New Perspectives in Prenatal Diagnosis of Sickle Cell Anemia

Hemoglobin disorders such as thalassemias and sickle cell anemias can be avoided by detecting carriers, ensuring genetic counseling and prenatal diagnosis. Nowadays Chorionic villus sampling (CVS amniocentesis, and cordocentesis are still the most widely used invasive sampling methods for prenatal diagnosis of the fetus. These traditional methods are associated with a risk of fetal loss. The revelation of cell-free fetal DNA (cffDNA) in maternal plasma and serum provides the opportunity of noninvasive prenatal diagnosis (NIPD). Different encouraging clinical applications have arose such as noninvasive identification of fetal sexing, fetal Rhesus D, and the determination of the paternal alleles in maternal plasma. The determination of the presence or absence of paternally inherited alleles in maternal plasma of sickle cell disease (SCD) and β-thalassemia would allow the diagnosis of autosomal dominant diseases or the exclusion of autosomal recessive diseases of the fetuses, respectively. prenatal diagnosis of genetic diseases. Analysis of cffDNA in maternal plasma for NIPD has the advantage of being safer versus the invasive methods. Different technologies were used since the discovery of cffDNA for NIPD—especially high-resolution melting (HRM) analysis is one of those methods. Genotyping can be done with HRM without using labeled probes and more complex regions can be analyzed with unlabeled hybridization probes. High-resolution melting is a rapid and useful method to detect paternal alleles for the NIPD of SCD and thalassemias when the fetus has a risk for double heterozygote

Laboratory Approach to Anemia

Anemia is a major cause of morbidity and mortality worldwide and can be defined as a decreased quantity of circulating red blood cells (RBCs). The epidemiological studies suggested that one-third of the world’s population is affected with anemia. Anemia is not a disease, but it is instead the sign of an underlying basic pathological process. However, the sign may function as a compass in the search for the cause. Therefore, the prediagnosis revealed by thorough investigation of this sign should be supported by laboratory parameters according to the underlying pathological process. We expect that this review will provide guidance to clinicians with findings and laboratory tests that can be followed from the initial stage in the anemia search

Invasive and Noninvasive Approaches in Prenatal Diagnosis of Thalassemias

Thalassemia is a significant health problem worldwide. There are two main classifications, α- and β-thalassemias, which are usually caused by the defective synthesis of the α-globin, and which are commonly caused by different mutations of the β-globin chain. Different hemoglobin mutations have been identified to date. Thalassemias can result in profound anemia from early life and, if not treated with regular blood transfusions, can lead to death in the first year. Prenatal diagnosis of thalassemia is the essential part of preventive medicine and is currently dependent on the use of invasive diagnostic tests within the first 2 months of pregnancy. These diagnostic techniques carry a small but significant risk of fetal loss up to 1%. Molecular diagnostic methods have been developed for genotyping thalassemias based on PCR techniques and high-throughput technologies. Noninvasive tests using cell-free DNA (cfDNA) from a maternal blood sample is also an alternative method, thus eliminating the risk of miscarriage. This chapter summarizes the current invasive approaches and the noninvasive methods using cell-free fetal DNA as new molecular diagnostic methods for genotypic diagnosis of thalassemia in clinical practice. Prevention strategies that encompass carrier screening, genetic counseling, and prenatal diagnosis are discussed

A New Alternative Approach for RhD Incompatibility; Determination Fetal RhD Status via Biosensor Technology

Prenatal detection of the fetal RHD status in early stage of pregnancy is observed to be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. The routine use of antenatal and postnatal anti-D prophylaxis reduces the incidence of hemolytic disease of the fetus and newborn. Cell-free fetal DNA in maternal plasma is in use today for routine genotyping fetal RHD status. Fetal RhD antigens can be detected in the blood of RhD-negative pregnant women using a nanopolymer-coated biosensor and could be an alternative method for medical diagnosis. We detected RhD-positive fetal antibodies with biosensor in maternal blood of RhD-negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat and corundum ceramic-based screen-printed gold electrode. The demonstrated method has a different view for the detection of fetal RhD status in early pregnancy. The biosensor technology is useful and can be carried out rapidly in clinical diagnosis. Biosensors are also reproducible methods which give results quickly compared to noninvasive fetal RHD genotyping with real-time PCR-based techniques. We suggest that this method could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility

Application of prenatal diagnosis with fetal nucleic acids in maternal blood.

TEZ8901Tez (Doktora) -- Çukurova Üniversitesi, Adana, 2010.Kaynakça (s. 86-94) var.xii, 95 s. : res. ; 29 cm.The discovery of cell free fetal DNA in the maternal circulation has driven developments in noninvasive prenatal diagnosis in the past decade. Detection of paternally derived alleles in cell free fetal DNA is becoming well established. Analysis of fetal DNA from maternal plasma by new techniques such HRM analysis offers great potential for noninvasive prenatal diagnosis. We examined 104 maternal plasma samples from 57 pregnant women carrier of sickle cell anemia, 32 pregnant women carrier of beta thalassemia and 15 healthy control subjects. Fetal DNA was extracted from 1 mL plasma samples. To detect male fetuses SRY and DYS14 markers specific to the Y chromosome analyzed by nested PCR and RT-PCR. DYS14 and beta globin were used for quantitation of fetal and total DNA levels respectively. Alternative to traditional methods in invasive prenatal diagnosis, we used HRM analysis for genotyping of fetal DNA in hemoglobinopathies. We detected Y chromosome specific DYS14 in maternal plasma of 100% women bearing male fetuses in early pregnancy. The value was 93.7% with SRY by nested PCR. The levels of total and fetal DNA increased with gestational age. We observed greater MoM values of fetal DNA in sickle cell anemia pregnancies compared to thalassemia and healthy groups. The MoM values were smaller in thalassemia pregnancies compared to other groups (p<0.001). In late period of pregnancy, we observed significantly increased levels of beta globin between sickle cell anemia, thalassemia and control groups. Genotyping by HRM we were able to distinguish paternal alleles of fetal DNA from maternal DNA. Problems were encountered when the fetus carries the same allele with mother. Our results show that investigation of the fetal and total DNA in early pregnancies with hemoglobinopathies is not found to be useful. HRM is an alternative method for invasive prenatal diagnosis to genotyping of fetal DNA when the fetus carries the paternal allele different from the mother's.Maternal dolaşımda serbest fetal DNA'nın bulunması ile geçtiğimiz on yılda girişimsel olmayan prenatal tanıda gelişmeler kaydedilmiştir. Fetüste paternal alellerin serbest fetal DNA'da tanımlanmasına yönelik çalışmalar artık kullanılmaya başlanmıştır. Maternal plazmada fetal DNA analizinde HRM eğrisi analizi girişimsel olmayan prenatal tanı için büyük bir potansiyel oluşturmaktadır. Elli yedisi orak hücre, 32'si beta talasemi taşıyıcısı ve 15'i sağlıklı kontrol olmak üzere toplam 104 primigravidanın maternal plazma örneğini çalıştık. Fetal DNA, 1 mL plazma örneğinden elde edildi; erkek fetüsleri saptamak için Y kromozomuna özgül SRY ve DYS14 belirteçleri ""nested"" PCR ve RT-PCR ile analiz edildi. Fetal ve total DNA kantitasyonu sırasıyla DYS14 ve beta globin genleriyle yapıldı. Girişimsel prenatal tanıdaki geleneksel yöntemlere seçenek olarak hemoglobinopatilerde HRM analizi ile fetal DNA genotiplendirilmesi yapıldı. Maternal plazmada gebeliğin erken döneminde fetal DNA (DYS14), RT-PCR ile % 100 oranında erkek taşıyan gebede saptandı; SRY dizisi kullanarak ""nested"" PCR ile bu oran % 93,7 olarak bulundu. Maternal plazma total (beta globin) ve fetal DNA düzeyleri gebelik haftası ile artış gösterdi. Orak hücre taşıyıcısı gebelerde fetal DNA düzeyi, kontrol ve talasemi grubuna göre artan MoM değerleri gösterdi. Talasemi taşıyıcısı gebelerde total DNA miktarında, kontrol ve orak hücre taşıyıcısı gruba göre azalan MoM değeri gözlendi (p<0,001). Gebeliğin geç döneminde, orak hücre taşıyıcısı, talasemi taşıyıcısı ve kontrol grupları arasında beta globin düzeylerinin anlamlı olarak arttığını saptadık. Fetal DNA'nın HRM ile genotiplemesi paternal alellerin maternal DNA'dan ayrımı ile yapıldı. Fetüsün anneyle aynı genotipi taşıdığı durumlarda kısmen de olsa sorunlar yaşandı. Sonuçlarımız hemoglobinopatili gebeliklerin erken döneminde, fetal ve total DNA düzeylerine bakmanın anlamlı olmadığını gösterdi. Hemoglobinopatilerin prenatal tanısında girişimsel yöntemlere seçenek olarak fetal DNA'nın genotiplendirilmesinde fetüsün anneden farklı paternal aleller taşıdığı durumlarda HRM analizinin kullanışlı olabileceğini göstermektedir.Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi tarafından desteklenmiştir. Proje No: TF2005D2

Koroner Arter Hastalarında Lipoprotein (a), Homosistein, Yüksek Duyarlı C-Reaktif Protein ve Fibrinojen Etkinliğinin Tanısal Değerlendirilmesi

Amaç: Koroner arter hastalığında (KAH) Lipoprotein (Lp) (a), homosistein (Hcy), yüksek duyarlıklı C-reaktif protein (hsCRP) ve fibrinojen gibi majör ve diğer risk faktörlerinin tanısal değerini araştırmak amaçlanmıştır. Materyal ve Metod: Çalışmaya koroner anjiyografi sonucuna gore 118'i KAH ve 105'i KAH olmadığı belirlenen toplam 223 kişi dahil edildi. Lipoprotein (a), Hcy, hs-CRP ve fibrinojen düzeyleri sırasıyla, immünoturbidometrik, flöresan polarizasyon immünoassay ve nefelometrik yöntemlerle ölçüm yapıldı. Açlık glukoz ve düşük dansiteli lipoprotein kolesterol (LDL-K) dışındaki lipid parametrelerine enzimatik kolorimetrik yöntemlerle bakılmış olup, LDL-K düzeyleri Fridewald formülüne göre hesaplanmıştır. Bulgular: Lojistik regresyon modelinde biyokimyasal değişkenlerden en önemlileri göre Lp (a), Hcy, hs-CRP ve fibrinojen olarak belirlenmiştir. Lipoprotein (a), Hcy, hs-CRP ve fibrinojenin herbir ünitesinin sırasıyla, 1.029, 1.177, 1.027 ve 1.013 kat KAH riskini artırdığı gösterilmiştir. Bu parametrelerden KAH için öngörüsü en hassas ve etkili olanın fibrinojen olduğu gösterilmiştir. Sonuç: Lipoprotein (a), Hcy, hs-CRP ve fibrinojen KAH için bağımsız risk faktörleri olması yanında bunların arasında en önemlisinin fibrinojen olduğu belirlenmiştir. Fibrinojen, klinik uygulamada KAH için güvenilir bir risk factör olarak kullanılabilir.Purpose: To evaluate the diagnostic value of major and other risk factors as lipoprotein (Lp) (a), homocysteine (Hcy), high sensitive C-reactive protein (hs-CRP) and fibrinogen in CAD patients. Materials and Methods: A total of 223 subjects (118 patients and 105 controls) were included in the study according to their coronary angiographic results. Lipoprotein (a), Hcy, hs-CRP and fibrinogen levels were measured using immunoturbidometric, florescent polarization immunoassay and nefelometric methods, respectively. Fasting glucose and lipid parameters, except low density lipoprotein cholesterol (LDL-C), are determined by enzymatic colorimetric methods and the LDL-C levels were calculated by the Fridewald formula. Results: Logistic regression analysis showed that when the biochemical variables in placed in a model, the most important variables were Lp (a), Hcy, hs-CRP and fibrinogen. We showed that each unit of Lp (a), Hcy, hs-CRP and fibrinogen increases the risk of CAD 1.029, 1.177, 1.027 and 1.013 fold, respectively. Among these, fibrinogen level was the most sensitive and efficient parameter in prediction of CAD. Conclusion: Although Lp (a), Hcy, hs-CRP and fibrinogen are independent risk factors for CAD, fibrinogen was the most important one. Fibrinogen can be used as a reliable risk factor for CAD in clinical practice

A new biosensor for noninvasive determination of fetal RHD status in maternal blood of RhD negative pregnant women.

Prenatal detection of the fetal RHD status can be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. Hemolytic disease causes morbidity and mortality of the fetus in the neonatal period. The routine use of antenatal and postnatal anti-D prophylaxis has reduced the incidence of hemolytic disease of the fetus and newborn. This study describe the detection of fetal RhD antigens in blood of RhD negative pregnant women using a nanopolymer coated electrochemical biosensor for medical diagnosis. Cell free fetal DNA in maternal plasma was also used to genotyping fetal RHD status using multiplex real-time PCR. Twenty-six RhD negative pregnant women in different gestational ages were included in the study. RhD positive fetal antibodies detected with a developed biosensor in maternal blood of RhD negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat, and corundum ceramic based screen printed gold electrode combined with the reference Ag/AgCl electrode, and the auxiliary Au/Pd (98/2%) electrode. Fetal RHD genotyping performed using fluorescence-based multiplex real-time PCR exons 5 and 7 of the RHD gene. The fetal RHD status of 26 RhD negative cases were detected 21 as RhD positive and 5 as RhD negative with electrochemical biosensor. Fetal RHD status confirmed with extracted fetal DNA in maternal plasma using multiplex real-time PCR RHD genotyping and by serological test after delivery. The new method for fetal RhD detection in early pregnancy is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to noninvasive fetal RHD genotyping from maternal plasma with real-time PCR-based techniques. We suggest the biosensor techniques could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility

Fetal hemoglobin seviyesinin orak hücre anemisi hastalarının kemik biyokimyası üzerine etkisi

Amaç: Bu çalışmanın amacı fetal hemoglobin seviyelerinin, orak hücre anemisi hastalarının kemik parametreleri üzerine olası etkisinin değerlendirilmesidir.Metod: 56'sı SS, 47'si kontrol grubu olmak üzere 103 kan örneği, fetal hemoglobin düzeylerine göre çalışmaya dahil edildi. Çalışmada fetal hemoglobin, kemik mineral yoğunluğu, kemik spesifik alkalen fosfataz, kalsiyum, osteokalsin, 25-OH vitamin D ve hematolojik parametreler analiz edildi. Bulgular: İstatistiksel analizler, düşük kemik mineral yoğunluğu ve biyokimyasal kemik parametrelerinin orak hücre anemisi hastalarında fetal hemoglobin seviyeleri ile yakından ilişkili olduğunu göstermiştir. Sonuç: Orak hücre anemisi hastalarının kemik durumunu için fetal hemoglobin seviyesinin iyi bir parametre olduğu belirlenmiştir.Objective: The aim of this study was to evaluate the possible roles of fetal hemoglobin levels on bone parameters in sickle cell anemia (SCA) patients.Methods: Blood samples taken from 56 SS and 47 control totally 103 subjects were included in this research work according to their fetal hemoglobin levels. Fetal hemoglobin, bone mineral density, bone specific alkaline phosphatase, calcium, osteocalcin, 25-OH vitamin D and hematological parameters were measured and analyzed in the study.Results: Statistical analysis showed that, lower bone mineral density and biochemical bone parameters significantly correlated with low fetal hemoglobin levels at SCA patients.Conclusion: It was concluded that fetal hemoglobin level is a good index for bone status in sickle cell anemia patient

Hemoglobinopatiler için ilk trimesterde fetal aortik Doppler

Amaç: Gebeliğin ilk üç ayında hemoglobinopatilerin prenatal tanısı için fetal aortik Doppler incelemesinin değerlendirilmesi amaçlanmıştır.Gereç ve Yöntemler: Ocak 2014 ve Kasım 2014 tarihleri arasında 108 hasta çalışmaya dahil edildi. Çiftler, Çukurova Üniversitesi Tıp Fakültesi Kadın Hastalıkları ve Doğum Bölümü Prenatal Tanı Merkezi'ne alfa/beta talasemi, orak hücre anemisi veya bunların kombine taşıyıcısı olup başvuran kişilerden oluşmakta idi. Koryonik villüs örneklemesinden (KVÖ) bir saat önce fetal aortik Doppler incelemesi yapıldı. Pulsatilite indeksi, tepe sistolik hız, kalp hızı not edildi.Bulgular: Farklı KVÖ sonucu grupları sağlıklı kontrollerle karşılaştırıldıklarında Doppler indeksleri açısından istatistiksel olarak anlamlı fark bulunmamıştır.Sonuç: Fetal aortik Doppler incelemesi hemoglobinopatilerin prenatal tanısında etkin bulunmamıştır.Objective: To evaluate fetal aortic Doppler for the prenatal diagnosis of hemoglobinopathies in the first trimester of pregnancy.Materials and Methods: Between January and November 2014, a total of 108 patients were enrolled in the study. The couples were carriers of either alpha/beta thalassemia, sickle cell disease or combined carriers of these and were admitted to Çukurova University Faculty of Medicine, Department of Obstetrics and Gynecology Prenatal Diagnosis Center. One hour before the chorionic villus sampling (CVS), patients were evaluated using fetal aortic Doppler. Pulsatility index, peak systolic velocity, and heart rate were noted.Results: There were no statistically significant differences in Doppler indices between different groups of CVS results when compared with the healthy controls.Conclusion: Fetal aortic Doppler investigation was found to be ineffective for the prenatal diagnosis of hemoglobinopathies

Investigation of the effects of vitamin D treatment on the uterine structural changes in the experimental model with polycystic ovary syndrome: an ultrastructural and immunohistochemical study

In this study, we aimed to investigate the structural changes seen in the endometrium in experimental PCOS rat model and the effects of vitamin D treatment on these changes at immunohistochemical and electron microscopic levels. 24 prepubertal female rats were divided into three groups. Two groups were injected with dehydroepiandrosterone and one of them was treated with 1,25(OH)2 D3 at the same time. The control group was injected with sesame oil. At the end of the 28th day, the blood samples were collected. Uterus tissues were prepared for light and electron microscopic examinations. Epithelial, stromal and endometrial thickness measurements were investigated. Immunohistochemical staining was applied against caspase-3 and Ki-67. Serum AMH and estradiol levels were higher in PCOS group compared to the control group. Serum progesterone levels were similar in all groups. Endometrial, epithelial and stromal thickness measurements were increased in PCOS group compared to the control group, and decreased in the vitamin D treatment group compared to the PCOS group. Light and electron microscopic results of PCOS group showed an increase in apoptosis and proliferation. In the PCOS group, immunohistochemical staining of caspase-3 and Ki-67 were found to be higher than in the control group, but stainings were decreased with vitamin D treatment compared to PCOS group. Structural changes observed in endometrium may be related to implantation problems seen in patients with PCOS. Our studies suggest that vitamin D therapy may be beneficial in these patients