Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms.

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.

Is prenatal HLA typing of uncultured amniocytes before the collection of related allogenic cord blood helpful?

The collection of related allogenic cord blood is gaining increasing importance in families with one child affected by haematopoietic disease. Within a family, there is only a 25% chance of a full HLA match between siblings. 50% of all collected cord blood samples cannot be used because of poor quality. Because of this, the determination of HLA type is useful for planning the collection of related allogenic cord blood transplants. We studied whether HLA typing is possible during late pregnancy if amniocentesis has not been performed during the first trimester. HLA -A, -B and -DRB loci were detected in amniotic fluid, as well as in corresponding cord blood and maternal blood using PCR-SSP. For the first time, HLA typing was performed from uncultured amniocytes. Unambiguous results were obtained from all samples. Fetal HLA-genotype in amniotic fluid was confirmed by typing results from corresponding cord blood. HLA typing of uncultured amniocytes during late pregnancy is a reliable and fast method. For the first time, prenatal HLA typing by amniocentesis after week 38 of gestation is possible in less than 8 h and without fetal risk.

Recurrent cytomegalovirus infection during pregnancy: ultrasonographic diagnosis and fetal outcome.

Fetal infection as a consequence of recurrent disease is uncommon. We present a case of recurrent cytomegalovirus infection in the second trimester of pregnancy. Fetal infection was detected through severely abnormal findings on ultrasound examination and verified by detecting cytomegalovirus DNA in the amniotic fluid and cytomegalovirus-specific immunoglobulin M antibodies in the fetal blood and associated pancytopenia. Because of the severity of the infection, a fatal outcome was predicted. A Cesarean section was performed at 33+5 weeks of gestation; the child died shortly after birth.

[Aspects of donation and processing of stem cell transplants from umbilical cord blood]. / Präparative und arzneimittelrechtliche Aspekte bei der Sammlung von Stammzellpräparaten aus Plazentarestblut 1.

BACKGROUND: In 1996, the Institute of Transfusion Medicine and Immunology started the banking of unrelated cord blood (CB) transplants in collaboration with the Department of Gynecology and Obstetrics at the Mannheim University Hospital. Up to now, more than 2000 placental blood samples have been cryopreserved. We analyze the results of CB collection and processing, and describe the legal background of CB collection in Germany. MATERIALS AND METHODS: CB is only collected with informed consent of the mothers, and without evidence of neither maternal infection nor deformity of the baby. Units with a volume of > 60 ml or > 5 x 10(8) total nucleated cells (NC) are processed as potential transplants. All specimens are evaluated for cell count, HLA typing, sterility testing, ABO/Rh blood groups and clonogenic capacity of hematopoietic progenitor cells. The maternal blood is screened for various infectious disease markers. To reduce the volume of the transplants, we perform buffy coat processing by centrifugation. Finally, we cryopreserve the transplant using a 5.5 percent final concentration of DMSO, and store it in the vapour phase of liquid nitrogen. Results We collected on average 86 +/- 21 ml CB containing 8.87 +/- 3.41 x 10(8) NC (mean +/- SD). 45 percent of the collected units had to be discarded for various reasons. We achieved a mean mononucleated cell recovery by the buffy coat preparation method of 93.4 percent. Defining a threshold dose of 3.0 x 10(7) NC per kg body weight (BW) for transplantation, the mean BW of patients to be sufficiently treated with the stored transplants showed to be 29.6 kg. Conclusions In a very close collaboration between transfusion medicine and obstetricians, a routine collection program for unrelated and related allogeneic CB transplants was established. The CB donation needs to be in accordance with the German drug law. On the other hand, in terms of evidence based medicine, it seems not to be reasonable to collect autologous placental blood for a future transplantation.

Demonstration of a recto-vaginal fistula with the ultrasound contrast medium Echovist.

The demonstration of a recto-vaginal fistula in a patient with Crohn's disease is described. The patient was examined by vaginal ultrasound using the contrast medium Echovist-200 (SHU 454, Schering AG, Berlin). This agent had not been used before under these circumstances and proved to be successful.

[Contraception in androgenised women with a low-dose cyproterone-acetate containing one-phase preparation (author's transl)]. / Die kontrazeptive Betreuung androgenisierter Frauen mit einem niedrig dosierten cyproteronacetathaltigen Einphasenpräparat.

101 hirsute women received Diane, a combination of 2 mg cyproterone acetate and 50 micrograms ethinyl oestradiol per capsule, from the fifth to twenty-fifth day during a total of 1105 cycles. The treatment was satisfactory in 96% of women with acne, 84% with androgenic alopecia, 79% with seborrhoea, and 50% of those with mild or moderate hirsutism. The effectiveness of treating hirsutism or alopecia depended on its severity and duration. There was no correlation between various measures of androgen levels before and during treatment and the clinical results. Side effects were similar to those after contraceptive one-phase preparations, both in kind and incidence. None of the women became pregnant. Diane is thus the drug of choice in hormonal contraception of patients with acne, seborrhoea, androgenic alopecia or mild hirsutism. It is also suitable in the follow-up or interval management of high-dose cyproterone acetate treatment of severe hirsutism, in order to maintain the effect obtained with lower hormone doses.

[Treatment of infertile women with epimestrol (author's transl)]. / Behandlung steriler Frauen mit Epimestrol. Erfahrungsbericht über 143 Patientinnen mit 30 Graviditäten

155 patients have been treated with epimestrol during 593 menstrual cycles in a daily dosage of 5, 10 or 2.5 mg from the 1st to the 10th or from the 5th to the 14th day of the cycle. 143 patients with secondary amenorrhoea, anovulatory cycles, ovulatory oligomenorrhoea, corpus luteum insufficiency, and normal cycles received epimestrol after an average period of 2.4 years of involuntary infertility. The pregnancy rate was 21% (n = 30), the spontaneous abortion rate 13% (n = 4). Twelve further patients not seeking pregnancy were treated with epimestrol because of secondary amenorrhoea and (or) anovulatory bleeding. The results were as follows: 1) In eugonadotropic secondary amenorrhoea 8 out of 12 patients ovulated, 5 out of 10 patients wanting children became pregnant. No patient with hypogonadotropism ovulated. 2) Out of 25 patients with anovulatory cycles or bleeding 13 ovulated and 4 became pregnant. 3) 17 out of 68 oligomenorrhoeic patients became pregnant. 4) In 12 out of 24 patients with corpus luteum insufficiency the hyperthermic phase improved and four patients became pregnant. 5) In 12 patients without anomalous cycles these remained unchanged and there were no pregnancies. 6) No side effects could be observed.

PIP: 155 women (593 cycles) were treated for sterility with daily doses of 5, 10, or 2.5 mg of epimestrol, from the 1st to the 10th or from the 5th to the 14th day of the cycle. 143 of these patients suffered from secondary amenorrhea, anovulatory cycles, ovulatory oligomenorrhea, corpus luteal insufficiency, and even normal cycles, which resulted in an average of 2.4 years of involuntary sterility. The pregnancy rate was 21% (n = 30) and the rate of spontaneous abortion 13% (n = 4). 12 other women were treated with epimestrol because of secondary amenorrhea or anovulatory bleeding. In those patients with eugonadotropic secondary amenorrhea, 8 of 12 ovulated and 5 of 10 desiring children became pregnant. No patients with hypogonadotropism ovulated. Of 25 patients with anovulatory cycles or bleeding, 13 ovulated and 4 became pregnant. Of those with oligomenorrhea, 17 out of 68 became pregnant. 12 out of 24 patients with corpus luteal insufficiency showed in improvement in the hyperthermic phase and 4 became pregnant. 12 patients with normal cycles showed no change and did not become pregnant. No side effects were experienced.