Aniza Puteri Mahyuddin

Separation of model mixtures of epsilon-globin positive fetal nucleated red blood cells and anucleate erythrocytes using a microfluidic device.

Microfluidic devices are capable of separating microparticles and cells. We developed and tested the efficiency of silicon cross-flow microfilters for the separation of primitive fetal nucleated red blood cells (FNRBCs) and adult anucleate red blood cell (AARBCs) from model mixtures. Stepwise improvements over three generations of device design resulted in an increasing trend in the recovery of FNRBCs. We obtained a recovery of FNRBCs (74.0+/-6.3%, p<0.05, n=5) using the third generation device, with a depletion of 46.5+/-3.2% AARBCs from the cell mixture. The purity of FNRBCs in the enriched fraction was enhanced by a factor of 1.7-fold.

Noninvasive prenatal diagnosis of fetal aneuploidies and Mendelian disorders: new innovative strategies.

The application of recent technical developments, such as digital PCR or shot-gun sequencing, for the analysis of cell-free fetal DNA, have indicated that the long-sought goal of the noninvasive detection of Down syndrome may finally be attained. Although these methods are still cumbersome and not high throughput, they provide a paradigm shift in prenatal diagnosis, as they could effectively pronounce the end of invasive procedures, such as amniocentesis or chorionic villous sampling for the detection of such fetal anomalies. However, it remains to be determined how suitable these approaches are for the detection of more subtle fetal genetic alterations, such as those involved in hereditary Mendelian disorders (e.g., thalassemia and cystic fibrosis). New technical developments, such as microfluidics and reliable automated scanning microscopes, have indicated that it may be possible to efficiently retrieve and examine circulating fetal cells. As these contain the entire genomic complement of the fetus, future developments may include the noninvasive determination of the fetal karyotype.

The promise of fetal cells in maternal blood.

Delaying childbirth increases the proportion of advanced maternal age pregnancies. This increases the number of pregnancies requiring invasive prenatal testing. Prenatal diagnosis of chromosomal aneuploidies and monogenic disorders requires fetal cells obtained through invasive procedures (i.e. chorionic villus sampling and amniocentesis). These procedures carry a risk of fetal loss, which causes anxiety to at-risk couples. Intact fetal cells entering maternal circulation have raised the possibility of non-invasive prenatal diagnosis. Rarity of fetal cells, however, has made it challenging. Fetal nucleated red blood cells are ideal candidate target cells because they have limited lifespan, contain true representation of fetal genotype, contain specific fetal cell identifiers (embryonic and fetal globins), and allow interrogation with chromosomal fluorescence in-situ hybridisation and possibly with array comparative genomic hybridisation. The utility of fetal nucleated red blood cells in non-invasive prenatal diagnosis has not reached clinical application because of the inconsistencies in enrichment strategies and rarity of cells.

SERS-based quantitative detection of ovarian cancer prognostic factor haptoglobin

Surface-enhanced Raman spectroscopy (SERS) is increasingly being used for biosensing because of its high sensitivity and low detection limit, which are made possible by the unique Raman ‘fingerprint’ spectra from the biomolecules. Here we propose a novel SERS method for the fast, sensitive, and reliable quantitative analysis of haptoglobin (Hp), an acute phase plasma glycoprotein that is widely gaining application as a prognostic ovarian cancer biomarker. We exploited the peroxidase activity of the hemoglobin–haptoglobin (Hb–Hp) complex formed by the selective and specific binding of Hp to free Hb to catalyze the reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and hydrogen peroxide to result in the final product of strongly SERS-active TMB2+. We observed a linear increase in the SERS signal of TMB2+ with increasing concentrations of Hb–Hp complex from 50 nM to 34 μM. Based on this concentration-dependent SERS spectrum, we quantified Hp in clinical samples. We observed that our inference about the prognosis of the disease coincided with the histology data and that our method was much more sensitive than the enzyme-linked immunosorbent assay method.

Membrane proteins of human fetal primitive nucleated red blood cells.

In humans, primitive fetal nucleated red blood cells (FNRBCs) are thought to be as vital for embryonic life as their counterpart, adult red blood cells (adult RBCs) are in later-gestation fetuses and adults. Unlike adult RBCs, the identity and functions of FNRBC proteins are poorly understood owing to a scarcity of FNRBCs for proteomic investigations. The study aimed to investigate membrane proteins of this unique cell type. We present here, the first report on the membrane proteome of human primitive FNRBCs investigated by two-dimensional liquid chromatography coupled with mass-spectrometry (2D-LCMS/MS) and bioinformatics analysis. A total of 273 proteins were identified, of which 133 (48.7%) were membrane proteins. We compared our data with membrane proteins of adult RBCs to identify common, and unique, surface membrane proteins. Twelve plasma membrane proteins with transmembrane domains and eight proteins with transmembrane domains but without known sub-cellular location were identified as unique-to-FNRBCs. Except for the transferrin receptor, all other 19 unique-to-FNRBC membrane proteins have never been described in RBCs. Reverse-transcriptase PCR (RT-PCR) and immunocytochemistry validated the 2D-LCMS/MS data. Our findings provide potential surface antigens for separation of primitive FNRBCs from maternal blood for noninvasive prenatal diagnosis, and to understand the biology of these rare cells.

Detection of aneuploidy from single fetal nucleated red blood cells using whole genome sequencing

The objective of the study was to detect aneuploidy in single fetal nucleated red blood cells (FNRBCs) from placental villi using whole genome amplification (WGA) and next generation sequencing.

In vivo model to determine fetal‐cell enrichment efficiency of novel noninvasive prenatal diagnosis methods

To develop an in vivo model to determine fetal‐cell enrichment efficiency of novel noninvasive prenatal diagnosis methods.

Biology of human primitive erythroblasts for application in noninvasive prenatal diagnosis

Human primitive erythroblasts produced during early embryogenesis have been found in maternal circulation at early gestation and are considered good target cells for noninvasive prenatal diagnosis. We aimed to gain a better understanding of the biology of primitive erythroblasts and maximize their potential utility for noninvasive prenatal diagnosis.

Diagnostic accuracy of haptoglobin within ovarian cyst fluid as a potential point-of-care test for epithelial ovarian cancer: an observational study

To investigate haptoglobin within ovarian cyst fluid (OCF) as a diagnostic biomarker for epithelial ovarian cancer (EOC) and develop an in vitro diagnostic point‐of‐care device test (IVDPCT) for use in the operating theatre.

Abstract POSTER-CTRL-1211: Haptoglobin identified within ovarian cyst fluid as an accurate intraoperative diagnostic biomarker for epithelial ovarian cancer

Introduction: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy, with a poor prognosis and low survival rate; most cases are diagnosed at a late stage due to the fact that symptoms at early stages are usually non-specific in nature. There is currently no screening method proven to be effective in improving the outcome of EOC patients; existing biomarkers for EOC, such as CA-125, generally suffer from a lack of specificity in early stage disease, which is the ideal time for therapeutic intervention. Aim: We hypothesized that a single protein biomarker within the ovarian cyst fluid (OCF) could be identified, isolated, characterized and validated for application in a point-of-care device (POC), which could subsequently be used in operating theaters for triage for frozen section (FS). Methods: We screened the OCF proteome by mass-spectrometry (MALTI-TOF/MS), confirmed the identity of the protein by western blot and SELDI immunocapture analysis. Next, demonstrated using tissue microarray (TMA) that cellular expression of haptoglobin varied in normal, benign and malignant ovarian tissue. We developed a simple ELISA and a rapid colorimetric assay that allowed semi-quantification of OCF haptoglobin intraoperatively. Finally, we validated a point-of-care test kit to accurately identify EOC using OCF with a higher predictive value than can be achieved using RMIs, and an equivalent accuracy to intraoperative FS. Results: The OCF haptoglobin concentration in benign tumors was 0.70±0.09 mg/ml compared to 6.22±0.53 mg/ml and 6.57±0.65 mg/ml in early- and late-stage EOCs, respectively (P Conclusion: This is the first study whereby an intraoperative tumor marker has been utilised in the differentiation between benign and malignant ovarian lesions. Its accuracy suggests that it could be utilised as a replacement or an adjunct to frozen section, particularly in situations where histopathological expertise is scarce. Citation Format: Mahesh Choolani, Loganath Annamalai, Lin Liu, Khalil Razvi, Changqing Zhao, Gregory Rice, Aniza P Mahyuddin, Arijit Biswas, Jerry Chan, Narasimhan Kothandaraman. Haptoglobin identified within ovarian cyst fluid as an accurate intraoperative diagnostic biomarker for epithelial ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1211.