Purpose: To compare various meibum collection methods and extraction techniques.
Methods: Sixty subjects were seen on two visits. Meibum was collected from the lower lid of the right eye with a glass microcapillary tube, and with a Dacron swab, cytology microbrush or spatula from the left eye. Extraction with 2:1 chloroform:methanol was done either immediately or after data collection was complete. Individual samples were divided into four equal aliquots for analysis of total lipids, cholesterol and inorganic phosphates via assay based techniques. Effects of collection method, extraction, and dry eye status were examined using repeated measures ANOVA and logistic regression. Mass spectrometry was used to evaluate for identification of lipid peaks typically found in human meibum for three representative samples from each collection technique group. Samples from nine normal subjects were selected for comparison, three from each of the three groups, and evaluated by mass spectrometric analysis. Each sample was analyzed with direct infusion electrospray time-of-flight mass spectrometry (Waters,Quadrupole-TOF II). Spectra obtained were examined for presence of nine peaks previously identified as typically in human meibum.
Results: Total lipids showed an impact of collection device (p<0.0001), but not for
extraction technique (p=0.13) or dry eye status (p=0.97). Dacron swab collection was associated with more total lipid on average than each other collection device (p<0.0001). The cholesterol assay showed an impact of collection device (p<0.0001) and extraction technique (p=0.0002), but not dry eye status (p=0.55). Spatula collection was associated with more cholesterol on average than each other collection device (p<0.0001). For inorganic phosphates, immediate extraction (p<0.0001), cytology microbrush collection(p<0.0001), and non-dry eye status (p=0.03) were associated with the greater likelihood of detection.
Mass spectrometric analysis results varied by collection device. Wax esters (mass to charge ratio m/z 641.62, 655.64, and 669.65) were identified in samples collected by microcapillary tube (100%), cytology microbrush (78%), and spatula (100%). Cholesterol esters (m/z 759.68, 773.72, and 787.73) were identified in samples collected by microcapillary tube (100%), cytology microbrush (22%), and spatula (78%). Triacylglycerols (m/z 907.77) were detected in 100% of samples collected by microcapillary tube, cytology microbrush and spatula. Diesters (m/z 1031.97 and 1150.05) were detected in samples collected by microcapillary tube (89%), cytology microbrush (50%), and spatula (83%). None of the peaks were identified in samples collected by Dacron swab and significant plastic polymer was visible in the spectra, masking the signal from lipid peaks.
Conclusions: Dacron swab collection was associated with the highest average amount
of total lipid detected, whereas spatula collection and immediate extraction was
associated with the highest average amount of cholesterol detected. Lastly, cytology microbrush collection with immediate extraction on non-dry eye subjects was associated with the highest probability of detection of inorganic phosphates. Microcapillary tube collection was associated with most frequent detection of each lipid class, with spatula collection a close contender. Dynamic range of the mass spectrometer was likely exceeded with resulting signal suppression by predominance of polymers in Dacron swab-collected samples, and to a considerable yet lesser degree with the cytology microbrush.