Impact of blood volume, tube shaking, and incubation time on reproducibility of QuantiFERON-TB gold in-tube assay.

Author(s): Gaur RL, Pai M, Banaei N

Publication: J Clin Microbiol, 2013, Vol. 51, Page 3521-6

PubMed ID: 23966505 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of blood volume, tube shaking, and duration of incubation on the sensitivity and specificity of the IFN-gamma ELISA determination of TB response. In addition, the effects of drawing specimens directly into assay tubes on specimen volume were evaluated, and TB skin test results were compared with ELISA results. Specimens were obtained from 33 uninfected individuals and 17 individuals infected with latent tuberculosis. Specimens were collected in 10 mL Monoject tubes, and blood was transferred to QFT-GIT or nil tubes (control), but a subset of 30 specimens were collected directly into the QFT-GIT and nil tubes. The control conditions for QFT-GIT testing included a blood volume of 1.0 mL, gentle inversion of tubes 10 times, and incubation at 37 degrees C for 24 h. After these procedures (or experimental variations), plasma was separated and held at room temperature for up to 8 h before assaying.

   Summary of Findings:

   The QFT-GIT and nil Vacutainer tubes were determined to have draw volumes of 0.785-1.0 mL and 0.810-1.010 mL, respectively. For specimens from uninfected volunteers, significantly higher TB responses were measured when 0.8 mL of blood was assayed than when 1.0 or 1.2 mL was assayed (p=0.03 and p=0.04, respectively), however, the percentage of positive results were not significantly different between blood volumes, although there was one false positive result at the 1.2 mL volume. For specimens from the 17 individuals with confirmed latent tuberculosis infection (positive skin test), significantly higher TB responses were also measured in 0.8 mL specimens than 1.0 or 1.2 mL specimens (p=0.002 and p<0.001, respectively), and the percentage of specimens with positive results was significantly lower for 1.2 mL specimens (10/17, 58.8%) than 0.8 mL specimens (15/17, 88.2%, p=0.05). Vigorous shaking of specimens led to significantly increased IFN-gamma levels in both nil tubes and QFT-GIT compared to gentle inversion (p<0.001 and p=0.004, respectively), but sensitivity and specificity of the TB responses (QFT-GIT levels - nil levels) were only significantly altered if the response was calculated using a QFT-GIT and a nil tube that were mixed in different ways. In general, incubation times at 37 degrees C of 16, 20, or 24 h did not affect TB responses or the sensitivity or specificity of the assay.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal
   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   Protein	ELISA
   Cell count/volume	Macroscopic observation

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Latent tuberculosis Normal
   Biospecimen Aliquots and Components	Aliquot size/volume	0.8 mL 1.0 mL 1.2 mL
   Biospecimen Aliquots and Components	Biospecimen mixing	Gentle inversion 10 times Vigorous shaking for 10 s
   Biospecimen Acquisition	Type of collection container/solution	Monoject tube QFT-GIT Vacutainer Nil Vacutainer
   Analyte Extraction and Purification	Incubation duration/condition	16 h 20 h 24 h
   ELISA Specific	Technology platform	Skin test
   ELISA Specific	Targeted peptide/protein	IFN-gamma

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