Initial cord blood unit volume affects mononuclear cell and CD34+ cell-processing efficiency in a non-linear fashion.

BACKGROUND AIMS: Umbilical cord blood (UCB) is a source of hematopoietic stem cells that initially was used exclusively for the hematopoietic reconstitution of pediatric patients. It is now suggested for use for adults as well, a fact that increases the pressure to obtain units with high cellularity. Therefore, the optimization of UCB processing is a priority. METHODS: The present study focused on parameters influencing total nucleated cell (TNC), mononucleated cell (MNC) and CD34+ cell (CD34C) recovery after routine volume reduction of 1553 UCB units using hydroxyethyl starch-induced sedimentation with an automated device, under routine laboratory conditions. RESULTS: We show that the unit volume rather than the TNC count significantly affects TNC, MNC and CD34C processing efficiency (PEf), and this in a non-linear fashion: when units were sampled according to the collection volume, including pre-loaded anticoagulant (gross volume), PEf increased up to a unit volume of 110-150 mL and decreased thereafter. Thus units with initial gross volumes < 90 mL and > 170 mL similarly exhibited a poor PEf. CONCLUSIONS: These data identify unit gross volume as a major parameter influencing PEf and suggest that fractionation of large units should be contemplated only when the resulting volume of split units is > 90 mL.

Accuracy of urethral swab and urine analysis for the detection of Mycoplasma hominis and Ureaplasma urealyticum in women with lower urinary tract symptoms.

OBJECTIVES: Our objective was to evaluate and compare the accuracy of urethral swabs and urine specimens in the detection of Mycoplasmas in women with lower urinary tract symptoms (LUTS). METHODS: During a urogynecological work-up, including cystometry, we obtained first-void urine, urethral and vaginal swabs in 207 consecutive women at our urogynecological division. Mycoplasma hominis and Ureaplasma urealyticum as well as other microorganisms were detected by standard culture methods. RESULTS: 131 of 207 women reported LUTS. The other 76 formed the controls. Of 207 women 50 (24.2%) had positive cultures for Mycoplasmas. The prevalence of Mycoplasmas in women with LUTS (30.3%) was statistically significant and higher in the group without LUTS (14.5%) (p = 0.011). The detection of M. hominis was most accurate using urethral swab (Specificity 99.9%, PPV 99.6%) compared to the urine specimen (96%, 75%) and vaginal swab (95.1%, 67%). Similar results could be achieved for U. urealyticum (urethral swab: specificity 98.7%, PPV 96.3%; urine specimen: 86.8%, 72%; vaginal swab: 80.5%, 65.2%). CONCLUSION: In the subgroup of women less than 50 years an (detectable) infection due to Mycoplasma or Ureaplasma leads typically to LUTS with normal filling cystometry, whereas no such findings were relevant for the elderly women.

Controversies in hybrid banking: attitudes of Swiss public umbilical cord blood donors toward private and public banking.

PURPOSE: Umbilical cord blood (UCB) stored in public inventories has become an alternative stem cell source for allogeneic stem cell transplantation. The potential use of autologous UCB from private banks is a matter of debate. In the face of the limited resources of public inventories, a discussion on "hybrid" public and private UCB banking has evolved. We aimed to explore the attitudes of the donating parents toward public and private UCB banking. STUDY DESIGN AND METHODS: A standardized, anonymous questionnaire was sent to the most recent 621 public UCB donors including items regarding satisfaction with recruitment process, the need for a second consent before release of the UCB unit for stem cell transplantation, and the donors' views on public and private UCB banking. Furthermore, we asked about their views on UCB research. RESULTS: Of the questionnaires, 48% were returned, and 16% were lost due to mail contact. Of our donors, 95% would donate to the public bank again. As much as 35% of them were convinced that public banking was useful. Whereas 27% had never heard about private UCB banking, 34% discussed both options. Nearly 70% of donors opted for public banking due to altruism and the high costs of private banking. Of our public UCB donors, 81% stated that they did not need a re-consent before UCB release for stem cell transplantation. In case of sample rejection, 53.5% wanted to know details about the particular research project. A total of 9% would not consent. CONCLUSIONS: Almost all donors would choose public banking again due to altruism and the high costs of private banking. Shortly after donation, mail contact with former UCB donors was difficult. This might be a relevant issue in any sequential hybrid banking.

Risk factors in prolonged postpartum urinary retention: an analysis of six cases.

PURPOSE: Prolonged first and second stage of labor, isolated prolongation of the second stage, forceps delivery or vacuum extraction, perineal laceration, nulliparity and epidural anesthesia are known risk factors for developing prolonged postpartum urinary retention (PUR). The aim of our study was to analyze number and constellations of these risk factors, in prolonged postpartum urinary retention (PPUR) in our own unit to facilitate the identification of patients at high risk and thus to prevent bladder overdistension by early intervention. METHODS: We performed a retrospective analysis of all our cases with PPUR between 2003 and 2008 including variables like age weight, height, body mass index, fetal birth weight and head circumference. RESULTS: The incidence of PPUR at our institution is low being 0.06%. No woman combined all six risk factors. The majority had five risk factors, all had at least four. An isolated prolonged second stage of labor was common to all patients with PPUR. Five women had an epidural anesthesia, three were nulliparous and only two women delivered spontaneously. All but one woman suffered from perineal tears. Interestingly, fetal head circumference was larger than 36 cm in four of six cases. CONCLUSION: In contrast to simple PUR, the prolonged form of PUR could be the result of a cumulative effect of different single risk factors.

Transplacental traffic after in utero mesenchymal stem cell transplantation.

Transplacental traffic of fetal progenitor and differentiated cells is a well-known phenomenon in pregnancies. We hypothesize that intrauterine stem cell transplantation leads to microchimerism in the dams and that this is gestational age-dependent. EGFP+ fetal liver-derived mesenchymal stem cell (MSC) (10(5) per fetus) were injected intraperitoneally into congeneic and allogeneic recipient fetuses at E12 versus E13.5 of murine pregnancy (56 dams). Engraftment in maternal organs was evaluated using TaqMan quantitative polymerase chain reaction (PCR) and fluorescence microscopy during pregnancy (1, 3, and 7 days after in utero transplantation [IUT]) and after delivery (1 and 4 weeks after delivery). One day after IUT donor cells were mainly found in the placenta (E12: 9/10 dams vs. E13.5: 4/8 dams) and laparotomy site (E12: 5/10 dams vs. E13.5: 4/8 dams). Three days after IUT these probabilities decreased significantly in the placenta to 3/8 and 1/3, respectively, whereas it was increased within the surgical wound to 8/8 and 2/4. One week after IUT donor cells could be detected in other single maternal organs, such as bone marrow or spleen. The surgical wound was chimeric in all dams. One week after delivery the surgical wound was still a major site of engraftment in both groups. E12 IUT resulted in detectable donor cell microchimerism in the maternal bone marrow (3/4), liver (2/4), lungs (1/4), spleen (1/4), and thymus (1/4), whereas engraftment probabilities were lower following E13.5 IUT (BM: 1/4, liver: 2/4, lungs: 1/4, spleen: 1/4, thymus: 0/4). At 4 weeks after delivery persistent microchimerism was found only after E12 IUT in various maternal organs (BM: 1/4, spleen: 1/4, lungs: 1/4) and within newly created surgical wounds (3/4), but completely not in the E13.5 group. Allogeneic IUT did also not result in any detectable long-term fetal microchimerism. An earlier IUT might lead to a higher transplacental traffic of donor MSC and persistent microchimerism within maternal tissues. Even 4 weeks after delivery, these cells are present in surgical wounds.

Bilateral papillary cystadenoma of the mesosalpinx: a rare manifestation of Von Hippel-Lindau disease.

We report a rare case of a woman with bilateral papillary cystadenomata of the broad ligament with von Hippel-Lindau disease (VHL) (other manifestations: capillary hemangioblastomas of the spinal cord). Patient surveillance is important, because in the course of VHL-associated tumors malignant lesions may arise that are relevant for the prognosis.

Cesarean section due to fetal distress increases the number of stem cells in umbilical cord blood.

BACKGROUND: Umbilical cord blood (UCB) can be used as hematopoietic stem cell source for transplantation. The success of a transplantation is highly correlated with the number of total nucleated cells (TNCs) and CD34+ cells in the UCB. Certain obstetric factors increase the yield of stem cells in the UCB. It is necessary to evaluate optimal conditions in labor to decrease the rate of sample rejection due to low cell count. No data exist regarding the difference between primary and secondary Cesarean sections in terms of efficacy of stem cell harvesting. STUDY DESIGN AND METHODS: Seventy-nine consecutive UCB units from women who had a Cesarean section between 1997 and 2003 were included. The number of TNCs, CD34+ cells, colony-forming units (CFUs), white blood cells (WBCs), nucleated red blood cells (NRBCs), and the total collection volume were compared between cases with primary and secondary Cesarean section. RESULTS: UCB obtained after a Cesarean section due to fetal distress has significantly higher numbers of TNCs, CD34+ cells, NRBCs, and WBCs compared to elective Cesarean section. Of the cases with secondary Cesarean section due to fetal distress, 67 percent resulted in UCB units with sufficient TNC numbers (> or =80 x 10(7) TNCs) compared to 42 percent of the cases with primary Cesarean section. CONCLUSION: Fetal distress increases the number of hematopoietic stem cells mobilized into UCB. Particular effort should be made to collect UCB from newborns who experienced fetal distress.

In utero haematopoietic stem cell transplantation. Experiences in mice, sheep and humans.

Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good long-term engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation.

In utero haematopoietic stem cell transplantation. Experiences in mice, sheep and humans.

Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good longterm engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation.

Disturbances in placental immunology: ready for therapeutic interventions?

Recent studies have provided new insight into aberrations in the immunological interplay between mother and fetus and their potential role in the development of recurrent fetal loss and preeclampsia. The action of anti-phospholipid antibodies in recurrent fetal loss is now proposed to involve the complement system, neutrophil activation and the production of TNFalpha by immune bystander cells. A clear involvement of the immune system is emerging in preeclampsia, involving mainly the innate arm, especially neutrophils. The activation of peripheral neutrophils by placentally released inflammatory debris triggers the induction of neutrophil extracellular traps (NETs), which may lead to an occlusion of the intervillous space, thereby further promoting a condition of placental hypoxia. It has, hence, been suggested that new therapeutic strategies be developed, including the possible use of TNFalpha antagonists in cases of recurrent miscarriage. These strategies need to be addressed with caution due to the possible induction of fetal congenital abnormalities.

Examination of maternal plasma erythropoietin and activin A concentrations with regard to circulatory erythroblast levels in normal and preeclamptic pregnancies.

OBJECTIVES: Preeclampsia has been shown to be associated with an increased number of fetal and maternal erythroblasts in the maternal circulation, suggesting that preeclampsia involves increased leakage of fetal cells across the placental barrier, as well as increased erythropoiesis. We examined the relationship between circulatory erythroblast levels with maternal plasma concentrations of erythropoietin and activin A. METHODS: In a case-control study, we examined 15 pregnancies affected by preeclampsia and 10 matched controls. Erythroblasts were enriched from maternal blood samples by magnetic cell sorting, enumerated and correlated with corresponding plasma activin A and erythropoietin concentrations. RESULTS: The proportion of erythroblast was elevated in preeclampsia (0.8 vs. 0.1%, p = 0.023). Erythropoietin and activin A concentrations were significantly elevated in preeclampsia (100.4 vs. 44.5 pg/ml, p = 0.023, and 7.4 vs. 1.85 ng/ml, p = 0.029, respectively). Circulatory erythroblast numbers were found to correlate with plasma activin A concentrations (r = 0.76, p = 0.01) in cases with preeclampsia. No such relationship existed for erythropoietin. CONCLUSIONS: Our data suggest that increased concentrations of activin A promote enhanced levels of erythropoiesis in preeclampsia. As the placenta is one of the major sources of activin A in pregnancy, this increase in activin A-dependent erythropoiesis in preeclampsia may be a reflection of an underlying placental hypoxic condition.

Engraftment kinetics of human cord blood and murine fetal liver stem cells following in utero transplantation into immunodeficient mice.

This study was undertaken to evaluate the kinetics of engraftment after in utero transplantation of murine fetal liver and human cord blood stem cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. NOD/SCID fetuses were injected with murine fetal liver or human cord blood CD34+ cells at day 13.5 of gestation. Frequencies of donor cells were analyzed by flow cytometry up to 48 h post transplantation and 4-16 weeks postnatally. Hematopoietic multilineage reconstitution capacity was assessed. Both types of donor cells home rapidly. However, the frequency of human cord blood stem cells rapidly diminished while the murine fetal liver stem cells expanded over time, resulting in multilineage hematopoietic reconstitution. Differences in long-term reconstitution of allogeneic versus xenogeneic donor cells were ascribed to the inability of the human cells to self-renew and differentiate in the fetal mouse environment, demonstrating the limitations of this commonly used xenograph.

Correlation of perineal ultrasound and lateral chain urethrocystography in the anatomical evaluation of the bladder neck.

Although perineal ultrasound has been widely used, no standard values have been published. In 52 women with urinary stress incontinence the following parameters were measured during resting and straining: a) with ultrasound: distance (Dy) between bladder neck and central line of the symphysis, distance (Dx) between bladder neck and lower border of the symphysis, and retrovesical angle beta; b) with urethrocystography (UCG): distance H between the bladder neck and the SCIPP line, inclination angle alpha and retrovesical angle beta. Dy and H correlated during resting ( r = 0.608; p<0.001) and straining ( r = 0.575; p<0.001). The distance Dy of 11 mm corresponded to a bladder neck position on the SQIPP line. A rotational descent was detectable when Dx increased from 13 mm (+/- 6.5) during resting to 16 mm (+/-7.8) during straining ( p=0.009). It was concluded that perineal ultrasound is valuable for the anatomical evaluation of the bladder neck. This is the first report to better define the position of the bladder based on ultrasonographic measurements.

Inability to detect cell free fetal DNA in the urine of normal pregnant women nor in those affected by preeclampsia associated HELLP syndrome.

OBJECTIVE: Recent reports have indicated that cell-free fetal DNA can be detected in the urine of pregnant women. We attempted to reproduce those data. METHODS: Urine samples were collected from 18 normal pregnant women (11 with a male fetus). Urinary DNA was examined by Y-chromosome-specific nested polymerase chain reaction (PCR) or real-time PCR. Samples were also examined from two pregnancies complicated by HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, which had very high levels of cell-free fetal DNA in the maternal plasma. To validate our data, a quantitative comparison of different DNA extraction procedures used in the previous reports was performed. RESULTS: In no instance were we able to detect any fetal DNA in maternal urine, although copious quantities of cell-free fetal DNA were present in the maternal plasma of those pregnancies affected by HELLP syndrome. Our quantitative analysis of the various extraction procedures used indicated that the commercial column elution method we used was comparable, if not superior, to the noncommercial methods used in previous reports. CONCLUSION: Our data strongly suggest that cell-free fetal DNA is not readily detectable in maternal urine, even under conditions known to increase kidney permeability.

The impact of intrapartum factors on umbilical cord blood stem cell banking.

AIMS: Umbilical cord blood can be used as an alternative source for related and unrelated allogeneic stem cell transplantation. This study was undertaken to determine whether intrapartum factors have an influence on the hematopoietic cell compartment of cord blood. METHODS: Cord blood samples were obtained from 102 normal full-term deliveries for the banking of stem cells. We analyzed the influence of intrapartum factors on the count of CD34+ cells, total nucleated cells, colony forming units and total volume of collection. Fluorescence-activated cell sorting was used to measure CD34+ cell numbers. Statistical analysis was undertaken using Pearson correlation test and multiple regression analysis. RESULTS: The higher the infants' birthweight the larger was the volume. A lower arterial umbilical pH and a larger blood volume resulted in an increased number of CD34+ cells. A large blood volume, long duration of labor, lower arterial and venous pH were correlated with more nucleated cells. A higher birthweight, larger blood volume and lower arterial pH resulted in an increased number of colony forming units. CONCLUSIONS: Some intrapartum factors have an impact on the characteristics of collected cord blood cells. Stress during delivery may influence the number of hematopoetic cells, through altered cytokine production. This knowledge may facilitate the selection of optimal cord blood samples for unrelated banking and the early discarding of suboptimal cord blood samples thus resulting in the saving of costs related to expensive further processing.

Spectral analysis of fetal heart rate variability in fetuses with supraventricular extrasystoles.

OBJECTIVE: Fetal heart rate (FHR) variability is an important indicator of fetal well-being. In fetal tachyarrhythmias, however, visual analysis of FHR variability is limited. We therefore applied power spectral analysis of FHR to evaluate the fetal state. METHODS: Fetal R-R intervals were detected by means of an external ECG in 3 fetuses with supraventricular extrasystoles after cardiac malformations had been excluded by fetal echocardiography. Using an autoregressive model, power spectral densities were calculated from 20 consecutive 256-beat segments for the following frequency bands: <0.03 Hz (very low frequency), 0.03-0.069 Hz (low frequency; LF), 0.07-0.129 Hz (mid-frequency) and 0.13-1.0 Hz (high frequency; HF). RESULTS: The FHR variability in fetal supraventricular extrasystoles mainly resulted from the HF component (63.91 +/- 6.97%). The sympatho-vagal balance (LF/HF) was decreased in the tracings with extrasystoles (0.13). CONCLUSION: The analysis of FHR variability in fetal supraventricular extrasystoles revealed an imbalance between sympathetic and parasympathetic regulation.

Attitudes of Swiss mothers toward unrelated umbilical cord blood banking 6 months after donation.

BACKGROUND: During the past decade, the use of umbilical cord blood (CB) as a source of transplantable hematopoietic stem cells has been increasing. Little is known about the psychosocial consequences that later affect parents after unrelated CB donation. The objective of this study was to evaluate the attitudes of mothers toward unrelated donation of umbilical CB for transplantation 6 months after giving birth. STUDY DESIGN AND METHODS: A prospective study was performed with a standardized, anonymous questionnaire distributed to 131 women 6 months after CB donation. The questionnaire included topics concerning views about the ethical accuracy of having donated CB, emotional responses after donation, concerns about genetic testing and research with CB samples, attitude toward anonymity between her child and possible unrelated CB recipient, and willingness to repeatedly donate umbilical CB in a next pregnancy. RESULTS: The vast majority (96.1%) stated that they would donate umbilical CB again, and all respondents were certain that their decision to have donated umbilical CB was ethical. With regard to the potential risks of genetic testing and "experimentation" of umbilical CB, a significant correlation (p = 0.01) was found between negative attitudes and the decision not to donate umbilical CB again. Additionally, it was observed that women who had a negative experience concerning the donation of CB would not donate again (p = 0.004). CONCLUSIONS: This study shows a high degree of satisfaction of unrelated umbilical CB donation for banking in women 6 months after delivery. Despite a well-performed and detailed informed consent procedure, one of the ongoing issues for the donators in CB banking involves the concern regarding of improper use of the cells, such as genetic testing or experimentation. Accurate and detailed counseling of pregnant women and their partners therefore maximizes the likelihood that they will donate CB for unrelated banking. These data provide a basis for the improvement of donor selection procedures and public education regarding the use of CB for banking and transplantation.

Influence of volume preloading on uteroplacental and fetal circulation during spinal anaesthesia for caesarean section in uncomplicated singleton pregnancies.

OBJECTIVE: Effects of volume preloading during spinal anaesthesia for elective caesarean section on maternal blood pressure, feto-maternal circulation and fetal outcome. PATIENTS AND METHODS: In a pilot study a randomised trial was performed in 22 healthy women with uncomplicated, singleton pregnancies at 36-40 weeks of gestation undergoing elective caesarean section under spinal anaesthesia. In the low volume group (group A) patients received 150 ml of crystalloid solution for preloading, in the high volume group (group B) they were given 15 ml/kg of crystalloid solution for preloading before the initiation of spinal anaesthesia. Maternal blood pressure was monitored intermittently. Hypotension was defined as a decrease in systolic pressure to less than 80% of the baseline value. The Doppler flow evaluation consisted of measurements from the uterine artery at the placental site, fetal umbilical artery and fetal middle cerebral artery. Pulsatility indices were derived before and after fluid preloading, and when spinal anaesthesia was established. The neonatal outcome was assessed by Apgar scores, arterial acid base status and neurologic and adaptive capacity scores (NACS). RESULTS: The incidence of maternal hypotension in both groups was 45.5% (n = 10); 3 cases occurred in group A compared to 7 cases in group B (n.s.). There was no evidence that the high dose volume is useful in preventing maternal hypotension. The pulsatility indices of uterine arteries, umbilical arteries and middle cerebral arteries were not altered. Statistical analysis showed no changes in neonatal outcome concerning umbilical arterial pH, Apgar score and NACS (n.s.) between groups A and B. CONCLUSIONS: Our preliminary results suggest that high dose crystalloid volume preloading has no preventive function in the avoidance of maternal hypotension in healthy parturients undergoing elective caesarean section under spinal anaesthesia, and shows no harmful effects on neonatal outcome as long as maternal hypotension is corrected immediately. However, the statistical significance may reflect the small sample size, and larger series are needed before changing the current management.