Opinion
Lipid accumulation in alveolar macrophages appears to occur
primarily through the phagocytosis of endogenous or exogenous
lipids and lipoproteins. Endogenous lipid accumulation within
alveolar macrophages is usually associated with bronchial
obstruction, with the lipid material derived from lysed cell
membranes of cells distal to the obstructive lesion (cholesterol
pneumonia). Exogenous sources of lipid within alveolar macrophages
result from aspiration of gastric contents, gastroesophageal reflux
disease or inhalation of lipids through vaping.Other factors such
as obesity and elevated serum cholesterol may also influence lipid
laden macrophage formation and prevalence, but their role is not
yet established.
In cytology, the lipid content in macrophages is determined
by applying a lipid stain, Oil Red O (1-[2,5-dimethyl-4-(2-5-
dimethylphenyl) phenyldiazenyl] azonapthalen-2-ol)) to a
formalin fixed smear of cells obtained from bronchoalveolar lavage
(BAL) fluid. Semi quantitative methods for assessing lipid laden
macrophages include scoring cytology samples based on a visual
assessment as originally proposed by Corwin and Irwin in 1985[1]
and slightly modified by Colombo and Hallberg [2,3].Specifically,
one hundred macrophages are scored on a 0 to 4 scale for presence
of lipid vacuoles. Out of a maximum lipid laden macrophage index
(LLMI) of 400, an LLMI of <40 would not suggest aspiration, 40-
90 would indicate an indeterminate result and >90 would be
suggestive of aspiration[2].A higher cut off of >165 was proposed
by Furuya et al.[4].
Another simplified method of semi quantification of lipid
laden macrophage quantification was developed by Collins
et al. [3].using cells obtained from tracheal aspirates of children
up to 3 years of age.The Oil Red O stained cytology specimen is
assigned a grade of absent (grade 0), low positive (grade 1: 1-25
lipid-laden macrophages), moderate positive (grade 2: 26-50 lipidladen
macrophages), and high positive (grade 3: > 50 lipid-laden
macrophages).
Theoretically, the presence of exogenous lipid scavenged form
the distal airways and alveoli would indicate aspiration of fatcontaining
food or liquid, either from dysfunctional swallowing
or gastro esophageal reflux.However, studies reach conflicting
conclusions regarding the accuracy of LLMI in predicting aspiration
[5-7].Furthermore, there are striking differences in LLMI results
across different studies, such that the control groups of some
studies had higher indices than other studies’ aspiration groups
[8,9].Thus, LLMI, as currently assessed appears to have insufficient
sensitivity and specificity to be clinically useful.
The lack of consensus might be due to procedural related
factors as well as the quantification method.Progress is being made
in this regard with the publication of American Thoracic Society
guidelines for improving the quality and reproducibility of flow
cytometry samples obtained from the lung [10].Quantification
factors influencing the assessment of LLMI in cytology include
marked interobserver variance due to the subjective nature of visual quantification.Most critically, the proposed indices do not take into
account the proportion of lipid-laden macrophages relative to the
whole specimen.
There are several ways in which lipid laden macrophages can
be better quantified.Marzahl et al. [11].used a deep learning-based
method of annotated cytology from whole slide digital images of BAL
to achieve high concordance in quantification of hemosiderophages.
Another approach would be to use mass spectrometry whereby the
lipid content can be precisely characterized, and its source identified
as food based (implying gastric contents). The concentration of
lipids in the specimen would serve as a measure of severity of the
aspiration.
Flow cytometry of BAL in inflammatory lung disorders has
proven to be clinically useful precisely because of the objective
quantification of immune cell subsets within the specimen.We have
used flow cytometry to assess lipid-laden macrophages by staining
alveolar macrophages with fluorochrome labeled CD68.Our
preliminary study utilized wide side scatter to demonstrate lipidladen
macrophages that correspond to Grade 3 index by Collins et
al.[3]. Flow cytometry would eliminate the influence of confounding
factors of pigment (hemosiderin, anthrocotic) laden macrophages,
adequacy of the Oil Red O stain and obscuring inflammation.
A critical need exists for a more objective, standardized method
to quantify lipid-laden alveolar macrophages before its value can
be determined in the differential diagnosis of disorders such as
asthma, oropharyngeal reflux and vaping.The dramatic increase in
vaping and the putative impact it has on lipid laden macrophages
has further underscored this need.Of the various options, flow
cytometry appears to hold the most promise of delivering such a
method but further research needs to be done to validate its utility.
References
- Corwin RW, Irwin RS (1985) The lipid-laden alveolar macrophage as a marker of aspiration in parenchymal lung disease. Am Rev Respir Dis 132(3): 576-581.
- Colombo JL, Hallberg TK (1987) Recurrent aspiration in children: lipid-laden alveolar macrophage quantitation. Pediatr Pulmonol 3(2): 86-89.
- Collins KA, Geisinger KR, Wagner PH, Blackburn KS, Washburn LK (1995) The cytologic evaluation of lipid-laden alveolar macrophages as an indicator of aspiration pneumonia in young children. Arch Pathol Lab Med 119(3): 229-231.
- Furuya ME, Moreno Córdova V, Ramírez Figueroa JL, Vargas MH, Ramón-García G, et al. (2007) Cutoff value of lipid-laden alveolar macrophages for diagnosing aspiration in infants and children. Pediatr Pulmonol 42(5): 452-457.
- Chang AB, Cox NC, Purcell J, Marchant JM, Lewindon PJ, et al. (2005) Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis. Respir Res 6: 72.
- Rosen R, Fritz J, Nurko A, Simon D, Nurko S (2008) Lipid-laden macrophage index is not an indicator of gastroesophageal reflux-related respiratory disease in children. Pediatrics 121(4): e879-e884.
- Nussbaum E, Maggi JC, Mathis R, Galant SP (1987) Association of lipid-laden alveolar macrophages and gastroesophageal reflux in children. J Pediatr 110(2): 190-194.
- Reid Nicholson M, Kulkarni R, Adeagbo B, Looney S, Crosby J (2010) Interobserver and intraobserver variability in the calculation of the lipid-laden macrophage index: implications for its use in the evaluation of aspiration in children. Diagn Cytopathol 38(12): 861-865.
- Gibeon D, Zhu J, Sogbesan A, Banya W, Rossios C, et al. (2014) Lipid-laden bronchoalveolar macrophages in asthma and chronic cough. Respir Med 108(1): 71-77.
- Tighe RM, Redente EF, Yu YR, Herold S, Sperling AI, et al. (2019) Improving the Quality and Reproducibility of Flow Cytometry in the Lung. An Official American Thoracic Society Workshop Report. Am J Respir Cell Mol Biol 61(2): 150-161.
- Marzahl C, Aubreville M, Bertram CA, Stayt J, Jasensky AK, et al. (2020) Deep Learning-Based Quantification of Pulmonary Hemosiderophages in Cytology Slides. Sci Rep 10(1): 9795.