Key words long-COVID - oxygen binding - COHb
Introduction
COVID-19, caused by the SARS-CoV-2 virus, is primarily a respiratory disease but can
also affect other organ systems, such as the hematological compartment. Several
studies reported an increase in white blood cell count, a decrease in red blood cell
(RBCs) count and hemoglobin level, an increase in ferritin and D-dimer levels and
other clotting markers [1 ]
[2 ]
[3 ]
[4 ]. Symptoms of COVID-19 vary
from mild to severe and may include fever, cough, shortness of breath, fatigue,
diffuse body pain, loss of taste or smell, and sore throat [2 ]
[3 ]. In
approximately 6–35% of all COVID-19 patients, these symptoms can
persist for months after the initial infection with SARS-CoV-2 [5 ]. This symptomatic persistence has been
termed Long-COVID or Post-COVID and can occur even in individuals who have had mild
or asymptomatic infections [3 ]. Symptomatic
constellations comprise chronic fatigue, shortness of breath, chest pain, and
cognitive dysfunction (“brain fog”) [6 ]. There is evidence that sex may have an impact on the development of
Long-COVID, as some studies report women at higher risk [7 ]
[8 ].
The pathophysiological mechanisms of Long-COVID are still under discussion and
include the effects of viral persistence, inflammation, excessive blood clotting,
and autoimmunity [9 ]. It was suggested that
the hematological system is altered in Long-COVID as lower hemoglobin levels,
increased D-dimers, anemia, thrombocytopenia, and lymphopenia have been reported in
Long-COVID patients [10 ]
[11 ]
[12 ].
In addition, changes in RBCs size and morphology have been demonstrated in
Long-COVID, which may contribute to impaired oxygen diffusion [13 ].
Previously, we and others identified RBC precursors as a direct target of SARS-CoV-2
and suggested that SARS-CoV-2 induces dysregulation in hemoglobin- and
iron-metabolism contributing to the severe systemic course of COVID-19 [14 ]
[15 ]
[16 ]. Therefore, in this study,
we put particular emphasis on the analysis of hematological parameters and blood gas
analysis of Long-COVID patients compared to healthy donors.
Material and Methods
Sample acquisition
Blood samples from healthy donors were provided by the German Red Cross Blood
Donation Service North-East, Institute for Transfusion Medicine Dresden and by
the Division of Pneumology, Medical Department I (University Hospital Carl
Gustav Carus, Dresden, Germany). Blood samples from Long-COVID patients were
provided by the Division of Pneumology, Medical Department I (University
Hospital Carl Gustav Carus, Dresden, Germany). The diagnosis of Long-COVID was
made according to the German national guideline for Long-COVID and included
documentation of previous SARS-CoV-2 infection, standardized symptom and
functional assessment and standardized exclusion of other causes [17 ]. Matching of Long-COVID patients and
healthy donors was not performed.
Blood collection and sampling
Three ml whole blood from healthy donors and from Long-COVID patients were
collected in S-Monovettes EDTA K3 (Sarstedt, Nümbrecht, Germany) by
venipuncture using a sterile disposable Safety-Multifly-Needle 21 G
(Sarstedt). Determination of RBCs, hemoglobin content and hematocrit were
performed on a Sysmex XN 1000 (Sysmex Deutschland GmbH, Norderstedt, Germany).
Measurement of pH value and blood gas analyzing were carried out with an ABL800
Flex (Radiometer Medical ApS, Brønshø, Denmark).
Plasma was collected by centrifugation of the blood at 2000×g for
10 minutes. Measurement of iron metabolism parameters was performed in
the Institute of Clinical Chemistry and Laboratory Medicine (Dresden,
Germany).
Statistical analysis
All graphic results are presented as mean±SEM. Graph Pad Prism v.6
(GraphPad Software, San Diego, USA) was used for statistical analysis and figure
preparation. All datasets were tested for normality. Clinical parameters were
analyzed using chi-square test. Two-sided p-values of less than 0.05 were
considered statistically significant.
Ethics approval
Blood samples from all donors and Long-COVID patients were used in anonymized
form and in accordance with the guidelines approved by the Ethics Committee of
the Technical University of Dresden [BO-EK-49012022]. Informed consent was
obtained from all donors and patients.
Results
Demographic data and clinical symptoms
Patient’s demographics are presented in [Table 1 ]. The mean age of Long-COVID patients was significantly lower
compared to healthy donors (46.1 vs. 35.3 years). However, there were no
significant differences in terms of gender distribution, with twice as many
women as men in both groups. Smoking status was also not statistically
significant different between the groups. The cohort of Long-COVID patients was
selected based on their clinical symptoms, including chronic fatigue syndrome,
cough, shortness of breath and cognitive dysfunction ([Table 2 ]). Furthermore, most of the
patients had only a mild course of COVID-19 disease and were treated in an
outpatient setting. Only 2 patients required inpatient treatment.
Table 1 Patient‘s demographics.
Healthy donors
Long-COVID patients
p-Value
Number of samples
24
40
Age, years
Mean (SD)
35.3±11.5
46.1±13.2
0.0018 **
Range
18–59
17–69
Sex
0.630
Female
17 (70.8%)
26 (65.0%)
Male
7 (29.2%)
14 (35.0%)
Number of smokers
0.320
Never
6 (25%)
12 (30.0%)
Non-smoker
10 (41.7%)
12 (30.0%)
Former
2 (8.3%)
7 (17.5%)
Current
4 (16.7%)
(0.45/22/38/36PY)
2 (5.0%) (5/32PY)
Unknown
2 (8.3%)
7 (17.5%)
Data are presented as mean and SD.
** p<0.01.
Table 2 Reported clinical symptoms of
Long-COVID.
Symptom
Number of Long-COVID patients
Chronic fatigue
28 (70.0%)
Shortness of breath
31 (77.5%)
Cough
25 (62.5%)
Cognitive dysfunction
7 (17.5%)
Hospitalization
2 (5.0%)
Total number of patients with reported symptoms
40
Lymphocyte count and plasma iron levels are significantly reduced in
Long-COVID
First, we analyzed the heme and iron metabolism in blood samples from Long-COVID
patients and healthy donors. We found no significant differences in hemoglobin
content, the amounts of RBCs and the hematocrit values in the blood samples of
Long-COVID patients compared to healthy donors ([Table 3 ]). However, the mean corpuscular
hemoglobin (MCH) as well as the mean corpuscular hemoglobin concentration (MCHC)
were significantly higher in Long-COVID patients compared to healthy donors
([Fig. 1a, b ]). In contrast, the
peripheral lymphocyte count was significantly decreased in Long-COVID, albeit
still within the physiological range. Regarding the iron metabolism, plasma iron
levels were marginally reduced, whereas transferrin saturation was significantly
lower in samples from Long-COVID patients compared to healthy donors.
Fig. 1 Blood gas analysis of arterial blood from Long-COVID
patients and healthy donors: (a ) MCH, (b ) MCHC, (c )
O2 Hb, (d ) COHb, (e ) pO2 ,
(f ) pCO2 , (g ) pH value, (h )
bicarbonate value, and (i ) base excess value. Grey doted boxes
indicate the physiological range. Data are presented as boxes with Min
and Max including single values. Tests were performed two-sided.
Mann–Whitney U-test was used for statistical analyses.
*p<0.05, **p<0.01.
Table 3 Hematological and iron metabolism parameters in
Long-COVID-19 patients compared to healthy donors.
Healthy donors
Long-COVID patients
p-Value
Blood parameters
n=24
n=40
Hemoglobin [mmol/l]
8.44±0.16
8.70±0.18
0.647
Red blood cells [1012 /l]
4.74±0.08
4.70±0.08
0.631
Hematocrit
0.412±0.01
0.416±0.01
0.723
MCV [fl]
87.2±1.47
88.2±0.80
0.948
Lymphocytes
[10
9
/l]
2.25±0.12
1.88±0.09
0.007
**
Thrombocytes [109 /l]
257.4±9.15
264.8±9.81
0.938
Monocytes [109 /l]
0.53±0.03
0.55±0.04
0.876
White blood cells [109 /l]
6.57±0.26
7.02±0.35
0.944
Iron metabolism
n=21
n=31
Plasma ferritin [µg/l]
84.8±12.4
135.4±24.1
0.236
Plasma iron [μmol/l]
11.94±1.01
9.22±0.72
0.05
Plasma transferrin [g/l]
2.40±0.08
2.35±0.07
0.72
Transferrin saturation [%]
20.2±1.78
15.6±1.34
0.04
*
Data are presented as mean±standard error of the mean (SEM).
Tests were performed two-sided. Mann–Whitney U-test was used for
statistical analyses. The bold entries represent significant differences
between the sample groups. * p<0.05.
** p<0.01.
Oxygen binding to hemoglobin is impaired in Long-COVID patients
Next, we performed clinical blood gas analysis to identify alterations in oxygen
binding to hemoglobin and the acid-base balance in Long-COVID patients compared
to healthy donors. The percentage of oxygen bound hemoglobin was significantly
lower in Long-COVID patients ([Fig. 1c ]).
Interestingly, this was accompanied by a concomitant increase in carbon monoxide
binding ([Fig. 1d ]). No differences were
observed regarding pO2 ([Fig.
1e ]), but the pCO2 was significantly lower in the arterial
blood of Long-COVID patients compared to healthy donors ([Fig. 1f ]). Despite the fact that blood pH
was within physiological limits in both groups ([Fig. 1g ]), the values for base excess and
bicarbonate were significantly lower in Long-COVID patients compared to healthy
donors ([Fig. 1h, i ]).
Discussion
Long-COVID is associated with a plethora of symptoms and can impact nearly all organ
systems, including the hematological compartment. Here, we show that Long-COVID
patients suffer from impaired oxygen binding to hemoglobin with concomitant increase
in carbon monoxide (CO) binding. Elevated carboxyhemoglobin (COHb) levels are common
in sepsis, hemolysis, and severe inflammatory conditions, and can cause profound
hypoxia and, ultimately, lead to neurocognitive deficits and myocardial depression
(reviewed in [18 ]). Furthermore, it was
proposed that the cell’s redox state and metabolic demands are regulated by
the hemoglobin oxygen saturation and deoxyhemoglobin binding to the cytosolic
N-terminus of band 3 (AE1) [19 ]
[20 ]. AE1 is known to be the most abundant
membrane protein in mature RBCs, with a role in chloride shift
(bicarbonate/chloride homeostasis) and as a docking site for several
structural proteins contributing to membrane integrity [20 ]
[21 ].
Elevated levels of COHb may affect the erythrocyte integrity by down-regulation of
oxygen saturation. This is in line with observations from Kubankova et al., who
reported changes in deformability, and heterogeneity of erythrocyte deformation and
size in COVID-19 patients that persisted for months after hospital discharge [22 ]. At low oxygen saturation, deoxyhemoglobin
outcompetes the glycolytic enzymes to bind to the AE1 N-terminus, thereby favoring
glycolysis and the generation of adenosine triphosphate (ATP) and
2,3-diphosphoglycerate (DPG) to promote further oxygen release and tissue
oxygenation, thus relieving hypoxia. Long-COVID patients regularly suffer from
shortness of breath, which in turn leads to a compensatory increase in respiratory
rate, which then could cause respiratory alkalosis. This is in part compensated by
kidney-dependent removal of circulating bicarbonate [23 ], which could explain lower bicarbonate levels and base excess in the
arterial blood of Long-COVID patients compared to healthy donors. This is further
supported by the observed lower pCO2 in Long-COVID patients, albeit pH
remained unchanged compared to healthy donors. Although, arterial plasma
[H+ ] changes have no effect on the O2 flow from
the lungs to peripheral tissues, they are important indicators of the actual degree
of a systemic acid-base disturbance [24 ].
Since MCH and MCHC were also elevated in Long-COVID patients, we suggest a potential
compensatory mechanism. Elevation of these hematological parameters were also
reported by other researchers focusing on this subject [25 ]. Higher MCHC can have multiple causes,
including autoimmune hemolytic anemia (AIHA), which is common during (chronic) viral
infections [26 ]
[27 ]. In case of AIHA, a rare autoimmune
disorder, the immune system creates autoantibodies to destroy red blood cells before
they can be replaced, leading to anemia and hemoglobin being present outside of the
red blood cells, which in turn results in higher MCHC values [28 ]. Thus, the moderate plasma iron deficiency
and transferrin saturation found in this study could indicate a certain degree of
hemolysis. Under hemolytic conditions, referring to an uncontrolled RBC breakdown,
iron is released and lost via the urine and thus transferrin saturation can be low
due to the resulting iron deficiency [29 ]. In
order to refine these hypotheses, it is important to measure DPG levels in
Long-COVID patients in future studies.
The lymphocyte count was significantly lower in Long-COVID patients compared to
healthy donors. Lymphocytes are crucial in regulating cellular immunity. Especially
in patients with severe COVID-19 disease, reduced numbers of all lymphocyte types
(T-, B- and natural killer cells) were measured [14 ]
[30 ], which has been shown to be
a direct effect of the coronavirus disease [31 ]. The decreased lymphocyte count in Long-COVID patients may be
explained by immune exhaustion, which is a phenomenon that is frequently associated
with chronic viral infections [32 ]. In both
lung-resident and circulating T cells from COVID-19 patients an escalation in the
expression of exhaustion markers, including PD-1, CTLA-4, TIGIT and Tim-3, was
observed [33 ]
[34 ]. There is evidence that SARS-CoV-2, similar to other chronic viral
infections, appears to severely impair the functional subsets of CD4+and
CD8+T cells [32 ]
[35 ]. Thus, weakening of the immune system could
favor the spread of SARS-CoV-2, which in turn could have yet unknown long-term
implications and could be a driver of Long-COVID. Moreover, the reactivation of
latent pathogens, including herpesviruses and others, may contribute to Long-COVID
[36 ]. Nevertheless, further investigation
is required to clarify the clinical relevance of reduced lymphocyte counts in
Long-COVID patients.
Potential limitations
There are certain limitations to this study, which have to be addressed in future
research on this topic. As healthy donors and Long-COVID patients were not
matched with regard to age, sex, comorbidities and co-medication a putative
confounding effect cannot be ruled out. In addition, no correlation with regard
to type and severity of symptomatic and functional impairment in Long-COVID
patients was performed. Furthermore, larger cohorts with complete information on
medical history will be necessary to validate our findings and to shed light on
disease-driving mechanisms.
Conclusions
Our study demonstrates for the first time, that Long-COVID patients show impaired
oxygen binding to hemoglobin potentially caused by elevated COHb concentration in
the arterial blood. Although the underlying pathomechanisms still need to be
determined in clinical as well as experimental studies, a potential immunodeficiency
and impaired erythrocyte function leading to disturbances of the acid-base balance,
could be plausible causes of Long-COVID. In conclusion, our study identified novel
facets of Long-COVID pathophysiology, which in turn could open up innovative
therapeutic avenues in the future.
Author contributions
R.K. designed, performed and analyzed experiments, interpreted data and wrote the
manuscript. K. T. recruited patients, recorded the medical history and interpreted
clinical data. M.C., M.T., and J.T. helped with performance of experiments. D.K.
helped with interpretation of data and contributed to manuscript writing. T.T.
initiated the study, acquired funding, contributed to data interpretation and edited
the manuscript. S.K. analyzed and interpreted data, oversaw statistical analysis and
wrote the manuscript.
