The precipitation of cryoglobulins, serum immunoglobulins, below 37 °C defines the clinical cryoglobulinemic syndrome, a systemic vasculitis usually characterized by purpura, weakness, and arthralgia. In most cases, this condition is associated with chronic infection by the hepatitis C virus (HCV) and can evolve into B-cell dysregulation and malignancies. The current literature on non-HCV-associated cryoglobulinemia is very limited, and little is known about the immunological and serological profile of affected patients. The cryoglobulinemic syndrome not associated with HCV infection is often found concomitantly with other infections, autoimmune diseases, and B-cell lymphoproliferative disorders. The cryoprecipitation of fibrinogen has been described as a rare disorder, perhaps underestimated and not fully understood, causing thrombotic occlusion and ischemia in different rheumatic disorders. Cold temperature plays a pathogenetic role in autoimmune hemolytic anemias, in which the presence of cold agglutinins produced by B cells at the lymphoplasmacytic cell stage may promote agglutination of red blood cells in the coldest parts of the circulation, even at mild room temperatures, undergoing hemolysis. Laboratory methods for the detection and quantification of cryoproteins are downright critical, and their concurrent detection is pivotal for the diagnosis. In this review, we summarize the clinical involvement of cryoglobulins, cryofibrinogen, and cold agglutinins in non-HCV autoimmune diseases, underlining the crucial steps of the most employed analytic methods.

The aims of this study were to investigate the prevalence of cryofibrinogenemia in a cohort of patients with systemic sclerosis (SSc) regardless of clinical manifestations, who were admitted to our hospital and determine the associations among CF positivity, disease features and ongoing therapies. This was a monocentric and retrospective study. The inclusion criteria were a diagnosis of SSc (according to the ACR/EULAR 2013 classification criteria), regular administration of i.v. prostanoids, and CF testing between February 2020 and February 2022. Data on demographic, clinical, and immunological features and ongoing treatments were retrospectively collected. Categorical data were compared with the chi-square test or Fisher's exact test, while quantitative variables comparisons were carried out with Student's t test or Mann‒Whitney test. In total, 101 SSc patients were ultimately enrolled. The majority of patients were female (92.1%) and had the limited cutaneous form of SSc (81.2%). CF positivity was observed in 69.3% of the patients, whereas only 9% presented cryoglobulins and CF. CF positivity was negatively associated to RNAP3 antibodies (p = 0.027). No direct associations with specific clinical phenotypes were observed. No associations with immunosuppressive treatments were identified, however a positive association with nifedipine administration (p = 0.040), and a negative association with endothelin receptor antagonists (ERAs) plus phosphodiesterase-5 (PDE5) inhibitors regimen (p = 0.031) were observed. Macitentan administration was also associated to CF cryocrit ≥ 1% (p = 0.045). Among patients who were not treated with ERAs, an estimated pulmonary artery systolic pressure ≥ 30 mmHg was significantly associated with CF positivity (p = 0.025). Moreover, a cryocrit ≥3% was associated with a relative risk of 3.44 (95% CI 1.26-9.39, p = 0.016) for digital amputation and 5.17 (95% CI 1.18-22.6, p = 0.029) for death. Isolated CF is a frequent phenomenon observed in SSc patients and is associated with a higher administration of vasoactive drugs, probably identifying a SSc clinical phenotype with a more severe microvascular involvement. Moreover, a higher cryocrit is associated with an increased risk of death and digital amputations. Screening SSc patients for CF would represent an opportunity to provide better therapeutic approaches by anticipating ERA administration in an earlier phase, thereby preventing the manifestation of severe microvascular involvement. Key Points • Cryofibrinogen is a cryoprotein that can cause microangiopathic damage. • Isolated cryofibrinogenemia is common in patients with systemic sclerosis. • SSc patients should be tested for cryofibrinogen because a high cryocrit (≥ 3%) is associated with death and/or digital amputation due to necrosis. • Cryofibrinogen is associated with indirect markers of pulmonary arterial hypertension in patients not treated with endothelin receptor agonists (ERAs). • ERAs could play a role in preventing cryofibrinogen damage.

Cryoglobulinaemia vasculitis can present with a variety of symptoms and there is limited data on the incidence and presentation of cryoglobulinaemia vasculitis in haemodialysis patients. We report a case of a 63-year-old male who had a series of presentations with rash, visual changes, abdominal pain, weight loss, fevers and digital ischaemia. This is on a background of a congenital single kidney with end-stage renal failure secondary to diabetes and hypertension, receiving haemodialysis for nearly 5 years. He initially experienced a leukocytoclastic vasculitis rash confirmed on skin biopsy, followed by multiple hospital presentations for undifferentiated abdominal pain and fever of unknown source. Jejunal biopsy revealed intestinal vasculitis. His peripheral blood flow cytometry and bone marrow biopsy were consistent with marginal zone lymphoma (indolent subtype, IgM kappa clone). Further testing revealed a type II cryoglobulinaemia consisting of an IgM kappa monoclonal band with polyclonal IgG (cryocrit 5%). A diagnosis of cryoglobulinaemia vasculitis was established and he was treated with pulsed methylprednisolone and rituximab therapy. However, after receiving three doses of rituximab the patient developed a presumed vasculitis-associated pulmonary haemorrhage for which he received treatment with five sessions of plasma exchange. His symptoms resolved and cryocrit reduced to < 1% after his final dose of rituximab. The clinical features of cryoglobulinaemia may be difficult to detect in chronic haemodialysis patients and vigilance is required.

HCV infection could have extrahepatic manifestations due to an aberrant immune response. HCV/HIV co-infection increases such persistent immune activation. Aim of the present study is to describe the evolution of inflammatory markers used in clinical practice, mixed cryoglobulinemia (MC) and autoantibody reactivity in co-infected individuals who achieved sustained virological response (SVR) after DAA treatment. This prospective, observational study included all HIV/HCV co-infected subjects who started any DAA regimen from 2015 to 2020. Samples for laboratory measurements (ferritin, C reactive protein, C3 and C4 fractions, rheumatoid factor, MC, anti-thyroglobulin Ab, anti-thyroid peroxidase Ab, ANCA, ASMA, anti-LKM, anti-DNA, AMA, ANA, T CD4+ and CD8+ cell count, and CD4/CD8 ratio) were collected at baseline, after 4 weeks, at end of treatment, and at SVR12. The analysis included 129 individuals: 51.9% with a F0-F3 fibrosis and 48.1% with liver cirrhosis. Cryocrit, C3 fraction, and rheumatoid factor significantly improved at week 4; ferritin, anti-thyroglobulin Ab, and C4 fraction at EOT; total leukocytes count at SVR12. MC positivity decreased from 72.8% to 35.8% (p < .001). T CD4+ cell slightly increased at SVR12, but with an increase also in CD8+ resulting in stable CD4/CD8 ratio. Autoantibody reactivity did not change significantly. ANA rods and rings positivity increased from 14.8% to 28.6% (p = .099): they were observed in three subjects without exposure to RBV. DAA therapy may lead to improvement in inflammatory markers and MC clearance but without significant changes in autoantibodies reactivity and CD4/CD8 ratio over a follow up of 12 weeks.

Our case details a 47-year-old female who presented to our cancer hospital with a petechial rash of the lower extremities as well as a headache and blurred vision for the prior two days. She was found to have systolic pressures in the 200s in the emergency department and was admitted for a hypertensive emergency. Notable medical history includes marginal zone lymphoma (MZL) status post-submandibular resection at an outside institution in 11/2017 (thought to be in remission). With her history in mind, she also reported subjective submandibular swelling on admission and an unintentional 25-pound weight loss over the eight weeks prior to admission. A PET scan was completed, which showed diffusely increased reticuloendothelial activity, and a follow-up bone marrow biopsy was without residual lymphoma activity. Creatinine was markedly elevated with significant proteinuria, and a renal biopsy revealed thrombotic microangiopathy, acute tubular injury, and moderate interstitial fibrosis. Remarkable laboratory tests included positive quantitative cryoglobulins ("cryocrit") and low complement 4 (C4). Qualitative cryoglobulins were never obtained, unfortunately. She was started on prednisone and transferred to a nearby academic hospital for formal rheumatologic evaluation. Importantly, testing at this facility showed elevated Sjögren's syndrome-related antigen A (SSA/Ro) antibodies. Also elicited at the academic hospital was that she had been experiencing symptoms of chronic dry eyes and mouth years even before her diagnosis of MZL. She was diagnosed with primary Sjögren's syndrome, which was thought to be the cause of her likely mixed cryoglobulinemia and the precipitant of her acute renal failure with hypertensive emergency, her skin changes, her anemia, and her hypocomplementemia. Of note, prior to discharge from the academic hospital, the patient's cryoglobulin testing was negative after prolonged steroid treatment, and she was placed on rituximab for maintenance. Our case is important as it helps illustrate one of the myriad precipitants of mixed cryoglobulinemia, in this case possibly untreated Sjögren's syndrome.

A considerable number of patients with high clinical suspicion for cryoglobulinaemic vasculitis either show negative results for the detection of cryoglobulins or show only trace amounts which cannot be characterized for composition. We aimed at establishing whether the failure to detect or the detection of trace amounts of cryoglobulin with conventional methods either identifies a peculiar subset of low level cryoglobulinaemia (from now on hypocryoglobulinaemia) or represents a separate entity. Using a modified precipitation technique in hypo-ionic medium, we prospectively identified between 2008 and 2021 237 patients (median age 60.8 years [22-97], 137 females) having < 0.5% cryocrit and clinical suspicion of autoimmune disorder. Of these 237 patients, only 54 (22.7%) had a history of HCV infection. One hundred and sixty-nine out of 237 patients (71%) had an established underlying disease, while 68 patients (28.6%) (median age 62.9 years [29-93], 35 females) did not show either laboratory markers or clinical symptoms consonant with an underlying aetiology. These 68 cases with only trace amounts of cryoglobulins were defined as having a putatively idiopathic hypocryoglobulinaemia. Nineteen of these 68 patients (27.9%) had a history of HCV infection. Twenty-four patients out of 68 (35.3%) were positive for rheumatoid factor (RF), while 25 (36.7%) patients had signs of complement consumption (i.e., C4 < 15 mg/dl and/or C3 < 80 mg/dl), and 36 (52.9%) had increased inflammatory indexes. Seven patients only had arthralgia and constitutional symptoms while 61 out of 68 (89.7%) presented with at least one of the three cardinal signs of cryoglobulinaemic vasculitis including skin lesions, peripheral nerve involvement, and glomerulonephritis. Seventy-five percent of the subjects had type III hypocryoglobulins. In patients with hypocryoglobulinaemia the histologic features of glomerulonephritis (also examined by electron microscopy) resembled those of mixed cryoglobulinaemia-associated glomerulonephritis. In conclusion, hypocryoglobulins are often polyclonal and are mainly unrelated to HCV infection. Patients who present high clinical suspicion for vasculitis, especially glomerulonephritis and yet test negative for cryoglobulinaemia detected by standard techniques, could require deeper investigation even in the absence of HCV infection, RF activity or signs of complement consumption.

In the countries where HCV infection is still endemic, about 90% of subjects with mixed cryoglobulinemia had previously been infected with HCV and about 80% are RNA positive. Remarkable results in severe HCV-related cryoglobulinemic vasculitis have been obtained with Rituximab. Details of the clinical characteristics and effective treatment of non HCV-related cryogloulinemic syndromes are presently lacking. This paper reports on a prospective single-Center open study aimed at evaluating the clinical presentation and effects of Rituximab administered alone in patients with severe non HCV-related cryoglobulinemic syndrome. The study group included 11 patients followed for at least 6 months. Three patients had type I cryoglobulinemia, 6 had type II and the remaining 2 patients had type III. Mean cryocrit was 2.5%. Four out of 11 patients had symptomatic sicca complex with anti-SSA (Ro)/anti SSB (La) antibodies. All 11 patients presented with biopsy-proven renal involvement, 4 out of 11 with leukocytoclastic vasculitis, and 8 with involvement of the peripheral nervous system. Renal biopsy revealed diffuse membranoproliferative glomerulonephritis (MPGN) in 9 out of 11 patients. Extracapillary proliferation and necrosis of the glomerular tuft was observed in 1 of these 9 cases. Interstitial nephritis together with mesangial expansion and capillary immune deposits were observed in 1 patient. Prevalent interstitial fibrosis and glomerular sclerosis were detected in the remaining case. Patients underwent treatment with rituximab alone. After 6 months we observed a remarkable improvement in the necrotizing skin ulcers and a substantial amelioration of the electrophysiological parameters of motor and sensory peripheral neuropathy. Improvement in both renal function (from 2.8 to 1.4 mg/dl, p < 0.001) and proteinuria (from 4.2 g/24 to 0.4 g/24 h, p < 0.001) was found in 10 out of 11 patients, while 1 could not be fully treated because of a severe infusion reaction and sudden development of anti-Rituximab antibodies. Good renal response was confirmed at the end of follow-up (38.4 months). Three patients had a relapse at 6, 12, and 48 months, respectively. In our cohort the administration of 4 once-weekly infusions of Rituximab followed by 2 more infusions after 1 and 2 months proved to be effective in the management of these rare patients.