Flow Cytometry Underestimates and Planimetry Overestimates Alveolar Epithelial Type 2 Cell Expansion after Lung Injury

Jansing, Nicole L.; Patel, Neesirg M.; McClendon, Jazalle; Redente, Elizabeth F.; Henson, Peter M.; Tuder, Rubin M.; Hyde, Dallas M.; Nyengaard, Jens Randel; Zemans, Rachel L.

doi:10.1164/rccm.201709-1838le

Cited by 18 publications

(28 citation statements)

References 14 publications

(29 reference statements)

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“…Similarly, in Sftpc ‐YFP mice, 23% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (2.1 million). Only one other study has reported the total number of AEC2 in mouse lungs using flow cytometry, where a number of 2.6 million AEC2 was estimated; however, the mouse age and strain background was not reported in that study (Jansing et al ., ). This number is in agreement with data that we report here for 7‐week‐old Sftpc ‐YFP mice on a mixed background (2.1 million AEC2).…”

Section: Discussionmentioning

confidence: 97%

“…The observations of that study are entirely in agreement with the data we present here, and documented that flow cytometry underestimated absolute AEC2 numbers in the lung, and it was suggested that pathological insult to the lung also impacted AEC2 recoverability from dissociated lung tissues. In sum, that study (Jansing et al ., ), together with the data we report here, highlight design‐based stereology as the model of choice for the estimation of the absolute number of a particular cell type in the lung.…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, stereology is appreciated as a means to quantify the parenchymal (Hyde et al ., ; Ochs et al ., , Knust et al ., ; Wulfsohn et al ., ; Herring et al ., ; Pozarska et al ., ; Hoang et al ., ) and vascular (Knust et al ., ; Willführ et al ., ; Mühlfeld et al ., ) structure of healthy immature, as well as adult lungs. To date, stereology has largely been applied to quantify elements of the lung architecture, and studies that employ stereology to quantify cells or cellular elements of the lung are few (Ochs et al ., , Jung et al ., ; Knudsen et al ., ; Bratu et al ., ; Knudsen et al ., ; Mühlfeld et al ., ; Jansing et al ., ).…”

Section: Discussionmentioning

confidence: 99%

“…During the conduct of this study, a manuscript emerged that addressed the comparison of absolute cell numbers in the lung by planimetry, design-based stereology, and flow cytometry, to study AEC2 expansion in response to lung injury (Jansing et al, 2018). The observations of that study are entirely in agreement with the data we present here, and documented that flow cytometry underestimated absolute AEC2 numbers in the lung, and it was suggested that pathological insult to the lung also impacted AEC2 recoverability from dissociated lung tissues.…”

Section: Discussionmentioning

confidence: 99%

“…Both of these approaches reflect a 'relative' cell number. However, in physiological situations where the tissue volume or cellular complexity rapidly changes, relative cell numbers might not be easily extrapolated to estimate absolute cell numbers (Jansing et al, 2018). In such cases, estimation of the absolute number of a particular cell-type may be more instructive.…”

Section: Introductionmentioning

confidence: 99%

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Estimation of absolute number of alveolar epithelial type 2 cells in mouse lungs: a comparison between stereology and flow cytometry

Dzhuraev

Rodríguez‐Castillo

Ruiz‐Camp

et al. 2019

Journal of Microscopy

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Summary Accurate estimation of the absolute number of a particular cell‐type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. The unbiased estimation of the absolute number of cells in an organ is complicated, and design‐based stereology remains the method of choice. This has led investigators to explore alternative approaches – such as flow cytometry – as a faster and less labour‐intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design‐based stereology was compared with microfluorosphere‐controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild‐type C57BL/6J mice and Sftpc‐YFP mice. Using design‐based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild‐type C57BL/6J mice and Sftpc‐YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild‐type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro‐surfactant protein C. Similarly, in Sftpc‐YFP mouse lungs, 23% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design‐based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. Lay Description There is much interest in studies on the pathological changes that accompany disease, to be able to count or estimate the number of a particular cell‐type in solid tissue, such as an organ. The easiest way to do this is to make liquid suspensions of single cells from solid tissue, and then to count the number of cells of interest, using either a microscope, or automated cell counting (for example, a flow cytometer). Alternatively, solid tissue may be examined microscopically, where the cell‐type of interest might also be counted ‘by eye’ or in an automated manner using software (called planimetry). All of these approaches to counting cells in solid organs come with serious drawbacks, and estimation of the cell number may thus be inaccurate. To overcome this, we have employed a combination of mathematical tools and statistical principles together with microscopy (called ‘design‐based stereology’) that permits the unbiased counting of cells in microscopic fields, which can then be extrapolated to the entire solid tissue volume, to accurately estimate the number of a cell‐type of interest in the solid tissue. We have compared this method with the estimation of cell number using a flow cytometer. Our data reveal that flow cytometry appreciably underestimates the total number of cells in solid tissue, where we used the lung as an example of solid...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Estimation of absolute number of alveolar epithelial type 2 cells in mouse lungs: a comparison between stereology and flow cytometry

Dzhuraev

Rodríguez‐Castillo

Ruiz‐Camp

et al. 2019

Journal of Microscopy

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Stereology as the 3D tool to quantitate lung architecture

Knudsen

Brandenberger

Ochs

2020

Histochem Cell Biol

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Stereology is the method of choice for the quantitative assessment of biological objects in microscopy. It takes into account the fact that, in traditional microscopy such as conventional light and transmission electron microscopy, although one has to rely on measurements on nearly two-dimensional sections from fixed and embedded tissue samples, the quantitative data obtained by these measurements should characterize the real three-dimensional properties of the biological objects and not just their “flatland” appearance on the sections. Thus, three-dimensionality is a built-in property of stereological sampling and measurement tools. Stereology is, therefore, perfectly suited to be combined with 3D imaging techniques which cover a wide range of complementary sample sizes and resolutions, e.g. micro-computed tomography, confocal microscopy and volume electron microscopy. Here, we review those stereological principles that are of particular relevance for 3D imaging and provide an overview of applications of 3D imaging-based stereology to the lung in health and disease. The symbiosis of stereology and 3D imaging thus provides the unique opportunity for unbiased and comprehensive quantitative characterization of the three-dimensional architecture of the lung from macro to nano scale.

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Fatal COVID-19 and Non–COVID-19 Acute Respiratory Distress Syndrome Is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State without Fibrosis

Ting

Aspal

Vaishampayan

et al. 2022

The American Journal of Pathology

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ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis.

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Flow Cytometry Underestimates and Planimetry Overestimates Alveolar Epithelial Type 2 Cell Expansion after Lung Injury

Cited by 18 publications

References 14 publications

Estimation of absolute number of alveolar epithelial type 2 cells in mouse lungs: a comparison between stereology and flow cytometry

Estimation of absolute number of alveolar epithelial type 2 cells in mouse lungs: a comparison between stereology and flow cytometry

Stereology as the 3D tool to quantitate lung architecture

Fatal COVID-19 and Non–COVID-19 Acute Respiratory Distress Syndrome Is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State without Fibrosis

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