Retrospective analysis of outcomes and complications after Korus hemiarthroplasty in elderly patients with neck femur fractures.

BACKGROUND: Femoral neck fractures (FNF) is one of the most common traumatic events in elderly patients: the choice of an appropriate treatment is necessary to decrease the related mortality and to achieve the best possible outcomes. Nowadays, it is still debated whether or not to cement the stem in hemiarthroplasty and above all, which stem to use to best respect the integrity of the elderly bone. METHODS: From January 2017 to December 2019, a bi-centric study utilizing prospectively collected databases of elderly patients with FNF treated with uncemented Korus stem hemiarthroplasty was performed. Patients were preoperatively classified according to ASA score. Patients' clinical and X-ray follow-up was at 1, 3, 6, 12 months. Harris Hip Score (HHS) was used for analysed clinical improvement. On the X-rays, we analysed iatrogenic fractures, osteolysis area and radiolucent lines in the stem region during follow up. RESULTS: 233 patients were identified. Median follow-up was 12 months. Over time, 51 patients died (21.88%). Mean age was 89,56 ± 6,25. 75 patients had ASA score of 2 (32.3%), 102 patients a score of 3 (43.7%), 56 an ASA score of 4 (24,0%). The main Harris hip score was 68,66 ± 8.53 at 1 month of follow-up, 71,74 ± 9.65 after 3 months, 72,50 ± 10.66 at 6 months and 75,61 ± 9.63 at 12 months control. CONCLUSIONS: Hydroxyapatite coated stem with an accurate design guarantee early fixation, good clinical and radiographic results, low rate of re-intervention and mortality rate and a satisfying return to pre-injury activities.

Droplet Digital PCR for Non-Invasive Prenatal Detection of Fetal Single-Gene Point Mutations in Maternal Plasma.

Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common ß thalassemia mutations in the Mediterranean area (ß+IVSI-110 and ß039), maternally and/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of ß+IVSI-110 and ß039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.

Performance evaluation of automated cell counts compared with reference methods for body fluid analysis.

OBJECTIVES: Cellular analysis of body fluids (BFs) can assist clinicians for the diagnosis of many medical conditions. The aim of this work is the evaluation of the analytical performance of the UF-5000 body fluid mode (UF-BF) analyzer compared to the gold standard method (optical microscopy, OM) and to XN-1000 (XN-BF), another analyzer produced by the same manufacturer (Sysmex) and with a similar technology for BF analysis. METHODS: One hundred BF samples collected in K3EDTA tubes were analyzed by UF-BF, XN-BF and OM. The agreement was evaluated using Passing and Bablok regression and Bland-Altman plot analysis. The receiver operating characteristic (ROC) curves were selected for evaluating the diagnostic agreement between OM classification and UF-BF parameters. RESULTS: Comparison between UF-BF and OM, in all BF types, showed Passing and Bablok's slope comprised between 0.99 (polymorphonuclear cells count, PMN-BF) and 1.39 (mononuclear cells count, MN-BF), the intercepts ranged between 26.47 (PMN-BF parameter) and 226.80 (white blood cell count). Bland-Altman bias was comprised between 7.3% (total cell count, TC-BF) and 52.9% (MN-BF). Comparison between UF-BF and XN-BF in all BF showed slopes ranged between 1.07 (TC-BF and PMN-BF) and 1.16 (MN-BF), intercepts ranged between 8.30 (PMN) and 64.78 (WBC-BF). Bland-Altman bias ranged between 5.8 (TC-BF) and 21.1% (MN-BF). The ROC curve analysis showed an area under the curve ranged between 0.9664 and 1.000. CONCLUSIONS: UF-BF shows very good performance for the differential counts of cells in ascitic, pleural and synovial fluids and therefore it is useful to screen and count cells in this type of BF.

Circulating microRNA-197-3p as a potential biomarker for asbestos exposure.

Asbestos is considered the main cause of diseases in workers exposed to this mineral in the workplace as well as an environmental pollutant. The association between asbestos and the onset of different diseases has been reported, but asbestos exposure specific biomarkers are not known. MicroRNAs (miRNAs) are small, single-strand, non-coding RNAs, with potential value as diagnostic, prognostic, and predictive markers in liquid biopsies. Sera collected from workers ex-exposed to asbestos (WEA) fibers were compared with sera from healthy subjects (HS) of similar age, as liquid biopsies. The expression of the circulating miRNA 197-3p was investigated employing two different highly analytical PCR methods, i.e. RT-qPCR and ddPCR. MiR-197-3p levels were tested in sera from WEA compared to HS. MiR-197-3p tested dysregulated in sera from WEA (n = 75) compared to HS (n = 62). Indeed, miR-197-3p was found to be 2.6 times down-regulated in WEA vs. HS (p = 0.0001***). In addition, an inverse correlation was detected between miR-197-3p expression level and cumulative asbestos exposure, being this miRNA down-regulated 2.1 times in WEA, with high cumulative asbestos exposure, compared to WEA with low exposure (p = 0.0303*). Circulating miR-197-3p, found to be down regulated in sera from WEA, is proposed as a new potential biomarker of asbestos exposure.

Epidemiology of paracoccidioidomycosis in Venezuela: a retrospective study from 1954 to 2019.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Etiological agents are Paracoccidioides species that diverge phylogenetically throughout South America. OBJECTIVES: This study aimed to document the epidemiology of PCM in Venezuela. METHODS: We have performed a retrospective cross-sectional descriptive study in 31,081 clinical records of patients from two reference centres during 65 years (1954-2019). FINDINGS: PCM diagnosis was confirmed in 745 patients. Chronic PCM was the most prevalent form (90.06% cases); 80.67% were male and the most affected age range was 41-60. Farming and construction were the most prevalent occupation and Miranda State had a higher prevalence. Lung and skin were the most affected organs, followed by oral manifestations. Direct examination, culture and serology showed a high sensibility, and no statistical difference was observed among the diagnostic tools. Out of 17 Paracoccidioides isolates genotyped from Venezuela, one was typed as Paracoccidioides americana and 16 as Paracoccidioides venezuelensis. MAIN CONCLUSIONS: Clinical manifestations observed, information about the epidemiology and molecular profile is essential not only for diagnosis but also for understanding therapeutic responses to mycotic drugs and prognosis. Therefore, it is necessary to sequence all positive isolated strains in order to confirm the dominance of P. venezuelensis in Venezuela.

Colorectal Carcinoma Affected Patients Are Significantly Poor Responders Against the Oncogenic JC Polyomavirus.

Background: Many investigations reported the association between human tumors and JCPyV, a polyomavirus with oncogenic potential. The association has been supported by studies that found JCPyV footprints in CRC and gliomas of different types. Indeed, JCPyV footprints including its nucleic acids and Tag oncoprotein have been revealed in CRC tissues. Methods: Herein, sera from colorectal carcinoma (CRC) affected patients and healthy individuals (HS), employed as control, were analysed for immunoglobulin G (IgG) antibodies against specific JCPyV viral capsid protein 1 (VP1) antigens. The investigation was carried out employing an innovative immunological assay. Indeed, an indirect enzyme-linked immunosorbent assay (ELISA) with JCPyV VP1 mimotopes was used. JCPyV VP1 mimotopes consisted of synthetic peptides mimicking VP1 epitopes. Results: Sera from CRC affected patients, evaluated using indirect ELISAs with synthetic mimotopes, showed a significant lower prevalence of IgG antibodies against JCPyV VP1 mimotopes (26%) compared to HS (51%), p<0.005. These data were confirmed by another method, the hemagglutination inhibition (HAI) assay. Altogether these results, i.e. the prevalence of serum IgG antibodies against JCPyV VP1 mimotopes from patients with CRC is approximately 50% lower than in HS, are of interest. Discussion: Our data suggest that patients with CRC are significantly poor responders against JCPyV VP1 antigens. It is possible that CRC patients are affected by a specific immunological deregulation. This immunological dysfunction, revelled in CRC patients, may account for their predisposition to the colorectal carcinoma onset.

Complete Blood Count as point of care testing QBC STAR™: Preliminary evaluation.

INTRODUCTION: Point of care testing (POCT) represents a valuable option when laboratory data shall be urgently available for timely clinical management, with a turnaround time (TAT) that is unfeasible using conventional laboratory instrumentation. This study was aimed to compare the performance of QBC STAR™ compared to Sysmex XN-module and the reference optical microscopy (OM) assessment. MATERIAL AND METHODS: One hundred peripheral blood samples, collected in K3 EDTA tubes, and 50 capillary blood samples obtained by finger stick were analyzed with QBC STAR™, Sysmex XN-module, and OM. Data were compared with Passing-Bablok regression and Bland-Altman plots. RESULTS: The Passing-Bablok regression analysis (QBC STAR™ capillary sample vs XN-module) yielded slopes comprised between 0.30 and 1.37, while the intercepts ranged between -17.57 and 232.6. Bland-Altman plots yielded relative bias comprised between -4.87% (for MN QBC STAR™ capillary sample vs XN-module) and 27% (PLT QBC STAR™ capillary sample vs XN-module). A significant bias was found for all parameters except MN and WBC, RBC in all and pediatric samples, and HB in adults samples. CONCLUSION: The results of this analytical evaluation suggest that QBC STAR™ may not be the ideal tool for performing complete blood count analysis for diagnostic purposes, while it could be more useful in urgent/emergent conditions, such as for rapid monitoring of some hematological parameters (eg, WBC and HB).

Surviving COVID-19 in Bergamo province: a post-acute outpatient re-evaluation.

Bergamo province was badly hit by the coronavirus disease 2019 (COVID-19) epidemic. We organised a public-funded, multidisciplinary follow-up programme for COVID-19 patients discharged from the emergency department or from the inpatient wards of 'Papa Giovanni XXIII' Hospital, the largest public hospital in the area. As of 31 July, the first 767 patients had completed the first post-discharge multidisciplinary assessment. Patients entered our programme at a median time of 81 days after discharge. Among them, 51.4% still complained of symptoms, most commonly fatigue and exertional dyspnoea, and 30.5% were still experiencing post-traumatic psychological consequences. Impaired lung diffusion was found in 19%. Seventeen per cent had D-dimer values two times above the threshold for diagnosis of pulmonary embolism (two unexpected and clinically silent pulmonary thrombosis were discovered by investigating striking D-dimer elevation). Survivors of COVID-19 exhibit a complex array of symptoms, whose common underlying pathology, if any, has still to be elucidated: a multidisciplinary approach is fundamental, to address the different problems and to look for effective solutions.

Epidemiology of paracoccidioidomycosis in Venezuela: a retrospective study from 1954 to 2019

BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis endemic to Latin America. Etiological agents are Paracoccidioides species that diverge phylogenetically throughout South America. OBJECTIVES This study aimed to document the epidemiology of PCM in Venezuela. METHODS We have performed a retrospective cross-sectional descriptive study in 31,081 clinical records of patients from two reference centres during 65 years (1954-2019). FINDINGS PCM diagnosis was confirmed in 745 patients. Chronic PCM was the most prevalent form (90.06% cases); 80.67% were male and the most affected age range was 41-60. Farming and construction were the most prevalent occupation and Miranda State had a higher prevalence. Lung and skin were the most affected organs, followed by oral manifestations. Direct examination, culture and serology showed a high sensibility, and no statistical difference was observed among the diagnostic tools. Out of 17 Paracoccidioides isolates genotyped from Venezuela, one was typed as Paracoccidioides americana and 16 as Paracoccidioides venezuelensis. MAIN CONCLUSIONS Clinical manifestations observed, information about the epidemiology and molecular profile is essential not only for diagnosis but also for understanding therapeutic responses to mycotic drugs and prognosis. Therefore, it is necessary to sequence all positive isolated strains in order to confirm the dominance of P. venezuelensis in Venezuela.

Specific antibodies reacting to JC polyomavirus capsid protein mimotopes in sera from multiple sclerosis and other neurological diseases-affected patients.

Published data support the hypothesis that viruses could be trigger agents of multiple sclerosis onset. This link is based on evidence of early exposure to viral agents in patients affected by this neurologic disease. JC (JC polyomavirus [JCPyV]), BK (BKPyV), and simian virus 40 (SV40) neurotropic polyomavirus footprints have been detected in brain tissue specimens and samples from patients affected by different neurological diseases. In this investigation, serum samples from patients affected by multiple sclerosis and other inflammatory and noninflammatory neurologic diseases, as well as healthy subjects representing the control, were investigated for immunoglobulin G (IgG) antibodies against JCPyV. To this end, an immunologic approach was employed, which consists of employing indirect enzyme-linked immunosorbent assay testing with synthetic peptides mimicking viral capsid protein 1 antigens. A significantly lower prevalence of IgG antibodies against JCPyV VP1 epitopes, with a low titer, was detected in serum samples from patients with multiple sclerosis (MS) and other neurologic diseases than in healthy subjects. Our study indicates that the prevalence of JCPyV antibodies from patients with multiple sclerosis is 50% lower than in healthy subjects, suggesting specific immune impairments. These results indicate that patients affected by neurological diseases, including MS, respond poorly to JCPyV VP1 antigens, suggesting specific immunologic dysfunctions.

Trend of Antitissue Transglutaminase Antibody Normalization in Children With Celiac Disease Started on Gluten-free Diet: A Comparative Study Between Chemiluminescence and ELISA Serum Assays.

BACKGROUND: The aim of this study is to compare the performance of antitissue transglutaminase (atTG) chemiluminescence immunoassay (CLIA) with the standard enzyme-linked immunosorbent assay (ELISA) methods in monitoring celiac children after the start of gluten-free diet (GFD). METHODS: Celiac children diagnosed between 2005 and 2016 at our centre were classified into 2 groups based on serum assay (ELISA vs CLIA) used for atTG monitoring, and were compared on percentage of decrease and time to normalization of atTG on GFD. RESULTS: Among 260 included children, the rate of normalization of atTG levels at 30 months' follow-up was 86% and 70% in ELISA and CLIA group, respectively (P < 0.01). Median time to normalization was 11.7 and 14.7 months in ELISA and CLIA group respectively (P = 0.003). Marsh score at diagnosis was not associated with time to atTG normalization (P = 0.770), whereas older age at diagnosis and higher baseline atTG predicted longer time to atTG normalization (P = 0.01, P < 0.01). CONCLUSIONS: The percentage and the time of the atTG normalization in celiac children on GFD should be interpreted according to the utilized assay: at 30 months' follow-up children tested by CLIA are less likely to normalize atTG levels compared to those tested by ELISA. Younger age at diagnosis and lower baseline atTG are predictors of earlier atTG normalization, regardless of the adopted assay.

Patellar and quadriceps tendons acute repair with suture anchors.

BACKGROUND AND AIM OF THE WORK: Quadriceps and patellar tendon rupture are relatively uncommon but can result in a disabling condition if untreated. We retrospectively review all our cases treated with suture anchors from 2014 to 2018, to evaluate midterm outcome of this technique. METHODS: Traumatic and atraumatic quadriceps and patellar tendon preinsertional lesions were acutely treated with Healix Ti and FaStin RC 5mm suture anchors and an aggressive rehabilitation protocol was prescribed to patients. RESULTS: Good to excellent results according to the Modified Cincinnati Rating System Questionnaire was obtained at a mean 12 months followup, without major complications. CONCLUSIONS: Suture anchors are a promising alternative to transosseus suture for acute repair of quadriceps and patellar tendon lesions, but longer followups are needed for detect long-term complications.

Antibodies reacting to mimotopes of Simian virus 40 large T antigen, the viral oncoprotein, in sera from children.

Recent data indicate that the Simian virus 40 (SV40) infection appears to be transmitted in humans independently from early SV40-contaminated antipolio vaccines. Serum antibodies against SV40 large T antigen (Tag) were analyzed in children/adolescents and young adults. To investigate antibodies reacting to SV40 Tag antigens, serum samples ( n = 812) from children and young adults were analyzed by indirect ELISAs using specific SV40 Tag mimotopes. Mimotopes were synthetic peptides corresponding to SV40 Tag epitopes. In sera ( n = 412) from healthy children up to 17 years old, IgG antibodies against SV40 Tag mimotopes reached an overall prevalence of 15%. IgM antibodies against SV40 Tag were detected in sera of children 6-8 months old confirming and extending the knowledge that SV40 seroconversion occurs early in life. In children/adolescents affected by different diseases ( n = 180) SV40 Tag had a prevalence of 18%, being the difference no significant compared to healthy subjects ( n = 220; 16%) of the same age. Our immunological data indicate that SV40 circulates in children and young adults, both in healthy conditions and affected by distinct diseases. The IgM detection in sera from healthy children suggests that the SV40 infection/seroconversion occurs early in life (>6 months). Our immunological data support the hypothesis that SV40, or a closely related still unknown polyomavirus, infects humans. The SV40 seroprevalence is lower than common polyomaviruses, such as BKPyV and JCPyV, and other new human polyomaviruses. In addition, our immunological surveillance indicates a lack of association between different diseases, considered herein, and SV40.

Non-invasive fetal sex diagnosis in plasma of early weeks pregnants using droplet digital PCR.

BACKGROUND: Fetal sex determination is useful for families at risk of X-linked disorders, such as Duchenne muscular dystrophy, adrenal hypoplasia, hemophilia. At first, this could be obtained through invasive procedures such as amniocentesis and chorionic villus sampling, having a 1% risk of miscarriage. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing permits the early diagnosis of fetal sex through analysis of cffDNA. However, the low amount of cffDNA relative to circulating maternal DNA requires highly sensitive molecular techniques in order to perform noninvasive prenatal diagnosis. In this context we employed droplet digital PCR (ddPCR) in order to evaluate the earliest possible fetal sex determination from circulating DNA extracted from plasma of pregnant women at different gestational ages. METHODS: We identified the fetal sex on cffDNA extracted from 29 maternal plasma samples at early gestational ages, several of them not suitable for qPCR determination, using ddPCR designed for SRY gene target. RESULTS: All maternal plasma samples were determined correctly for SRY gene target using ddPCR even at very early gestational age (prior to 7 weeks). CONCLUSIONS: The ddPCR is a robust, efficient and reliable technology for the earliest possible fetal sex determination from maternal plasma.

Serum IgG antibodies from healthy subjects up to 100 years old react to JC polyomavirus.

JC polyomavirus (JCPyV) was identified in 1971 in the brain tissue of a patient (J.C.) affected by the progressive multifocal leukoencephalopathy (PML). JCPyV encodes for the oncoproteins large T antigen (Tag) and small t-antigen (tag). These oncoproteins are responsible of the cell transformation and tumorigenesis in experimental animals. JCPyV is ubiquitous in human populations. After the primary infection, which is usually asymptomatic, JCPyV remains lifelong in the host in a latent phase. Its reactivation may occur in heathy subjects and immunocompromised patients. Upon reactivation, JCPyV could reach (i) the CNS inducing the PML, (ii) the kidney of transplant patients causing the organ rejection. Association between JCPyV, which is a small DNA tumor virus, and gliomas and colorectal carcinomas has been published. In the present investigation, we report on a new indirect ELISA with two specific synthetic peptides mimicking JCPyV VP1 immunogenic epitopes to detect specific serum IgG antibodies against JCPyV. Serum samples of healthy subjects (n = 355) ranging 2-100 years old, were analyzed by this new indirect ELISA. The linear peptides VP1 K and VP1 N resemble the natural JCPyV VP1 capsidic epitopes constituting a docking site for serum antibodies. Data from this innovative immunologic assay indicate that the overall prevalence of JCPyV-VP1 antibodies in healthy subjects is at 39%. The innovative indirect ELISA with JCPyV VP1 mimotopes seems to be a useful method to detect specific IgG antibodies against this virus, without cross-reactivity with the closely related SV40 and BKPyV polyomaviruses.

Patient specific instrumentation in total knee arthroplasty.

BACKGROUND AND AIM OF THE WORK: The international literature and analysis of the prosthetic registers highlight a significant relationship between the alignment of the components and the survival of prosthetic implants of the knee. The patient specific instrumentation (PSI) technology exploits the data obtained with the MRN for the production of cutting blocks (CB) useful to a TKA. Revisiting the recent international literature, comparing the results of the conventional method and PSI, numerous studies confirm a statistically significant difference of inliers (± 3 degrees) for HKA. The purpose of this retrospective study was to investigate whether these statistically significant difference is also present in our group. METHODS: Postoperative radiographic measures of alignment based on a mechanical limb axis (hip-knee-ankle angle, HKA) of 180° were sought. A range of 180° ± 3° varus/valgus was defined as optimal for mechanical axis. RESULTS: The percentage of knees that had a HKA within ±3° of the desired value was 92.2. CONCLUSION: the CB did accurately produce the desired HKA. The PS system is an effective and reproducible, whose organizational effort is fully justified.

Specific IgG Antibodies React to Mimotopes of BK Polyomavirus, a Small DNA Tumor Virus, in Healthy Adult Sera.

BK polyomavirus (BKPyV) was isolated in 1971 from the urine of a kidney transplant patient. Soon after its identification, BKPyV was characterized as a kidney-tropic virus, which is responsible of a significant fraction of the rejection of transplant kidney in the host. Moreover, in experimental conditions, BKPyV is able to transform different types of animal and human cells and to induce tumors of different histotypes in experimental animals. BKPyV DNA sequences have been detected in healthy individuals and cancer patients using polymerase chain reaction/Shouthern blot hybridization methods. Serum antibodies against this polyomavirus were revealed using immunological techniques, which, however, cross-react with other polyomaviruses such as JC (JCPyV) and Simian Virus 40. These non-specific data indicate the need of novel immunological methods and new investigations to check in a specific manner, BKPyV spread in humans. To this aim, mimotopes from BKPyV structural capsid protein 1 (VP1) were employed for specific immunological reactions to IgG antibodies of human serum samples. An indirect enzyme-linked immunosorbent assay with synthetic peptides mimicking immunogenic epitopes of BKPyV VP1 was set up and employed to test sera of healthy adult subjects. Data from this innovative immunological assay indicate that serum antibodies against BKPyV VP1 mimotopes are detectable in healthy subjects ranging from 18 to 90 years old. The overall prevalence of serum samples that reacted to BKPyV VP1 mimotopes was 72%. The strong points from this investigation are the novelty of the immunological method, its simplicity of the approach, and the specificity of BKPyV antibody reaction to VP1 mimotopes.

Postnatal and non-invasive prenatal detection of ß-thalassemia mutations based on Taqman genotyping assays.

The ß-thalassemias are genetic disorder caused by more than 200 mutations in the ß-globin gene, resulting in a total (ß0) or partial (ß+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for ß-thalassemia are: ß039, ß+IVSI-110, ß+IVSI-6 and ß0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human ß-globin gene. Four TaqMan® genotyping assays for the most common ß-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 ß-thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations ß039, ß+IVSI-110, ß+IVSI-6, ß0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of ß-globin gene mutations, starting from the 9th gestational week.