Leena Gole

Same-day prenatal diagnosis of common chromosomal aneuploidies using microfluidics-fluorescence in situ hybridization

Rapid molecular prenatal diagnostic methods, such as fluorescence in situ hybridization (FISH), quantitative fluorescence‐PCR, and multiplex ligation‐dependent probe amplification, can detect common fetal aneuploidies within 24 to 48 h. However, specific diagnosis or aneuploidy exclusion should be ideally available within the same day as fetal sampling to alleviate parental anxiety. Microfluidic technologies integrate different steps into a microchip, saving time and costs. We have developed a cost‐effective, same‐day prenatal diagnostic FISH assay using microfluidics. Amniotic fluids (1–4 mL from 40 pregnant women at 15–22 weeks of gestation) were fixed with Carnoys before loading into the microchannels of a microfluidic FISH‐integrated nanostructured device. The glass slides were coated with nanostructured titanium dioxide to facilitate cell adhesion. Pretreatment and hybridization were performed within the microchannels. Fifty nuclei were counted by two independent analysts, and all results were validated with their respective karyotypes. Of the 40 samples, we found three cases of fetal aneuploidies (trisomies 13, 18, and 21), whereas the remaining 37 cases were normal. Results were concordant with their karyotypes and ready to be released within 3 h of sample receipt. Microfluidic FISH, using 20‐fold less than the recommended amount of probe, is a cost‐effective method to diagnose common fetal aneuploidies within the same day of fetal sampling. Copyright

Analysis of cystic hygroma, ascitic, and pleural fluids by conventional lymphocyte culture and fluorescent in situ hybridization

Serous fluids from cystic hygromas, pleural effusions, and ascites are an easily accessible and plentiful source of lymphocytes. The feasibility and reliability of using these as alternative sources to conventional amniotic fluid or fetal blood cultures have been studied here. In some cases of prenatal diagnosis, especially in pregnancies complicated by the presence of cystic hygromas and fetal hydrops, obtaining amniotic fluid or fetal blood can be difficult due to obstruction by the cyst or oligohydramnios. A total of 14 cases with fetal hydrops detected ultrasonigraphically between 15 and 33 weeks of pregnancy over a period of 1 year have been subjected to conventional amniotic fluid or fetal blood karyotyping, along with samples of fluids from cystic hygromas, ascites or pleural effusions as obtained. Pleural fluids (n=4), cystic hygroma fluids (n=5), and ascitic fluids (n=6) were obtained. The culture failure rate was low, 2/14. Karyotypically, two of the fluids, both from cystic hygromas, were 45,X; the rest were normal. A rapid 1‐day additional test of fluorescent in situ hybridization (FISH) was carried out on uncultured cells of the alternative fluids using probes for the most commonly occurring aneuploidies, 13, 18, 21, X and Y, with good results.

Modified cIg-FISH protocol for multiple myeloma in routine cytogenetic laboratory practice

The International Myeloma Working Group recommends that fluorescence in situ hybridization (FISH) be performed on specifically identified plasma cells (PC). This is because chromosomal abnormalities are not frequently detected by traditional karyotyping due to the low proliferative rate of PC in multiple myeloma (MM). Conventional FISH enhances the sensitivity but lacks the specificity, as it does not distinguish PC from other hematopoetic cells. To fulfill this recommendation, PC need to be selected either by flow cytometry or immunomagnetic bead-based PC sorting or by concomitant labeling of the cytoplasmic immunoglobulin light chain, which allows for unambiguous identification. These techniques require expertise, time, and funding and are not easily incorporated into the routine workflow of the cytogenetic laboratory. We have modified and refined the technique using fixed cell pellets to achieve nicely separated and easily identifiable PC. With immunostaining and subsequent FISH (i.e., cytoplasmic immunoglobulin FISH, cIg-FISH), this technique can be easily incorporated into every cytogenetic laboratory. Twenty samples from patients with MM were subjected to routine FISH, cIg-FISH, and chromosomal karyotyping and the results were compared. Three FISH probes, which enabled detection of the t(4;14), t(14;16) and deletion of TP53, were used to validate this modified technique successfully.

Multiple copies of a rare rearrangement of Philadelphia chromosome in a chronic myeloid leukemia patient: a case report

Chronic myeloid leukemia (CML) is a stem cell neoplasm characterized by the uncontrolled proliferation of myeloid cells in the bone marrow. The observed incidence of CML in Singapore is markedly lower compared to the United States of America, at 0.7 compared to 1.48 per 100,000 patients [1]. The BCR-ABL fusion oncogene occurs in more than 95% of CML patients, and is seen as a Philadelphia (Ph) chromosome, where the ABL1 gene on 9q34 is juxtaposed to the BCR gene on chromosome 22q11.2. The resulting fusion oncogene is a tyrosine kinase, due to the originating nature of the ABL gene, which in turn results in the uncontrolled proliferation of the cells in which this mutation occurs [2]. Most of the isoderivatives (ider) of chromosome 22 or the isodicentric (idic) Ph chromosomes reported in the literature have been described to be a fusion occurring at the p arms, resulting in multiple copies of this fusion oncogene. We describe here a rare case of an isoderivative Philadelphia chromosome formed by a fusion in the q arm, in a 74-year-old Indian woman diagnosed with de novo CML in the hematologic chronic phase. She presented with loss of appetite, accompanying loss of weight, upper respiratory tract infection, and a history of diabetes mellitus. Upon admission, the patient’s total white cell count was 46.438 10/L, with neutrophils accounting for 82% and myelocytes at 2%. Peripheral blood film showed moderate anisopoikilocytosis, marked neutrophilia with moderate left shift, with occasional myelocytes, metamyelocytes, and promyelocytes noted. This was accompanied by a mild increase in basophils, and blast cells accounted for around 2% of cells seen. At the time of diagnosis, bone marrow investigation indicated a hypercellular marrow with markedly increased granulopoiesis and megakaryopoiesis. Erythropoiesis was quantitatively reduced. Myelocytes were increased in number, and mild basophilia was observed. Myeloblasts comprised 1% of total nucleated cells. Features were consistent with that of a CML in chronic phase. Flow cytometry of the bone marrow was also consistent with that of a myeloproliferative disorder. The patient passed away before the start of treatment. Cytogenetic investigation on GTL-banded chromosomes revealed Philadelphia chromosome rearrangements in all the cells analyzed. Of 20 cells analyzed, 16 cells had an

Prenatal Detection of Isochromosome 21 by QF-PCR

Objective: To compare rapid aneuploidy diagnostic tests with traditional karyotyping in the prenatal detection of Down syndrome due to isochromosome 21. Methods: Quantitative fluorescence PCR (QF-PCR) and fluorescent in situ hybridization (FISH) for chromosomes 13, 18, 21, X and Y were performed on uncultured amniotic fluid, followed by routine karyotyping. Chromosomal and microsatellite analysis of peripheral blood from parents was also carried out. Results: The QF-PCR screening showed a trisomic diallelic pattern for 5 of 6 markers spanning the long arm of chromosome 21. FISH showed 3 signals in the interphase cells for the region 21q22.13-q22 during LSI 21 probe mapping. Cultured amniotic fluid revealed an isochromosome 21 resulting in a 46,XX,i(21)(q10),+21 karyotype. Parental microsatellite analysis proved that the isochromosome was paternal in origin. Conclusion: The most informative analytical tool in this case appears to be QF-PCR, although a combination of QF-PCR and karyotyping provided the most evidence.

Characterization of breakpoints in the GABRG3 and TSPY genes in a family with a t(Y;15)(p11.2;q12)

We report the clinical, cytogenetic, and molecular findings in a family in which a t(Y;15)(p11.2;q12) is segregating. The Y chromosome breakpoint disrupts the DYZ5 sequence containing the TSPY genes that are exclusively expressed in the testes while the chromosome 15 breakpoint is within the GABRG3 gene. The father and his son who both carried the balanced form of the translocation are clinically normal. A daughter who carried the der Y had the clinical features of Prader–Willi syndrome while a son who carries the der 15 has mild developmental delay and hypogonadism. The relationship of the translocation to the clinical phenotypes is discussed.

Authors' Reply: The assessment of combined first trimester screening in women of advanced maternal age in an Asian cohort.

Dear Sir, We thank Lai and Yeo(1) for their comments regarding our study on the performance of first trimester screening (FTS) in women of advanced maternal age in a Southeast Asian population. We are delighted to hear that the performance of FTS at KK Women’s and Children’s Hospital is similar to that at National University Hospital and Singapore General Hospital, since it suggests that high standards are being maintained throughout Singapore. Our data was not selected from a high-risk population, but was derived from consecutive cases presenting for FTS at the two clinics. The prevalence rate for the trisomies within this unselected population is very similar to that seen in another study of over 10,000 all-risk Caucasian women carried out in Western Australia.(2) FTS is offered to all women in Singapore, but not taken up by all; intuitively, it seems logical that as women get older, they will be more likely to wish to screen for aneuploidy following counselling from their doctor. Therefore, the percentage of older women undergoing FTS is likely to be higher than the total percentage of women in that age group giving birth at any particular centre, causing a self-selection bias in the screening population. We stated in our study that the pregnancy outcome was known either by the karyotype data or birth outcome; we later went on to explain that the birth outcome data was a limitation of the study, since we are relying on a feedback loop from the delivering hospitals to notify us if there is a case of aneuploidy discovered at birth. We acknowledge that there may be some shortcomings in this system, and we will be keen to enlist the help of the National Birth Defects Registry as suggested for future studies. Yours sincerely,

Discrepancy between Cytogenetic and FISH Results on an Amniotic Fluid Sample of 45,X/46,X,idic(Y)(p11)

The presence of abnormal ultrasound markers showing a thick nuchal fold with short middle phalanx of the fifth finger in an otherwise normal-appearing female fetus led to the sampling of amniotic fluid at 16 weeks gestation. Cytogenetic analysis with routine G-banding showed a 45,X karyotype in all 20 cells analysed from two flasks. However, fluorescent in situ hybridization on uncultured cells showed presence of a Y signal in 9 cells, 11 cells showing a single signal for the X. A cytogenetic analysis of the fetal blood at 23 weeks confirmed the presence of two cell lines, 45,X and 46,X,idic(Y)(p11). The couple opted to have the pregnancy terminated. However, the fetus was not available to carry out confirmatory tests.