Clinical Potential of Effective Noninvasive Exclusion of <b><i>KEL1</i></b>-Positive Fetuses in <b><i>KEL1</i></b>-Negative Pregnant Women

Abstract

Background: The clinical importance of assessing the fetal KEL genotype is to exclude ‘K'-positive fetuses (genotype KEL1/KEL2) in ‘K'-alloimmunized pregnant women (genotype KEL2/KEL2). Noninvasive assessment of the fetal KEL genotype is not yet available in the Czech Republic. Objective: The aim of this study was to assess the fetal KEL1/KEL2 genotype from cell-free fetal DNA in the plasma of KEL2/KEL2 pregnant women. Methods: The fetal genotype was assessed by minisequencing (a dilution series including control samples). A total of 138 pregnant women (between the 8th and 23rd gestational week) were tested by minisequencing. The fetal genotype was further verified by analysis of a buccal swab from the newborn. Results: Minisequencing proved to be a reliable method. In 2.2% (3/138) of the examined women, plasma sample testing failed; 94.8% (128/135) had the KEL2/KEL2 genotype, and a total of 3.1% of fetuses (4/128) had the KEL1/KEL2 genotype. Sensitivity and specificity reached 100% (p < 0.0001). Conclusion: Minisequencing is a reliable method for the assessment of the fetal KEL1 allele from the plasma of KEL2/KEL2 pregnant women.

Risk Minimization of Hemolytic Disease of the Fetus and Newborn Using Droplet Digital PCR Method for Accurate Fetal Genotype Assessment of RHD, KEL, and RHCE from Cell-Free Fetal DNA of Maternal Plasma

“…RHD tests using real-time PCR and RHCE and KEL using minisequencing have already been introduced at our workplace [ 26 , 27 , 28 ]. One of the aims of the study was to methodically unify both procedures and, in the case of RHCE and KEL , to simplify and speed up the examination.…”

“…To avoid the real-time PCR limitation, the minisequencing methodology was optimized and validated. The minisequencing method is able to determine fetus specific SNP if the amount of cffDNA fraction is as low as 1% [ 27 ]. Compared to ddPCR, the minisequencing methodology is more laborious.…”

“…Comparison of minisequencing with ddPCR methodology: Noninvasive genotyping of SNPs using real-time PCR may be limited due to the non-specific fluorescent background [27]. To avoid the real-time PCR limitation, the minisequencing methodology was optimized and validated.…”

“…All of the subjects enrolled in the study signed an informed consent form approved by the Ethics Committee of the University Hospital Olomouc (approval code: 150/10). The samples that had already been tested by validated TaqMan real-time PCR and minisequencing methods [ 26 , 27 , 28 ] were used to validate and assess the ddPCR methodology. The correlation between fetal genotype and phenotype was further verified in the newborn using an immunoassay.…”

An innovative test for non‐invasive Kell genotyping on circulating fetal DNA by means of the allelic discrimination of K1 and K2 antigens

“…Otherwise, sequencing methods have higher detection limit (around 20%), increasing the possibility of false‐negative results. Moreover, the larger amount of maternal DNA in plasma sample compared to fetal DNA concentration could make it difficult to discriminate the fetal peak from the background in sequencing ferogram …”