Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparison of Methodologies to Detect Low Levels of Hemolysis in Serum for Accurate Assessment of Serum microRNAs.

Author(s): Shah JS, Soon PS, Marsh DJ

Publication: PLoS One, 2016, Vol. 11, Page e0153200

PubMed ID: 27054342 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare four different methods of detecting hemolysis and to determine if levels of hemolysis-sensitive microRNAs (miRNA, miR) differ between serum spiked with different dilutions of hemolysate. Hemolysate was prepared by sonication of a clotted blood specimen collected from a healthy woman. A hemolysis dilution series was prepared by dilution of the hemolysate in serum to achieve 100%, 20%, 4%, 1%, 0.25%, 0.062%, 0.016%, 0.004% and 0.001% hemolyzed serum.  RNA was extracted using the miRCURY RNA Isolation kit. Hemolysis was assessed using the Coulter1AcT diff Analyzer and the Tainer Reagent Kit, visually, by absorbance at 414 nm using a spectrophotometer, and by real-time PCR amplification of miR-451a and miR-23a-3p. Additional blood was collected from 56 women with high grade serous ovarian cancer and 30 age-matched healthy women. Blood collection was via 21-gauge needle or from the peripherally inserted central catheter (PICC line). Blood was allowed to clot for 15-30 min at 4°C before separation of serum by centrifugation at 3000 rpm for 15 min at 4°C.  

   Summary of Findings:

   The sensitivity of hemolysis detection assays was measured by serial dilution using isolated hemolysate and was determined to be method dependent, with the lowest levels of hemolysis (0.001%) detectable only by real-time PCR. Conversely, spectrophotometry could detect ≥0.004% hemolysate, visual inspection detected hemolysis at ≥ 0.025% hemolysate, while the Coulter1AcT diff Analyzer detected hemolysis at ≥1% hemolysate. Using the miRNA ratio of real-time PCR amplicons (miR-451a to miR-23a-3p), 16%, 48% and 36% of the specimens were classified as having low (ratio of miR-451a to miR-23a-3p <5), moderate (5< ratio < 7), or severe (ratio > 7) hemolysis.  Visual inspection proved to be accurate only when hemolysis was severe, as specimens with the lowest visually detectable level of hemolysis had an elevated absorbance value at 414 nm and a miR-451 to miR-23a-3p ratio of 7.67 (by real-time PCR), both of which indicated hemolysis was severe. The absorbance at 414 nm was comparable in specimens classified as having low and moderate hemolysis based on real-time PCR ratio but the absorbance was 1.855-fold higher in specimens that were severely hemolyzed (P<0.001, both). Nevertheless, specimens with low hemolysis based on real-time PCR results (ratio of miR-451a to miR-23a-3p <5) were detected by spectrophotometry with a sensitivity of 25% but a specificity of 96.6% when an absorbance threshold of 0.072 at 414 nm was applied. Levels of miR-16-5p and miR-15b-3p were 5.9-fold and 4.5-fold higher, respectively, in specimens with severe hemolysis than those with low hemolysis (P<0.0001) based on the real-time PCR ratio (miR-451a to miR-23a-3p) and approximately 2-fold higher in specimens with moderate hemolysis than low hemolysis.

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Spectrophotometry
   RNA	Real-time qRT-PCR
   Protein	Macroscopic observation
   Protein	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-451a miR-23a-3p miR-16-5p miR-15-3p
   Real-time qRT-PCR Specific	Technology platform	Spectrophotometer Coulter1AcT diff Analyzer and the Tainer Reagent Kit Real-time PCR of miR-451a to miR-23a-3p Visual

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