Page 89 - Haematologica May 2022

Complement dysregulation & COVID-19
Table 1. COVID-19 patient characteristics.
Age (years), median Sex
Female
Male Race
Black White Other
Ethnicity Hispanic Non-hispanic
BMI, median
Days from COVID-19 diagnosis
to serum sample collection, median Days from hospital admission
to serum sample collection, median Length of hospitalization (days), median
Oxygen (N=32)
55 (26-78)
18 14
11 11 10
23
9
33.6 (20.5-58.2) 4 (1-13)
2 (1-13) 9 (2-45)
Hi-Flow Oxygen (N=7)
58 (49-69)
3 4
4 2 1
6
1
34.2 (27.1-51.9) 8 (2-16)
6 (1-10) 11 (7-15)
Ventilation (N=17)
59 (27-80)
7 10
9 4 4
14
3
30.4 (16.2-46.3) 13 (3-34)
13 (2-32) 28 (3-70)
Death (N=2)
57 (56-57)
1 1
1 0 1
1
1 65.3 18
13 17
Figure 1. Complement-mediated cell killing induced by COVID-19 patient serum is associated with disease severity. TF1PIGAnull cells were treated with 20% COVID- 19 patient serum, and cell killing was measured using the modified Ham (mHam) test. Complement-mediated cell killing (%) was significantly elevated in COVID-19 patients requiring mechanical ventilation (vent), as compared to pooled normal human serum (NHS) and COVID-19 patients who needed minimal oxygen support (P<0.01). The dotted line at 20% non-viable cells represented the threshold for a positive mHam based on a previously established receiver operator curve. All exper- iments were run in triplicate. Sia: sialidase (used as a positive control); stx1: Shiga toxin subunit 1 (used as a positive control). Hi-Flow: High-Flow Oxygen.
test was positive (> 20% cell killing) in 41.2% (7 of 17) of patients who required intubation (WHO score 7), com- pared to 6.3% (2 of 32) of those who only needed minimal oxygen support (WHO score 4) (P=0.002) (Figure 1). Serum from COVID-19 patients who required mechanical venti- lation induced significantly higher cell killing compared to those on minimal oxygen support (mean 17.3% vs. 7.7%, P<0.01), suggesting that SARS-CoV-2 infection impairs the ability to regulate complement on cells.
COVID-19 patient serum increased C5b-9 and C3c deposition on cell surface
C3c and C5b-9 deposition, biomarkers for complement activation, on the surface of TF1PIGAnull cells after expo- sure to COVID-19 patient serum was evaluated. All com-
plement pathway buffer (GVB++) and alternative pathway specific buffer (GVB0 MgEGTA) were used. In all pathway buffer, C5b-9 deposition was increased compared to con- trol normal human serum in virtually all COVID-19 patients regardless of WHO score (Figure 2A). In APC spe- cific buffer, the deposition of C5b-9 was significantly ele- vated in patients requiring minimal oxygen support, intu- bation and those who died (Figure 2B) compared to con- trol serum. C3c deposition was significantly increased in all patient groups in APC specific buffer (Figure 2D) but not in all complement pathway buffer (Figure 2C).
We selected patient samples with positive mHam and increased C5b-9 deposition to perform blocking experi- ments. Representative examples of blocking with the complement inhibitors in two patient sera are shown in
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