Diagnosis of acute foetal distress does not preclude banking of umbilical cord blood units.

Clinical diagnosis of acute foetal distress (AFD) is based on several parameters such as abnormal foetal heart rate (FHR) pattern and/or meconium liquid staining (MLS). Standards for cord blood (CB) banking indicate that AFD should be considered as exclusion criteria for CB collection, but precise guidelines on how to proceed with CB collection in the presence of AFD signs during labour are not available. We evaluated whether the presence of FHR abnormality and/or MLS during labour 1) reduced the CB collection activity; 2) were associated with the infant's acidaemia or asphyxia and 3) deteriorated the biological characteristics of CB units. Thirty-three units of CB were evaluated for biological parameters, gas values and newborn's Apgar score. The results were compared with a control group of 33 consecutive units previously banked. No differences were observed between the two groups and all but one newborn showed normal Apgar score and absence of metabolic acidaemia. The results showed that 1) AFD reduced the CB collection activity by 10% each year; 2) the majority of CB units collected in the presence of abnormal FHR and/or meconium have biological characteristics eligible for banking; 3) FHR alterations or meconium in the presence of normal gas analysis do not represent certain diagnosis of AFD.

Evaluation of biological features of cord blood units collected with different methods after cesarean section.

INTRODUCTION: Cord blood banks are established worldwide as a result of the increased use of umbilical cord blood (UCB) transplantation. The outcomes of this procedure relate to the cell dose of the UCB unit and the UCB collection. The aim of this study was to evaluate whether the mode of collection influenced the biological features of the UCB units. MATERIALS AND METHODS: We studied 151 UCB units consecutively collected in the cesarean setting with two different methods: in utero after infant delivery and before delivery of the placenta, and ex utero after delivery of placenta. RESULTS: Sixty-nine UCB units were collected in utero and 82 ex utero. The two groups were comparable for maternal and obstetric factors. The proportion of banked UCB units was similar in the two groups (38% vs 40%, respectively). No statistically significant differences were observed between the methods of collection in term of volume, white blood cell count, total nucleated cell content, CD34(+) cells, and CFU-GM. CONCLUSION: This preliminary study showed that the two methods of collection in the cesarean setting were overlapping and valid if performed according to standard operating procedures.

Optimizing umbilical cord blood collection: impact of obstetric factors versus quality of cord blood units.

INTRODUCTION: The main limitation factor for wide use of umbilical cord blood units (UCBs) as a source of hematopoietic progenitors for transplantation is cell dose. International standard guidelines recommend 2 x 10(7)/kg as the minimal nucleated cell dose for UCB transplantation for adults and 3.7 x 10(7)/kg for children. Therefore it is important to the optimize donor selection and the collection method so as to achieve high cell doses. In this study our main purpose was to determine whether obstetric factors influence UCBs collected. STUDY DESIGN: The study involved 304 UCBs collected from January to December 2004. The UCBs were collected after donor selection based on international criteria for cord blood banking. We analyzed UCB biological features such as collected volume, total nucleated cells (TNC), and CD34-positive cells, and obstetric factors. RESULTS: First, our study showed by multivariate analysis that infant weight was the main factor that influenced biologic features of UCB collected such as total volume (P = .000), TNC (P = .000), CD34 total count (P = .003), and CFU-GM (P = .004). Placental weight > 600 g produced a better volume (P = .007) and increased TNC (P = .056). Gestational age > 39 weeks enhanced CD34% (P = .016). Regarding route of delivery, we found that cesarean section produced higher volume and reduced WBC count compared to vaginal delivery, regarding cord length, it increased TNC (P = .037). And last, we noticed that female infants increased WBC (P = .013) and CD34(+) total count (P = .019) more than male ones. CONCLUSIONS: Our results confirm that volume and TNC are influenced by several obstetric factors, such as greater infant and placental weight, predicting a better collection.

Placental/umbilical cord blood: experience of st. Eugenio Hospital collection center.

BACKGROUND: Umbilical cord blood (UCB) contains stem cells and may be an alternative source to bone marrow and peripheral blood stem cells for allogeneic transplantation for the treatment of hematologic malignancies and genetic disorders. Many UCB banks have been established worldwide to quickly provide high quality materials for transplantation. Moreover, a detailed set of standards for cord blood banking have been drawn to guarantee the quality of the products. STUDY DESIGN AND METHODS: The aim of this study was to analyze our activity between 1998 and 2001, seeking to improve the number of suitable UCB units. Written informed consent was obtained from healthy women with an uncomplicated pregnancy. RESULTS: During the study period, we collected 1060 UCB units; 328 (31%) were banked, whereas 732 (69%) were discarded. The collections were performed in 73% vaginal deliveries and 27% of cesarean sections. The main reason for the exclusion was biological; in fact, a low number of nucleated cells and a low volume accounted for 71% of the total discarded units. Moreover, 20% of the units were discarded because of parental clinical reasons. CONCLUSIONS: Our study shows that maternal history must be completely reviewed before labor and obstetrical factors should be considered to improve the number of UCB units.

Umbilical cord blood: the role of apoptosis in the control of CD34+ cell counts.

BACKGROUND AND OBJECTIVE: Cord blood obtained at delivery can be used for hematopoietic precursor cells (HPC) transplantation. The major limit for its success is represented by the low cellular yield of the stem cell population. The objective of this study was to determine the role played by apoptosis in the numerical control of CD34+ cell counts. DESIGN AND METHODS: Umbilical cord blood samples were collected from 15 women at the time of the delivery and cord blood units processed. Cells, collected following 24h and 48h of incubation, were analysed by flow cytometry using the gating strategy. RESULTS: Remarkable levels of apoptosis were detected in the stem cell population and a significant difference between apoptosis mean values at 24h and 48h within CD34+ cells were found. The difference between the percentage of apoptosis in CD34+ cells and that in the remaining population was significant both at 24h and at 48h. CONCLUSIONS: CD34+ cells have a higher likelihood to undergo apoptosis in comparison to the remaining ones present in umbilical cord blood. This process of cellular death plays a major role in the control of CD34+ cell counts in placental blood and influence, for this reason, the possibility of success of a cord blood transplantation.

Red blood cell depletion of cord blood using hydroxyethylstarch double sedimentation: analysis of 40 cases.

Cord blood has been shown to successfully reconstitute haematopoiesis following allogeneic transplantation in a variety of disorders. A major drawback of cord blood has been the risk of transfusion reaction secondary to ABO incompatibility and reduction in the stem cell pool if cord blood is manipulated to remove red cells. We report our experience on red blood cell depletion of cord blood (CB) with hydroxyethylstarch (HES) double sedimentation. The nucleated and mononucleated cell recovery passed from 78.4% at 90 min to 92.9% at 180 min and from 85% at 90 min to 96% at 180 min, respectively. The overall recovery of CCD34+ cells and of haemopoietic progenitors (CFU-GM) was 90.5% and 83.8%, respectively. The data indicate that HES double sedimentation is a simple and effective technique for cord blood manipulation, but further studies are necessary to evaluate the clonogenic progenitor recovery after thawing.

Detection of cellular heterogeneity by DNA ploidy, 17 chromosome, and p53 gene in primary carcinoma and metastasis in a case of ovarian cancer.

An unusual case of a patient with ovarian carcinoma carrying the p53 point mutation in both metastases (omentum and lymph node), but not in the primary tumor, is described. The presence of a p53 single mutation (G:A) at the second base of codon 248 was examined by polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) analysis. This case was examined also by fluorescent in situ hybrization (FISH) analysis and flow cytometry (FCM) to obtain further information at the single cell level and to detect heterogeneity within a population of cells. FCM analysis evidenced the same multiple aneuploid cell subpopulations in primary and in metastatic samples showing the presence of a cellular heterogeneity. FISH analysis showed a disomic condition for the 17 chromosome in the primary and in one metastasis, while in the other metastasis a monosomic together with a disomic subpopulation was revealed. Our results confirm the independent clonal evolution of the metastasis. The late mutation event observed only in metastatic specimens suggests the hypothesis that in the primary tumor the wild-type gene either does not perform its control role for unknown genetic structural events or the p53 gene in this case does not play a critical role in carcinogenesis.

Effects of 50 Hz magnetic fields on mouse spermatogenesis monitored by flow cytometric analysis.

Flow cytometry (FCM) was performed to monitor the cellular effects of extremely-low-frequency magnetic field on mouse spermatogenesis. Groups of five male hybrid F1 mice aged 8-10 weeks were exposed to 50 Hz magnetic field. The strength of the magnetic field was 1.7 mT. Exposure times of 2 and 4 h were chosen. FCM measurements were performed 7, 14, 21, 28, 35, and 42 days after treatment. For each experimental point, a sham-treated group was used as a control. The possible effects were studied by analyzing the DNA content distribution of the different cell types involved in spermatogenesis and using the elongated spermatids as the reference population. The relative frequencies of the various testicular cell types were calculated using specific software. In groups exposed for 2 h, no effects were observed. In groups exposed for 4 h, a statistically significant (P < 0.001) decrease in elongated spermatids was observed at 28 days after treatment. This change suggests a possible cytotoxic and/or cytostatic effect on differentiating spermatogonia. However, further studies are being carried out to investigate the effects of longer exposure times.

Cellular effects of image diagnostic ultrasound on murine spermatogenesis monitored by flow cytometry.

A study has been carried out to evaluate the possible cellular effects induced by image diagnostic ultrasound on murine spermatogenetic cells. Exposure to ultrasound was carried out using a commercial diagnostic instrument that operates in B-mode. Male hybrid F1 mice, aged 8-10 weeks, were exposed to ultrasound for 30 min and observed from 7 to 35 days after treatment. Flow cytometric analysis has been used to monitor the relative frequency of the different types of spermatogenetic cells. This analytical approach showed changes in cell frequency in the compartment containing elongated spermatids which was used as an endpoint. A statistically significant decrease in the frequency of this cell type was observed 21, 28 and 35 days after exposure. These changes suggest that there may be a cytotoxic and/or cytostatic effect on spermatocytes and spermatogonia. These results showed that image diagnostic ultrasound induces effects on murine spermatogenesis at cellular level and that the flow cytometric approach makes it possible to identify quantitative cellular changes with reference to specific cell type.

Aneuploid cells in benign breast-lesions monitored by flow-cytometry.

It has been observed that various types of benign breast disease are associated to an increased risk of breast cancer. The biological significance of this association remains unclear: both benign and malignant lesions could independently have a common set of risk factors. The cellular DNA content of biopsy samples from 47 breast benign lesions was analyzed by flow cytometry. Flow cytometric measurements evidenced that 11/47 cases showed at least one aneuploid cell subpopulation. The presence of aneuploid subpopulations in benign lesions could be related to an unknown cellular alteration predisponding the developement of benign and malignant lesions independently.

Flow cytometric and cytogenetic analyses in human spontaneous abortions.

Cytogenetic and flow cytometric analyses were performed on 38 human spontaneous abortions in an attempt to obtain information on karyotype abnormalities and to compare the two approaches of analysis. In 19 cases, it was not possible to perform cytogenetic analysis because too long a time had passed between surgical sampling and cell culture, and in vitro culture failed. Of the 19 cases analyzed, 10/19 showed a normal karyotype and 5/19 showed a single trisomy (2/5 trisomies involved chromosome 16, 1/5 trisomy involved chromosome 18, 1/5 trisomy involved chromosome 20, and 1/5 was Klinefelter syndrome). Of the remaining 4/19 cases, 2/19 showed a polyploid condition (1 tetraploidy and 1 triploidy), 1/19 a double trisomy (chromosomes 13 and 21), and 1/19 a pentasomy of the sex chromosomes (49,XXXXY). Flow cytometric analysis was performed on all abortive samples. The samples were subdivided, when possible, into two portions conventionally named "amniotic" and "chorionic", using the amniotic membrane as an anatomical reference. Maternal blood lymphocytes were used as a diploid standard for each sample. In the 19 cases not analyzed by the cytogenetic approach, flow cytometric analysis showed 9 diploid and 10 aneuploid DNA distributions. In the remaining 19 cases, analyzed with both approaches, the comparison of DNA estimations using cytogenetic and flow cytometric analyses showed good agreement. In the cases with karyotype abnormalities, flow cytometric measurement provided evidence of an alteration of DNA content with respect to the diploid standard. Flow cytometric analysis showed a diploid distribution, whereas cytogenetic analysis revealed chromosomal abnormalities in only 4/19 cases. These discordant results could be related to mosaic conditions or maternal cell contamination. Moreover, cytogenetic and flow cytometric analyses were performed on 2 amniotic cell cultures, and concordant results were obtained. The results obtained suggest that a combination of these techniques is beneficial in attempts to obtain information about DNA content alterations, even when cultures fail, and in screening studies of human abortions.

Cellular effects of diagnostic Doppler ultrasound on murine spermatogenic cells monitored by flow cytometry.

A study has been carried out to investigate the possible effects caused by Doppler diagnostic ultrasound on murine spermatogenesis. The frequency of the different types of cells has been analyzed using flow cytometry. Exposure to ultrasound was carried out using a commercial device used in diagnostic conditions. Male hybrid mice were exposed to ultrasound for 30 min and observed from 7 to 35 days after treatment. Flow cytometrical analysis showed changes in the relative frequency of the elongated spermatids and this was used as an end-point. A statistically significant decrease in the frequency of this cell type was observed after 7 and 35 days with both pulsed and continuous ultrasound. With the former, a decrease was also seen in this compartment after 14 and 21 days. Our results have shown that diagnostic ultrasound used in the Doppler technique induces effects on specific cell types of murine spermatogenesis.

Flow cytometric nuclear DNA content of fresh and paraffin-embedded tissues of breast carcinomas and fibroadenomas.

Flow cytometric analysis of DNA ploidy level in fresh tissue samples of human breast tumors has been carried out extensively. Recently, investigations regarding the prognostic value of nuclear DNA content have been facilitated by using nuclei isolated from paraffin-embedded tissues. The aim of our study was to monitor the possible differences between the analysis in fresh and fixed paraffin-embedded tissues in the same tumor and to investigate the possible prognostic implications obtained with this new approach. Nuclei suspensions were obtained, according to the method proposed by Hedley et al. with minor modifications, from 45 carcinomas and 5 fibroadenomas. Flow cytometric analysis revealed diploidy in 57% of carcinomas, while the remaining 43% showed cytometric aneuploidy. Corresponding results were observed between fresh and paraffin-embedded tissue in 26/35 cases. Moreover, a fairly good correlation between the DNA indices of fresh and paraffin-embedded carcinoma samples was observed. Furthermore, the frequency of recurrence was higher in the aneuploid group. Finally, 4 fibroadenomas were diploid and one was aneuploid. Our results confirm that this approach permits retrospective studies to evaluate the potential prognostic significance of nuclear DNA content monitored by flow cytometry.

Flow cytometric DNA analysis of the human cervix affected by human papillomavirus and/or intraepithelial neoplasia.

The relative DNA contents of 164 cellular samples from 59 patients affected by the viral cytopathic effects (VCE) of human papillomavirus (HPV) infection and/or by cervical intraepithelial neoplasia (CIN) and 12 cellular samples from 12 normal donors were analyzed by flow cytometry (FCM) with the aim of correlating the cytometric measurements with the morphologic and etiologic parameters. The unselected group of 59 patients was found to be characterized by statistically significant differences in average ages in the VCE and CIN (31.4 years) and CIN only (44.8 years) subgroups. Of the pathologic samples, 32 (54%) exhibited at least one cell subpopulation with an abnormal DNA content; in all but 2 of those cases, a diploid cell subpopulation was also present. The results indicate a relationship between the FCM ploidy and the morphologic classification, as shown by the increase in the occurrence of subpopulations with abnormal DNA contents from VCE only (38%) to VCE + CIN 1 (57%), to VCE + CIN 2/3 (70%). These results suggest that cytometric parameters, in association with the determination of the HPV types and in parallel to the colpocytohistopathologic criteria, can contribute to a more accurate characterization of cervical lesions in diagnostic and prognostic terms.

Effects of heat on mouse spermatogenesis monitored by flow cytometry.

The effects of heat on mouse spermatogenesis have been determined using both testis weight and flow cytometrically determined DNA content distribution as experimental end-points. Temperatures of 38-42 degrees C and exposure times of 20-60 min have been tested. The results concerning the testis weight substantially confirm those reported by other authors (Hand et al. 1979). The measurement of DNA content distributions shows a relatively higher depletion, 14 days after treatment, of the cytometric compartment containing elongated spermatids in respect to that containing round spermatids. The analysis of the cytotoxic effects, monitored 14 vs. 28 days after treatment, as a function of the exposure time at a given temperature, or of the temperature for a fixed exposure time, indicates that, in the course of spermatogenesis, late spermatocytes are more sensitive to heat than differentiated spermatogonia. Following the approach based on flow cytometry, the effect of exposures as low as 20 min at 38 degrees C can be appreciated.

Flow cytometrically determined DNA content of breast carcinoma and benign lesions: correlations with histopathological parameters.

The relative DNA content of cellular samples from 54 patients affected by breast carcinomas and 20 affected by benign breast lesions (including 11 fibroadenomas) was measured by flow cytometry. All normal tissue samples and 17/20 (85%) specimens from benign lesions exhibited a cytometrically diploid DNA distribution, 3/20 (15%) benign lesions an abnormal DNA content, and 35/54 (65%) carcinomas at least one aneuploid cell subpopulation. Furthermore, 9/54 (17%) tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results also indicate that flow cytometry can be used to recognize lymph nodes infiltrated by aneuploid cells. Statistically significant correlations were evidenced between the occurrence of aneuploidy or the ploidy level measured as DNA index and the nodal infiltration status. The percentage of S cells can also be extracted from DNA content distribution histograms. Statistically significant differences (p less than 0.01) were also observed for the percentage of S cells between normal tissues (6.2 +/- 3.2 SD) and benign lesions (11.1 +/- 6.6 SD), normal tissues (6.2 +/- 3.2 SD) and aneuploid tumors (19.7 +/- 10.3 SD), benign lesions (11.1 +/- 6.6 SD) and aneuploid tumors (19.7 +/- 10.3 SD), and diploid (7.9 +/- 4.0 SD) and aneuploid tumors (19.7 +/- 10.3 SD).

Flow cytometry and sizing for routine andrological analysis.

Flow fluorometry and Coulter type sizing analysis of sperm have been applied separately in order to improve human semen analysis. Different methods of sample preparation were evaluated and a protocol involving prestaining pepsin treatment of sperm samples is proposed for fluorometric analysis. The data obtained with fluorometry and sizing analysis result in different kinds of information: Coulter counting allows to automate sperm counting and fluorometry yields more detailed information about normozoospermia and oligozoospermia by determining the proportion of mature spermatozoa and immature germ-cells. These two methods, together with light microscopy, may help to explore the correlation of fertility and pathology of spermatozoa. The aim of these investigations is to yield the preconditions for simultaneous two-parameter analysis of DNA content and cellular size distributions.