First and Second Level Haemoglobinopathies Diagnosis: Best Practices of the Italian Society of Thalassemia and Haemoglobinopathies (SITE).

The purpose of this best practice paper is to review the current recommendations for the identification and prenatal diagnosis of hemoglobinopathies. METHODS: The management committee of SITE selected and gathered a multidisciplinary team in order to formulate recommendations based on the available scientific evidence integrated with the opinions of experts, with the purpose of supporting clinicians. RESULTS: We provide recommendations for first level tests (complete blood count, hemoglobin separation and iron balance), second level tests (molecular diagnosis) and prenatal diagnosis. Five Italian experts in hemoglobinopathies were consulted regarding the orientation of prenatal diagnosis, and for each indication, the degree of agreement among the experts has been specified. CONCLUSIONS: Best practice recommendations are the final outcome of this translational research and allow transfer to daily clinical practice.

Early prenatal diagnosis of Hb Lepore Boston-Washington and ß-thalassemia on fetal celomatic DNA.

INTRODUCTION: Analysis of fetal DNA in at risk couples for thalassemia is performed from fetal trophoblast or amniotic fluid cells. Although these procedures are in common use, the main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity develops around 4 weeks of pregnancy within the extraembryonic mesoderm and contains embryonic erythroid precursor cells as a source of fetal DNA that can be used to perform invasive prenatal diagnosis. METHODS: Celomatic fluids were obtained at 8 weeks of gestation in thirteen women with high-risk pregnancies. Twelve of these couples were at risk for Hb Lepore disease and ß-thalassemia and one couple represented a rare case in which both parents were carriers of Hb Lepore Boston-Washington. Fetal cells were isolated by micromanipulator and nested polymerase chain reactions were performed. RESULTS: The analysis was successfully performed in all examined cases. Two fetuses were found to have a compound heterozygosity for ß-thalassemia and Hb Lepore Boston-Washington, three fetuses were found to be carriers of ß-thalassemia, three fetuses of Hb Lepore, five were found without parental mutations. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. CONCLUSION: Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in celomatic fluid and demonstrate for the first time that prenatal diagnosis of ß-thalassemia and Hb Lepore may be feasible in an earlier time of pregnancy than other procedures.

Celomic Fluid: Laboratory Workflow for Prenatal Diagnosis of Monogenic Diseases.

BACKGROUND: Celomic fluid can be considered as an ultra-filtrate of maternal serum, containing a high protein concentration, urea, and many other molecules. It is an important transfer interface and a reservoir of nutrients for the embryo. Celomic fluid contains fetal cells that can be used for prenatal diagnosis of monogenic diseases in an earlier gestational period than villocentesis and amniocentesis. OBJECTIVE: The purpose of this study was to evaluate the characteristics of celomic fluid and to establish a workflow laboratory procedure for very early prenatal diagnosis of monogenic diseases. METHODS: Three hundred and eighty-five celomatic fluids were collected between the seventh and tenth week of gestation. We sampled 1 mL of celomic fluid in all cases. The embryo-fetal erythroid precursor cells were selected by the anti-CD71 microbead method or by a direct micromanipulator pick-up on the basis of their morphology. We amplified the extracted DNA using a nested polymerase chain reaction. Primers for short tandem repeat amplification were used to perform a quantitative fluorescent polymerase chain reaction evaluation to control maternal contamination. RESULTS: We observed maternal contamination in 95% of celomic fluids with a range between 5 and 100%. No fetal cells were observed in 0.78% of celomic fluids. The number of fetal cells ranged from a few units to several hundred. Isolation of embryo-fetal erythroblasts selected by the micromanipulator made diagnosis feasible in all cases. CONCLUSIONS: The selection of fetal cells by a micromanipulator and nested polymerase chain reaction analysis made celomatic fluid suitable for early prenatal diagnosis of monogenic disorders even in the presence of high maternal contamination and few fetal cells. The procedure reported in this study provides the opportunity for the use of celomic fluid sampled by celocentesis as an alternative to chorionic villi sampling and amniocentesis, to allow invasive prenatal diagnosis at a very early stage of pregnancy.

Lentiviral globin gene therapy with reduced-intensity conditioning in adults with ß-thalassemia: a phase 1 trial.

ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

Very early prenatal diagnosis of Cockayne's syndrome by coelocentesis.

Cockayne's syndrome (CS) is a rare autosomal recessive multisystem disease characterised by early severe progression of symptoms. This study reports the feasibility of earlier prenatal diagnosis of CS by coelocentesis at 8 weeks of gestation respect to amniocentesis or villocentesis. Three couples at risk for CS asked to perform prenatal diagnosis by coelocentesis. Coelomic fluid was aspired from coelomic cavity in four singleton pregnancy at 8 weeks of gestation and 40 foetal cells were recovered by micromanipulator. Maternal DNA contamination was evaluated by quantitative fluorescent PCR (QF-PCR) and target regions of foetal DNA containing parental mutations of ERCC6 gene were amplified and sequenced. In all these cases, molecular analysis was possible. One foetus resulted affected of CS and the diagnosis was confirmed on placental tissue after voluntary abortion. In three cases, foetuses resulted carrier of a parental mutation and the results were confirmed after the birth. This study suggests that reliable prenatal diagnosis of CS could be performed using foetal cells present in coelomatic fluid in earlier pregnancy. Coelocentesis could be applied in prenatal diagnosis of CSs as well as for other monogenic diseases, at very early stage of pregnancy, if parental mutations are already known.Impact StatementWhat is already know on this subject? Previous studies utilising coelocentesis for prenatal determination of foetal sex reported variable success ranging from 58% to 95%, because of low total DNA content and presence of maternal cell contamination. This procedure has never been reported for early prenatal diagnosis at 8 weeks of gestation for rare genetically transmitted diseases such as Cockayne's syndrome.What do the results of this study add? This study demonstrates that coelomic fluid sampling combined with well-standardised laboratory procedures can be applied for prenatal diagnosis at eight weeks of gestation for any rare monogenic disease if molecular defects are known.What are the implications of these findings for clinical practice and/or further research? The findings of this study in at risk couples for monogenic diseases investigated by coelocentesis demonstrate that embryo-foetal cell selection from CF allows reliable and early prenatal diagnosis of diseases. This technique is attractive to parents because it provides prenatal diagnosis of genetic disease at least 4 weeks earlier than what can be achieved by the traditional procedures reducing anxiety of parents and provides the option for medical termination of affected cases at 8-10 weeks' gestation, which is less traumatic and safer than second-trimester surgical termination. Further research concerns the possibility to obtain foetal karyotype at eight weeks of gestation and the possibility of intrauterine corrective therapy.

Celocentesis for Early Prenatal Diagnosis in Couples at-Risk for ß-Thalassemia and Sicilian (δß)0-Thalassemia.

The procedures commonly used for prenatal diagnosis (PND) of thalassemia are villocentesis or amniocentesis, respectively, at the 11th and 16th weeks of gestation. Their main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity is accessible between the 7th and 9th weeks of gestation and it has been demonstrated that it contains embryonic erythroid precursor cells as a source of fetal DNA for earlier invasive PND of thalassemia and other monogenic diseases. In this study, we report the use of celomatic fluids obtained from nine women with high-risk pregnancies for Sicilian (δß)0-thalassemia [(δß)0-thal] deletion (NG_000007.3: g.64336_77738del13403) and ß-thalassemia (ß-thal). Fetal cells were isolated by a micromanipulator, and nested polymerase chain reaction (PCR) and short tandem repeats (STRs) analysis were performed. Prenatal diagnosis was successfully performed in all examined cases. One fetus was a compound heterozygote for (δß)0- and ß-thal, three fetuses were found to be carriers of ß-thal, four fetuses carriers of a Sicilian 뫧 deletion, and one fetus without parental mutations. Accidentally, a rare case of paternal triploidy was observed. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in the celomatic fluid and demonstrate, for the first time, that PND of Sicilian (δß)0-thal and ß-thal is feasible at an earlier time in pregnancy than other procedures.

Incidental Detection of a Chromosomal Aberration by Array-CGH in an Early Prenatal Diagnosis for Monogenic Disease on Coelomic Fluid.

BACKGROUND: Turner syndrome is a rare genetic condition in which a female is partly or completely missing an X chromosome. Signs and symptoms vary among those affected. In fetuses that survive at birth and without congenital malformations, the prognosis is usually positive, but it has high lethality in utero, especially in the first trimester of pregnancy. METHODS: We report a case of monosomy X detected during a prenatal diagnosis for beta thalassemia on coelomic fluid (CF) at the VIII week of gestation. Beta globin gene analysis, whole genome amplification (WGA), quantitative fluorescent PCR and array comparative genomic hybridization (array-CGH) were performed on DNA extracted from CF. RESULTS: A monoallelic pattern of all Short Tandem Repeats mapped on the X chromosome was found and array-CGH performed on WGA from a few fetal erythroblasts confirmed monosomy X. CONCLUSION: This report underlines the importance of an early prenatal diagnosis and the countless potentialities of array-CGH that could make definition of molecular karyotype possible from a few fetal cells, unlike conventional cytogenetic techniques that require a greater cellular content. This is the first report of a molecular karyotype obtained from two cells selected by micromanipulation of CF and defined at such an early gestational age.

Rasburicase-induced Methemoglobinemia: A Case Report and Literature Review.

Rasburicase is a recombinant urate oxidase enzyme indicated for tumor lysis syndrome, a potential life-threatening oncologic emergency that occurs most commonly during initial chemotherapy for hematological malignancies. As a result of the defects in the physiological antioxidant pathway, erythrocytes of patients with glucose-6-phosphate dehydrogenase deficiency are not protected against the oxidizing stress exerted by hydrogen peroxide generated with the administration of rasburicase. The authors report a 14-year-old patient, diagnosed with T-cell acute lymphoblastic leukemia, who developed methemoglobinemia and hemolytic anemia with low oxygen saturation after starting steroids, hyperhydratation, and rasburicase administration. The complications resolved with supportive therapy only.

Genetic disorder prenatal diagnosis and pregnancy termination practices among high consanguinity population, Saudi Arabia.

The prevalence of consanguineous marriage and genetic disorders are high in Saudi Arabia. There were records on the practices of Saudis toward prenatal diagnosis (PND) and termination of pregnancy (TOP), however the sample sizes are small. This study has targeted the Saudi Arabian community and family history of genetic disorders to determine the practices toward PND and TOP. The cross-sectional survey was conducted among Saudis (n = 2761) to determine their practices toward reproductive-decision making. Regression analysis was conducted to identify the association of the limiting factors, relative merits and family history on the outcomes. Total of 2507 participants returned completed questionnaire. The practice towards PND (68%) were more favorable than TOP (33%). PND was found to be a good opportunity for early diagnosis and gives parent's choice. Education, history with affected baby, prior knowledge and religious belief were significant deciding factors of PND and TOP. Down syndrome (n = 161) and sickle cell anemia (n = 152) were commonly available genetic disorder among participant's family. Respondents with autistic cases in their family have higher acceptance rate for TOP. Non-consanguineous are more willing to consider TOP than consanguineous. Participants with abnormal fetus, aged of > 36 years, married and educated Saudis were more likely consider TOP. Though, religion is the most influencing factor for not accepting TOP, comparatively willingness to PND and TOP have increased recently. Awareness campaigns about PND and TOP may increase the chances of accepting prenatal genetic diagnosis.

Double Heterozygosity for Hb Durham-N.C. (HBB: c.344T>C) [ß114(G16)Leu→Pro] and the IVS-I-110 (HBB: c.93-21G>A) Causing a Severe ß-Thalassemia Phenotype.

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two ß-globin gene defects, a ß-thalassemia (ß-thal), mutation at IVS-I-110 (HBB: c.93-21G>A) and a variant hemoglobin (Hb) mutation at codon 114 (HBB: c.344T>C) on the other allele, reported as Hb Durham-N.C. (also known as Hb Brescia) [ß114(G16)Leu→Pro] in the HbVar database. A very low Hb level (Hb 3.5 g/dL), microcytosis [mean corpuscular volume (MCV) 63.2 fL] and hypocromia [mean corpuscular Hb (MCH) 19.6 pg], increased red blood cell (RBC) distribution width (RDW) (36.0%), higher reticulocytes (6.2%), anisocytosis, poikilocytosis, hypocromia, basophilic stippling and inclusion body formation, were present in the affected subject. Analysis of other family components showed the presence of HBB: c.93-21G>A defect in the mother and in her brother, while Hb Durham-N.C. was absent in all other relatives, thus, this mutation has arisen as a de novo defect. This is the first case described as a severe thalassemic phenotype in a compound heterozygote carrier of this unstable Hb and a common ß-thalassemic allele. The important information gained from this case is that a rare dominant or recessive mutation may arise in every individual, even if this is a very rare event.

Human coelomic fluid investigation: A MS-based analytical approach to prenatal screening.

Coelomic fluid (CF) is the earliest dynamic and complex fluid of the gestational sac. CF contains maternal cells and proteins produced by embryonic cells, tissues and excretions. The biochemical composition of CF is modified throughout the first trimester of pregnancy and its protein profile reflects both physiological/pathological changes affecting the embryo and mother. Identification of variations in the balance of proteins might indicate particular types of pathologies, or ascertain specific genetic disorders. A platform utilizing protein enrichment procedures coupled with shotgun identification and iTRAQ differentiation provided the identification and quantitation of 88 unique embryonic proteins. It is relevant to note that chromosome X protein CXorf23 was found suggesting the embryo sex. Foetal sex was determined by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) on coelomic cells, foetal tissues and maternal white blood cells, with a 100% concordance rate between iTRAQ-MS/MS and QF-PCR data. The functional associations among the identified proteins were investigated using STRING database. Open Targets Platform showed as significant the following therapeutic areas: nervous, respiratory, eye and head system disease.

ß-Thalassemia heterozygote state detrimentally affects health expectation.

BACKGROUND: Thalassemia minor (Tm) individuals, are generally considered healthy. However, the prognosis of Tm individuals has not been extensively studied. The aim of this study was to evaluate the prognosis of Tm versus controls without ß-thalassemia carrier state. METHODS: A total of 26,006 individuals seeking thalassemia screening at the AOOR Villa Sofia-V. Cervello, Palermo (Italy) were retrospectively studied. Logistic penalised regression model was used to estimate risk of potential complications and survival techniques were used to study mortality. RESULTS: We identified a total of 4943 Tm and 21,063 controls. Tm was associated with significantly higher risks of hospitalisation for cirrhosis (OR 1·94, 95% CI 1·30 to 2·90, p = 0·001), kidney disorders (OR 2·11, 95% CI 1·27 to 3·51, p = 0·004), cholelithiatis (OR 1·39, 95% CI 1·08 to 1·79, p = 0·010), and mood disorders (OR 2·08, 95% CI 1·15 to 3·75, p = 0·015). No statistically difference in life expectancy between thalassemia minor and control group was found (HR 1·090, 95% CI 0·777 to 1·555, p < 0·590; log-rank test p = .426). CONCLUSION: This study shows that Tm affects the prognosis of Tm carriers regarding health expectation. Probably, iron overload and anaemia for several years may be at the basis of these effects.

Fetal aneuploidy diagnosed at celocentesis for early prenatal diagnosis of congenital hemoglobinopathies.

INTRODUCTION: Currently, prenatal diagnosis of genetic disorders requires chorionic villus sampling or amniocentesis carried out after 11 and 16 weeks of gestation, respectively. Celocentesis is a procedure for prenatal diagnosis that could be used from as early as 7 weeks. The present investigation evaluated the feasibility of performing diagnosis for monogenic diseases using celomic fluid containing cells of fetal origin. MATERIAL AND METHODS: Analysis consisted of 489 singleton pregnancies undergoing celocentesis for the prenatal diagnosis of hemoglobinopathies (n = 367) or before surgical termination of pregnancy for social indications (n = 122). Embryo-fetal cells were isolated from celomic fluid using CD71 antibodies or by micromanipulation. Quantitative fluorescent polymerase chain reaction of short tandem repeat sequences of chromosomes 13, 18, 21, X and Y were used to determine the presence of maternal DNA. RESULTS: 357/489 (73%) of celomic fluid samples were contaminated with maternal cells. In two cases, diagnosis was not possible due to the high contamination of celomic fluid. Eighty-seven (23.8%) fetuses were affected by hemoglobinopathies and, in five cases, chromosomal aneuploidies were found, including three cases of trisomy 21, one of trisomy 13 and one of triploidy. In all cases, the diagnosis of hemoglobinopathies and chromosomal abnormalities was confirmed by molecular and traditional cytogenetic analysis after amniocentesis, chorionic villus or placental tissue collection following pregnancy termination. CONCLUSIONS: The findings of this study demonstrate that embryo-fetal cell selection from celomic fluid allows reliable and early prenatal diagnosis of hemoglobinopathies and can give more information on any fetal aneuploidy following the control of maternal contamination by quantitative fluorescent-PCR.

Co-inheritance of HBB:c.-106G > C, a rare single nucleotide variation at position -56 relative to transcription initiation site, with other known mutations in the globin clusters.

OBJECTIVES: We performed molecular analysis to evaluate clinical implications of a rare nucleotide change, particularly when co-inherited with other known mutations in the globin clusters, in order to conduct an appropriate genetic counselling. METHODS: Complete blood cell counts and high-performance liquid chromatography were the routine first level analysis for patients referred to our Hospital Center in Palermo to undergo the screening test for haemoglobinopathies. Sequencing analysis was the selected method for the phenotypic characterization, especially in case of new or very rare mutations in globin genes. RESULTS: We report data of a rare single nucleotide variation at position -56 relative to transcription initiation site (NM_000518.4(HBB):c.-106G > C), identified in ten patients of Italian origin during the screening programme of the 'Sicilian population'. It was found in simple heterozygosity (n = 8), in association with beta haemoglobin variant Hb S (n = 1) and in heterozygosity with beta-thalassaemic allele IVS-I-1 G->A [(HBB):c.92 + 1G > A] and ααα anti3.7 rearrangement (n = 1). DISCUSSION: Heterozygous subjects for this substitution showed normal haematological and electrophoretic features. Heterozygotes for this mutation and other defect in globin genes showed the classical phenotype of a healthy carrier, therefore it can be considered a benign variant that does not alter the production and function of haemoglobin. CONCLUSION: This is another example of rare or new nucleotide variations whose identification and characterization is crucial in order to carry out appropriate genetic counselling to a potential risk couple.

HBB: c.316-125A>G and HBB: c.316-42delC: Phenotypic Evaluations of Two Rare Changes in the Second Intron of the HBB Gene.

We report two very rare changes in the second intron of the HBB gene, a substitution at nucleotide (nt) 726 [IVS-II-726 (A>G) (ß+), NM_000518, HBB: c.316-125A>G] and a deletion of a cytosine at nt 809 [IVS-II-809 (-C) (ß), NM_000518, HBB: c.316-42delC] identified during the screening program for hemoglobinopathies in the resident Sicilian population. The purpose of this study was to evaluate the clinical implication of these rare changes, particularly in coinheritance with known mutations in the globin clusters, in order to conduct an appropriate genetic counseling for at-risk couples. Molecular analysis detected the first rare nt substitution in two cases in simple heterozygosity and in two cases in association with other known mutations on globin genes, while the deletion was identified in a pregnant woman, carrier of ß-thal, and in her fetus at prenatal diagnosis (PND) for hemoglobinopathies. The present study emphasizes the importance of sharing the observed changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression and possible interactions with known molecular defects.

Phenotypic evaluations of HBB:c.93-23T>C, a nucleotide substitution in the IVS I nt 108 of ß-globin gene.

BACKGROUND: Thalassaemia and variant haemoglobin are the most common severe monogenic disorders worldwide. AIMS: To develop prenatal diagnosis programmes for the prevention of the most important haemoglobin disorders and identify healthy carriers of thalassaemia. METHODS: Sequencing analysis was used to obtain complete data on gene structure and to correlate specific phenotypic expression with mutations, especially for new or very rare mutations in globin genes. RESULTS: A rare single nucleotide variation, HBB:c.93-23T>C, located in nucleotide 108 of the first intervening sequence of the HBB gene, was identified. This variation was previously reported but its clinical significance was not known. Six heterozygous patients had this nucleotide variation and eight further cases co-inherited it together with other defects in the globin genes. Heterozygous subjects for this substitution showed normal haematological and electrophoretic features, whereas subjects who were compound heterozygotes for this mutation and another defect in globin genes showed the classic phenotype of a healthy carrier. CONCLUSION: This nucleotide can be considered a single nucleotide polymorphism and not a thalassaemic mutation that reduces the production of haemoglobin. This is another example of a very rare nucleotide variation. Knowledge of this is important so that appropriate genetic counselling can be carried out of a couple potentially at risk, where one of the partners is a carrier of ß-thalassaemia and the other is carrier of a nucleotide variation.

Phenotypic Evaluation of a Novel Nucleotide Substitution (HBD: c.442T>C) on the δ-Globin Gene.

HBD: c.442T>C is a new mutation at the stop codon (TGA>CGA) of the δ-globin gene, which produces a new codon for arginine. This substitution causes a 51 nucleotides longer open reading frame determining the synthesis of a potential larger δ subunit, which is a probable target of mechanisms for the degradation of aberrant proteins as well as the defective synthesized mRNA molecules, and may also be rapidly degraded by a variety of RNA surveillance pathways. We identified this molecular defect in four patients: three women with a reduced HbA2 level and a 37-year-old male showing the typical phenotype of an α-thalassemia (α-thal) carrier with reduced values of red cell indices and normal HbA2 level (2.5%). The mutation on the δ-globin gene was found to have been coinherited with a ß-globin gene defect leading to a normalized HbA2 level. These data support the necessity of investigating these cases at a molecular level, particularly if the partner is also a ß-thalassemia (ß-thal) carrier. The present data emphasizes the importance of a careful evaluation of correlation between genotypes resulting from DNA analysis and phenotypes, especially in cases of atypical hematological parameters, in order to carry out an adequate diagnostic process finalized to appropriate genetic counseling.

Identification of embryo-fetal cells in celomic fluid using morphological and short-tandem repeats analysis.

OBJECTIVE: The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo-fetal cells. METHOD: Celomic fluids were aspired by ultrasound-guided transcervical celocentesis at 7-9 weeks' gestation from singleton pregnancies before surgical termination for psychological reasons. DNA extracted from celomic fluid cells showed the same morphology, and quantitative fluorescent polymerase chain reaction (PCR) assay was performed to evaluate their fetal or maternal origin. RESULTS: Six different types of non-hematological maternal and four different types of embryo-fetal cells were detected. The most common maternal cells were of epithelial origin. The majority of embryo-fetal cells were roundish with a nucleus located in an eccentric position near the wall. These cells were considered to be erythroblasts, probably derived from the yolk sac that serves as the initial site of erythropoiesis. CONCLUSIONS: The combined use of morphology and DNA analysis makes it possible to select and isolate embryo-fetal cells, even when maternal contamination is high. This development provides the opportunity for the use of celocentesis for early prenatal diagnosis of genetic diseases and application of array comparative genomic hybridization. © 2016 John Wiley & Sons, Ltd.