Insulin Lispro in pregnancy--retrospective analysis of 33 cases and matched controls.

AIMS/HYPOTHESIS: To evaluate if the application of Lispro insulin in pregnancy increases the risk for malformations or unusual pregnancy courses. METHODS: Diabetes specialists were contacted in Germany and Austria and asked to report women with diabetes who had been treated with Lispro during pregnancy. Furthermore they were asked to report another pregnant diabetic woman treated with regular insulin with similar HbA1c and age for each Lispro case. Following data were requested: age, first HbA1c in pregnancy and time of analysis, start and duration of Lispro treatment, ultrasound examination, chorionic villi biopsy, amniocentesis, unusual pregnancy courses, standard examination of the new-born for any malformation. Two-sided 95% confidence limits (95%-CI) for risk differences of proportions of malformations or unusual pregnancy courses were calculated. RESULTS: 33 pregnant diabetic women with Lispro and 27 with regular insulin treatment were analysed (mean age 28.3 years (17-41)and 30.1 (19-40); mean HbA1c 6.9% (4.5-10.7) and 6.8% (4.7-9.8), respectively). There were four malformations or unusual pregnancy course (spontaneous abortion; elective interruption because of multiple malformations; heart malformations and hyaline membrane syndrom; premature birth) in the Lispro and one malformation (dyplastic hip) in the regular insulin group. Risk differences in proportions of malformation or unusual pregnancy courses were 8% (95%-CI: - 5% to 21%). CONCLUSIONS/INTERPRETATION: Risk differences for malformations or unusual pregnancy courses were not higher in the insulin Lispro group compared to the controls. However, we observed four malformations or unusual pregnancy courses in the Lispro group. A case-control study is suggested to get a precise risk estimate.

[Reversible multifocal encephalopathy in fulminant eclampsia]. / Reversible multifokale Enzephalopathie bei fulminanter Eklampsie.

Fulminant eclampsia characterised by the onset of focal neurological signs and convulsions is a rare complication of normal pregnancy in the third trimenon. We report on eight women (22-35 years old) with fulminant eclampsia who were admitted to our hospital due to seizures and central pareses. The patients presented with multifocal lesions involving grey and white matter preferentially of the occipital lobes, as evidenced by neuroimaging. Transcranial Doppler sonography revealed increased cerebral blood flow velocities. Treatment consisted of antihypertensive and anticonvulsive medication and operative termination of pregnancy. The patients normalised within 14 days. Our data show that fulminant eclampsia can induce a severe multifocal encephalopathy that can be reversible when treatment and termination of pregnancy are initiated immediately.

Simultaneous cord blood transplantation of ex vivo expanded together with non-expanded cells for high risk leukemia.

In the absence of a donor alternative a stem cell transplantation consisting of two cord blood components originating from the haploidentical brother was performed in a 2-year-old girl with c-ALL, early CNS relapse and 7% of blast cells in the BM 14 days before transplantation. Because of various ongoing infectious complications at that time, 1/8 of the immunogenetically acceptable sibling cord blood was ex vivo expanded 10 days before the transplantation date. The total CB consisting of 1.17 x 10(9) NC was cryopreserved in four separate bags. The one containing 1/8 of the total CB with 1.4 x 10(8) NC CliniMACS selected CD34+ cells was expanded in the presence of 100 ng/ml G-CSF, 100 ng/ml TPO and 100 ng/ml flt3-L in 10% autologous CB plasma and X-VIVO 10 medium at day -10 before transplantation. This expanded cell population was sterile and consisted of about 60% granulocytic cells (CD13+, CD15+), about 30% myelomonocytic cells (CD14, HLA-DR+), 5.2% megakaryocytes (CD61+) and 1.2% CD34+ cells. The proportion of T (CD3+), NK cells (CD56+) as well as dendritic cells (CD83+) was below 0.2%. The unseparated CB infused at day 0 and +1 consisted of a total of thawed 4.4 x 10(7) NC/kg BW, 5.8 x 10(4) CFU-GM/kg BW, 1.54 x 10(5) CD34+cells/kg BW and 7. 73 x 10(2) LTC-IC/kg BW. In addition, the 1 x 10(7) NC/kg BW ex vivo expanded cells representing 1.9 x 10(4) CFU-GM/kg BW, 1.13 x 10(5) CD34 cells/kg BW and 4.37 x 10(2) LTC-IC/kg BW, were infused at day +1. At day +2 after transplantation the patient revealed a focal pneumonia on X-ray with generalized sepsis and became catecholamine dependent. From day +4 the patient received 280 microg/m2 G-CSF. At day +5 she developed an erythroderma, which could not be identified as acute GVHD by biopsy. Early engraftment with leukocyte counts at days 8 and 14 were 350 and 700/microl, ANC 310 and 410/microl, respectively. Donor cells determined by chimerism analysis were 97% and 98% in the periphery at this early time. Most importantly, the pneumonia as well as the septicemia subsided within a few days. Notably, as well is the clearly shortened aplastic phase observed after this simultaneous CB cell component transplantation. The patients T cell and NK cell reconstitution could be detected at day +37 with 330 CD3+ cells/microl and 40 CD56+ cells/microl, respectively. The time to reach an absolute platelet count of 20 000 (50 000)/microl was 75 (103) days. The disease-free survival now exceeds 1 year in complete remission without chronic GVHD or any other health problems. These data show that the applicability of ex vivo expanded committed progenitors and LTC-IC, even in high risk leukemia at the time of transplantation, is feasible and can provide sufficient myeloid progenitors resulting in rapid engraftment able to clear bacterial pneumonia and sepsis. In addition, accelerated hematopoietic reconstitution apparently served as a well functioning platform for definitive graft-versus-leukemia activity. This transplantation of defined ex vivo generated components presents a feasible and generally applicable approach and may open a promising new avenue for cell therapy in malignant diseases.

Haematopoietic transplant potential of unrelated and related cord blood: the first six years of the EUROCORD/NETCORD Bank Germany.

To date, human umbilical cord blood (CB) has been employed successfully in well over 1000 allogeneic (unrelated and sibling) stem cell transplantations. Because of primary limitations in volume and cell numbers, over 90% of these transplantations were performed in children. Therefore requests for well standardised cord blood units of high quality are now increasing constantly. Examination and standardisation of unrelated and related cord blood stem cell preparations and banking as well as their biological characterisation was already initiated in Düsseldorf in 1992. Hitherto a total of 3236 CB samples with a mean volume of 89 +/- 25 ml, a mean total number of nucleated cells (NC) of 10 +/- 5 x 10(8) and a mean number of CFU-GM of 6 +/- 5 x 10(5) have also been validated by haematological, immunological and microbiological criteria. In addition to that, 97 directed CB donations of siblings with a clinical indication have been characterised and banked along the same lines. All CB units were collected from the umbilical cord vein immediately after vaginal full term delivery or caesarean section, then frozen and stored in liquid nitrogen. 1940 CB units were stored unseparated, the other 1296 were volume reduced using Hetastarch (HES) with a mean recovery of 85 +/- 13% of the nucleated cells, 86 +/- 12% and 84 +/- 13% for CFC and CD34+ cells, respectively. Only 5.0 ml of a CB sample is required for routine laboratory testing as there are HLA-class I typing, HLA-class II typing by sequence specific oligonucleotide probes (PCR-SOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony forming assays, and CD34+ status as well as certain viral infections such as CMV, Hepatitis B, C, HIV, Parvo B19 by PCR technology before releasing the CB unit for transplantation. For apparent viral infections, maternal sera obtained at birth were tested for HBsAg, anti-HBc, anti-HCV, -HAV-(IgG, IgM), -HIV-1-2, -EBV- (IgG, IgM), -HTLVI-II, -CMV (IgM, IgG), toxoplasmosis and syphilis. Within the last three years a total of 4860 preliminary searches and 680 extended unit reports were submitted to the CB bank Düsseldorf by fax or World Wide Web. So far 68 unrelated and 3 related CB units were delivered. From these 70 have been transplanted in 30 different transplant centres world-wide. Until now the evaluation of the first 53 unrelated CB-transplantations was performed together with the EUROCORD transplant registry. Three patients were excluded from the analysis, since they received an unrelated CB-transplant for non-engraftment after previous allotransplants. The median patient age of these 50 patients was 5.0 years (range 0.3-44), the median weight 18 kg (range 4-70 kg). The majority of the patients transplanted for malignancies (66%) suffered from ALL (n = 19), AML (n = 7), CML (n = 4) and lymphoma (n = 2) with two third (75%) in an intermediate (2nd CR) or advanced stage of disease (> 2nd CR); 13 patients had metabolic diseases and immunodeficiencies and three had aplastic anaemia. All CB samples as well as the patients' blood samples were typed in Düsseldorf for HLA-class I by serology confirmed by PCR-SSP and by high resolution DNA typing for HLA-DRB1 and HLA-DQB1 alleles. 96% of the 50 patients receiving unrelated CB were mismatched at one or more HLA-antigens. 41 of the 50 patients transplanted with unrelated CB from Düsseldorf were evaluable for engraftment with an overall engraftment rate of 83%. According to the defined criteria of EUROCORD, 9 of the 50 patients were not evaluable for engraftment, since they died before day 60. The present median follow-up time is 14 months (1.4-38). The Kaplan-Meier estimate of survival at one year is 42 percent. The three paediatric patients after sibling CB transplantations (ALL, amegakaryocytic thrombocytopenia and CML) are alive with a follow-up time of 350, 379 days and 531 days. (ABSTRACT TRUNCATED)

[Pattern of various cytokeratins of normal vulva, vulvar intraepithelial neoplasia (VIN) and vulvar carcinoma]. / Muster verschiedener Zytokeratine der normalen Vulva, der vulvären intraepithelialen Neoplasia (VIN) und des Vulvakarzinoms.

The presence of specific keratins can be of diagnostic value for studying normal and neoplastic epithelium of the vulva. The aim of the present study was to investigate normal, preneoplastic and neoplastic epithelium of the vulva. Keratins 5, 6 and 18, identified by a polyspecific anti-human CK antibody (clone LP 34, DAKO), and the keratin subtypes 7, 10, 14, 18, 19 and 20 of normal, dysplastic and malignant vulval epithelium (paraffin-embedded sections) were detected by immunohistochemical APAAP staining. Keratins 5, 6, and 18 (clone LP 34) and keratin subtype 10 are expressed in the upper third of the normal vulval epithelium. In mild and moderate intraepithelial neoplasia only a few cells express these keratins. In patients with severe intraepithelial neoplasia (VIN III) the expression of these keratins seems to be associated with recurrence of the disease. In biopsy specimens of patients without recurrence we find positive results for keratins 5, 6 and 18 (clone LP 34) and keratin 10. If patients have a recurrence of the disease, expression of these keratins is only diffuse or is absent. The expression of these keratin subtypes in vulval carcinomas is mostly seen in differentiated cells. There was no association between recurrence and keratin pattern. We have not found any other expression of the tested keratin subtypes in VIN and in vulval carcinoma.

Products of the Major Histocompatibility Complex and CD4/CD8 Cell Infiltration in Cervical Intraepithelial Neoplasia and in Cervical Carcinoma.

OBJECTIVES: Major histocompatibility complex (MHC) products and the CD4/CD8 cell ratio in the lymphocyte infiltrate in cervical preneoplastic and neoplastic lesions are investigated to determine the correlation between the infiltration of lymphocytes in subepithelial stromal tissue and the expression of MHC class I and MHC class II antigens on epithelial cells. MATERIALS AND METHODS: The expression of MHC products and the lymphocyte infiltration were studied by an indirect immunohistochemical staining (alkaline phosphataseanti-alkaline phosphatase) in 6 biopsies of normal cervical squamous epithelium, in 22 biopsies of cervical intraepithelial neoplasia (CIN), and in 9 biopsies of cervical carcinoma. Immunohistochemically positive lymphocytes were counted in highpower fields. The density of MHC expression in CIN and cervical carcinoma was described semiquantitatively in the histological slides. RESULTS: We have seen a number of differences in the expression of MHC products between normal cervical squamous epithelium and CIN or cervical carcinoma. Our results also show that the CD4/CD8 cell ratio in normal, in dysplastic, and in malignant cervical tissues differs from peripheral blood. We found statistically significant differences (Mann Whitney U test, p < .05) between the CD4/CD8 ratio of the normal epithelium when compared to the preneoplastic and neoplastic cervical lesions. In the histological sections tested, CD8 cells exceeded the number of CD4 cells in CIN and cervical carcinoma. Expression of MHC products is independent of the infiltration of CD4 and CD8 cells in the tissue of low-grade and high-grade cervical squamous intraepithelial lesions (SILs) and in cervical carcinoma. CONCLUSIONS: We found a significant decrease of the CD4/CD8 cell ratio in low-grade SILs, high-grade SILs, and squamous cell carcinoma compared to normal cervical epithelium. The loss of MHC class I antigen expression in poorly differentiated tumor cells does not correlate with the amount of lymphocyte infiltration.

Risk factors for recurrent VIN. Role of multifocality and grade of disease.

OBJECTIVE: To evaluate possible risk factors for recurrence of vulvar intraepithelial neoplasia (VIN): menopausal status, smoking, method of treatment (local excision with cold knife, CO2 laser vaporization, CO2 laser excision), grade and focality. STUDY DESIGN: At the dysplasia clinic of the Department of Obstetrics and Gynecology, Medical Center, Heinrich Heine University, Dusseldorf, Germany, we treated 102 patients with VIN with a mean follow-up of 3.5 years. VIN was demonstrated for all these patients by histologic examination of biopsies taken under colposcopic control. Follow-up was performed by clinical examination and colposcopy. Statistical evaluation was done by the chi 2 test. RESULTS: VIN recurrence developed in 36.6% of patients. We could not prove any correlation between recurrence of disease and menopausal status, smoking behavior or treatment method used. Recurrent VIN correlated with the grade (P < .001) and focality (P < .05) of the disease. CONCLUSION: In the prediction of possible risk of recurrence of disease, not only the grade of VIN but also the multifocality is important. Treatment should be done very carefully considering the functions of the vulva; therefore, mutilating operations ought to be avoided as much as possible.

Platelet PGI2 receptor affinity is reduced in pre-eclampsia.

Prostacyclin (PGI2) receptors were studied in platelet membrane preparations from women with normal pregnancy, pregnancy-induced hypertension (PIH) or pre-eclampsia. Patient groups showed no differences in gestational week at delivery. A markedly lower birth weight, however, was found in pre-eclampsia. No differences between groups could be detected in platelet PGI2 receptor number. In contrast, the binding affinity to the PGI2 mimetic iloprost was considerably reduced in pre-eclampsia, whereas receptor affinity between PIH and normal pregnancy did not differ significantly.

[Thrombocyte prostacyclin receptors in gestational hypertension and pre-eclampsia]. / Thrombozytäre Prostacyclin-Rezeptoren bei Gestationshypertonie und Präeklampsie.

OBJECTIVE: To determine prostacyclin (PGI2) receptor characteristics in pregnancies complicated by hypertension and to assess any relation to the clinical outcome. METHODS: Radioligand binding studies with [3H]-Iloprost were performed to measure receptor capacity (Bmax) and affinity (Kd-1) using platelet membranes from patients with preeclampsia, gestational hypertension or normal pregnancy. RESULTS: PGI2 receptor capacity did not differ between the patient groups. In contrast, PGI2 receptor affinity was diminished in gestational hypertension and considerably reduced in preeclampsia compared to normal pregnancy. A similar pattern was found in fetal growth (normal pregnancy > gestational hypertension > preeclampsia). Furthermore, the rate of low Apgar scores and acidosis was increased in preeclampsia. CONCLUSIONS: In preeclampsia reduced platelet PGI2 receptor affinity was found as well as poor pregnancy outcome in comparison with normal pregnancy, whereas these differences were less pronounced in gestational hypertension. This suggests a role of PGI2 and its receptor in gestational hypertension and in particular in preeclampsia.

Hematopoietic transplant potential of unrelated cord blood: critical issues.

To date, hematopoietic stem and progenitor cells from human umbilical cord blood (CB) have been employed in approximately 90 allogeneic (56 sibling and 34 unrelated) matched and mismatched transplantations worldwide with easy and successful restoration of hematopoiesis. Requests for stem cell preparations from CB will continue to increase. Thus, as a pilot study, the examination and standardization of unrelated cord blood-derived stem cell preparations and banking as well as their biologic characterization were initiated. Up to October 1995, a total of 574 samples [mean volume 79 +/- 26 ml, total nucleated cells (NC) 8.5 +/- 5 x 10(8), BFU-E 9.5 +/- 8.6 x 10(5), CFU-GM 5.7 +/- 6.3 x 10(5), CFU-GEMM 1.6 +/- 1.9 x 10(5)] from cord-derived or placental-derived residual blood have been defined by hematologic, immunologic, and microbiologic criteria. These CB samples were collected from the umbilical cord vein immediately after vaginal full-term delivery (n = 450) or cesarean section (n = 124) and stored frozen in liquid nitrogen. Seven percent of all samples collected could not be considered for potential transplants because of volumes < 40 ml. Only 5.0 ml of a CB sample is required for routine laboratory testing, consisting of HLA class I typing, HLA class II typing by sequence-specific oligonucleotide probes (PCR-SSOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony-forming and LTC-IC assays, and CD34+ status. To assess the potential problem of contaminating maternal cells, a PCR was performed on 7 representative samples. During the initial 6 months of the unrelated CB collection program, a median bacterial contamination rate of 18% (20% skin flora species, 80% perineal flora species) was encountered, which has since been reduced to < 1% through practical experience. With regard to viral infections, maternal sera was tested for HBsAg (0.6% positive), anti-HCV (0%), anti-HAV (IgG 18%, IgM 0%), anti-HIV-1-2 (0%), anti-EBV (IgG 98%, IgM 0%), anti-HTLVI-II (0%), anti-CMV (IgG 43%, IgM 0.4%), toxoplasmosis (46%) and syphilis (0%). In addition, all cord blood samples were tested by PCR for CMV infection. With regard to its clinical relevance, it is important that only 0.3% of all the samples were positive for CMV by this sensitive method. This may represent a critical advantage of CB grafts over bone marrow (BM) since, in contrast, > 40% of the unrelated BM donors have been identified to be positive for CMV. An additional advantage of CB is that since 20% of CB samples were collected from ethnic minorities, it appears possible to balance common HLA types and uncommon HLA types represented in this group. In summary, with the extensive practical experience of the obstetric collection team as well as the stem cell-processing laboratory, it appears feasible to obtain a 90% yield of unrelated CB-derived stem cell preparations for banking, which clearly should meet the medical and regulatory qualification criteria required for clinical transplantation. To test the feasibility of hematopoietic transplant potential of unrelated CB for adult patients, ex vivo expansion of CD34+-enriched stem/progenitor cell populations isolated from fresh or frozen CB was attempted in the presence of rh-IL-3, rh-IL-6, rh-EPO, rh-GM-CSF, and rh-SCF with or without fit 3. At varying time points (days 0, 2, 4, 7, 14, 21), the contents of these cultures were analyzed for the numbers of cells, CFC (BFU-E, CFU-GM, CFU-GEMM), and LTC-IC. In this setting, the increase of cells was 200-fold, that of CFC 70-fold, and most importantly that of LTC-IC was 4.5-fold after 7 days in culture in the presence of flt3. In conclusion, LTC-IC derived from CB can be maintained and considerably expanded ex vivo from highly enriched CD34 + CB cell populations from fresh or frozen CB samples.

Pregnancies in women with diabetic nephropathy: long-term outcome for mother and child.

In order to improve the basis upon which to advise women with diabetic nephropathy about pregnancy, we studied the effect of diabetic nephropathy on the course of pregnancy, perinatal outcome, infant development and long-term outcome of the mothers. All pregnancies of women with diabetic nephropathy (defined as proteinuria > 400 mg/day (n = 26), creatinine clearance < 80 ml/min and hypertension in the first trimester (n = 10)) followed at our centre from 1982 to 1992 were identified (34 White class F and 2 White class T) and the women and their children re-examined in the spring 1993. From the first to the third trimester the percentage of women with proteinuria over 3 g/day increased from 14 to 53% and those treated with antihypertensive medication from 53 to 97%. There were no intrauterine or perinatal deaths, but one child died suddenly 4 weeks postpartum. Of 36 newborns (gestational week at birth 36(3), birth weight 2384(834) g)), 11 were born before week 34 and 8 had respiratory distress syndrome. Renal function in the first trimester, diastolic blood pressure in the third trimester and an HbA1c above normal were predictive of gestational age at delivery and low birth weight (stepwise regression analysis). At follow up of the children (n = 35, age 4.5 (0.4-10) years) the majority (n = 27) were normally developed but seven had psychomotor retardation (four of them major). One child had a severe motor retardation due to a congenital anomaly. At follow up, 21 of the 29 mothers had preserved renal function (creatinine 1.3 (0.8-4.3) mg/dl and 8 had developed end stage renal disease and required dialysis (2 of whom were White class T) within 3 (1-9) years postpartum.(ABSTRACT TRUNCATED AT 250 WORDS)

[Treatment and after-care of HIV patients with cervix dysplasia]. / Behandlung und Nachsorge von HIV-Patientinnen mit einer Zervixdysplasie.

We have been observing an increased prevalence of cervical intraepithelial neoplasia in HIV-infected women in our department of obstetrics and gynaecology. 10 HIV-infected patients with cervical intraepithelial neoplasia were treated by CO2-laser - 6 were subjected to laser excision conisation, and 4 patients to laser vaporisation. After laser vaporisation and laser excision conisation, no postoperative complications were observed. Intraoperative bleeding was rarely seen. The preoperative examination of the CD4-cell count had no influence on the choice of the applied method of laser treatment, laser vaporisation or laser excision conisation. But all the 4 patients with a CD4-cell count < or = 200/microliters experienced recurrence of disease shortly after the first laser treatment. Only one out of five patients with CD4-cell counts between 200-499/microliters had a recurrence of disease after laser treatment. All in all, a recurrence of disease was seen in five out of ten HIV-infected patients with cervical intraepithelial neoplasia. The recurrence rate is possibly increased because HIV-infected women have multifocal cervical, vulval and vaginal dysplasia more frequently than HIV-negative patients. In our study, we observed multifocal cervical, vulval and vaginal dysplasia in four HIV-infected patients. Therefore, an accurate short-term follow-up with colposcopy and cytological smears should be carried out after the treatment of HIV-infected patients with cervical intraepithelial neoplasia.

Excretion of prostacyclin and thromboxane metabolites before, during, and after pregnancy-induced hypertension.

The aim of the present study was to assess whether changes in prostacyclin (PGI2) and thromboxane (TXA2) generation precede the manifestation of pregnancy-induced hypertension (PH). The metabolites 6-oxo-PGF1 alpha and TXB2 were measured in the urine of 69 randomly selected pregnant women from 16-20 weeks of gestation (wg) until delivery and more than 6 weeks postpartum. Between 16-20 and 21-24 wg 6-oxo-PGF1 alpha excretion did not change in patients who later developed PIH (n = 6) but increased significantly in the control group (n = 63). In contrast, a marked rise in TXB2 excretion was found in the PIH group but not in controls. Thereafter significant differences between both groups persisted from 25 wg until delivery. The 6-oxo-PGF1 alpha/TXB2 ratio was below the 10th percentile from 21-24 wg until delivery in patients with developing PIH. The excretion of both metabolites was substantially lower in the non-pregnant state without any difference between patient groups. These results show an altered urinary excretion of both 6-oxo-PGF1 alpha and TXB2 preceding the onset of the disease. A pathophysiological role of PGI2 deficiency and increased TXA2 formation in PIH appears substantiated.

[The advantages of CO2 laser use in treatment of cervix dysplasia]. / Die Vorzüge der CO2-Laseranwendung bei der Behandlung der Zervixdysplasie.

We use two kinds of laser treatment, namely, laser vaporisation and laser excision conisation. Laser vaporisation is a widely used method of localised ablative treatment based on both histologically confirmed cervical intraepithelial neoplasia and clear colposcopic visualisation of the upper limit of the lesion. Laser excision conisation is performed, if cervical intraepithelial neoplasia reaches the endocervix, being out of colposcopic view, and the squamocolumnar junction is localised in the endocervical canal. 116 patients with cervical intraepithelial neoplasia were treated with the CO2 laser. 81 (70%) were subjected to laser vaporisation, 35 (30%) to laser excision conisation. Intraoperative bleeding was rarely seen. After laser vaporisation, no complications were observed, but laser excision conisation complications occurred in one patient who developed postoperative cervical stenosis, and in another patient, who had postoperative bleeding requiring surgical examination. Follow-up with colposcopy and cytologic smears was carried out every 6 months. Recurrence of disease 6 months or later after laser treatment was seen in 6 patients (7.4%) after laser vaporisation and in 2 patients (5.7%) after laser excision conisation. This report shows the advantages of the carbon dioxide laser in comparison to cold knife conisation in the treatment of cervical intraepithelial neoplasia.

Evaluation of the Ceprate LC34 affinity system for the enrichment of CD34+ hematopoietic stem cells from cord blood.

Umbilical cord blood (CB) has been shown to contain sufficient hematopoietic stem cells for allogeneic bone marrow transplant (BMT) and to contain progenitor cells susceptible to retrovirally mediated gene transduction. Enrichment of CD34+ cells from fresh unseparated or thawed unseparated CB could offer several advantages for (1) the storage of CB samples in an unrelated stem cell bank, which includes a decrease in volume and thus less storage space and (2) gene transfer into these cells. Cord blood was collected from the umbilical cord vein immediately after vaginal full-term delivery and samples of 5-12 ml (total leucocytes: 100 +/- 50 x 10(6)) of fresh unseparated (n = 6) and thawed unseparated (n = 8) CB were processed to enrich CD34+ cells using the Ceprate LC Biotin-Avidin affinity system. CD34+ cells, present at a frequency of 1.2 +/- 0.8% among leukocytes from CB (calculated by immunophenotyping and fluorescence-activated cell sorter (FACS) analysis), can be rapidly enriched to 54.4 +/- 12.3% (range, 38.6-87.1%) from fresh unseparated CB and 44.6 +/- 28% (range, 13.6-76%) from thawed unseparated CB. Hematopoietic progenitor assays for unseparated (cell concentration 3 x 10(4), 1 x 10(5), 3 x 10(5)) and CD34-enriched cells (cell concentration 1 x 10(3), 3 x 10(3), 1 x 10(4)) were performed in the presence of 5 U human interleukin-3 (hu IL-3), 30 U hu-IL-6, 10 U human granulocyte colony-stimulating factor, 6 U erythropoietin, and 15 ng human stem cell factor.(ABSTRACT TRUNCATED AT 250 WORDS)

The rapid detection of the quantity (genotype) and cell lineage (immunophenotype) of contaminating maternal white cells in cord blood samples by fluorescence in situ hybridization combined with confocal laser scanning microscopy.

The role of maternal blood cell contamination in cord blood as hematopoietic stem cell transplant could be an important factor driving the graft vs. host reactivity. Therefore, the efficiacy of fluorescence-labeled chromosome probes for the X and Y chromosomes to study contaminating maternal cells (XX+, Y-) in nucleated cells of male cord blood specimen (X+, Y+) by fluorescence in situ hybridization (FISH) was evaluated here. Unfortunately, the FISH technique evaluated by standard indirect immunofluorescence was not sufficiently sensitive to safely detect maternal contamination below 2-3%. Thus, the analysis of FISH had to be adapted with confocal laser scanning microscopy, which then gave no false-negative reading values. A Bio-Rad MRC-600 laser scanning microscope with a very high sensitivity for low-intensity and hidden signals was used for the observation of cord blood cells. Sixteen samples from the whole cord blood (CB), 10 analyses of mononuclear cells, 6 of the granulocytic fraction, 6 of picked erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) colonies and 5 of enriched CD34+ cells were performed. Only in 1 of 16 cases was a contamination by maternal cells detected: 10% of all nucleated cells from the whole CB, 5% of the CD3+ T-cell fraction, 15% of the myelomonocytic cells from mononuclear cells (MNCs), and 15% of picked BFU-E and CFU-GM colonies were of maternal origin.(ABSTRACT TRUNCATED AT 250 WORDS)