An Evaluation of Nucleated Red Blood Cell Count by an Automated Haematology Analyser- A Tertiary Centre Experience.

nRBCs also referred to as normoblasts are seen in the peripheral blood films invariable numbers; both in physiological as well as pathologic states. Enumeration ofthese cells by modern day automated analysers remains a challenge. We wanted toassess the precision of the Beckman Coulter LH 755 & 780 haematology analysersTMin enumerating the nRBC count.METHODSThis is a retrospective study conducted in the Clinical Laboratory and HaematologyDivision of Kasturba Hospital, Manipal over a 3-month period on the BeckmanCoulter LH 755 & 780 haematology analysersTM (Beckman Coulter Inc., Miami, FL,USA) after obtaining requisite clearance from the Institutional Ethics Committee. Adata of 47,332 random blood samples run on the analysers was collected. Bothdescriptive and analytical statistics were performed using the SPSS softwareversion 22.0TM (Chicago, IL, USA). The sensitivity, specificity and kappa agreementwere calculated using the same.RESULTSA total of 797 cases from the 47,332 samples showed a “flag” for nRBCs. TwoTwenty of these cases were confirmed microscopically to have had nRBCs in theperipheral smear (true positives). 137 cases had nRBCs in the smear but were notdefinitively evaluated by the instrument (false negatives). A vast majority of thecases (577) did not reveal any nRBC on microscopic examination despite a flaggenerated by the machine (false positives). Additionally, a sensitivity of 27.6% andspecificity of 99.7% were also noted. The kappa agreement was 0.376 whichshowed a fair agreement between the two methods.CONCLUSIONSThe automated haematology analysers Beckman Coulter LH 755 & 780 were foundto be less sensitive in adequately enumerating the nucleated red blood cells. Thenumber of false positives can be reduced by noting the ‘cellular interference’ flag. Amanual review of such smears is necessary to confirm or refute such instrument

Prenatal diagnosis by isolation of fetal nucleated RBCs in maternal peripheral blood / 대한산부인과학회지

OBJECTIVE: To identify prenatal fetal sex and chromosomal aneuploidies by FISH using isolation of fetal nucleated RBCs. METHODS: peripheral blood samples was collected from 37 women between 11 and 24 weeks of gestation. we tried to enrich nucleated RBCs morphologically by Kleihaur-Betke staining after double gradient centrifugation and magnetic activating cell sorting (MACS) from maternal blood. Fluorescence in situ hybridization (FISH) analyses with CEP X and CEP Y probes for K-B positive nucleated RBCs were performed to detect whether fetal cells were existed among nucleated RBCs by observation of sex chromosomes. RESULTS: The average number of K-B positive nucleated RBCs separated from 10ml of maternal blood was 17.3 (+/-17.2) and the maximum number of nucleated RBCs was 54. We observed FISH signals in nucleated RBCs separated from 18 pregnant women, and Y probe signals were observed in 67.3% of nucleated RBCs separated from 10 pregnant women. CONCLUSION: We confirmed that separated nucleated fetal RBCs can be used to identify fetal sex and chromosomal aneuploidies by FISH. Since nucleated RBCs from maternal origin were not excluded, further studies are needed to overcome this limitation.

Prenatal Diagnosis of Fetal Trisomy 21 from Maternal Peripheral Blood

This study was undertaken to establish a noninvasive prenatal genetic diagnostic method for trisomy 21 using the fetal nRBCs that is rarely present in maternal circulation. Peripheral venous blood samples were collected from 30 women with an advanced maternal age, abnormal triple marker test results, or abnormal ultrasound findings such as an increased nuchal translucency. The blood samples were treated with heparin. The triple density gradient centrifugation, and MACS using CD45 and CD71 were used to isolate the fetal cells. FISH analysis using probe 21 was performed with GPA-immunostaining. The study population consisted of 30 patients from 13 to 25 weeks of gestation, and nRBCs were separated in all cases. In GPA-immuno FISH analysis using probe 21, 3 cases of trisomy 21 were diagnosed and these results were confirmed by the amniocentesis. In conclusion, a prenatal diagnosis of trisomy 21 through GPA- immuno fluorescence in situ hybridization (FISH) analysis using separated fetal nRBCs is a useful, innovative, accurate, rapid and non-invasive diagnostic method.

A New Prenatal Diagnosis of Fetal Cells Isolation from Maternal Peripheral Blood -Using Comparative Genomic Hybridization by Microdissection / 대한산부인과학회잡지

OBJECTIVE: The objective of this study was to determine the clinical use of CGH (comparative genomic hybridization) for detection of fetal aneuploidy from fetal cells (nucleated red blood cells, nRBCs) isolated from maternal peripheral blood. METHODS: Maternal peripheral venous blood sample was collected and treated by heparin. Triple density gradient centrifugation, and MACS (magnetic activated cell sorting) using CD45 and CD 71 were used to isolated the fetal nRBCs. With microdissection, DOP (degenerate oligonucleotide primed)-PCR (polymerase chain reaction), and nick translation, CGH was performed. RESULTS: Fetal nRBCs were successfully isolated from maternal peripheral blood. After microdissection of fetal nRBCs, DOP-PCR. and nick translation, DNA size was suitable for hybridization. In CGH analysis, we can confirm normal female and trisomy 21 male fetus. CONCLUSION: Prenatal diagnosis from fetal cells in maternal peripheral blood by comparative genomic hybridization shows clinical promise in terms of speed, accuracy, and non-invasiveness. To enable widespread use of this method, further studies involving many cases are warrented.

Isolation of Fetal Cells in Maternal Peripheral Blood and Genetic Analysis of Trisomy 21 by GPA-immuno FISH(Fluorescence in situ hybridization / 대한산부인과학회잡지

INTRODUCTION: Down's syndrome is the most common congenital chromosomal anomalies which occurs 1 out of 700-1000 births. Until now, for prenatal diagnosis of Trisomy 21, invasive techniques such as amniocentesis, chorionic villus sampling(CVS) and cordocentesis were used, but they encompass the rare possibility of morbidity to the mother and fetus. Triple marker using maternal serum is a currently used noninvasive method, but it only shows the accuracy of 60%. Accordingly, a noninvasive method, using fetal cells from maternal blood is under extensive investigation. This study was undertaken to establish a noninvasive prenatal genetic diagnostic method of trisomy 21 using fetal nRBCs rarely present in maternal circulation. MATERIALS AND METHODS: Peripheral venous blood samples were collected from 76 women and treated by heparin. For the isolation of fetal cells, we used a triple density gradient centrifugation, and Vario-MACS and Mini-MACS using CD45 and CD71, and then, the morphological differentiation of the fetal nRBC was performed by Kleihaur-Betke stain. With GPA immunostain, nRBCs were identified by cytoplasm and GPA attatchment, and after marking the site, a FISH was performed. RESULTS: This study population included 76 patients from 8 to 41 weeks of gestation, and nRBC was separated from all cases. The morphological differentiation was achieved by K-B stain. The mean number of nRBC collected from 20 ml of maternal peripheral blood was 15. The number of nRBCs retrieved reached its peak in 12-18 gestational weeks(18.9 6.0) which decreased from 20 gestational weeks and thereafter. Fetal sex was determined by FISH analysis using probe X, Y with GPA-immunostained cells. GPA-immuno FISH analysis using probe 21 in 30 cases of advanced maternal age or positive triple markers, we confirmed 3 cases of Down's syndrome. These results were also confirmed using the CVS or amniocentesis. CONCLUSIONS: Fetal nRBCs were separated from all cases after 8 gestational weeks. Prenatal diagnosis of trisomy 21 through GPA-immuno FISH analysis of chromosome 21 using separated fetal nRBCs is an useful, innovative, accurate, rapid and non-invasive diagnostic method. But for clinical use, more cases of experiments will be needed.