Cryofibrinogen-Characteristics and Association with Cryoglobulin: A Retrospective Study Out of a Series of 1,712 Samples over 7 Years

Abstract

Objective: Cryofibrinogens (CFs) and cryoglobulins (CGs) are cryoproteins responsible for obstructive vasculopathy and vasculitis. The aim of this study was to compare the characteristics of CF and CG, and to define the conditions of their association.

Methods and results: This retrospective study was conducted at the Lyon University Hospitals, and included patients with at least one sample tested for CF and/or CG between September 2013 and April 2021. Serum and plasma samples were analyzed in very strict conditions of temperature. After cold precipitation, CF and CG were characterized and quantified in the cryoprecipitates. CRP and plasma fibrinogen levels were also investigated. Over this 7-year period, 1,712 samples for CF detection and 25,650 samples for CG detection were sent to the laboratory. Simultaneous testing of CF and CG was performed in 1,453/1,712 samples (85%). CF was less often positive than CG (8.3 vs. 13.5%, p < 0.0001). In positive CF samples, CG was associated in 28.9% of cases. In CF, fibrinogen was associated with fibronectin in 98/142 (69%) samples, especially in highly concentrated CF. CF concentration was independent of C-reactive protein and plasma fibrinogen concentrations.

Conclusion: The simultaneous detection of CF and CG is essential for the diagnosis of vasculitis or thromboembolic events and their treatment.

Fig. 1

Examples of appearance of cryofibrinogen (CF) and cryoglobulin (CG) precipitates in plasma and serum. ( A ) Positive CF in plasma (A1) and negative CG in serum (A2) of the same patient. In this case of positive CF, the precipitate formed a pellet at the bottom of the conical tube containing plasma (A1) and no precipitation in the serum (A2) when the samples are placed for 7 days at +4°C. ( B ) Example of aspects of CG precipitates in serum, such as a large pellet in B1, fine flakes deposited on the wall of the tube and forming a pellet in B2, thicker flakes on the wall or at the bottom of the tube (B3). Cryogel formation (B4) is more common with CG than with CF.

Fig. 2

Specific electrophoresis-immunofixation for the characterization of cryofibrinogen (CF) and cryoglobulin (CG) in cryoprecipitates. C: purity control (total protein antiserum); Fib: antifibrinogen antiserum; Fn: antifibronectin antiserum; G: anti-gamma chain antiserum; A: anti-α chain antiserum; M: anti-mu chain antiserum; K: anti-kappa chain antiserum; L: anti-lambda chain antiserum. Line ( A ): (1) detection of fibrinogen not associated with fibronectin (class 0); (2) detection of fibrinogen associated with fibronectin+ (class 1); (3) detection of fibrinogen associated with fibronectin + + (class 2). In this last case (A-3), the band observed in lane C corresponds to an interference linked to the high concentration of fibrinogen and fibronectin in this sample, certainly forming complexes, which become blocked in the meshes of the agarose gel. Line ( B ): (1) IgM kappa type I CG; (2) IgM kappa and polyclonal IgG type II CG; (3) polyclonal IgG and IgM type III CG. Line ( C ): presence of a type III CG (1: polyclonal IgM and IgG) associated with a CF (2). CG has precipitated in the plasma and is visible on the CF gel associated with fibrinogen (2). Line ( D ): presence of a type II CG (1: IgM lambda and polyclonal IgG), without a CF (2). CG has precipitated in the plasma and is visible on the CF gel in the absence of fibrinogen and fibronectin (2).

Fig. 3

Cryofibrinogen (CF) quantification method ( A ) and concentration according to the presence of fibronectin ( B ). (A) Radial immunodiffusion for fibrinogen antigenic quantification in cryoprecipitates. After 48 hours of cryoprecipitate diffusion in the gel, circles of precipitation ( white arrows ) appear and their diameters are measured to quantify fibrinogen according to an abacus. (B) CF concentration according to fibronectin semi-quantification (class 0–1–2) on electrophoresis-immunofixation (staining intensity). Class 0: fibronectin negative in the cryoprecipitate; class 1: fibronectin positive+ in the cryoprecipitate; class 2: fibronectin positive + + in the cryoprecipitate; ** p  = 0.002; ns = not significant.

Fig. 4

Cryofibrinogen and inflammation. ( A ) CF concentration in mg/L in function of inflammatory state, noninflammatory group with CR p  < 10 mg/L and inflammatory group with CRP ≥ 10 mg/L. ( B ) CF concentration in mg/L in function of inflammatory state, noninflammatory group with plasma fibrinogen <4.2 g/L and inflammatory group with plasma fibrinogen ≥4.2 g/L. ( C ) CRP concentration in patients with transient CF (positive CF and negative CF). ( D ) Plasma fibrinogen concentration in patients with transient CF (positive CF and negative CF). CF, cryofibrinogen; CRP, C-reactive protein; ns, not significant. * p  = 0.02.