Morphological features of breast cancer circulating tumor cells in blood after physical and biological type of isolation.

BACKGROUND: Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different isolation tech-niques based on their biological or physical features were established. Currently, the most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology. MATERIALS AND METHODS: The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix®) and biological (MACS®) separation technique was performed. RESULTS: In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer patient cohort, morphological features of isolated CTCs were dependent on the separation technique. After physical separation, CTCs with preserved cell morphology were detected. After biological separation the majority of the isolated CTCs were so degenerated that their identity was difficult to confirm. CONCLUSIONS: Taken together, physical separation is a suitable technique for detection of CTCs with preserved cell morphology for the use in a routine cytopathological laboratory.

Evaluation of the training program for p16/ Ki-67 dual immunocytochemical staining interpretation for laboratory staff without experience in cervical cytology and immunocytochemistry.

Background p16/Ki-67 dual immunocytochemical staining (DS) is considered easy to interpret if evaluators are properly trained, however, there is no consensus on what constitutes proper training. In the present study we evaluated a protocol for teaching DS evaluation on students inexperienced in cervical cytology. Methods Initial training on 40 DS conventional smears was provided by a senior cytotechnologist experienced in such evaluation. Afterwards, two students evaluated 118 cases. Additional training consisted mainly of discussing discrepant cases from the first evaluation and was followed by evaluation of new 383 cases. Agreement and accuracy of students' results were compared among the participants and to the results of the reference after both evaluations. We also noted time needed for evaluation of one slide as well as intra-observer variability of the teacher's results. Results At the end of the study, agreement between students and reference was higher compared to those after initial training (overall percent agreement [OPA] 81.4% for each student, kappa 0.512 and 0.527 vs. OPA 78.3% and 87.2%, kappa 0.556 and 0.713, respectively). However, accuracy results differed between the two students. After initial training sensitivity was 4.3% points and 2.9% points higher, respectively compared to the reference, while specificity was 30.6% points and 24.4% points lower, respectively, compared to the reference. At the end of the study, the sensitivity reached by one student was the same as that of the reference, while it was 2.6% points lower for the other student. There was a statistically significant difference in specificity between one student and the reference and also between students (16.7 and 15.1% points). Towards the end of the study, one student needed 5.2 min for evaluating one slide while the other needed 8.2 min. The intra-observer variability of the senior cytotechnologist was in the range of "very good" in both arms of the study. Conclusions In teaching DS evaluation, the students' progress has to be monitored using several criteria like agreement, accuracy and time needed for evaluating one slide. The monitoring process has to continue for a while after students reach satisfactory results in order to assure a continuous good performance. Monitoring of teacher's performance is also advisable.

Clinical relevance of the borderline results of the Hybrid Capture 2 High-Risk HPV DNA assay with cervical samples collected in Specimen Transport Medium.

Background The Hybrid Capture 2 (HC2) High-Risk HPV DNA assay serves as a triage test in the Slovenian national cervical cancer screening programme ZORA. To improve the limited analytical accuracy of HC2 test results near the cut-off value (1.0 relative light units/cut-off (RLU/CO)), we follow an internal protocol of repeating the test on all samples with borderline results within the 0.7-2.0 RLU/CO interval. The aim of the study was (i) to determine the clinical relevance of HC2 test results within three different "grey zones" for samples stored in Specimen Transport Medium (STM) and (ii) to determine whether the current algorithm of retesting "grey zone" STM specimens with the HC2 assay is clinically relevant. Patients and methods The study included 594 women between 20 and 65 years of age. All participating women were referred for colposcopy, and in cases of abnormal results, biopsy was performed. We assessed the distribution of HC2 test results and the corresponding proportion of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) lesions in three different "grey zones" (1.0-2.5, 0.4-4.0 and 0.7-2.0 RLU/CO), retested specimens with results within a 0.4-4.0 RLU/CO interval and calculated the sensitivity and specificity for HC2 at different RLU/CO values. Results The proportion of specimens within 1.0-2.5, 0.4-4.0 and 0.7-2.0 RLU/CO intervals was 3.9%, 10.8% and 4.5%, respectively. The proportion of CIN2+ lesions within these "grey zones" was 2.5%, 5.6% and 1.2%, respectively. Retesting the samples did not detect any additional CIN2+ cases. Within the 1.0-2.5 RLU/CO interval, the sensitivity decreased from 93.8% to 91.4%, while the specificity increased from 63.3% to 67.5%; for the 0.4-4.0 RLU/CO interval, the sensitivity decreased from 95.1% to 89.5%, while the specificity increased from 56.8% to 69.4%; and for the 0.7-2.0 RLU/CO interval, the sensitivity remained nearly constant (94.4 vs. 93.2%), while the specificity increased from 60.6% to 66.4%. Conclusions Our results show that retesting STM samples within the "grey zones" is not necessary. Retesting samples in the negative "grey zone" does not increase sensitivity, and retesting in the positive "grey zone" is not followed by a less intensive management of women, since these women are recalled regardless of the results of the retest. Furthermore, the majority of samples retain the original HC2 results after retest, and the number of CIN2+ lesions among women with "grey zone" HC2 results is low.

Isolated polyhydramnios in the third trimester or polyhydramnios secondary to late-onset gestational diabetes: is it worth distinguishing?

SUMMARY OBJECTIVE: The aim of this study was to compare pregnancy outcomes of patients with polyhydramnios due to late-onset gestational diabetes mellitus and patients with isolated polyhydramnios. METHODS: Of the women who fully participated in prenatal examinations at Etlik Lady Zübeyde Hospital between January 1, 2018, and December 31, 2019, women with polyhydramnios of nonfetal-placental origin manifesting in the third trimester were retrospectively reviewed. Women with normal 75-g oral glucose tolerance test results between 24 and 28 weeks gestation who met the inclusion criteria were enrolled in the study and divided into two groups based on the results of rescreening with the 75-g oral glucose tolerance test for polyhydramnios in the third trimester: women with isolated polyhydramnios (group 1) and women with late-onset polyhydramnios due to gestational diabetes mellitus (group 2). RESULTS: There were a total of 295 participants, of whom 35 (11.8%) were diagnosed with polyhydramnios due to late-onset gestational diabetes mellitus. There were no differences in the main outcomes. Birthweight and gestational age at birth were identified as independent risk factors for predicting composite maternal outcome {[odds ratio (OR)=1.273, 95% confidence interval (CI) 1.063-1.524, p=0.009]} and composite neonatal outcome (OR=0.606, CI 0.494-0.744, p<0.001), respectively. CONCLUSION: Polyhydramnios in late pregnancy without evidence of pregnancy-related causes leading to polyhydramnios may be a sign of late-onset gestational diabetes mellitus in women with a normal prior oral glucose tolerance test. As pregnancy outcomes and management were indifferent, it does not seem necessary or useful to diagnose whether or not late-onset gestational diabetes mellitus is present.

Effect of preoperative anxiety level on postoperative pain, analgesic consumption in patients undergoing laparoscopic sleeve gastrectomy: an observational cohort study

Abstract Backgraund: This prospective observational cohort study aimed to investigate the relationship between preoperative anxiety levels and postoperative pain and analgesic requirement in patients undergoing laparoscopic sleeve gastrectomy. Methods: Forty two female patients with body mass index ≥ 35, who underwent laparoscopic sleeve gastrectomy for treatment of obesity were included in the study. Spielberger's state and trait anxiety scales were used in this study. Demographic data of the patients, anesthetic and analgesic drugs during the surgery, pain levels measured with verbal analog scale at the postoperative 1st, 4th, 12th, and 24th hour, sedation levels measured with the Ramsay sedation scale, and the amount of analgesic consumed were recorded. Anesthesiologist, surgeon, and patient were not informed of the anxiety level results. The relationship between preoperative anxiety and postoperative pain and analgesic consumption was evaluated by Spearman's correlation analysis. Stepwise multiple linear regression analysis was applied. Normal Distribution control was performed by applying the Shapiro-Wilk test to residual values obtained from the final model. Results: There was no relationship between trait anxiety level and postoperative pain and analgesic consumption. A correlation was found between state anxiety level and pain level up to 24 hours and analgesic consumption (p < 0.05). According to the obtained model it had been observed that the university graduates consumed more analgesic compared to other education level groups. Conclusion: In this study, a relationship was found between preoperative state anxiety level and 24-hour pain scores and analgesic consumption in patients who underwent laparoscopic sleeve gastrectomy under general anesthesia.

Mothers' views of the health visiting service in the UK: a web-based survey.

User and client views are an important source of information for providers and commissioners of public services. This paper reports on a user-led UK wide survey of mothers' views about sources of support and experiences accessing the health visiting service. Netmums, a family of websites run by local mothers, undertook the survey following concerns that the health visiting service was proving increasingly difficult to access. 4,665 mothers responded to questions about a) who they would seek help from, b) views of the local health visiting service and c) perceived change in it over the past two years. The results show that the majority of respondents rate their service as quite or very good, valuing most the characteristics of a) expertise in child health development and parenting and b) time and willingness to listen on the part of health visitors. In all areas but one, the service was seen as changing for the worse. Following the re-affirmation of a need for a universal health visiting service to be able to pick up families in need, the challenge is now to design local services that have sufficient resources so that mothers can maintain relationships and confidently access the services when they need them.

Yellow sac spider (Cheiracanthium punctorium) bites in Slovenia: case series and review.

In Central Europe, reports of human envenomation by Cheiracanthium punctorium, commonly known as the yellow sac spider, are sporadic, despite the fact that this species is widespread in Europe. However, in recent years, C. punctorium has been repeatedly described globally in medical and toxicological literature. Its venom was found to possess insecticidal, haemolytic, cytotoxic, and membrane-damaging activities. Its bite is often very painful, frequently associated with local and transient cutaneous and neurotoxic effects, but sometimes also with systemic symptoms which require medical help. The main objective of this article is to introduce more details about C. punctorium, the clinical manifestations and circumstances of its bite, the characteristics of its venom and proposed clinical management. The authors provide case reports of patients bitten by C. punctorium during the 10-year observational period. All patients presented in this article showed generally mild clinical manifestations and recovered completely without sequelae. No further treatment in terms of hospital surveillance or specific clinical measures was necessary in any of the reported cases.

Detection of electrocardiographically imperceptible ventricular pre-excitation by phase imaging.

The sequence of ventricular contraction was studied by radionuclide phase imaging in 25 patients with Wolff-Parkinson-White syndrome. The studies were performed when no signs of precontraction were present in the electrocardiogram; in these cases pre-excitation was either intermittent or suppressable by injection of ajmaline. In 11 of the 16 patients with free wall accessory pathways, precontraction could be detected in spite of electrocardiographically absent pre-excitation. Discrete precontraction was seen also in 2 of the 9 patients with paraseptal accessory pathways. We conclude that antegrade conduction through the accessory pathway does not need to be completely blocked if signs of pre-excitation are absent on the electrocardiogram, and that phase imaging is, at least in some patients (especially those with free wall accessory pathways), a more sensitive technique for detection of pre-excitation (precontraction) than the electrocardiogram.

Computer simulation of ciprofloxacin pharmacokinetics in patients on CAPD.

Continuous ambulatory peritoneal dialysis (CAPD) has become an accepted alternative to chronic hemodialysis in the treatment of end-stage renal disease. The method utilizes the diffusion of drugs from the blood through the peritoneal membrane to the peritoneal cavity if administered intravenously (IV) and perorally (PO) and in the opposite direction if applied intraperitoneally (IP). The present work uses an open, two-compartment pharmacokinetic model reversibly linked with the compartment representing the peritoneal cavity and an analog-hybrid computer to simulate drug levels in sampled and unsampled compartments under conditions of various routes of administration (IV, IP and sequential IV, IP and PO) and different clinical status (presence and absence of peritonitis). The drug chosen for simulation was ciprofloxacin (CIP), a new synthetic antibacterial agent of the 4-quinolone group. Eight patients were included in the study, and CIP concentrations in plasma and dialysate were obtained by HPLC analysis to assess the reliability of the model and the efficiency of the sequential dosing scheme. CIP plasma and dialysate levels were adequate for the majority of microbes causing CAPD peritonitis. The proposed regimen was efficient in 85% of cases.

Subcutaneous erythropoietin in the treatment of renal anaemia.

Erythropoietin was applied subcutaneously to 49 patients, 41 have been treated by hemodialysis, 3 by continuous ambulatory peritoneal-dialysis, 5 had chronic progressive renal failure. Mean initial dose of erythropoietin was 139.4 U/kg/week and maintenance dose 115.9 U/kg/week. In 43% of patients serum ferritin was decreasing during treatment, and in 20% it was low before the commencing of the treatment. During erythropoietin therapy vitamin B12 was decreasing in 22% of the patients, and the substitution was necessary in 18%. Only in 1 patient it was necessary to substitute also folic acid. There were no nonresponders among erythropoietin treated patients. Elevation of blood pressure was observed in half of the patients, hypertensive encephalopathy in 1, and thrombosis of arterio-venous fistula in 3.

Treatment of anemia with epoetin in kidney transplant recipients.

The aim of this study was to analyze the prevalence and efficacy of renal anemia treated with epoetin in maintenance kidney transplant recipients in Slovenia. By the end of 2009, 107 out of 537 patients (19.9%) had been treated with epoetin. A cohort of 49 patients (45.8%) were analyzed in detail: 11 patients received epoetin alpha, 18 epoetin beta, 10 darbepoetin alpha, and 10 patients received methoxy polyethylene glycol-epoetin beta. The median epoetin dose was 0.36 µg/kg body weight per week. The median serum laboratory parameters were as follows: hemoglobin 120 g/L, hematocrit 0.36, ferritin 332 ng/mL, transferrin saturation 34%, serum creatinine 145 µmol/L, serum albumin 41 g/L, intact parathyroid hormone 79 ng/L, and C-reactive protein 3 mg/L. We concluded that the prevalence of renal anemia in kidney transplant recipients treated with epoetin was approximately 20%, and laboratory parameters suggested that the treatment of renal anemia in this study cohort was optimal.

Membrane plasma exchange for the treatment of thrombotic thrombocytopenic purpura.

The aim of our report is to present our 11-year experience with therapeutic membrane plasma exchange therapy for the treatment of idiopathic thrombotic thrombocytopenic purpura syndrome (TTP). In 56 patients, membrane plasma exchange therapy was initiated immediately and performed once or twice daily until the platelet count normalized. During each plasma exchange procedure, 1-1.5 plasma volumes (3606 +/- 991 mL) were replaced with fresh frozen plasma. In 37 females and 19 males (44 +/- 21 years), 1066 plasma exchange procedures were performed. The average duration of treatment was 23 +/- 17 days. The average number of plasma exchanges was 19 +/- 17 per patient. Renal impairment was detected in 36% of patients. At the initiation of plasma exchange treatment, the average platelet count was 31 +/- 30 x 10(9)/L and reached 199 +/- 95 x 10(9)/L thereafter. Fifty-two of 56 (93%) patients demonstrated an excellent response to plasma exchange therapy, of whom 48 patients (86%) attained complete remission with a platelet count of more than 100 x 10(9)/L. Four patients died soon after the initiation of plasma exchange therapy, when only 1-3 procedures had been performed. During the follow-up period, six patients with complete remission had 1-5 subsequent relapses each year. One of them died of acute hemolytic reaction during the tapering of plasma exchange procedures. Three patients underwent additional splenectomy. Our experience with primary TTP supports the plasma exchange treatment with fresh frozen plasma as a mandatory, up-to-date therapy. Close monitoring during all 1066 procedures showed no serious side-effects.

Correlation between thyroxine, triiodothyronine and thyrotropin in geriatric patients with goitre.

Serum TSH, T4 and T3 have been assayed in three groups of older persons (greater than 60 years): in 28 normal subjects (A), in 80 patients with nontoxic goitre (B), in 22 patients with hyperthyroidism (C). Group B was divided in group B-1 (TSH 0.20-0.30 mU/l) and group B-2 (TSH greater than 0.30 mU/l). The test characteristics were determined by comparison of group C patients with group A patients and the former with group B patients. The specificity and posterior probability of TSH test calculated by the first comparison was 1.0 for both characteristics, and by the second comparison TSH test specificity was 0.75 and posterior probability 0.62. Specificity of thyroid hormones calculated in the second comparison was 0.96 for T4-test and 0.94 for T3-test. Therefore TSH test is the most adequate in clinical diagnostic of thyrotoxic goiter. The particularities of several groups were: B-2 group had significantly lower mean value of T4 than Group A, but there was no significant difference between mean values of TSH found. There were no correlations between TSH and T4 and between T4 and T3. Group B-1 had significantly lower mean TSH value than Group A and Group B, but the groups did not differ in mean T4 value. The correlation between TSH and T4 in group was positive as the correlation between T4 and T3. It is suggested that the value of serum T4 of Group B-1 may be inappropriately low due to defective immunological activity of T4 from autonomous follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

[Early prediction of hypothyroidism after subtotal thyroidectomy (author's transl)]. / Früherkennung von Hypothyreose nach Strumaresektion.

30 patients were classified euthyroid (group A, n = 13) and hypothyroid (group B, n = 17) 4 years after subtotal thyroidectomy. A retrospective analysis of the hormonal status was then performed. In both groups (A and B) a significant decrease of serum T3 was found 3 days after surgery. 3 months later T3 levels had returned to normal. Serum T4 showed no significant change, neither at day 3 nor 3 months after the operation. Serum thyrotropin (TSH) also showed no significant change 3 days after surgery in both groups. However, 3 months later serum TSH levels were found to be significantly increased in group B (greater than 4.8 microU/ml, Mean = 13.0 +/- 6.7 microU/ml, p less than 0.001) while in group A TSH remained normal (normal range: 0--3.4 microU/ml). Assuming a serum TSH of 5.0 microU/ml as critical level at 3 months after subtotal thyroidectomy 25% of eventually hypothyroid patients will be missed and 1% will be classified hypothyroid. It is therefore suggested that all patients with serum TSH of more than 4.8 microU/ml 3 months after surgery should be considered chronically hypothyroid. The patients with a serum TSH between 3.4--4.8 microU/ml should be kept on prophylactic thyroid hormone therapy until further 3 months later the final diagnosis is established.

Localization of accessory pathways in Wolff-Parkinson-White syndrome by phase imaging.

Phase image analysis of blood pool scintigrams was performed in 6 patients with Wolff-Parkinson-White (WPW) syndrome to determine the relationship of phase changes and abnormalities of atrioventricular conduction. The site of preexcitation was located by invasive endocardial mapping. The phase advance generally correlated with the electrophysiologically determined location of the accessory pathway. Factors which increase or decrease the amount of preexcitation also increased or decreased the advance of mean phase angles. Phase imaging is a useful noninvasive means for tentative location of accessory pathways in WPW syndrome.

A comparison between epoetin omega and epoetin alfa in the correction of anemia in hemodialysis patients: a prospective, controlled crossover study.

The objective of the study was to evaluate and compare the safety and effectiveness of epoetin omega (produced in baby hamster kidney cells) and epoetin alfa (produced in Chinese hamster ovary cells) in sustaining the correction of anemia in maintenance hemodialysis patients. The study, a prospective and controlled crossover, was completed in 38 stable patients treated with both epoetins for 24 weeks. Group A (17 patients) started with epoetin omega, and Group B (21 patients) started with epoetin alfa. After 24 weeks, a 4 week crossover (wash out) was made: Group A was switched to epoetin alfa and group B to epoetin omega for the next test period of 24 weeks. Both epoetins were administered subcutaneously after each dialysis. Doses were adjusted with the aim of maintaining a target hemoglobin level between 10 and 12 g/dl (hematocrit 30% to 35%). The mean weekly dose of epoetin omega/kg body weight (BW) was 67 +/- 43 U. The mean weekly dose of epoetin alfa/kg BW was 86 +/- 53 U. The average of all mean values of hemoglobin (Hb) during treatment with epoetin omega was 11.4 +/- 0.7 g/dl (hematocrit 34 +/- 2%), and during treatment with epoetin alfa was 11.3 +/- 0.7 g/dl (hematocrit 33 +/- 2%) (not significant). Thromboses of vascular access occurred in 3 patients during an epoetin omega treatment and in 3 patients during epoetin alfa treatment. At the site of injection, only 1 patient described a mild pain when treated with epoetin omega and only 6 patients when treated with epoetin alfa. In conclusion, both epoetin omega and epoetin alfa were effective in correcting the anemia of all studied patients. However, lower doses of epoetin omega were needed to maintain the same target hemoglobin level. No serious side effects with either epoetin were noted. The authors believe that additional comparisons of different epoetin preparations should be performed and will provide better insight into their biological activity and clinical responsiveness.

Indices of iron status in patients treated by chronic haemodialysis.

Iron deficiency in patients with end stage renal disease (ESRD) treated by haemodialysis (HD) is difficult to diagnose. The reticulocyte hemoglobin content (CHr) and the percentage of hypochromic red cells (%hypo) are sensitive novel assays for the detection of functional iron deficiency in patients treated with erithropoietin (EPO). In our study thirty-nine chronically hemodialyzed patients were evaluated to determine the value of these two parameters in comparison to the conventional biochemical indicators of iron metabolism. There were significant correlations between CHr and transferrin saturation, CHr and weekly dosage of EPO, and also between %hypo and weekly dosage of EPO. Our data represent superior value of %hypo and CHr to the transferrin saturation and ferritin concentration in detecteng of iron deficiency in HD patients.