Serum samples were collected from October 2022 to May 2023 from patients with known or newly diagnosed cryoglobulinemic vasculitis, other vasculitides, connective tissue diseases, and from patients without a diagnosis of rheumatic disease who were sent for routine CG testing and analyzed at the Immunology Laboratory of the Department of Rheumatology, University Medical Centre Ljubljana, Slovenia. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the National Medical Ethics Committee, Ljubljana, Slovenia (0120–133/2022/4).

Samples were collected in pre-warmed 4 mL tubes without gel or anticoagulant at 37 °C, transported in a pre-warmed thermostable transport box, and kept in the incubator at 37 °C for a two-hour clotting period. Blood samples were centrifuged three times for 10 min at 37 °C (1800 g). Sera were transferred to conical-bottomed test tubes (3 tubes of 600 μL each) and stored at 4 °C.

After 5–8 days of incubation at 4 °C, the cryoprecipitate was separated by cold centrifugation (30 min, 1800 g) and washed three times in 2 mL cold PBS. After the last wash, samples were treated with 600 μL PBS and placed in an incubator at 37 °C for up to one hour to dissolve the precipitate.

The presence of proteins from the dissolved cryoprecipitate was confirmed using Folin-Ciocalteu (FC) reagent. In the first step, 3 mL of reagent D (2%Na2CO3 in 0.1 M NaOH, 1% CuSO4 × 5 H2O, 2% K, Na tartrate) was added to 200 μL of the sample and incubated for 10 min. Then 600 μL of the reagent FC was added and after 30 min of incubation, the absorbance was measured at 720 nm. A concentration greater than 100 mg/L was considered a positive result, while clinically significant concentrations were defined as concentrations greater than 300 mg/L [28]. At concentrations greater than 100 mg/L, CG isotypes (IgG, IgM, IgA) were determined from stored sera by radial immunodiffusion according to the manufacturer`s instructions (Siemens Healthcare).

According to Kolopp-Sarda et al. [27], we introduced the new protocol for detection and characterization of CG – with sampling, visual observation, isolation of CG, classification of CG, quantification of CG and in the last step additional analyses of the activity of RF in cryoprecipitate and serum.

Samples were collected in pre-warmed 4 mL-tubes without gel or anticoagulant at 37 °C, transported in pre-warmed thermostable transport box and kept in the incubator at 37 °C for a two-hour clotting period. After the two-hour clotting time at 37 °C, blood samples were centrifuged at the same temperature (2200 g). The sera were transferred to conical-bottomed test tubes and stored at 4 °C for seven days (Fig. 1-1).

Graphical representation of sampling, visual observation and isolation of CG. Legend: CG – cryoglobulins, PBS – phosphate buffered saline. The figure was created with BioRender

Visual analysis of serum was performed after incubation at 4 °C for 7 days (Fig. 1-2). Samples with visible precipitate were further analyzed, and others were considered negative.

The volume of serum supernatant was carefully measured to calculate the final concentration of CG. Cold centrifugation (15 min, 2200 g, 4 °C) was performed to isolate CG. The cryoprecipitate was then purified in 2-mL tubes by washing three times in 1 mL of cold PBS. After each wash, samples were centrifuged at 4500 g for 15 min. After the final wash, 500 μL of PBS with Fluidil® (2%, Sebia, Lisses, France) was added to the cryoprecipitate and placed in an incubator at 37 °C for one hour to dissolve the precipitate (Fig. 1-3).

Immunofixation of dissolved isolated CG was performed according to the manufacturer`s instructions (Hydragel 4IF, Sebia, Lisses, France) to determine the type of CG, isotypes (IgG, IgA, or IgM; kappa or lambda), monoclonality or polyclonality (Fig. 2). Samples were first applied to an agarose gel and then CG were separated by electrophoresis. The sample is simultaneously electrophoresed in six lanes to find and identify the CG. Then antisera were applied to the gel and the process of immunofixation began. One lane is used as a reference to show the overall electrophoretic pattern of the isolated proteins in the sample after electrophoresis. The remaining five lanes allow identification of the isotype of CG - gamma (Ig G), alpha (Ig A) and mu (Ig M) heavy chains and kappa and lambda light chains. When the appropriate immunoglobulins are present, the antisera diffuse into the gel and precipitate them. Blotting and washing of the gel removes the soluble, non-precipitated immunoglobulins. The gel matrix contains the precipitin of the antigen-antibody combination and staining with acid blue was used to identify the precipitated CG.

Examples of immunofixation gels used to determine cryoglobulin type (A-C: Type II CG, polyclonal IgG and monoclonal IgM kappa, D-F: Type III CG, polyclonal IgG and IgM (D) and polyclonal IgG and IgA (E-F))

After identification of immunoglobulin isotypes by immunofixation, the concentration of isolated immunoglobulins was determined with the Atellica NEPH 630 nephelometer (Siemens, Heatlhineers, Erlangen, Germany) using reagents for low Ig concentrations. The final concentration of CG is adjusted to the initial serum volume and expressed in mg/L. A threshold CG concentration of 30 mg/L was defined as a clinically important value [29], whereas concentrations between 20 and 30 mg/L were defined as a grey zone.

For positive samples, RF activity (not isotype specific) was measured in cryoprecipitate using the Atellica NEPH 630 nephelometer and, if positive, also in serum at 37 °C.

For immunofixation, we determined analytical sensitivity, between-run precision, and within-run precision for immunoglobulin detection in CG. Serial dilutions of samples were prepared and analyzed on the gel. The last visible dilution was defined as the analytical sensitivity after calculating the concentration from the concentration measured with nephelometer. For between-run precision, a commercial control was used and tested on three different gel lots on different days, whereas for within-run precision, a patient sample was used and tested 4 times on one gel.

To determine the precision of IgG, IgM, and IgA concentration by immunonephelometry, a commercial control was used and within- and between-run precision was calculated. Control samples were tested 5 times per day for 5 days to obtain 25 results for each sample.

RF and the concentrations of complement fractions C3c and C4 were measured in the serum of all patients using an Atellica NEPH 630 nephelometer (Siemens Heatlhineers, Erlangen, Germany).

The overall agreement between the results of the two protocols was calculated. Spearman’s rank correlation coefficient was used to analyze quantitative correlation and Cohen’s kappa test was used to determine qualitative agreement between the results. The Mann-Whitney U test and Kruskal-Wallis test were used to compare groups because of the nonnormal distribution of the data. P values of less than 0.05 were considered significant. Data are presented as median (25th -75th percentile). Statistical analyses were performed using Analyse-IT for Microsoft Excel (Analyse-IT Software Ltd., Leeds, UK).

The CHAID decision tree was calculated to find predictors (SPSS IBM). Specifically, the overall accuracy of the CHAID decision tree model for predicting cryoglobulinemia and the percentage of patients correctly classified as negative or positive were calculated. Using these results, the index of Gains for nodes was calculated as the ratio of the response percentage for the target category compared to the response percentage for the entire sample. An index greater than 100% indicated significant prediction accuracy.