected donors were subjected to double density gradient centrifugation followed by incubation under capacitating conditions for 3 h and SPA. Apoptosis signalling was monitored by assessment of EPS, disruption of MMP and activation of caspase-3 by flow cytometry. Semen samples with subnormal SPA values (<20% penetrated oocytes) contained significantly higher amounts of spermatozoa with EPS, disrupted MMP and activated caspase-3 compared with those samples with normal SPA values (>20% penetrated oocytes, p < 0.01). All three apoptosis markers showed a significantly negative correlation with the percentage of penetrated oocytes (p < 0.01). Apoptosis-related signalling appears to have a negative association with sperm-oocyte penetration. The exclusion of sperm presenting with those apoptosis-related features during assisted reproduction may improve success rates of procedures such as intrauterine insemination and in vitro fertilization.

A Comparative Study of 2 Methods for Obtaining Platelet-Rich Plasma

Double and single centrifugation are the most commonly used techniques for obtaining platelet-rich plasma (PRP) in dentistry. In this study, we used and compared 2 methods for obtaining PRP: double centrifugation (ACE system; Surgical Supply and Surgical Science Systems, Brockton, MA) and single centrifugation (Nahita system; Nahita, Navarra, Spain). Blood samples were obtained from 30 random patients. Each blood sample was treated using the ACE system and Nahita system methods, after which the obtained material was analyzed by flow cytometry for platelet counts and by transmission electron microscopy (TEM) for ultrastructural analysis of the PRP gel. Platelet count analysis of the PRP obtained from both methods revealed that the ACE and Nahita systems accomplished platelet concentrations of (336%) and (227%), respectively. The platelet counting results obtained from the ACE system samples were more dispersed than their Nahita system counterpart. The ultrastructural (ie, TEM) study showed considerable alterations of the platelet aggregates in the ACE's PRP, especially when the samples were not mixed in the final stage of the procedure, whereas the Nahita aggregates always had a normal physiological appearance. The ACE double-centrifugation method is able to achieve higher platelet concentrations than the single-centrifugation Nahita system, although the results obtained by ACE were more dispersed. Nevertheless, the ACE system provoked alterations in the PRP ultrastructure, and it was more sensitive to small errors during preparation.

Development of prenatal genetic screening test using fetal cells in maternal blood

Prenatal genetic diagnosis using invasive techniques such as amniocentesis and chorionic villus sampling reveal significant risks to the fetus. The triple marker screening test has low sensitivity and cannot confirm sex chromosomal abnormalities. Prenatal genetic diagnosis using the fetal nucleated RBCs in maternal blood has been studied to overcome this shortage. However, it is difficult to use because of extremely small numbers of fetal RBC in maternal blood. Therefore, we tried to collect nucleated RBCs morphologically by Kleihaur-Betke staining after double gradient centrifugation and magnetic activating cell sorting from maternal blood. Fluorescent in situ hybridization (FISH) analyses with CEP X and CEP Y probes for K-B positive nucleated RBCs were performed to detect whether fetal cells were existed among nucleated RBCs by observation of sex chromosomes. The average number of K-B positive nucleated RBCs separated from 10 ml of maternal blood was 17.3, and the maximum number of nucleated RBCs was 54. We observed FISH signals in nucleated RBCs separated from 18 pregnant women, and Y probe signals were observed in 67.3% of nucleated RBCs separated from 10 pregnant women. We confirmed that separated nucleated fetal RBCs can be used to identify fetal sex and chromosomal aneuploidies by FISH. Since nucleated RBCs form maternal origin were not excluded, further studies are needed to overcome this limitation.

Human herpesvirus 8 genoprevalence in populations at disparate risks of Kaposi's sarcoma

The prevalence of human herpesvirus 8 (HHV-8) in populations at different risks of developing Kaposi's sarcoma (KS) was assessed using a protocol involving immunomagnetic fractionation of CD45+ blood cells prior to detection of the HHV-8 genome by nested PCR. Preliminary studies using blood of eight gay men infected by human immunodeficiency virus-1 (HIV-1) revealed that, for the detection of HHV-8 DNA derived from open reading frame (ORF) 26 of the HHV-8 genome, this protocol provided substantially higher rates (100%) compared to one involving red blood cell (RBC) lysis (0%) and to another requiring double density gradient centrifugation (DDGC) of leukocytes (13%). When the CD45+ fractionation protocol was applied to samples from 103 other HIV-1-infected patients (the vast majority of whom were gay men) and 100 blood donors, the ORF 26 DNA detection rates obtained were 37% and 8%, respectively. When DNA from the variable region 1 of ORF K1 was additionally amplified from samples of the blood donors, a detection rate of 9% was achieved. This rate was highly concordant with the ORF 26 DNA detection rate. Furthermore, the ORF K1 sequences were predominantly unique, assignable to genotypes A1, A4, and C3. When assays for anti-HHV-8 and anti-herpes simplex viruses (HSV) 1 and 2 were applied, significant concordances between HHV-8 DNA detection rates and those relating to anti-HHV-8 and to anti-HSV 1 and 2 were more frequently observed for HIV-1-infected patients than for blood donors. The higher-than-expected HHV-8 genoprevalence rate in blood donors requires further confirmation in view of its implications for post-transfusion HHV-8 transmission.

Random Healthy Donor Sera Show Varying Effectiveness in Hemolyzing ABO Incompatible Red Blood Cells.

Mixing of allogeneic serum with RBC that are ABO-incompatible is well known to cause lysis of the RBC. However, the severity and extent of such hemolysis may not be the same for every mismatched pairing and is not documented in the literature. We were interested in quantitating the lytic potential of a large panel of normal sera when exposed to ABO-incompatible RBC. We collected 80mL of whole blood in red-top tubes from 110 random healthy subjects (84 female 26 male) with blood Groups A (n=6), AB (n=5), B (n=10) and O (n=89). Sera were prepared by clotting at room temp, frozen in aliquots, stored at −80°C. CPD whole blood (N=14) from healthy subjects were obtained commercially, shipped and stored per standard methods (A1: 8, A2: 2, O: 4). Forward ABO typing on all serum subjects and whole blood was with Immucor-Gamma murine monoclonal antibody reagents. Within 48 h of donation, packed RBC were prepared by centrifugation of an aliquot at 900xg for 5 min. RBC were resuspended in AS-3 to a final hematocrit of 10%. Total Hb was measured on a BayerAdvia120 analyzer, and hematocrit was determined by capillary tube centrifugation. Aliquots of 125μL of this RBC suspension were incubated with 250μL aliquots of the serum panel for 30 min at 37°C followed by double centrifugation at 900xg for 5 min. Supernatant (sup) was assayed for Hb by the cyanmethemoglobin method reading on a spectrophotometer at 540nm with a turbidity correction for 680nm absorbance. Results were corrected for test serum and RBC plasma absorbances. Percent hemolysis was calculated as 100xHb(sup, mg/dL)x(1-hct)/Hb(total, mg/dL). A repeated measures analysis of variance mixed-effects model was used to test the effect of serum-donor sex, serum-donor ABO group and RBC ABO group on hemolysis. Sup Hb was categorized into visually detectable hemolysis (>56 mg/dL; Elliot et al, Transfusion 2003; 43:297) or not. A total of 1462 plasma-RBC incubations were carried out, with each of the 110 test sera tested versus the test RBC. As expected, no significant hemolysis was detected for ABO-compatible combinations (p>0.8). Complete hemolysis was not observed in any combination. Of the 89 O-sera, median hemolysis with A1-RBC was 3.9% (max 33.4%), but only a median of 0.9% (max of 17.5%) with A2-RBC. There was no significant effect of serum-donor gender on hemolysis (p=0.8). Group O-sera caused higher average hemolysis than B sera for both A1 and A2-RBC. A1-RBC had a larger hemolytic response to both sera types. Notably, not all sera of the same ABO-group caused equivalent hemolysis. We observed a larger variation in hemolysis between serum donors, with an intraclass correlation coefficient ρ=0.55, than between RBC units (ρ=0.028). Therefore, in vitro testing of hemolysis potential should be against a large panel of sera to capture the inherent person-to-person variability.Hemolysis of ABO Incompatible Mixtures of RBC and SeraPhenotypeHemolysis (%)Hb (mg/dL)Visual LysisRBCSerumNMean±SE (min-max)pMean±SE (min-max)> 56 mg/dL (p<0.0001)A1O6866.2±0.2 (0–33.4)<0.0001264±10 (0–1276)526/686 (77%)A1B793.3±0.5 (0–14.9)0.0057142±20 (0–570)43/79 (54%)A2O1782.5±0.3 (0–17.5)0.0011113±13 (0–790)70/178 (39%)A2B91.3±0.6 (0–5.20.516060±28 (0–236)3/9 (33%)

P-988: Association of intermediate staining patterns using the Toluidine blue test with late apoptosis and necrosis in human spermatozoa

P-988: Association of intermediate staining patterns using the Toluidine blue test with late apoptosis and necrosis in human spermatozoa

P-989: Comparison of DNA contents of apoptotic sperm cells in neat and processed semen

Semen processing is used during assisted reproductive techniques to eliminate unwanted, damaging cells and debris from semen samples. To date little is known about the sperm DNA content and its relation to apoptosis or DNA fragmentation in neat semen and following density gradient centrifugation. Sperm DNA content can be evaluated by flow cytometry using propidium iodide (PI) stain. Flow cytometric analysis could quantitatively differentiate between intensities of haploid spermatid, spermatozoa with condensed chromatin and sub-haploid with low DNA content. Our objective was to investigate the DNA content of the late apoptotic and necrotic sperm cells in neat and processed semen following density gradient centrifugation. Prospective-controlled study. Semen samples were collected from 22 donors. The neat samples were divided into mature and sperm immature fractions using double density gradient centrifugation (90% and 47% layers). Both fractions as well as an aliquot of the neat sample were evaluated for the incidence of early apoptosis and necrosis using the annexin V/ PI assay using flow cytometry. To evaluate the DNA content, PI-positive cells were gated and subjected to analysis. In addition, the DNA fragmentation index (%DFI) and high DNA stainability index (%HDS) were measured using the sperm chromatin structure assay. Mature sperm fractions showed significantly lower abnormal DNA content parameters compared to neat samples: sub-haploid (p=0.003) and spermatid (p=0.03), while the number of sperm with condensed chromatin was comparable (Table). Neat semen and mature fractions showed significantly higher proportion of spermatozoa with condensed chromatin compared with immature fraction (p=0.02 and p=0.01, respectively). There was no significant difference between mature and immature fractions regarding the number of sub-haploid cells. In neat semen, there was a significant negative correlation between haploid apoptotic cells and %HDS (r=-0.48, p=0.04), while positive correlation was found between immature and sub-haploid apoptotic cells with %DFI (r=0.6, p<0.01). In mature and immature fractions there was strong positive correlation between each apoptotic haploid and spermatid with DFI (r=0.5, p<0.01 and r= 0.6, p=0.005, respectively).Tabled 1 Mature sperm fractions in processed semen contain more normal DNA content than neat and mature semen. The %DFI in neat semen could be related to immature and low DNA content cells, while %HDS could be related to diploid immature cells.

P-595: Modified processing of teratozoospermic specimens with a high incidence of pinhead forms for ICSI

Although it is argued that sperm morphology does not influence the fertilization outcome in ICSI, embryo development and implantation may be compromised. Processing methods are available to isolate selected sperm populations based on motility and morpholy. However, in cases of severe teratozoospermia, modifications are required to isolate morphologically normal sperm for ICSI. In this report, we describe a modified processing method to obtain sperm for ICSI in a subject with consistently <1% normal morphology characterized by a high incidence of pinhead spermatozoa. Prospective clinical study. A series of semen analyses on a subject revealed a high incidence (> 78% total count) of pinhead spermatozoa. In order to identify possible modifications that will maximize the recovery of sperm without pinhead defects, the semen sample was divided into two aliquots and processed using either (1) double layer mini-density gradient centrifugation (DGC) with centrifugation at 100-300 g for 10min, and (2) swim-up for 60min (SU). The recovered sperm populations from these two methods (DGC-Proc and SU-Proc), as well as the left over raw samples (DGC-Raw and SU-Raw) were individually washed twice at 100 g for 2.5 min and assessed. On the day of oocyte retrieval, a semen specimen was collected and processed according to the methods tested. A total of 12 MII oocytes were retrieved of which 6 were injected via ICSI with sperm from the DGC-Proc sample and the remaining 6 oocyte were injected with sperm from the SU-Raw sample. The characteristics of the semen specimens processed via mini-DGC and SU are described in the table below. The semen specimen (Control-Raw) was totally teratozoospermic with 98% of abnormalities corresponding to pinhead spermatozoa and the remaining 2% corresponded to other head abnormalities. Pinhead spermatozoa showed the highest forward progression in comparison to the rest of the sperm population. Morphological abnormalities other than pinhead sperm consisted of variations in the size of the sperm head. Three embryos derived from the SU-Raw specimens were transferred on day 3 and resulting in an ongoing pregnancy.Tabled 1 Both the DGC-Proc and SU-Raw methods reduced the number of pinheads after preparation. In the swim-up method, the pinhead spermatozoa were able to reach the upper layer first due to their forward progression, leaving a lesser proportion of sperm without pinhead defects in the bottom layer, which is usually discarded in conventional swim-up. Furthermore, the SU-Raw sample showed a higher fertilization rate and better embryo quality when compared to the DGC method. Although sperm with normal morphology were not available, selection of sperm for ICSI was relatively simple since the ratio of sperm without pinhead defects increased in those specimens. This report demonstrates a novel approach for the isolation of sperm without pinheads from a completely teratozoospermic sample for use in ICSI.

P-986: Evaluation of seminal fractions using the Toluidine blue test and sperm chromatin structure assay

Over the past decade, several techniques have emerged as potential protocols for evaluating the sperm chromatin integrity. Such integrity is of utmost importance for successful fertilization. Most of the techniques used for sperm chromatin evaluation require extensive instrumentation and special expertise. Toluidine blue (TB) test has been reported as an easily accessible alternative test for sperm chromatin assessment. Spermatozoa may either stain as dark violet (DV, abnormal chromatin conformation/DNA integrity) or light blue (LB, normal chromatin conformation). The sperm chromatin structure assay (SCSA) is the one of the most standardized methods for assessment of fragmented sperm DNA and the percentage of sperm with immature nuclear development (high DNA stainability index, %HDS). The objective of our study was to evaluate the Toluidine blue test and SCSA for assessment of sperm DNA damage in different seminal fraction. Prospective-controlled study. Semen samples were collected from 19 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation (90% and 47% layers). Both fractions as well as an aliquot of the neat sample were evaluated using the Toluidine blue test. In addition, the %HDS was measured using the SCSA. The means ± standard errors of the percentages of sperm with normal (light blue) and abnormal (dark violet) chromatin in neat semen, mature and immature fractions are given in the table below. Using the Toluidine blue test, mature sperm fractions showed higher incidence of spermatozoa with normal DNA compared to the immature fractions and neat semen samples (p=0.04 and p=0.006, respectively). Similarly, the mature sperm fractions showed lower incidence of spermatozoa with abnormal DNA compared to the immature fractions and neat semen samples (p=0.01 and p<0.0001, respectively). The %HDS showed a significant positive correlation with the number of DV sperm (r=0.51, p=0.0002) and a significant negative correlation with the number LB sperm (r=-0.59, p<0.0001).Tabled 1 Mature sperm fractions show a lower incidence of abnormal DNA as measured by the Toluidine blue test. The association of %HDS with cells that are stained dark violet suggests that Toluidine blue can identify immature nuclear chromatin with abnormal chromatin conformation.

Identification of fetal gender in maternal blood is a helpful tool in the prenatal diagnosis of haemophilia

Fetal DNA identification in maternal circulation has provided a new approach for non-invasive prenatal diagnosis. However, fetal DNA can persist in maternal blood long after the delivery, severely hampering this possibility. We addressed the issue of fetal DNA persistence in maternal blood. Thus, we investigated cell-free fetal DNA as a reliable approach in prenatal diagnosis of haemophilia. Forty non-pregnant women, who had had at least a male fetus, 29 control pregnant women, and 14 pregnant women, carriers of hemophilia A or B. The assessment of Y-chromosomal sequences was performed by analysing SRY and amelogenin genes using PCR-based techniques. A protocol consisting of double centrifugation at full speed followed by plasma filtration hampered the detection of Y chromosome-specific sequence in non-pregnant women. In 29 control pregnant women, blinded determination of fetal sex confirmed the specificity and sensitivity of the method applied. In 14 pregnant carriers of hemophilia, the investigation revealed a male fetus in nine pregnancies. Excluding the three cases in which a spontaneous miscarriage occurred, the sensitivity and specificity of fetal sex prediction by SRY and amelogenin gene analyses were both 100% as compared with the invasive approach and the fetal sex outcome at birth (six males and five females). Because of its high accuracy in prediction, fetal gender determination with cell-free fetal DNA in maternal plasma may be a useful tool in prenatal diagnosis of haemophilia allowing for the avoidance of invasive procedures for female fetuses.

Evaluation of single and double centrifugation tube methods for concentrating equine platelets

The aim of this study was to evaluate single and double centrifugation tube methods for concentrating equine platelets. Whole blood samples were collected from clinically normal horses and processed by use of single and double centrifugation tube methods to obtain four platelet concentrates (PCs): PC-A, PC-B, PC-C, and PC-D, which were analyzed using a flow cytometry hematology system for hemogram and additional platelet parameters (mean platelet volume, platelet distribution width, mean platelet component concentration, mean platelet component distribution width). Concentrations of transforming growth factor beta 1 (TGF-β 1) were determined in all the samples. Platelet concentrations for PC-A, PC-B, PC-C, and PC-D were 45%, 44%, 71%, and 21% higher, respectively, compared to the same values for citrated whole blood samples. TGF-β 1 concentrations for PC-A, PC-B, PC-C, and PC-D were 38%, 44%, 44%, and 37% higher, respectively, compared to citrated whole blood sample values. In conclusion, the single and double centrifugation tube methods are reliable methods for concentrating equine platelets and for obtaining potentially therapeutic TGF-β 1 levels.

多血小板血漿の血小板凝集能とＴＧＦ‐β含有量に関する解析

Recently, platelet-rich plasma (PRP) has been used clinically to promote bone regeneration and wound healing. However, no study has clearly reported on the activity of PRP after preparation. We established a protocol for the safe production of PRP from small quantities of blood.Various conditions were used for anticoagulants and centrifugal separation speed for blood samples obtained from 23 healthy subjects to investigate optimal concentration conditions. Our results showed that when 20% ACD-A liquid was used as the anticoagulant, PRP was most efficiently obtained by double centrifugation at 800 r. p. m. and 1300 r.p.m. The effectiveness of PRP was confirmed by platelet aggregation reaction and the TGF-β content of PRP after double centrifugation.

Sperm Recovery Evaluation Following Magnetic Cell Sorting

Double density gradient centrifugation (DGC) is being recognized as a method of choice for sperm preparation for assisted reproduction techniques. However, the sperm density in DGC does not correlate with apoptosis, which may be a cause of fertilization failure. Magnetic cell sorting (MACS) using annexin V-conjugated microbeads effectively separate apoptotic and non-apoptotic spermatozoa based on the externalization of phosphatidylserine (PS) to the outer membrane leaflet. MACS in combination with DGC can be used as a sperm preparation technique to enhance the sperm quality and function.