Page 209 - 2021_12-Haematologica-web

Letters to the Editor
Table 1. In vitro effects of plasma or sera from patients or healthy subjects on parameters of platelet function in whole blood or washed platelet suspensions from healthy subjects.
Plasma* or serum* donor subjects
Healthy subjects
(n. of subjects)
HS+Ig 3.66±1.5 17.75±4.2 2.0±0
Thrombus formation in microchannels Surface coverage (%)
Thrombus formation in microchannels Mean thrombus area (mm2)
PAT
Light transmission (%)
Flow cytometry Platelets/monocytes heteroaggregates (%)
11.30 ± 0.1 (n=6) ND
ND ND
22.06 ± 0.0 (n=2) ND
ND
ND
12.02 ± 0.1 (n=2)
86.68 ± 0.04 (n=2) ND
ND
Flow cytometry Annexin V binding (%)
0.70 ± 0.0 (n=6) ND
ND ND
0.90 ± 0.0 (n=2) ND
ND
ND
0.77 ± 0.0 (n=2)
16.01 ± 0.1 (n=2) ND
ND
5.35 ± 1.5 (n=18)
23.14 ± 5.3 (n=18)
1.86 ± 0.7 (n=7)
(n. of subjects) HS + aspirin (n. of subjects) HS + cangrelor (n. of subjects)
Patient 1 before IVIg
(n. of experiments)
Patient1beforeIVIg+Ig 2.97±0.77 18.00±6.1
(n=4) 5.20 ± 2.2 (n=4) 4.61 ± 1.36 (n=4)
(n=4) 16.75 ± 3.8 (n=4) 18.75 ± 2.2 (n=4)
(n=2) ND
ND
35 ± 13.8 (n=7) 1±0 (n=2) 1±0 (n=2) 1±0 (n=2) 1±0 (n=2)
0±0 (n=2) ND
ND
7.06 ± 3.7 (n=3)
24.67 ± 11.6 (n=3)
(n. of experiments)
Patient 1 before IVIg + aspirin (n. of experiments)
Patient 1 before IVIg + cangrelor (n. of experiments)
Patient 1 after IVIg
(n. of experiments)
Patient 2 before IVIg
(n. of experiments) Patient 2 before IVIg + Ig
(n. of experiments)
Patient 2 before IVIg + aspirin (n. of experiments)
(n=3) 7.25 ± 2.2 (n=3) 5.28 ± 2.4 (n=3) 5.40 ± 2.1 (n=3)
10.86 ± 2.1 (n=3) 5.45 ± 0.5 (n=3) 9.48 ± 1.2 (n=3) 6.86 ± 2.7 (n=3)
(n=3) 19.67 ± 4.7 (n=3) 18.67 ± 6.5 (n=3) 25.33 ± 11.7 (n=3)
34.33 ± 8.3 (n=3) 18.67 ± 1.5 (n=3) 23.00 ± 1.7 (n=3) 21.33 ± 6.8 (n=3) 25.50±6.1 (n=3)
19.0 ±1.4 (n=2)
Patient 2 before IVIg + cangrelor
(n. of experiments)
Patient2afterIVIg 7.17±1.0
ND
1±0 8.86±0.0 0.55±0.0
(n. of experiments)
Patient 3
(n. of experiments)
(n=3)
4.63 ± 0.4
(n=2)
(n=2)
ND
ND (n=2)
ND (n=2)
6.29 ± 0.0 (n=2)
0.51 ± 0.0 (n=2)
*Citrate plasma samples were used in experiments of thrombus formation in microchannels and of platelet activation test (PAT),while serum samples were used in flow cytometry experiments.Blood withdrawal for after-IVIg experiments was performed on day 15 for patient 1 and on day 13 for patient 2.Aspirin= 100 μmol/L;cangrelor= 1 μmol/L; Ig= 5 mg/mL. HS: healthy subjects; Ig:immunoglobulin; IVIg: intravenous immunoglobulin; pt: patient.
nal CT scan showed thrombosis of the portal, superior mesenteric and splenic veins, not associated with liver cirrhosis, occult malignancy or JAK2 V617F. Both patients had normal platelet counts before vaccination.
Experiments for the confirmation of TTS diagnosis, the evaluation of platelet activation in such patients and its modulation by IVIg and anti-platelets were performed as follows. Anti-PF4/polyanions antibodies were measured by an enzyme-linked immunosorbent assay (ELISA, PF4 Enhanced Test, Immucor), which contains immunoglob- ulin G (IgG), IgA and IgM antibodies and is more sensi- tive than non-ELISA rapid immunoassays.9 The platelet activation test (PAT) was measured (i) by light transmis- sion aggregometry (LTA) using normal washed platelet suspensions (WPS) prepared by the method described by Mustard et al.10 in the Platelet Aggregation Profiler-8E (Bio/Data, Milan, Italy), and (ii) by whole blood imped- ance aggregometry (HIMEA)11 using normal whole blood
(WB) in a Multiplate ECC (F. Hoffmann-La Roche). Platelets in WPS and WB were normally reactive to phys- iological agonists; patients’ sera were tested in parallel in the same experimental sessions. For flow cytometry experiments, normal citrate-anticoagulated WB was incu- bated with anti-CD14-PE or annexin V-PE and anti- CD42b-FITC at room temperature (RT) for 20 minutes. Subsequently, samples for platelet-monocyte hetero- aggregates were fixed, and red cells lysed. A total of 2,000 events of CD14+ or 10,000 events of CD42b for annexin V were acquired at medium flow rate by FACS Verse Cytometer (BD Biosciences, San Jose, CA, USA). In some experiments, patients’ sera were incubated with normal WB at RT for 20 minutes before staining. Experiments of in vitro thrombus formation were per- formed as previously described,12 perfusing normal WB anticoagulated with lepirudin (450 ATU/mL) (Refludan, Pharmion) on collagen-coated (100 mg/mL) microchan-
haematologica | 2021; 106(12)
3229