[The influence of peripheral blood sample storage and delivery on the quantitative detection result of BCR-ABL (P210) transcript levels].

Objective: To explore the influence of storage and delivery conditions of the peripheral blood samples from patients with chronic myeloid leukemia (CML) on the real-time quantitative PCR (RQ-PCR) detection of the BCR-ABL (P210) transcript levels. Methods: The peripheral blood samples of 84 CML patients were collected. The same sample was divided into different groups according to storage time (0, 6, 12, 24, 48, and 72 h) , temperature (room temperature, 18-24 ℃; low temperature, 2-8 ℃) , and vibration conditions (3, 6, and 12 h) . RQ-PCR was used to detect BCR-ABL (P210) transcript levels of the different groups. This study logarithmically transformed (log(10N)) the original data [BCR-ABL copy number, ABL copy number, and BCR-ABL (P210) transcript levels]. Results: ①Agarose gel electrophoresis showed significant RNA degradation of samples after storage for 48 and 72 h at room temperature. ②Among the overall samples, the BCR-ABL copy number of the samples stored at room temperature for 48 and 72 h was significantly lower than that of the samples stored at low temperature (P<0.05) . However, the BCR-ABL (P210) transcript levels had no significant difference between samples stored at low temperature and room temperature. ③No significant changes were noted in the BCR-ABL (P210) transcript levels at different storage times (6, 12, 24, 48, and 72 h) regardless of storage temperature (P>0.05) compared with that at baseline (0 h, -0.56±1.51) . ④ The BCR-ABL copy number of the overall sample only decreased significantly (P<0.05) at 48 h (2.93±1.59) and 72 h (2.79±1.42) compared with that at baseline (0 h, 3.35±1.60) when stored at room temperature. The ABL copy number in the overall sample decreased significantly at 48 and 72 h (whether low and room temperature; P<0.05) . However, no significant changes were noted in the BCR-ABL (P210) transcript levels after vibration for 3 h (-1.29±1.81) , 6 h (-1.24±1.72) , and 12 h (-1.18±1.68; P>0.05) compared with that at baseline (0 h, -0.60±1.37) . Conclusion: Sample storage time, storage temperature, and vibration can interfere with the results of BCR-ABL and ABL copy number but have no significant effect on the quantitative determination of BCR-ABL (P210) transcript levels. This study provides strong support for the feasibility of transregional transportation of peripheral blood samples from patients with CML.

Prognostic model for identifying candidates for hepatectomy among patients with hepatocellular carcinoma and hepatic vein invasion.

BACKGROUND: Hepatic vein tumour thrombus (HVTT) is a major determinant of survival outcomes for patients with hepatocellular carcinoma (HCC). An Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT model was established to predict the prognosis of patients with HCC and HVTT after liver resection, in order to identify optimal candidates for liver resection. METHODS: Patients with HCC and HVTT from 15 hospitals in China were included. The EHBH-HVTT model with contour plot was developed using a non-linear model in the training cohort, and subsequently validated in internal and external cohorts. RESULTS: Of 850 patients who met the inclusion criteria, there were 292 patients who had liver resection and 198 who did not in the training cohort, and 124 and 236 in the internal and external validation cohorts respectively. Contour plots for the EHBH-HVTT model were established to predict overall survival (OS) rates of patients visually, based on tumour diameter, number of tumours and portal vein tumour thrombus. This differentiated patients into low- and high-risk groups with distinct long-term prognoses in the liver resection cohort (median OS 34·7 versus 12·0 months; P < 0·001), internal validation cohort (32·8 versus 10·4 months; P = 0·002) and external validation cohort (15·2 versus 6·5 months; P = 0·006). On subgroup analysis, the model showed the same efficacy in differentiating patients with HVTT in peripheral and major hepatic veins, the inferior vena cava, or in patients with coexisting portal vein tumour thrombus. CONCLUSION: The EHBH-HVTT model was accurate in predicting prognosis in patients with HCC and HVTT after liver resection. It identified optimal candidates for liver resection among patients with HCC and HVTT, including tumour thrombus in the inferior vena cava, or coexisting portal vein tumour thrombus.

ANTECEDENTES: La trombosis tumoral de la vena hepática (hepatic vein tumour thrombus, HVTT) es un determinante importante de los resultados de supervivencia en pacientes con carcinoma hepatocelular (hepatocellular carcinoma, HCC). Se desarrolló el modelo llamado Eastern Hepatobiliary Surgery Hospital (EHBH)-HVTT para predecir el pronóstico de los pacientes con HCC y HVTT después de la resección hepática (liver resection, LR), con el fin de identificar los candidatos óptimos para LR entre estos pacientes. MÉTODOS: Se incluyeron pacientes con HCC y HVTT de 15 hospitales en China. El modelo EHBH-HVTT con gráfico de contorno se desarrolló utilizando un modelo no lineal en la cohorte de entrenamiento, siendo posteriormente validado en cohortes internas y externas. RESULTADOS: De 850 pacientes que cumplieron con los criterios de inclusión, hubo 292 pacientes en el grupo LR y 198 pacientes en el grupo no LR en la cohorte de entrenamiento, y 124 y 236 en las cohortes de validación interna y externa. Los gráficos de contorno del modelo EHBH-HVTT se establecieron para predecir visualmente las tasas de supervivencia global (overall survival, OS) de los pacientes, en función del diámetro del tumor, número de tumores y del trombo tumoral de la vena porta (portal vein tumour thrombus, PVTT). Esto diferenciaba a los pacientes en los grupos de alto y bajo riesgo, con distinto pronóstico a largo plazo en las 3 cohortes (34,7 versus 12,0 meses, 32,8 versus 10,4 meses y 15,2 versus 6,5 meses, P < 0,001). En el análisis de subgrupos, el modelo mostró la misma eficacia en la diferenciación de pacientes con HVTT, con trombo tumoral en la vena cava inferior (inferior vena cava tumour thrombus, IVCTT) o en pacientes con PVTT coexistente. CONCLUSIÓN: El modelo EHBH-HVTT fue preciso para la predicción del pronóstico en pacientes con HCC y HVTT después de la LR. Identificó candidatos óptimos para LR en pacientes con HCC y HVTT, incluyendo IVCTT o PVTT coexistente.

[The correlation between gastric bubble size and laryngopharyngeal reflux pattern in patients with laryngopharyngeal reflux disease].

Objective: To investigate the correlation between gastric bubble size and laryngopharyngeal reflux pattern in patients with laryngopharyngeal reflux disease(LPRD). Methods: A total of 52 LPRD patients who underwent Dx-pH monitoring and anteroposterior chest radiography at the same time from February 2016 to November 2018 were retrospectively studied. Patients were devided into three position-related groups according to the Ryan score of upright and supine: isolated upright reflux(IUR), isolated supine reflux(ISR) and bipositional reflux(BR) groups. In addition, 13 healthy volunteers with negative pH monitoring were selected as the control group. Gastric bubble size and pH monitoring data among the four groups were compared. SPSS 24.0 was used for statistical analysis. Results: In all the 52 patients, 35 cases (67.3%) were classified as IUR, 9 cases (17.3%) as ISR, and 8 cases (15.4%) as BR. The height of gastric bubbles in the four groups were: IUR (26±14) mm, ISR (9±8) mm, BR (20±13) mm, control (17±15) mm, and statistical difference was found among the four groups(P=0.004). Post Hoc Multiple Comparisons found that IUR group had statistical difference between ISR group and control group (P=0.001, P=0.034 respectively). There was no statistical difference of gastric bubble width and area among the four groups(P=0.340, P=0.186 respectively). The ROC curve of the gastric bubble height with isolated upright and supine reflux patterns was obtained, and the optimal cutoff value of the gastric bubble height was 11 mm. Accordingly, we divided the patiens into two groups with high and low gastric bubble. LPRD reflux pattern distribution was significantly different between the two groups(P<0.001). The comparison of reflux parameters in pH monitoring also showed that the supine reflux parameters in the lower group were significantly higher than those in the higher group, and the upright reflux parameters in the higher group were significantly higher than those in the lower group(P<0.001). Conclusions: The height of gastric bubble is significantly correlated with the reflux patterns in LPRD patients. The gastric bubble of patients with IUR is significantly higher than that of patients with ISR. Taking 11 mm as the cutoff value, patients with higher gastric bubble are more prone to upright laryngopharyngeal reflux, while those with lower gastric bubble are more prone to supine laryngopharyngeal reflux.

[The establishment and application of internal quality control system for real-time quantitative PCR detection of BCR-ABL (P210) transcript levels].

Objective: To set internal quality control system of BCR-ABL (P210) transcript levels for real-time quantitative PCR (RQ-PCR). Methods: Using K562 cells and HL-60 cells, we prepared high- and low-level BCR-ABL internal quality control substance. The BCR-ABL (P210) transcript levels of internal quality control substance have been determined for 184 times together with clinical samples from August 2013 to October 2015. The slope rate, intercept and correlation coefficient of standard curve were calculated according to different reagent lots (lots number 20130303, 20131212, 20140411 and 20150327 are called R1、R2、R3 and R4 for short respectively), and the detection results of quality control substance were calculated according to different reagent lots and quality control substance lots (lots number 20130725, 20140611 are called Q1、Q2 for short respectively). Then the results were analyzed by Levey-Jennings quality control chart combined with Westgard multi-rules theory. Results: ①We analyzed the slope rate and intercept of standard curve. Fifty-three times of the R1 reagent detection, 80 times of the R3 reagent detection and 14 times of the R4 reagent detection were all under control. For 37 times detection of R2 reagent, the slope rate was out of control for 6 times. It was lower than x-s for the 2-8 tests and upper the average for the 12-37 tests. The intercept was out of control for 9 times, upper the x+s for the 1-8 tests and lower the average for the 12-37 tests. ② According to the detection results of quality control substance, for Q1 quality control substance, 49 tests by R1 reagent were under control, and 1 out of 23 tests by R2 reagent was out of control. For Q2 quality control substance, 14 tests by R2 reagent detection, 72 tests by R3 reagent detection and 14 tests by R4 reagent were all under control. Conclusion: The preparation of high- and low-level quality control substance using K562 and HL-60 cells was convenient and the detection results were reliable and stable. The application of quality control substance combined with slope rate and intercept in the internal quality control may contribute to quality assurance for quantitative detection of BCR-ABL (P210) transcript levels.

Research progress in the radioprotective effect of superoxide dismutase.

Irradiation from diverse sources is ubiquitous and closely associated with human activity. Radiation therapy (RT), an important component of the multiple radiation origins, contributes significantly to oncotherapy by killing tumor cells. On the other hand, RT can also cause some undesired normal tissue injuries that afflict numerous cancer patients. Although many promising radioprotective agents are emerging, few of them have entered the market successfully due to various limitations. At present, the most accepted hypothesis for the radiation-caused injury involves reactive oxygen species (ROS) generation. Superoxide dismutase (SOD), the unique enzyme responsible for the dismutation of superoxide radicals, is expected to occupy an indispensable position in the treatment of ROS-mediated tissue injuries originating from exposure to radiation. This review focuses on the mechanism of radioprotection by SOD at the tissue or organ level, cellular level, and molecular level, respectively, in order to provide references for further investigation of radiation injury and development of new radioprotectors.

Detection of altered acrosomal physiology of cryopreserved human spermatozoa after sperm residence in the female reproductive tract.

At least some of the spermatozoa that remain motile following cryopreservation have sustained sublethal damage that reduces their functional capacity in vivo. Although it is believed that acrosomal damage is partly responsible for impaired sperm function in vivo, direct evidence for this hypothesis is lacking because spermatozoa have not been collected from the female reproductive tract for evaluation. In the study reported here, cervical mucus was collected from women 24 h after artificial insemination by cervical cup. For both cryopreserved and nonfrozen inseminates, spermatozoa within the cervical mucus and spermatozoa that migrated out of mucus into culture medium (t = 1 h) were viable and had intact acrosomes. However, although nonfrozen spermatozoa did not initially respond to induction of the acrosome reaction with follicular fluid, a significant proportion of cryopreserved spermatozoa did respond. These results demonstrate that cryopreservation increases the acrosomal lability of spermatozoa residing in the female reproductive tract. An in vitro test was developed to detect this form of cryodamage. Sperm-free mucus was collected before insemination and spermatozoa from the inseminate were allowed to swim into this column of mucus in vitro. Spermatozoa recovered from this mucus sample were compared with spermatozoa from the paired sample collected from the cervix 24 h later. This in vitro test could detect acrosomal lability in cryopreserved semen samples, and this approach may prove valuable for studying sublethal cryodamage to the acrosome.

Separation of cryopreserved human semen using Sephadex columns, washing, or Percoll gradients.

The following methods were evaluated for their ability to separate motile cryopreserved sperm from semen after thawing: single washing, Percoll separation followed by a single washing, and Sephadex column separation. For Sephadex separation, washing, and Percoll separation, percent recovery of motile sperm was 65%, 76%, and 28%, and motility was 81%, 39%, and 60%, respectively. Percoll separation and washing were the best methods for removing seminal constituents, but sperm velocity and linearity were lower after Percoll separation and washing than after Sephadex separation. During 3 hours of incubation, there was an additional decrease in the motility, viability (exclusion of supravital dye), velocity, linearity, and intact acrosomes of Percoll-separated sperm, indicating that Percoll separation may not be suitable for cryopreserved sperm. Motile, washed sperm also had lower velocities and higher spontaneous acrosome reactions than Sephadex-separated sperm, but velocity and linearity were maintained during incubation. When semen was separated with Sephadex followed by washing, motility was well maintained (84%). The Sephadex method is a promising technique for selecting and concentrating motile cryopreserved sperm.

Study on sperm function in men long after cessation of gossypol treatment.

Sperm function was assessed in 19 men 3-10 years after cessation of gossypol treatment and 2-9 years after recovery of normal sperm density. Nineteen normal fertile men of similar age served as the controls. The zona-free hamster egg-sperm penetration assay (SPA) revealed a highly significant difference (P less than 0.01) in the penetration rates between the treated and the control groups. Hormone assays indicated that there were no significant differences in circulating levels of luteinizing hormone (LH) and testosterone (T) between the two groups, but the follicle stimulating hormone (FSH) level of the treated group was significantly higher than that of the controls (P less than 0.05). The results showed that sperm function in the treated group was lower than that in the control, which may be a result of persistent gossypol-mediated damage to the testes.

Serum bioactive follicle-stimulating hormone levels in girls with precocious sexual development.

We studied the serum immunoreactive (immuno) and bioactive (bio) FSH concentrations in 16 prepubertal children (1.3-9 yr old), 6 girls with premature thelarche (0.8-2 yr old), and 9 girls with central precocious puberty (2.5-9.3 yr old). The serum bio-FSH was measured by the granulosa cell aromatase bioassay. The basal serum bio-FSH levels were not significantly different in patients with central precocious puberty (6.4 +/- 1.5 IU/L), premature thelarche (7.5 +/- 0.5 IU/L), and prepubertal controls (4.4 +/- 0.7 IU/L). However, the peak responses of both serum immuno- and bio-FSH levels to iv GnRH were higher in patients with premature thelarche (immuno-FSH, 29.3 +/- 2.3 IU/L; bio-FSH, 100.7 +/- 12.2 IU/L) than in those with central precocious puberty [immuno-FSH, 17.5 +/- 3.1 IU/L (p less than 0.05); bio-FSH, 42.4 +/- 9.8 IU/L (p less than 0.01)]. This suggests that in children with premature thelarche, there is a predominant immuno- as well as bio-FSH response to GnRH. After 12 months of GnRH agonist therapy, both serum immuno- and bio-FSH levels were suppressed in patients with central precocious puberty. The differences in clinical presentation between central precocious puberty and premature thelarche cannot be explained by the differences in FSH bioactivity.

[Relationship between malignant transformation of ovarian mucinous cystadenoma and intestinal epithelial metaplasia].

Ninety cases of ovarian mucinous tumors were studied histologically and histochemically. Intestinal metaplasia was found in 48.2% (14/29) of benign. 73.6% (14/19) of borderline and 92.9% (39/42) of malignant mucinous cystadenomas. The differences between these three groups are statistically significant (P less than 0.01). Among 67 cases of intestinal metaplastic mucinous tumors, 43 contained argyrophil cells, and 36 contained argentaffin cells. The coexistence of intestinal metaplasic and uterocervical canal type epithelia was observed in 2/3 of borderline and 1/3 of malignant intestinal mucinous cystadenomas. In addition, there were 5 cases of borderline and 3 cases of malignant uterocervical canal type mucinous cystadenomas among the 90 cases. It is evident that the malignant transformation of ovarian mucinous cystadenoma was closely related to intestinal metaplasia. Anyhow, it seems not necessary for malignant transformation of all ovarian mucinous cystadenomas to pass through a stage of intestinal metaplasia: some of the malignant mucinous cystadenomas were considered probably originating from the uterocervical canal type epithelium.

[Studies on sperm function in man long after cessation of gossypol administration].

PIP: 19 volunteers comprising the treated group who had terminated use of gossypol between 3-10 years previously were observed and compared to 19 healthy, fertile men of similar age who comprised the control group. Sperm penetration assay (SPA) showed that the difference between the penetration rates of the treated and control groups was quite remarkable (P0.01). The penetration rates in the repetition of 6 cases were similar to each other. Using the Spearman rank correlation method, there proved to be a significant difference between sperm density of insemination and sperm penetration rates (r8=0.94258, P0.05). The test of 2 X C linear regression also showed that there was a significant correlation between the penetration rates and the insemination density (X2=80.6124, P0.001). The sperm chromatin heterogeneity test showed no difference (P0.05) between both groups. Sperm ATP contents of the treated group was notably lower than that of the control (P0.01). There was a correlation between the penetration rates and ATP contents of sperm (r=0.5984, P0.05). There was a significant difference between the LH and T levels in the blood of both groups, while FSH in the treated group was higher than in the control group (P0.05). The results indicate that the sperm fertilization function of man long after gossypol treatment has been terminated was lower than that of normal, which may be due to factors other than the decrease in the blood T level. (author's modified)^ieng

[The morphology and formation of micronuclei of blood lymphocytes in man after cessation of gossypol administration].

PIP: 2 groups of men were compared under controlled experimental conditions. Group A consisted of men who were administered gossypol over a period of 2 months (subgroup I), and 9 months (subgroup II); those in group B were never administered gossypol. This experiment was performed in order to determine the levels of formation of micronuclei of blood lymphocytes in males after the administration of gossypol is terminated. The results indicate that subgroup I had a slightly higher evidence of micronuclei than subgroup II, but remarkably higher than group B. Among the blood lymphocytes, scientists discovered 1 with 22 micronuclei capable of generating more micronuclei. It is suggested that gossypol might possibly cause genes to break up.^ieng

Effect of Tripterigium wilfordii Hook. f. on the fertility of rats.

Studies were undertaken to assess the effect of the total glycosides extracted from Tripterigium wilfordii Hook. f. (GTW), a ready-made Chinese herb medicine, on the fertility of both male and female rats. In male rats, GTW was given by gastric gavage at a dose of 10 mg/kg per day, 6 days a week for 8 weeks. All the treated animals became infertile. The body weight growth was normal and the mating behavior was present. There was a drastic decrease in the viability of the epididymal spermatozoa, while the decrease in the sperm density, though significant, was far less marked. Seminiferous tubular damage was minimal and no perceptible change was observed in the Leydig and the Sertoli cells. All other parameters examined, including the serum testosterone level and the histology of various organs, were normal. In female rats, GTW given orally at a dose of 50 mg/kg per day on days 1-4 or 7-9 of pregnancy did not significantly affect the fertility. It is thus concluded that non-toxic dose of GTW can cause infertility in male rats, the mechanism of which may involve and interference in the function of the epididymal and/or testicular spermatozoa; in female rats, the dose regimens employed do not have significant anti-implantation or early-pregnancy termination effect.