A total of 47 patients with CSU (8 males and 39 females, median age 44 years, range 14-71 years), from outpatient unit of the Internal Medicine Service, Allergology and Clinical Immunology clinics of the Teaching Hospital “Duilio Casula”, A.O.U. of Cagliari, from December 2017 to November 2019, were enrolled in this study. A group of 53 HC (16 males and 37 females, median age 43 years, range 16-70 years) was also studied.
In a total of 23 out of 47 patients (4 males and 19 females, median age 43 years, range 19-70 years) we collected plasma samples on a scheduled planning:
• T0 before first Omalizumab infusion (300 mg subcutaneously, every 4 weeks)
• T1 after 4 weeks as second Omalizumab infusion
• T2 after 8 weeks as third Omalizumab infusion
• T3 after 24 weeks as sixth Omalizumab infusion
• T4 after 28 weeks as 4 weeks after the last Omalizumab infusion.
They fulfilled the inclusion criteria:
• Diagnosis of CSU lasting 6 weeks or more, based on “The EAACI/GA²LEN/EDF/WAO guidelines for the definition, classification, diagnosis and management of urticarial” [1].
• On-going therapy with anti H1 at the maximum dose
• UAS score > 35 at the first visit.
We did not include patients previously treated with Omalizumab or cyclosporin, younger than 16 years old or having known cardiovascular diseases or active cancer.
We collected for each patient demographic and clinical data. (Table 1)
Table 1
Demographic and clinical data in 47 CSU patients at T0 (before first Omalizumab infusion). Data are expressed as median and range.
Demographic and clinical characteristics at T0
|
Median, range
|
Years from diagnosis
|
6.00, 2.00-21.00
|
IgE total
|
172.00, 8.00-2131.00
|
Eosinophils (x103 /µL)
|
0.1, 0.1-0.6
|
Eosinophils (%)
|
2.00, 0.50-10.00
|
Basophils (x103 /µL)
|
0.00, 0.00-0.1
|
Basophils (%)
|
0.00, 0.00-1.00
|
UAS7 at first Omalizumab infusion
|
35.00, 18.00-42.00
|
We divided the patient group in three subgroups based on the response to Omalizumab treatment:
• Early responders: UAS7 < 14 at T1
• Late responders: UAS7 < 20 at T2
• Not responders: UAS7 unchanged at T2
The other group of controls were CIA patients: 11 patients (7 males and 4 females) with Chronic Spontaneous Urticaria on anti-H1 therapy.
Informed consent was obtained from all patients and controls. The Investigations were carried out following the rules of the Declaration of Helsinki of 1975 revised in 2013. Approval of this study was obtained by the local Ethical Committee (Prot. PG/2021/153).
Blood was collected in citrate vacuum tubes (VenoSafe, Terumo Europe) made of polyethylene terephthalate, containing 0.109 M sodium citrate. Citrated blood was centrifuged at 2020 x g for 20 minutes at room temperature and the separated plasma was placed in polypropylene tubes. Plasma samples for analysis of Activated Partial Thromboplastin (aPTT) ratio, CWA, D-Dimer, Fibrinogen, thrombin generation and F1+2 assays were stored at -80°C after being kept in liquid nitrogen and were used for each patients and controls after thawing at 37°C.
Activated Partial Thromboplastin (aPTT) ratio, CWA, D-Dimer and Fibrinogen were determined on an ACL TOP 550 CTS (Werfen, Barcelona, Spain) using the reagents SynthAsIL, D-Dimer HS, QFA HemosIL (Werfen, Barcelona, Spain) respectively. In particular, the method used for describing CWA was a turbidimetric method for clot detection. In this type of coagulometer, 0% absorbance defines the pre-coagulation phase then the absorbance (mAbs) increases after the initiation of clotting.
CWA investigation has been previously described in other studies of ours [9, 10]. Briefly, the first derivative was the time at which maximum velocity of clot formation was reached (expressed as mAbs/s). The second derivative was the time at which maximum change in acceleration of clot formation was reached (expressed as mAbs/s2). Delta was the total change in optical density, that is, the maximum density of the clot (expressed as mAbs). CWA was derived from an implemented software dedicated to the ACL TOP 550 CTS [11]. This topic has been the subject of a communication by the subcommittees of the International Society of Thrombosis and Haemostasis (ISTH) aimed at the standardization of this procedure and the recommendations for its clinical application [12]. An example of CWA is shown in Figure 1.
For F1+2 an enzyme immunoassay was used (Enzygnost F1+2, Siemens Healthcare S.r.l., Milano, Italy). It consists of an immuno-enzymatic method for the quantitative in vitro determination of the human F1+2 prothrombin fragment in plasma based on the sandwich principle with mouse monoclonal antibodies in microplate.
In 23 out of 47 patients with CSU Thrombin Generation (TG) was determined in Platelet Poor Plasma (PPP) using the Calibrated Automated Thrombogram (CAT) method (Diagnostica Stago, Asnières sur Seine France) [13]. Eighty µl of PPP were pipetted into the well of a microtiter plate together with 20 µl of PPP-Reagent +/- TM (with and without Thrombomodulin) (Thrombinoscope BV, Maasticht, The Netherlands), PPP-Reagent contains a mixture of Tissue Factor (5pM final concentration) and synthetic phospholipids (4 µM final concentration), PPP-Reagent with Thrombomodulin contains a mixture of Phospholipids, Tissue Factor and Thrombomodulin (in the range of 4–6 nM). The reaction was started with 20 µl of a mixture composed of the fluorogenic thrombin substrate (Z-GlyGly-Arg-AMC, Thrombinoscope BV, 417 µM final concentration) and CaCl2 (15 mM final concentrations). The substrate is cleaved by the thrombin formed and liberates a fluorophore, which is converted to thrombin-equivalent concentrations (nM) using a reference curve [12]. To obtain a thrombin generation (TG) curve from the conversion of added fluorogenic substrate, thrombin concentrations are to be derived from the observed velocity of increase of fluorescence (dF/dt). Fluorescence was read in a Fluoroscan Ascent® reader (Thermo Fisher Scientific Corporation, Vantaa, Finland) and TG curves were calculated using the Thrombinoscope Software. Endogenous thrombin potential (ETP, area under the curve, nM*min was expressed as with/without thrombomodulin ratio. This ratio represents the resistance to the anticoagulant activity of TM and should be considered as an index of the procoagulant imbalance. The higher the ratio, the greater the procoagulant activity. The ETP is the area under de curve (AUC) that represents all the enzymatic activity of thrombin when is activated. Therefore, it is the parameter that properly represents the coagulation phase. Finally, the platelet count and the Mean Platelet Volume were carried out using an automated cell counter (Yumizen HS2500, Horiba ABX SAS, Parc Euromédecine, Montpellier, France).