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Non-invasive fetal RHD genotyping for RhD negative women stratified into RHD gene deletion or variant groups: comparative accuracy using two blood collection tube types.

Author(s): Hyland CA, Millard GM, O'Brien H, Schoeman EM, Lopez GH, McGowan EC, Tremellen A, Puddephatt R, Gaerty K, Flower RL, Hyett JA, Gardener GJ

Publication: Pathology, 2017, Vol. , Page

PubMed ID: 29096879 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of processing delays and collection tube type on cfDNA concentration and genotype determination. Blood was collected into EDTA and Streck Cell-Free DNA BCT tubes from 665 pregnant women and was transported in insulated containers to a centralized processing location. Upon arrival, specimens were stored at 2-8˚C before plasma isolation by centrifugation at 3300 X the g for 10 min.  The time between specimen collection and plasma isolation ranged from 2.9-187.5 h (mean 61.7 h). DNA was isolated using DSP Virus/Pathogen Kit and Cell Free 1000 v6 protocol on the QIAsymphony SP module. RHD genotype was determined by automated real-time PCR using a Rotor-Gene Q. CCR5 was used to compare levels of background cfDNA and white blood cell DNA contamination. Maternal DNA was prepared from the buffy coat of EDTA blood and genotype was determined by microarray and, when not resolvable by sequencing, on a MiSeq.

   Summary of Findings:

   The mean concentration of CCR5 was higher in plasma from blood collected in EDTA tubes (mean 4.99 ng/µL) than BCT tubes (mean 0.12 ng/µL). Along with the higher values, specimens in EDTA tubes had a much larger range of values than those in BCT tubes (0.03–138 ng/µL versus 0.01-3.24 ng/µL). Further analysis revealed that the increased values occurred in specimens stored in EDTA tubes for more than 48 h. RHD exon 5 was detected in BCT tubes in a case genotyped after a processing delay of 24 h, 72 h, and 120 h; but was detected only in specimens stored in EDTA tubes for 24 h.

   Of the 647 specimens with maternal deleted RHD, two produced inconclusive results as only the fetal RHD exon was detected. Importantly, genotyping results were concordant between EDTA and BCT tubes for 644 of the 645 specimens with maternal deleted RHD and the remaining specimen was positive in all six signals using plasma from BCT tubes but only one of six signals in plasma from EDTA tubes. Potentially contributing to the discrepancy, this discordant specimen was subjected to a processing delay of 63 h and had higher CCR5 levels in the EDTA tubes compared to the BCT tube (0.05 versus 14.7 ng/µL) resulting in an overall positive predictive value (PPV) of 99.7% for both tube types and a negative predictive value (NPV) for specimens in BCT and EDTA tubes of 100% and 99.6%, respectively. The detection of fetal RHD*D-CE-D hybrids was possible in all six cases when collected in BCT tubes but only in three of six cases when collected in EDTA tubes. All three discordant cases had higher than expected maternal cfDNA levels and processing delays of 71-139 h. Amplification of fetal RHD did not occur in one RHD negative case or in the case of maternal RHCE*CE-D-CE hybrid, regardless of collection tube.  

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Streck/BCT
   • None (Fresh)

   Diagnoses:

   • Pregnant

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	BCT tube EDTA tube
   Storage	Storage duration	<24 h 24-48 h 48-72 h 72-96 h 96-120 h >120 h 24 h 72 h 120 h
   Biospecimen Preservation	Type of fixation/preservation	EDTA Blood collection tube additive

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