key: cord-316631-um0olqet
authors: Park, Kyung Chan; Donovan, Killian; McKechnie, Stuart; Ramamurthy, Narayan; Klenerman, Paul; Swietach, Pawel
title: Single‐cell oxygen saturation imaging shows that gas exchange by red blood cells is not impaired in COVID‐19 patients
date: 2020-08-01
journal: Br J Haematol
DOI: 10.1111/bjh.17025
sha: 
doc_id: 316631
cord_uid: um0olqet

SARS-CoV-2 coronavirus infection is characterised by a marked inflammatory state and viral pneumonitis. A striking clinical feature is severe hypoxaemia, often in the presence of near-normal lung mechanics.

SARS-CoV-2 coronavirus infection is characterised by a marked inflammatory state and viral pneumonitis. A striking clinical feature is severe hypoxaemia, often in the presence of near-normal lung mechanics. Several hypotheses have been put forward to explain these findings, 1,2 including pulmonary microvascular thrombosis, dysregulated hypoxic pulmonary vasoconstriction and dysfunctional gas transport by red blood cells (RBCs). Derangement in convective O 2 transport is an attractive hypothesis as this would explain why COVID-19 hypoxaemia is often refractory to supplemental oxygen. A controversial in silico prediction postulated that the virus attacks haemoglobin (Hb), 3 and despite subsequent criticism, 4 a number of hypotheses have emerged linking Hb with COVID-19, such as the association between thalassaemias or fetal Hb with disease severity. [5] [6] [7] Notwithstanding these opinions, studies in China have confirmed modestly lower Hb levels in severe COVID-19 8, 9 and greater heterogeneity in terms of RBC volume, quantified as RBC Distribution Width-Standard Deviation (RDW-SD). 10 In a recent letter to this Journal, Hb oxygen affinity was shown to be unaltered in a cohort of 14 patients infected with SARS-CoV-2. 11 However, steady-state measurements of affinity cannot predict the kinetics of gas exchange by RBCs, which may become rate-limiting in COVID-19 due to impaired perfusion of the injured lung and inflammationtriggered RBC deformations that expand intracellular diffusion path length. Moreover, measurements on whole blood report an ensemble population average, which cannot resolve the presence of small subpopulations of dysfunctional RBCs, if these emerge in COVID-19. Indeed, given that RDW-SD increases in COVID-19, O 2 handling must be interrogated with cellular resolution.

We recently designed single-cell oxygen saturation imaging to assess O 2 unloading kinetics and O 2 storage capacity on a cell-by-cell basis. 12 We now applied this technique to study blood from COVID-19 patients at the John Radcliffe Hospital, Oxford, UK. Ten SARS-CoV-2-positive patients [9/10 confirmed by polymerase chain reaction (PCR) result, remaining patient diagnosed clinically] were recruited to this study through the Oxford GI Biobank (ethics 16/YH/0247). In half of the patients, blood was sampled within the first two weeks of diagnosis, and for the other half, sampling was in the subsequent fortnight. Three patients were asymptomatic healthcare workers, identified by voluntary PCR testing, and the remaining seven presented with COVID-19 symptoms. None of the patients had a history of RBC disorders or haemoglobinopathy. For reference measurements, healthy donors were recruited. Venous blood samples were spun down (1200 RCF for 3 min at room temperature) to collect 10 µl of red cells, which were re-suspended in 4 ml of HEPES-buffered Tyrode solution containing 10 mmol/l ascorbate and then treated with UV-C light (4000 µW/cm 2 at a distance of 6 cm for 15 min, UVS-18 EL Series UV lamp, Analytik-Jena, Germany) to inactivate the virus. Ascorbate was included to protect Hb from oxidative damage by UV light. After resuspending in fresh Tyrode solution (containing no ascorbate), cells were loaded with CellTracker DeepRed and Green (Ther-moFisher Scientific, Waltham, MA, USA) to produce HbO 2sensitive fluorescence on a confocal imaging system. 12 Rapid solution switching between an oxygenated and deoxygenated microstream triggered O 2 exchange, which was imaged at high temporal resolution for each RBC individually (Fig 1A) . Repeating this several times yielded a measure of O 2 unloading kinetics (time constant s), normalised O 2 binding capacity (j) and cell radius in the horizontal plane. Data were analysed in terms of their frequency distribution (Fig 1B) .

RBC s, j and radius were no different in COVID-19 patients, compared to controls (Fig 1C-E) . There was also no evidence for a change in the distribution of these functional variables, arguing against the emergence of dysfunctional subpopulations over the course of infection. When expressed in terms of the fraction of cells that release >95% of stored oxygen in a given time (equivalent to capillary transit), there was no effect of SARS-CoV-2 infection on RBC O 2 handling (Fig 2) .

Our findings add new evidence that SARS-CoV-2 infection does not lead to dysfunctional convective transport. The cause of hypoxaemia in COVID-19 patients is therefore unlikely to relate to impaired O 2 handling by RBCs, either as a result of direct coronavirus infection or a consequence of the inflammatory state. Our findings argue against a mechanistic link between Hb variants and disease outcomes. 

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