CI: Blue Dye Use (2006)

Study Design:
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Quality Rating:
Research Purpose:

To determine the extent to which a mixture of human gastric juice and enteral formula stained with two concentrations of FD&C Blue No. 1 food dye (0.8 and 1.5ml dye per L formula) was visible in suctioned tracheobronchial secretions following three forced small-volume pulmonary aspirations over a six-hour period in an animal (rabbit) model.

Inclusion Criteria:
  • 90 New Zealand white rabbits, 3kg each
  • 90 acutely ill humans furnished gastric juice:
    • Fasting for at least four hours
    • No medications by tube or mouth within the preceding hour
    • Did not previously receive dye stained formula.


Exclusion Criteria:

Not described.

Description of Study Protocol:


  • 90 New Zealand Rabbits were purchased
  • 90 acutely ill subjects were used (recruitment not described).


Diagnostic test:

  • On the day preceding each experiment, human gastric juice was collected from each adult, fasting at least four hours, with no medication or feeding via tube for one hour. No subjects had received dye-stained formula
  • Gastric juice was refrigerated and on the next morning mixed half and half with one of eight enteral formulas stained with one of two dye concentrations. Dye used was 2.5% FD&C Blue No. 1 (Novartis Nutrition) to provide 100mg tint per 4ml. Formulas were Glucerna, Jevity, Osmolite, Pediasure, Pulmocare, TwoCal HN, Isocal and Traumacal.
  • Animals were intubated, ventilated and received gastric juice plus formula intratracheally in three separate boluses. Each bolus was infused over a 30-minute period. The infusion was then stopped and 90 minutes elapsed before suctioning.
  • By the end of the six-hour experiment, each animal received a volume of juice plus dye-stained formula equivalent to 1.2ml per kg body weight
  • Tracheobronchial secretions (half from each concentration of dye) were obtained by suctioning each animal three times. Research assistants evaluated the secretions for presence or absence of blood and dye and independently recorded observations.

Statistical Analysis

  • Comparisons of two dye concentrations groups on dependent variables of dye visibility and presence or absence of blood: Mann-Whitney test
  • Changes in proportions over time: Cochran Q and McNemar (non-parametric tests)
  • Changes in volume of two dye concentrations over time: 2x3 repeated-measures analysis of variance
  • Relationships between dye visibility and volume: Series of two sample T-tests, using 0.05 level.
Data Collection Summary:

Timing of Measurements

  • Tracheobronchial secretions (half from each concentration of dye) were obtained by suctioning each animal three times (two hours, four hours and six hours after start of the test)
  • Research assistants evaluated the secretions for presence or absence of blood and dye and independently recorded observations.

Dependent Variables

  • Dye visibility
  • Presence or absence of blood.

Independent Variables

Dye concentration.

Description of Actual Data Sample:
  • Initial N: 90 acutely ill subjects; 90 New Zealand Rabbits
  • Anthropometics: Rabbits were 3kg each
  • Location: St. Louis, Missouri.
Summary of Results:


  • Dye visibility changed significantly over time with both concentrations; least visible at six hours (P< 0.05)
  • When concentrations were examined separately, significant decrease in dye visibility for each concentration was not until six hours (P<0.05)
  • Volumes of suctioned secretions were significantly increased at four and six vs. two hours
  • Volume of suctioned tracheobronchial secretions at four hours when dye was not visible was significantly (P<0.01) greater than volume of secretions when dye was visible; differences at two and six hours was not statistically significant
  • Blood was a significant factor on the ability to visualize the dye.

Not Significant

  • Dye concentration effect on dye visibility
  • Volumes of secretion at four and six hours
  • Volume of secretions at two and six hours when dye was not visible.
Author Conclusion:
  • Findings from this animal model study do not support the use of the dye method to detect repeated small-volume aspirations. Dye method was only 46.3% overall sensitive in detecting multiple forced aspirations.
  • When only non-bloody secretions were considered sensitivity was 78.8%.
  • Drop in dye visibility from first to third aspiration events casts serious doubt on ability of this approach to detect repeated aspirations.
  • For clinicians who choose to use the dye method in selected situations, dye concentration of 0.8ml per L may be as effective in detecting aspiration as a 1.5ml per L concentration.
Funding Source:
Government: National Institute of Nursing Research
University/Hospital: St. Louis University, Oregon Health Sciences University, Ohio State University, Washington University School of Medicine
Reviewer Comments:
  • Author notes that extent of extrapolation to humans is unknown
  • Percentage of bloody secretions was much higher than typically observed in humans
  • Observers were not blinded to dye concentrations
  • Dye visibility in human secretions may be even lower than in this study since rabbit goblet cells secrete less mucous than human cells
  • Variety of formulas may have been confounding variable.