Non-invasive Prenatal Diagnostics Research Articles

CONGENITAL AND HEREDITARY PATHOLOGY IN WOMEN CONTRACTED COVID-19 DURING PREGNANCY

The study aimed to investigate the frequency and structure of congenital and hereditary pathologies identified through non-invasive and invasive methods of prenatal diagnostics in women who contracted COVID-19 during pregnancy, among the population of the Lviv region. The data from prenatal diagnostics results of congenital developmental defects in non-invasive prenatal diagnostics and hereditary diseases in invasive prenatal diagnostics were analyzed in women who contracted COVID-19 during pregnancy. The study demonstrated that the frequency of diagnosing congenital anomalies (CA) in the fetus among 159 pregnant women in non-invasive prenatal diagnostics (PND) was 13.8%. In the structure of congenital anomalies, facial anomalies were the most prevalent, accounting for 5.0% in the first position, followed by anomalies of the cardiovascular system at 2.5% in the second position, anomalies of the skeletal system at 2.5% in the third position, and multiple congenital anomalies at 1.9% in the fourth position. Invasive prenatal diagnostics were performed on 12.6% of women in this group, with chromosomal pathology detected in 5.0% of women: trisomy 21 was diagnosed in 3.8%, Turner syndrome in 1.3%, and a normal karyotype was identified in 7.5% of patients. The frequency of chromosomal pathology in women who contracted COVID-19 during pregnancy was 5.0%. The results of this study will contribute to the improvement of effective medical-genetic counseling and the prediction of healthy offspring.

A noninvasive method for the detection of foetal DNA in early pregnancy based on differential methylation pattern of Ras association domain family member 1A

Background The detection of foetal DNA and extravillus trophoblasts (EVTs) in early pregnancy in cervical and uterine samples offers a potential pathway for non-invasive prenatal diagnostics. However, the challenge lies in effectively quantifying these samples. This study introduces a novel approach using the Ras association domain family 1 A (RASSF1A), which exhibits hypermethylation in foetal cells and hypomethylation in maternal cells. The differential methylation pattern of RASSF1A provides a unique biomarker for quantifying foetal cells in cervical and intrauterine samples. Methods This study was conducted between September 2022 and May 2023. In total, 23 samples (12 cervical cell samples and 11 intrauterine samples) were collected from women in the Sichuan Jinxin Women & Children Hospital, Jingxiu District, Chengdu, China. The cervical cell samples were collected via lavage and brush techniques, and the intrauterine cell samples were obtained via uterine lavage. These samples were collected as part of a broader effort to advance our understanding of foetal cell dynamics during early pregnancy. The sampling methods were chosen for their minimally invasive nature and their potential in capturing a representative cell population from the respective sites. After digestion of the cell samples using a methylation-sensitive restriction enzyme cocktail, a critical step to differentiate between maternal and foetal DNA, the quantitative polymerase chain reaction (qPCR) of RASSF1A and β-actin (ACTB) were employed to measure foetal DNA and cell concentrations. Immunofluorescence techniques targeting histocompatibility complex, class I G (HLA-G) and GATA binding protein 3 (GATA-3) were employed to detect EVTs in the cell samples and in decidual tissue, with the latter providing an additional layer of confirmation for the presence of foetal cells. Results The results showed no hypermethylated RASSF1A was detected in any of the cervical samples, irrespective of whether the samples were obtained by brush or lavage. However, an average of 17,236 ± 7490 foetal cells per sample were detected in the uterine lavage samples. Foetal cells accounted for approximately 0.14% ± 0.10% of the total cell population in these samples. The presence of EVTs in these samples was confirmed by their expression of both HLA-G and GATA-3. Conclusion The detection of foetal cells in uterine cavity samples based on hypermethylation of RASSF1A and quantification of foetal cells can be used to prenatal screening. GATA-3 can be used to label EVTs.

The Next Frontier in Noninvasive Prenatal Diagnostics: Cell-Free Fetal DNA Analysis for Monogenic Disease Assessment.

With the widespread clinical adoption of noninvasive screening for fetal chromosomal aneuploidies based on cell-free DNA analysis from maternal plasma, more researchers are turning their attention to noninvasive prenatal assessment for single-gene disorders. The development of a spectrum of approaches to analyze cell-free DNA in maternal circulation, including relative mutation dosage, relative haplotype dosage, and size-based methods, has expanded the scope of noninvasive prenatal testing to sex-linked and autosomal recessive disorders. Cell-free fetal DNA analysis for several of the more prevalent single-gene disorders has recently been introduced into clinical service. This article reviews the analytical approaches currently available and discusses the extent of the clinical implementation of noninvasive prenatal testing for single-gene disorders.

Prediction of fetal blood group antigens from maternal plasma using Ion AmpliSeq HD technology.

Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.

RhD-induced immunogenetic disparity between mother and fetus: medical importance and economic effect of using molecular genetic technology

Introduction. Here we discuss the problem of timely diagnostics and prevention of Rh-immunization of pregnant women as well as fetal hemolytic disease, which remains currently relevant, despite the existence of proven diagnostic, therapeutic and preventive methods.Aim: to assess the medico-economic efficiency of non-invasive prenatal diagnostics of using fetal Rh factor (rhesus D antigen, RhD) in maternal blood – a fetal RhD-genotyping.Materials and Methods. A retrospective observational study was conducted to analyze determining fetal Rh-factor in the blood samples from 4109 Rh-negative pregnant women observed in the 41 medical facilities of the Ulyanovsk region in the years 2018–2020. The fetal RhD level was determined by polymerase chain reaction at gestational age of ≥ 10 weeks. To assess testrelated medical effectiveness, there were examined sensitivity, specificity, predictive value of positive and negative data as well as diagnostic accuracy. The data collected during the study were compared with those obtained after delivery. To assess the economic efficiency, the difference between the cost of immunization and the cost of determining the fetal Rh factor level was determined.Results. A positive and negative fetal Rh-factor was detected in 67.26 % (n = 2793) and 32.74 % (n = 1316) cases, respectively. Diagnostic accuracy of the test system "Test-RhD" was 99.40 %, sensitivity – 99.84 %, specificity – 97.51 %, the prognostic value of a positive result was 99.43 %, the predictive value of a negative result – 99.28 % with low rate of false positive and false negative data. It was shown that our study allows to avoid unnecessary immunization costs for all Rh-negative pregnant women.Conclusion. Analysis of the diagnostic characteristics and cost-effectiveness of the RhD test evidences about high medical significance of the method described and allows to recommend its wider application.

Non-invasive prenatal diagnostics (NIPD) in the system of medical care. Ethical and legal issues.

The procedures of prenatal and preimplantation diagnostics are discussed critically again and again in our community. In addition to the permanently controversial issues of embryo protection and abortion, considerations that discrimination on the basis of disability could occur with problematic consequences also for already born people with disabilities and their relatives now play a central role.

Cell-free fetal DNA coming in all sizes and shapes.

Cell‐free fetal DNA analysis has an established role in prenatal assessments. It serves as a source of fetal genetic material that is accessible non‐invasively from maternal blood. Through the years, evidence has accumulated to show that cell‐free fetal DNA molecules are derived from placental tissues, are mainly of short DNA fragments and have rapid post‐delivery clearance profiles. But questions regarding how they come to being short molecules from placental cells and in which physical forms do they exist remained largely unanswered until recently. We now know that the distributions of ending sites of cell‐free DNA molecules are non‐random across the genome and bear correlations with the chromatin structures of cells from which they have originated. Such an insight offers ways to deduce the tissue‐of‐origin of these molecules. Besides, the physical nature and sequence characteristics of the ends of each cell‐free DNA molecule provide tell‐tale signs of how the DNA fragmentation processes are orchestrated by nuclease enzymes. These realizations offered opportunities to develop methods for enriching cell‐free fetal DNA to facilitate non‐invasive prenatal diagnostics. Here we aimed to collate what is known about the biological and physical characteristics of cell‐free fetal DNA into one article and explain the implications of these observations.

The isolation and analysis of fetal nucleated red blood cells using multifunctional microbeads with a nanostructured coating toward early noninvasive prenatal diagnostics.

Prenatal diagnostics holds great significance for pregnant women desiring healthy babies. Fetal nucleated red blood cells (fNRBCs), bearing the complete genome of the fetus, have been regarded as an important biomarker for noninvasive prenatal diagnostics (NIPD). The high-performance detection and enrichment of fNRBCs from maternal blood, especially during early pregnancy, is urgently needed for NIPD, which, unfortunately, remains a big challenge for early-pregnancy fNRBC isolation. In this study, we developed an innovative platform based on silica microbeads for fNRBC isolation and release in early pregnancy. Microbeads were coated with self-assembled MnO2 nanoparticles (SiO2@MnO2) and then modified with a specific antibody. Benefiting from the three-dimensional nanostructure of the MnO2 nanoparticles, the isolation efficiency of the fNRBCs was enhanced. Subsequently, fNRBCs were released via dissolving the MnO2-nanoparticle coating using oxalic acid. We successfully isolated fNRBCs from the maternal peripheral blood samples of 20 pregnant women in the early pregnancy period, ranging from 41 to 62 gestational days. More importantly, the fetal origin of isolated cells was confirmed via fluorescent in situ hybridization and short tandem repeat analysis. This platform based on SiO2@MnO2 microbeads has verified the existence of fNRBCs in early-pregnancy maternal blood and is a promising approach for NIPD in early pregnancy.

A Biocompatible Nanofibers‐Based Microchip for Isolation and Nondestructive Release of Fetal Nucleated Red Blood Cells

AbstractThe enrichment and analysis of fetal nucleated red blood cells (fNRBCs) with fetal intact genome provide an entry for noninvasive prenatal diagnostics (NIPD) as well as intervention of birth defects. Although efficiently fishing and counting fNRBCs from maternal peripheral blood is gaining tremendous interest for decades, it has been impeded recently by its low abundance. In this study, a compatible chitosan nanofiber microchip is developed to specifically capture and nondestructively release fNRBCs from maternal blood. The substrate has 3D nanostructures formed by electrospun chitosan nanofibers, which integrates with anti‐CD147 to capture fNRBCs with an efficiency of 90%. Subsequently, dithiothreitol is employed to release the captured cells with the efficiency up to 90% while the cell viability is kept at 91.2%. Using the chip, the fNRBCs are successfully isolated from peripheral blood of ten pregnant women in the first trimester ranging from 7 to 13 weeks. Overall, this nanofibrous bioplatform combined with functionalized interface molecules offers a promising strategy to isolate fNRBCs from early gestation period, and provides new prospects for advanced studies to facilitate NIPD in future.

Nanostructured Substrates for Detection and Characterization of Circulating Rare Cells: From Materials Research to Clinical Applications.

Circulating rare cells in the blood are of great significance for both materials research and clinical applications. For example, circulating tumor cells (CTCs) have been demonstrated as useful biomarkers for "liquid biopsy" of the tumor. Circulating fetal nucleated cells (CFNCs) have shown potential in noninvasive prenatal diagnostics. However, it is technically challenging to detect and isolate circulating rare cells due to their extremely low abundance compared to hematologic cells. Nanostructured substrates offer a unique solution to address these challenges by providing local topographic interactions to strengthen cell adhesion and large surface areas for grafting capture agents, resulting in improved cell capture efficiency, purity, sensitivity, and reproducibility. In addition, rare-cell retrieval strategies, including stimulus-responsiveness and additive reagent-triggered release on different nanostructured substrates, allow for on-demand retrieval of the captured CTCs/CFNCs with high cell viability and molecular integrity. Several nanostructured substrate-enabled CTC/CFNC assays are observed maturing from enumeration and subclassification to molecular analyses. These can one day become powerful tools in disease diagnosis, prognostic prediction, and dynamic monitoring of therapeutic response-paving the way for personalized medical care.

Analysis of fetal DNA in maternal plasma with markers designed for forensic DNA mixture resolution

PurposeWith the description of circulating fetal DNA in maternal blood,noninvasive prenatal diagnostics became theoretically possible. As the presenceof background maternal DNA interferes with the detection of fetal DNA,analytical methods require genetic markers capable of distinguishing byquantitative or targeted approaches the minor population of DNA molecules of thefetus. Here we evaluate the feasibility of analyzing fetal DNA with novelDIP-STR genetic markers, designed for the investigation of forensic mixedbiological evidence. MethodsThe DIP-STR molecular approach is based on sequence-specificanalysis of paternally inherited fetal alleles. These sequences are biallelicdeletion/insertion polymorphisms (DIPs) located very close to short tandemrepeat (STR) markers, for combined analysis. In this study, 48 women were testedwith 28 DIP-STRs during the first, second, and third trimester ofpregnancy. ResultsPositive results were obtained across markers, including longer ones(386 base-pairs) and with blood samples collected during early pregnancy, suchas 10 weeks of gestational age. ConclusionThese data show that DIP-STR markers can be used to amplify specificgenomic regions of circulating fetal DNA to obtain targeted genetic information.This method may contribute to developments in noninvasive prenatal paternitytesting and diagnosis of certain genetic diseases.

Expanded non-invasive prenatal diagnostics

Non-invasive prenatal diagnostics allow for the successful identification of paternally inherited and de novo mongenic diseases using circulating cell-free DNA.

Noninvasive Prenatal Diagnostics: Recent Developments Using Circulating Fetal Nucleated Cells

The purpose of this review is to highlight recent research advances in noninvasive prenatal diagnostic methods. Recent studies developing noninvasive prenatal diagnostic (NIPD) methods have been focused on either fetal nucleated red blood cells (fNRBCs) or circulating trophoblasts (cTBs). Enriched cTBs were successfully utilized for whole genome profiling and short tandem repeat (STR) identification to confirm feto-maternal relationship. However, further analysis of isolated fNRBCs remains confined to examining fetal cytogenetics. Invasive prenatal diagnostic procedures, amniocentesis and chorionic villus sampling, are the gold standard for the diagnosis of fetal chromosomal abnormalities and genetic disorders. Meanwhile, noninvasive techniques of analyzing circulating cell-free fetal DNA (cffDNA) have been limited to screening tools and are highly fragmented and confounded by maternal DNA. By detecting circulating fetal nucleated cells (CFNCs) we are able to noninvasively confirm fetal chromosomal abnormalities, truly realizing the concept of "noninvasive prenatal diagnostics". The primary technical challenge is the enrichment of the low abundance of CFNCs in maternal peripheral blood. For any cell-based NIPD method, both fetal whole genome profiling and confirmation of the feto-parental relationship are essential. This has been successfully performed using enriched and isolated cTBs, making cTB a better candidate for NIPD. cTB enumeration also correlates with abnormal fetal or placental development. On the other hand, downstream analysis of fNRBCs remains limited to examining fetal sex and aneuploidies. Furthermore, trophoblast-based NIPD via an endocervical sample is also promising because of reduced dilution from hematologic cells.

Enhanced isolation and release of fetal nucleated red blood cells using multifunctional nanoparticle-based microfluidic device for non-invasive prenatal diagnostics

Fetal nucleated red blood cells (fNRBC) in maternal peripheral blood has the potential for non-invasive prenatal diagnostics (NIPD), given its intrinsic nature carrying total genetic information of the fetus. Unfortunately, the scarcity of fNRBCs in maternal blood circulation greatly hinders the effective isolation of fNRBCs and its further uses for clinical prenatal diagnostics. Herein, we developed a gelatin nanoparticles (GNPs) decorated microchip that modified with anti-CD147 as specific capture antibody to efficiently isolate fNRBCs from maternal peripheral blood. The corrugated GNP nanocoating on the walls of the channel, together with herringbone grooves in the continuous curved channel design, produced enhanced interactions between fNRBC and the device for better cell capture performance. Furthermore, the captured cells could be gently released for subsequent off-chip analyses, using an enzymatic treatment to dissolve the biodegradable GNP nanocoating. Significant target cell capture efficiency (>80%), release efficiency (∼89%) and purity (∼85%) as well as a high viability of >90% were achieved using simulated spiked samples. fNRBCs were detected from a series of maternal peripheral blood samples ranging from 7 to 13 weeks of gestation, and the diagnostic application for fetal chromosomal disorders was demonstrated. Our strategy may provide new insights into developing an approach to recover fNRBCs from early pregnancy for improved cell-based NIPD.

Maternal plasma metabolic fingerprint indicative for fetal Down syndrome

The objective of the study was to perform maternal plasma metabolic fingerprinting to evaluate differences in plasma metabolites between healthy and Down syndrome (DS) pregnancies and to indicate novel non-invasive markers for DS prenatal diagnostics. This was a case-control study of pregnancies between 15th and 18th gestational week. LC-MS-based metabolic fingerprinting of plasma samples was performed. Levels of five metabolites were significantly lower in the plasma of DS pregnancies. The majority of the statistically significant metabolites may be connected with fetal brain and central nervous system development (eg, fatty acid amides). According to the receiver operating characteristic (ROC), the combination of linoleamide and piperine has the highest diagnostic potential: area under the curve (AUC)=0.878, sensitivity of 100%, and specificity of 73.3%. The study indicates disturbances in maternal metabolic pathways evoked by fetal DS. Novel potential maternal plasma metabolomic markers for non-invasive prenatal diagnostics of fetal DS are proposed.

Highly sensitive and rapid isolation of fetal nucleated red blood cells with microbead-based selective sedimentation for non-invasive prenatal diagnostics

Non-invasive prenatal diagnostics (NIPD) has been an emerging field for prenatal diagnosis research. Carrying the whole genome coding of the fetus, fetal nucleated red blood cells (FNRBCs) have been pursued as a surrogate biomarker traveling around in maternal blood. Here, by combining a unique microbead-based centrifugal separation and enzymatic release, we demonstrated a novel method for FNRBC isolation from the blood samples. First, the gelatin-coated silica microbeads were modified with FNRBC-specific antibody (anti-CD147) to capture the target cells in the blood samples. Then, the density difference between microbead-bound FNRBCs and normal blood cells enables the purification of FNRBCs via an improved high-density percoll-based separation. The non-invasive release of FNRBCs can then be achieved by enzymatically degrading the gelatin film on the surface of the microbeads, allowing a gentle release of the captured target cells with as high as 84% efficiency and ∼80% purity. We further applied it to isolate fetal cells from maternal peripheral blood. The released cells were analyzed by real-time polymerase chain reaction to verify their fetal origin and fluorescent in situ hybridization to detect fetal chromosome disorders. This straightforward and reliable alternative platform for FNRBC detection may have the potential for realizing facile NIPD.

Correction to Imprinted NanoVelcro Microchips for Isolation and Characterization of Circulating Fetal Trophoblasts: Toward Noninvasive Prenatal Diagnostics.

Circulating fetal nucleated cells (CFNCs) in maternal blood offer an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics (NIPD). We developed a class of nanoVelcro microchips to effectively enrich a subcategory of CFNCs, i.e., circulating trophoblasts (cTBs) from maternal blood, which can then be isolated with single-cell resolution by a laser capture microdissection (LCM) technique for downstream genetic testing. We first established a nanoimprinting fabrication process to prepare the LCM-compatible nanoVelcro substrates. Using an optimized cTB-capture condition and an immunocytochemistry protocol, we were able to identify and isolate single cTBs (Hoechst+/CK7+/HLA-G+/CD45-, 20 μm > sizes > 12 μm) on the imprinted nanoVelcro microchips. Three cTBs were polled to ensure reproducible whole genome amplification on the cTB-derived DNA, paving the way for cTB-based array comparative genomic hybridization (aCGH) and short tandem repeats analysis. Using maternal blood samples collected from expectant mothers carrying a single fetus, the cTB-derived aCGH data were able to detect fetal genders and chromosomal aberrations, which had been confirmed by standard clinical practice. Our results support the use of nanoVelcro microchips for cTB-based noninvasive prenatal genetic testing, which holds potential for further development toward future NIPD solution.

Unravelling the biological secrets of microchimerism by single-cell analysis.

The presence of microchimeric cells is known for >100 years and well documented since decades. Earlier, microchimeric cells were mainly used for cell-based non-invasive prenatal diagnostics during early pregnancy. Microchimeric cells are also present beyond delivery and are associated to various autoimmune diseases, tissue repair, cancer and immune tolerance. All these findings were based on low complexity studies and occasionally accompanied by artefacts not allowing the biological functions of microchimerism to be determined. However, with the recent developments in single-cell analysis, new means to identify and characterize microchimeric cells are available. Cell labelling techniques in combination with single-cell analysis provide a new toolbox to decipher the biology of microchimeric cells at molecular and cellular level. In this review, we discuss how recent developments in single-cell analysis can be applied to determine the role and function of microchimeric cells.

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.

Imprinted NanoVelcro Microchips for Isolation and Characterization of Circulating Fetal Trophoblasts: Toward Noninvasive Prenatal Diagnostics.

Circulating fetal nucleated cells (CFNCs) in maternal blood offer an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics (NIPD). We developed a class of nanoVelcro microchips to effectively enrich a subcategory of CFNCs, i.e., circulating trophoblasts (cTBs) from maternal blood, which can then be isolated with single-cell resolution by a laser capture microdissection (LCM) technique for downstream genetic testing. We first established a nanoimprinting fabrication process to prepare the LCM-compatible nanoVelcro substrates. Using an optimized cTB-capture condition and an immunocytochemistry protocol, we were able to identify and isolate single cTBs (Hoechst+/CK7+/HLA-G+/CD45-, 20 μm > sizes > 12 μm) on the imprinted nanoVelcro microchips. Three cTBs were polled to ensure reproducible whole genome amplification on the cTB-derived DNA, paving the way for cTB-based array comparative genomic hybridization (aCGH) and short tandem repeats analysis. Using maternal blood samples collected from expectant mothers carrying a single fetus, the cTB-derived aCGH data were able to detect fetal genders and chromosomal aberrations, which had been confirmed by standard clinical practice. Our results support the use of nanoVelcro microchips for cTB-based noninvasive prenatal genetic testing, which holds potential for further development toward future NIPD solution.