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Cryoglobulins: An update on detection, mechanisms and clinical contribution
Autoimmunity Reviews 17 (2018) 457–464
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Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev
Review
Cryoglobulins: An update on detection, mechanisms and clinical contribution Marie-Nathalie Kolopp-Sarda a,b, Pierre Miossec a,⁎ a b
Department of Immunology and Rheumatology, Immunogenomics and inflammation research Unit EA 4130, University of Lyon, Lyon, France Immunology Laboratory, University Hospital Lyon, France
a r t i c l e
i n f o
Article history: Received 15 November 2017 Accepted 20 November 2017 Available online 8 March 2018
a b s t r a c t Cryoglobulins are immunoglobulins precipitating in cold condition. They are classified in 3 types according to the Brouet classification and may lead to vasculitis of small and medium size vessels. Vasculitis is related to vessel obstruction by monoclonal cryoglobulin aggregates in type I cryoglobulins and immune complex deposition in type II and III mixed cryoglobulins. This phenomenon is favored by low temperature, especially in skin, joints, and peripheral nerves, or increased cryoglobulin concentration in kidneys. For their detection, collection and clotting at 37 °C are critical pre-analytical conditions. Cryoglobulin characterization and quantification are important to identify the underlying disease. Since detection and identification of cryoglobulins lack standardization, a protocol for such detection, characterization and quantification is proposed. © 2018 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Pre-analytical conditions and analytical characterization of cryoglobulins 2.2.1. Pre-analytical conditions . . . . . . . . . . . . . . . . . . 2.2.2. Analytical aspects of cold precipitation . . . . . . . . . . . . 2.2.3. Characterization: typing and quantification. . . . . . . . . . 2.2.4. Additional analysis of the cryoprecipitate and serum . . . . . 3. Mechanisms of cryoglobulin precipitation . . . . . . . . . . . . . . . . . . 3.1. Type I cryoglobulins . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Type II and III mixed cryoglobulins. . . . . . . . . . . . . . . . . . 4. Clinical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Diagnosis approach of cryoglobulinemia and biological follow-up . . . . 4.2. Clinical manifestations of cryoglobulinemia . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Cryoglobulins are immunoglobulins (Ig) that precipitate in vitro at cold temperature and dissolve at 37 °C. Their precipitation in vivo could lead to vasculitis of small and medium-size vessels. Maxwell
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Wintrobe and Mary Buell in 1933 first described an abnormal protein precipitating in cold condition and found in the serum of a patient diagnosed with multiple myeloma associated with a unilateral retinal vein thrombosis and Raynaud's phenomenon [1]. Lerner and Watson in 1947 named these cold insoluble proteins, cryoglobulins [2]. Then,
Abbreviations: AA, amino acid; Ab, antibodies; HCV, hepatitis C virus; HBV, hepatitis B virus; HIV, human immunodeficiency virus; Ig, immunoglobulin; RF, rheumatoid factor. ⁎ Corresponding author at: Clinical Immunology Unit, Department of Immunology and Rheumatology, Hospital Edouard Herriot, 69437 Lyon, France. E-mail address: [email protected] (P. Miossec).
https://doi.org/10.1016/j.autrev.2017.11.035 1568-9972/© 2018 Elsevier B.V. All rights reserved.
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Lospalluto et al. described two proteins of 19S and 7S in a cryoprecipitate, associated by an antibody-antigen mechanism [3]. These proteins were IgM with a Rheumatoid Factor (RF) activity associated with IgG [4–6]. These complexes are now defined as mixed cryoglobulins [7]. Vasculitis involving skin, joints, nerves and kidneys are the main clinical manifestations of cryoglobulinemia. However, the presence of cryoglobulin is not always symptomatic and the contribution of a cryoglobulin to the pathophysiology of a patient presenting with vasculitis is not always obvious. The quality of detection and quantification of cryoglobulins is critical, but the conditions of detection, identification and quantification often differ between laboratories. In this review, we will define the method of detection and characterization of the cryoglobulins. Then we will describe the mechanisms and biological consequences of cold precipitation and the clinical aspects of the cryoglobulinemia. 2. Technical aspects 2.1. Classification In 1974, Brouet et al. proposed a classification of cryoglobulins, which is widely used because of its correlation with clinical presentation [7]. They are classified in 3 types (Fig. 1). Type I cryoglobulins are monoclonal Ig of IgM or IgG, rarely IgA isotype. They often result from the monoclonal expansion of a clone that can be malignant (multiple myeloma, Waldenström's macroglobulinemia, lymphoma) or indolent (monoclonal gammopathy of unknown significance), or secondary to lymphoproliferative disorders (chronic lymphocytic leukemia). The cryoglobulin concentration is often greater than 2 g/L and frequently of the IgM than IgG type with kappa or lambda light chains. Only a few studies report monoclonal IgA cryoglobulinemia [8–16]. Type II and III are mixed cryoglobulins. The type II cryoglobulins associate a monoclonal component with polyclonal Ig. The rheumatoid factor (RF) activity of the monoclonal cryoglobulin leads to the binding of polyclonal IgG to their crystallizable fragment (Fc). Type III cryoglobulins are composed of immune complexes of polyclonal Ig
with RF activity. The IgM and IgG are the most frequent Ig isotypes found in mixed cryoglobulins, but monoclonal or polyclonal IgA can also be associated. Mixed cryoglobulins are often found secondary to chronic infectious diseases, including viral diseases (hepatitis B and C, HIV), bacterial or parasitic infections. They are also found in auto-immune diseases such as Sjogren's syndrome, systemic lupus erythematous or rheumatoid arthritis. 2.2. Pre-analytical conditions and analytical characterization of cryoglobulins The ability to detect the presence of a cryoglobulin in the serum depends on the quality of the pre-analytical stage. The most critical point is the need to maintain samples at 37 °C (± 2 °C) from the collection to the laboratory [17,18]. 2.2.1. Pre-analytical conditions For optimal cryoglobulin detection, strict conditions of sample collection and transport must be followed. Blood samples are collected in dry test tubes without separation gel. Test tubes containing calcium (Ca) chelators, such as EDTA, should not be used, because some cryoglobulins need Ca to precipitate [19]. A sufficient volume of serum is required to detect small amounts of cryoprecipitate: 3 test tubes of 5 mL are collected to obtain a total volume of 4–5 mL of serum. Blood samples need to be rapidly collected and maintained in an incubator at 37 °C for 2 h to clot and to avoid cryoglobulin precipitation. Most of the cryoglobulins start precipitating at a temperature between 25°–30 °C [20–22]. The transport to the laboratory needs to be carried out in containers that maintain the temperature at around 37 °C. Sending the test tubes immediately after sampling to the laboratory via a pneumatic system will allow a rapid transfer, without impact on the amount of cryoprecipitate compared to manual transport at 37 °C. 2.2.2. Analytical aspects of cold precipitation Once clotted, the samples are centrifuged for 15 min at 2200g, at ambient temperature or at 37 °C [18]. The sera are decanted in conical bottom test tubes and placed for 7 days at 4 °C. A 500 μL aliquot of serum is kept at 4 °C to confirm the dissolution of the precipitate when put back
Fig. 1. Types of cryoglobulins according to Brouet et al. classification [7].
M.-N. Kolopp-Sarda, P. Miossec / Autoimmunity Reviews 17 (2018) 457–464
at 37 °C. This aliquot is also used for the detection of RF activity, complement fractions or electrophoresis. Variable cold conservation durations are reported in the literature ranging from 12 h to 9 days, even 1 month [23,24]. Cryoprecipitation may occur within hours for type I and some type II cryoglobulins, or several days for mixed cryoglobulins [5,25–27]. A visual observation at day 7 allows the detection of a cryoprecipitate [24]; however, if no cryoprecipitate is observed, this negative result must be confirmed with two other blood samples. The cryoprecipitate seen at the bottom of the tubes may vary in volume (Fig. 2a–b) and in aspect with fine scroll flakes or cloudy white precipitate (Fig. 2c) or formation of a cryogel (Fig. 2d). If the cryoprecipitate looks like a lipid layer at the surface of the serum, the centrifugation of the sample is essential for differentiation. The presence of lipids, hemolysis or both makes it difficult or sometimes impossible to interpret the results. A new sample should be tested to confirm the presence or not of cryoglobulins. The cryoprecipitate is isolated by cold centrifugation (2200g, 15 min, 2–8 °C) and purified using 3 washes with cold PBS (phosphate buffered saline, pH = 7.4, 2–8 °C) to remove other serum proteins (albumin and not precipitating Ig). After each wash, the samples are centrifuged at 4500g for 15 min at 2–8 °C. Following the last wash, 500 μL PBS or more (in function of the precipitate volume) is added to the sample with Fluidil® 2% (Sebia, Lisses, France) to facilitate immune complex dissociation. This sample is placed at 37 °C for up to 2 h to dissolve the precipitate for further analyses. The detection of a cryoprecipitate in a tube kept at 2–8 °C for 7 days must be confirmed by its identification and quantification.
2.2.3. Characterization: typing and quantification The classification of cryoglobulins takes into account the clonality of the Ig constituting the precipitate [7]. Electrophoresis-immunofixation is the most accurate and easiest technique to determine cryoglobulin isotype (IgG, IgA or IgM, kappa or lambda), monoclonality (defined by a clear band with an anti-heavy chain antiserum at the same level as a band with an anti-light chain antiserum) or polyclonality (homogeneous aspect spread out in the zone of the gammaglobulins) (Fig. 3). In our laboratory, cryoglobulins are first identified by electrophoresisimmunofixation of the dissolved cryoprecipitate. Then the identified Ig is quantified by nephelometry (BNprospec®, Siemens, Germany, reagents for low concentrations). The electrophoresis-immunofixation indicates the degree of purity of the cryoprecipitate by the absence of other serum proteins. The concentration of Ig in the cryoprecipitate is adjusted to the initial volume of serum from which the cryoglobulin was isolated. The results are expressed as mg/L of serum. The characterization steps are summarized in Fig. 4, based on the literature [23,24] and our own experience. Combining an electrophoresisimmunofixation to assess the isotype and nature of the cryoglobulin and the quantification of Ig in the cryoprecipitate is the most sensitive
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and specific approach to confirm the diagnosis of cryoglobulinemia [24,28]. 2.2.4. Additional analysis of the cryoprecipitate and serum Mixed cryoglobulins frequently have a RF activity, either linked to the monoclonal Ig for type II or to the polyclonal Ig for type III. The RF activity measured by nephelometry or turbidimetry detects only IgM anti-IgG RF. The quantification of the RF activity in the cryoprecipitate and in the remaining serum aliquot dissolved at 37 °C is important to best define the cryoglobulins and their activity. The mixed cryoglobulins with RF activity are more likely to form immune complexes and to contribute to disease. Measurement of the complement functional activity (CH50), C3 and C4 fractions in the serum are also indicators of a cryoglobulin activity. Hypocomplementemia is often observed in the serum of cryoglobulinemic patients: decreased C1q, C2 and C4 fractions, normal or below limit C3, decreased or below limit CH50, and normal post-C5 fractions. Of those, C4 is the most sensitive fraction. The diminution of complement fractions results from the fixation of complement fractions in vivo to the immune complexes or the aggregates of monoclonal cryoglobulin, rather than an activation through the classical pathway [29–31]. 3. Mechanisms of cryoglobulin precipitation Cryoglobulins have a general structure of normal cold-soluble Ig, with electrophoretic mobility similar to those of whole serum monoclonal or polyclonal Ig [5,32–34]. The reversible cryoprecipitability of Ig depends on environmental conditions (temperature, protein and salt concentrations, pH). 3.1. Type I cryoglobulins Type I cryoglobulin cold aggregation is due to a decrease in solubility at low temperature and not to an immune complex formation. Highly concentrated monoclonal Ig may self-associate reversibly in vivo and contribute to disease [35,36]. In whole serum in vitro, monoclonal cryoglobulins precipitate more readily and at higher temperature than mixed cryoglobulins. As the concentration of protein increases, the precipitation temperature also increases. Structure and amino acid (AA) composition are other parameters promoting precipitation. The AA residues as well as their sequential and steric arrangements are the most important factors determining protein solubility [13,37,38]. The comparison of structure and charge of cold-insoluble cryoglobulins and cold-soluble normal Igs have shown that monoclonal cryoglobulins were more negatively charged as a consequence of AA substitution [37–42], favoring their insolubility. The carbohydrate and sialic acid content is a controversial factor involved in the decrease of the Ig solubility [41–45]. All these factors may modify surface properties of
Fig. 2. Different aspects of cryoprecipitate in serum left 7 days at +4 °C. (a–b) precipitates of different volume and aspect at the bottom of the tube; (c) flakes within serum or cloudy white precipitate; (d) cryogel.
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Fig. 3. Electrophoresis-immunofixation allowing cryoglobulin classification [7]. a: Type I cryoglobulin (monoclonal IgG λ); b: type II cryoglobulin (monoclonal IgMκ and polyclonal IgG); c: type III cryoglobulin (polyclonal IgG and IgM). (ATS: anti-total serum protein antibodies (Ab), G: anti-γ chain Ab, A: anti-α chain Ab, M: anti-μ chain Ab, K: anti-κ chain Ab, L: anti-λ chain Ab).
monoclonal Igs, favoring weak intermolecular bonds and their aggregation at low temperature. Physicochemical properties of cryoprecipitates (variation of solubility in function of temperature, ionic strength or pH)
confirm the involvement of intermolecular non-covalent, weak interactions such as hydrogen bonds, hydrophobic and electrostatic interactions in the aggregation of monoclonal cryoglobulins [13,20,21,39–41].
Fig. 4. Flowchart for detection, characterization and quantification of cryoglobulins and their activity.
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Table 1 Diagnosis approach of cryoglobulinemia and biological interferences. Clinical context
Cutaneous vasculitis, Raynaud's phenomenon, peripheral neuropathy, arthralgia-weakness-purpura Important: healthy individuals can have cryoglobulin at low concentration Cryoglobulin detection Good pre-analytic conditions: Blood collected and transported at ~37 °C ⇨ If Negative: to be confirmed by 2 other detections in blood ⇨ If Positive: see ↓ → In case of positive cryoglobulin In the cryoprecipitate • Typing: to find the associated disease detection after purification • Quantification: to evaluate the risk of vasculitis, to follow evolution and response to treatment • RF activity: to confirm the risk of cold precipitation in mixed cryoglobulin In the serum • Complement exploration:
Cutaneous or renal biopsy Diagnosis of underlying pathology
- Complement profile with decreased C4 frequently associated with active cryoglobulin • RF activity of serum collected in warm condition Leukocytoclastic vasculitis, … Type 1 membranoproliferative glomerulonephritis, …. • Type I: - Lymphoproliferative diseases (MGUS, myeloma, Waldenström macroglobulinemia, chronic lymphoid leukemia, lymphoma, …) • Type II or III mixed cryoglobulins:
Biological interferences associated with the presence of cryoglobulin
- Infectious diseases: frequent with HCV, or HBV, HIV, bacterial or parasitic diseases autoimmune diseases: Sicca syndrome, systemic erythematosus lupus, rheumatoid arthritis - Possibility of lymphoproliferative disease if monoclonal IgM cryoglobulin has RF activity (often in Waldenström macroglobulinemia) • If no underlying disease: essential/primitive cryoglobulin • Interferences with complete blood count: leukocytosis, thrombocytosis or thrombocytopenia • Serum gel formation: prevents serum/plasma sampling by automated systems • Nephelometry or turbidimetry analysis: immune complexes due to RF activity increase nonspecific turbidity • Ig cold precipitation: - False hypogammaglobulinemia and hypoproteinemia, - Decreased monoclonal peak on electrophoresis - Risk of non-detection of specific antibodies, especially with ELISA techniques
Van der Waals interactions are also involved for IgM molecules where large surface area and pentameric structure favor these interactions [21]. Different structures of cryoprecipitates were shown in vitro for IgG [25– 27,42] and IgM [26,43] monoclonal cryoglobulins: amorphous, cryogel or crystals. These different precipitation morphologies depend on the isotype, Ig concentration and precipitating temperature and are associated with disease severity [42]. The most frequent aggregate structure is amorphous and the most severe is a crystal responsible for occlusion of small and mediumsize vessels, leading to tissue necrosis [26,42]. All these properties have clinical consequences. The higher precipitation temperature for high concentrated cryoglobulins could explain the renal involvement of type I cryoglobulin in part due to the high protein concentration in the glomeruli. The slow type I cryoglobulin crystallization due to a nucleation phenomenon could explain the delayed apparition of cutaneous signs after an extended time in the cold. The surface property of monoclonal cryoglobulins is also altered at physiological temperature suggesting that cryoglobulins may represent the outer edge of solubility distribution of total serum Ig [44]. Pathologic signs may develop when these abnormal Ig are produced at high concentration during various disease states. This could explain why a low concentration of cryoglobulin is commonly detected in the serum of healthy subjects and that all monoclonal Ig are not cryoprecipitable, even at high concentration. 3.2. Type II and III mixed cryoglobulins The mechanism of cold precipitation of mixed cryoglobulins combines immune complex formation and precipitation in small and medium-size vessels. These immune complexes are formed by the association of Ig with RF activity, more often monoclonal IgM and polyclonal IgG. For mixed cryoglobulins, the size of immune complexes is temperature dependent with more IgG bound to IgM below 15 °C and increase of immune complex size [45]. As for the monoclonal cryoglobulin
aggregates, immune complex precipitation is inhibited by ionic strength increase and extreme pH [13]. This suggests the formation of one or few electrostatic contacts between IgM and IgG, allowing the size increase of the immune complexes. Cold insolubility of mixed cryoglobulins is due to the insolubility of RF complexes rather than extensive lattice formation [45]. Immune complex formation is not sufficient to explain cold precipitability of mixed cryoglobulins. As for type I cryoglobulins, Igs involved in mixed cryoglobulin immune complexes must have cold precipitation properties. This is linked to RF IgM rather than associated IgG [13,46]. These IgM preferentially bind with IgG1, less with IgG2 and IgG4, and not with IgG3 subclasses [13,47]. The predominance of IgM Kappa is described particularly for Ig with RF activity [48,49]. The preferential use of an uncommon V region subgroup VkIII and sub-subgroup VkIIIb genes among the light chain of human monoclonal IgM kappa was documented through sequence analyses of cryoglobulins and RF of different origin (rheumatoid arthritis, Sjogren's syndrome) [47,50,51]. The RF specificity of monoclonal cryoglobulins is acquired by somatic hypermutation of malignant or non-malignant B-cell clones [48,52] and the major cross idiotype WA on RF IgM is described in 60% of mixed cryoglobulins [53]. These IgM, synthetized by WA B cell clones, are clearly involved in IgM cryoprecipitability and cryoglobulin pathogenicity. In HCV chronic infection, clonal WA B cells are found preferentially in patients with cryoglobulinemic vasculitis and associated with higher risk of B cell malignancies [50,54]. 4. Clinical aspects 4.1. Diagnosis approach of cryoglobulinemia and biological follow-up In Table 1, we propose an approach of cryoglobulinemia diagnosis and the risks of biological error associated with the presence of
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Fig. 5. A: Proposed pathological mechanisms of type I cryoglobulins. Monoclonal cryoglobulins form aggregates that can increase in size in cold temperature. Obstruction of medium and small sized vessels by these aggregates leads to vasculitis responsible for purpura, livedo reticularis or necrosis. B: Proposed pathological mechanisms of mixed cryoglobulins Mixed cryoglobulins (type II cryoglobulin) form immune complexes. When the temperature decreases, the complexes' size increases and may precipitate in small and medium sized vessels. Complement is activated, and this leads to leukocytoclastic vasculitis responsible for purpura, livedo reticularis or necrosis.
cryoglobulin in the serum. The presence of a cryoglobulin should be monitored regularly, especially in mixed cryoglobulinemia containing monoclonal Igs. Even in small quantities, their presence indicates a monoclonal clone of B cells that lead to a lymphoma, particularly in patients with HCV infection and Sjögren's syndrome [50,55–57].
4.2. Clinical manifestations of cryoglobulinemia The presence of cryoglobulin may be asymptomatic or be associated with severe clinical manifestations such as kidney failure. Vasculitis is the main symptom of cryoglobulinemia due to obstruction of small or medium-size vessels, by cryoglobulin aggregate or immune complex precipitation (Fig. 5A and B). The skin temperature depending on the environment (air and humidity) is about 28–30 °C vs. 37 °C for the body, especially in the extremities, explaining the frequency of cutaneous vasculitis responsible for purpura, livedo and ulceration. General symptoms of cryoglobulinemia were first described as the Meltzer's triad associating arthralgia–purpura–weakness [10]. Cryoglobulin most frequent clinical manifestations are intermittent purpuric lesions affecting the lower limbs. The lesions regress spontaneously leaving scar patches on the skin (hemosiderin deposition) and may lead to livedo reticularis. The purpura may be accompanied by Raynaud's phenomenon [58–60]. Ulcers may occur, usually around malleoli, as well as necrosis especially at the extremities, and are more frequent in type I cryoglobulinemia [61–65]. Arthralgia affects the hands, knees and hips. Peripheral neuropathies either sensory, motor or mixed, are seen in 30–50% of patients with cryoglobulins, they result in paresthesia and pain usually of the lower limbs [60]. Renal involvement such as type 1 membranoproliferative glomerulonephritis is seen in 10–30% of patients, which can progress to acute or chronic renal failure after several years [62,66–68]. Other organs may be affected such as the lungs (hemorrhagic alveolitis), heart or intestines.
An association between mixed cryoglobulinemia and HCV infection was first described in 1989 [50,55]. In these patients, vasculitis, kidney failure, neurologic impairment and non-Hodgkin B-cell lymphoma are more frequent [55,65]. Immunosuppressive therapy added to their treatment by interferon and ribavirin, often did not lead to total disappearance of cryoglobulins [69,70]. New antiviral drugs have shown remission of cryoglobulinemic vasculitis associated with viral clearance, confirming the link between anti-HCV chronic response and cryoglobulinemia [71,72]. 5. Conclusion Cryoglobulins can lead to vasculitis of small and medium-size vessels. Precipitation of aggregates of monoclonal cryoglobulin or immune complexes is favored by cold temperature and by environmental chemical conditions. For their detection, good pre-analytical conditions are critical for transport and coagulation at 37 °C. Cryoglobulin typing and quantification are important to confirm the role of cryoglobulins in disease and response to treatment. References [1] Wintrobe M, Buell M. Hyperproteinemia associated with multiple myeloma. Bull Johns Hopkins Hosp 1933;52:156–65. [2] Lerner AB, Watson CJ. Studies of cryoglobulins; unusual purpura associated with the presence of a high concentration of cryoglobulin (cold precipitable serum globulin). Am J Med Sci 1947;214:410–5. [3] Lospalluto J, Dorward B, Jr Miller W, Ziff M. Cryoglobulinemia based on interaction between a gamma macroglobulin and 7S gamma globulin. Am J Med 1962;32: 142–7. [4] Costanzi JJ, Coltman CA, Clark DA, Tennenbaum JI, Criscuolo D. Cryoglobulinemia associated with a macroglobulin; studies of a 17.5S cryoprecipitating factor. Am J Med 1965;39:163–72. [5] Meltzer M, Franklin EC. Cryoglobulinemia–a study of twenty-nine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med 1966;40: 828–36.
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Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev
Review
Cryoglobulins: An update on detection, mechanisms and clinical contribution Marie-Nathalie Kolopp-Sarda a,b, Pierre Miossec a,⁎ a b
Department of Immunology and Rheumatology, Immunogenomics and inflammation research Unit EA 4130, University of Lyon, Lyon, France Immunology Laboratory, University Hospital Lyon, France
a r t i c l e
i n f o
Article history: Received 15 November 2017 Accepted 20 November 2017 Available online 8 March 2018
a b s t r a c t Cryoglobulins are immunoglobulins precipitating in cold condition. They are classified in 3 types according to the Brouet classification and may lead to vasculitis of small and medium size vessels. Vasculitis is related to vessel obstruction by monoclonal cryoglobulin aggregates in type I cryoglobulins and immune complex deposition in type II and III mixed cryoglobulins. This phenomenon is favored by low temperature, especially in skin, joints, and peripheral nerves, or increased cryoglobulin concentration in kidneys. For their detection, collection and clotting at 37 °C are critical pre-analytical conditions. Cryoglobulin characterization and quantification are important to identify the underlying disease. Since detection and identification of cryoglobulins lack standardization, a protocol for such detection, characterization and quantification is proposed. © 2018 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Pre-analytical conditions and analytical characterization of cryoglobulins 2.2.1. Pre-analytical conditions . . . . . . . . . . . . . . . . . . 2.2.2. Analytical aspects of cold precipitation . . . . . . . . . . . . 2.2.3. Characterization: typing and quantification. . . . . . . . . . 2.2.4. Additional analysis of the cryoprecipitate and serum . . . . . 3. Mechanisms of cryoglobulin precipitation . . . . . . . . . . . . . . . . . . 3.1. Type I cryoglobulins . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Type II and III mixed cryoglobulins. . . . . . . . . . . . . . . . . . 4. Clinical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Diagnosis approach of cryoglobulinemia and biological follow-up . . . . 4.2. Clinical manifestations of cryoglobulinemia . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Cryoglobulins are immunoglobulins (Ig) that precipitate in vitro at cold temperature and dissolve at 37 °C. Their precipitation in vivo could lead to vasculitis of small and medium-size vessels. Maxwell
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Wintrobe and Mary Buell in 1933 first described an abnormal protein precipitating in cold condition and found in the serum of a patient diagnosed with multiple myeloma associated with a unilateral retinal vein thrombosis and Raynaud's phenomenon [1]. Lerner and Watson in 1947 named these cold insoluble proteins, cryoglobulins [2]. Then,
Abbreviations: AA, amino acid; Ab, antibodies; HCV, hepatitis C virus; HBV, hepatitis B virus; HIV, human immunodeficiency virus; Ig, immunoglobulin; RF, rheumatoid factor. ⁎ Corresponding author at: Clinical Immunology Unit, Department of Immunology and Rheumatology, Hospital Edouard Herriot, 69437 Lyon, France. E-mail address: [email protected] (P. Miossec).
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Lospalluto et al. described two proteins of 19S and 7S in a cryoprecipitate, associated by an antibody-antigen mechanism [3]. These proteins were IgM with a Rheumatoid Factor (RF) activity associated with IgG [4–6]. These complexes are now defined as mixed cryoglobulins [7]. Vasculitis involving skin, joints, nerves and kidneys are the main clinical manifestations of cryoglobulinemia. However, the presence of cryoglobulin is not always symptomatic and the contribution of a cryoglobulin to the pathophysiology of a patient presenting with vasculitis is not always obvious. The quality of detection and quantification of cryoglobulins is critical, but the conditions of detection, identification and quantification often differ between laboratories. In this review, we will define the method of detection and characterization of the cryoglobulins. Then we will describe the mechanisms and biological consequences of cold precipitation and the clinical aspects of the cryoglobulinemia. 2. Technical aspects 2.1. Classification In 1974, Brouet et al. proposed a classification of cryoglobulins, which is widely used because of its correlation with clinical presentation [7]. They are classified in 3 types (Fig. 1). Type I cryoglobulins are monoclonal Ig of IgM or IgG, rarely IgA isotype. They often result from the monoclonal expansion of a clone that can be malignant (multiple myeloma, Waldenström's macroglobulinemia, lymphoma) or indolent (monoclonal gammopathy of unknown significance), or secondary to lymphoproliferative disorders (chronic lymphocytic leukemia). The cryoglobulin concentration is often greater than 2 g/L and frequently of the IgM than IgG type with kappa or lambda light chains. Only a few studies report monoclonal IgA cryoglobulinemia [8–16]. Type II and III are mixed cryoglobulins. The type II cryoglobulins associate a monoclonal component with polyclonal Ig. The rheumatoid factor (RF) activity of the monoclonal cryoglobulin leads to the binding of polyclonal IgG to their crystallizable fragment (Fc). Type III cryoglobulins are composed of immune complexes of polyclonal Ig
with RF activity. The IgM and IgG are the most frequent Ig isotypes found in mixed cryoglobulins, but monoclonal or polyclonal IgA can also be associated. Mixed cryoglobulins are often found secondary to chronic infectious diseases, including viral diseases (hepatitis B and C, HIV), bacterial or parasitic infections. They are also found in auto-immune diseases such as Sjogren's syndrome, systemic lupus erythematous or rheumatoid arthritis. 2.2. Pre-analytical conditions and analytical characterization of cryoglobulins The ability to detect the presence of a cryoglobulin in the serum depends on the quality of the pre-analytical stage. The most critical point is the need to maintain samples at 37 °C (± 2 °C) from the collection to the laboratory [17,18]. 2.2.1. Pre-analytical conditions For optimal cryoglobulin detection, strict conditions of sample collection and transport must be followed. Blood samples are collected in dry test tubes without separation gel. Test tubes containing calcium (Ca) chelators, such as EDTA, should not be used, because some cryoglobulins need Ca to precipitate [19]. A sufficient volume of serum is required to detect small amounts of cryoprecipitate: 3 test tubes of 5 mL are collected to obtain a total volume of 4–5 mL of serum. Blood samples need to be rapidly collected and maintained in an incubator at 37 °C for 2 h to clot and to avoid cryoglobulin precipitation. Most of the cryoglobulins start precipitating at a temperature between 25°–30 °C [20–22]. The transport to the laboratory needs to be carried out in containers that maintain the temperature at around 37 °C. Sending the test tubes immediately after sampling to the laboratory via a pneumatic system will allow a rapid transfer, without impact on the amount of cryoprecipitate compared to manual transport at 37 °C. 2.2.2. Analytical aspects of cold precipitation Once clotted, the samples are centrifuged for 15 min at 2200g, at ambient temperature or at 37 °C [18]. The sera are decanted in conical bottom test tubes and placed for 7 days at 4 °C. A 500 μL aliquot of serum is kept at 4 °C to confirm the dissolution of the precipitate when put back
Fig. 1. Types of cryoglobulins according to Brouet et al. classification [7].
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at 37 °C. This aliquot is also used for the detection of RF activity, complement fractions or electrophoresis. Variable cold conservation durations are reported in the literature ranging from 12 h to 9 days, even 1 month [23,24]. Cryoprecipitation may occur within hours for type I and some type II cryoglobulins, or several days for mixed cryoglobulins [5,25–27]. A visual observation at day 7 allows the detection of a cryoprecipitate [24]; however, if no cryoprecipitate is observed, this negative result must be confirmed with two other blood samples. The cryoprecipitate seen at the bottom of the tubes may vary in volume (Fig. 2a–b) and in aspect with fine scroll flakes or cloudy white precipitate (Fig. 2c) or formation of a cryogel (Fig. 2d). If the cryoprecipitate looks like a lipid layer at the surface of the serum, the centrifugation of the sample is essential for differentiation. The presence of lipids, hemolysis or both makes it difficult or sometimes impossible to interpret the results. A new sample should be tested to confirm the presence or not of cryoglobulins. The cryoprecipitate is isolated by cold centrifugation (2200g, 15 min, 2–8 °C) and purified using 3 washes with cold PBS (phosphate buffered saline, pH = 7.4, 2–8 °C) to remove other serum proteins (albumin and not precipitating Ig). After each wash, the samples are centrifuged at 4500g for 15 min at 2–8 °C. Following the last wash, 500 μL PBS or more (in function of the precipitate volume) is added to the sample with Fluidil® 2% (Sebia, Lisses, France) to facilitate immune complex dissociation. This sample is placed at 37 °C for up to 2 h to dissolve the precipitate for further analyses. The detection of a cryoprecipitate in a tube kept at 2–8 °C for 7 days must be confirmed by its identification and quantification.
2.2.3. Characterization: typing and quantification The classification of cryoglobulins takes into account the clonality of the Ig constituting the precipitate [7]. Electrophoresis-immunofixation is the most accurate and easiest technique to determine cryoglobulin isotype (IgG, IgA or IgM, kappa or lambda), monoclonality (defined by a clear band with an anti-heavy chain antiserum at the same level as a band with an anti-light chain antiserum) or polyclonality (homogeneous aspect spread out in the zone of the gammaglobulins) (Fig. 3). In our laboratory, cryoglobulins are first identified by electrophoresisimmunofixation of the dissolved cryoprecipitate. Then the identified Ig is quantified by nephelometry (BNprospec®, Siemens, Germany, reagents for low concentrations). The electrophoresis-immunofixation indicates the degree of purity of the cryoprecipitate by the absence of other serum proteins. The concentration of Ig in the cryoprecipitate is adjusted to the initial volume of serum from which the cryoglobulin was isolated. The results are expressed as mg/L of serum. The characterization steps are summarized in Fig. 4, based on the literature [23,24] and our own experience. Combining an electrophoresisimmunofixation to assess the isotype and nature of the cryoglobulin and the quantification of Ig in the cryoprecipitate is the most sensitive
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and specific approach to confirm the diagnosis of cryoglobulinemia [24,28]. 2.2.4. Additional analysis of the cryoprecipitate and serum Mixed cryoglobulins frequently have a RF activity, either linked to the monoclonal Ig for type II or to the polyclonal Ig for type III. The RF activity measured by nephelometry or turbidimetry detects only IgM anti-IgG RF. The quantification of the RF activity in the cryoprecipitate and in the remaining serum aliquot dissolved at 37 °C is important to best define the cryoglobulins and their activity. The mixed cryoglobulins with RF activity are more likely to form immune complexes and to contribute to disease. Measurement of the complement functional activity (CH50), C3 and C4 fractions in the serum are also indicators of a cryoglobulin activity. Hypocomplementemia is often observed in the serum of cryoglobulinemic patients: decreased C1q, C2 and C4 fractions, normal or below limit C3, decreased or below limit CH50, and normal post-C5 fractions. Of those, C4 is the most sensitive fraction. The diminution of complement fractions results from the fixation of complement fractions in vivo to the immune complexes or the aggregates of monoclonal cryoglobulin, rather than an activation through the classical pathway [29–31]. 3. Mechanisms of cryoglobulin precipitation Cryoglobulins have a general structure of normal cold-soluble Ig, with electrophoretic mobility similar to those of whole serum monoclonal or polyclonal Ig [5,32–34]. The reversible cryoprecipitability of Ig depends on environmental conditions (temperature, protein and salt concentrations, pH). 3.1. Type I cryoglobulins Type I cryoglobulin cold aggregation is due to a decrease in solubility at low temperature and not to an immune complex formation. Highly concentrated monoclonal Ig may self-associate reversibly in vivo and contribute to disease [35,36]. In whole serum in vitro, monoclonal cryoglobulins precipitate more readily and at higher temperature than mixed cryoglobulins. As the concentration of protein increases, the precipitation temperature also increases. Structure and amino acid (AA) composition are other parameters promoting precipitation. The AA residues as well as their sequential and steric arrangements are the most important factors determining protein solubility [13,37,38]. The comparison of structure and charge of cold-insoluble cryoglobulins and cold-soluble normal Igs have shown that monoclonal cryoglobulins were more negatively charged as a consequence of AA substitution [37–42], favoring their insolubility. The carbohydrate and sialic acid content is a controversial factor involved in the decrease of the Ig solubility [41–45]. All these factors may modify surface properties of
Fig. 2. Different aspects of cryoprecipitate in serum left 7 days at +4 °C. (a–b) precipitates of different volume and aspect at the bottom of the tube; (c) flakes within serum or cloudy white precipitate; (d) cryogel.
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Fig. 3. Electrophoresis-immunofixation allowing cryoglobulin classification [7]. a: Type I cryoglobulin (monoclonal IgG λ); b: type II cryoglobulin (monoclonal IgMκ and polyclonal IgG); c: type III cryoglobulin (polyclonal IgG and IgM). (ATS: anti-total serum protein antibodies (Ab), G: anti-γ chain Ab, A: anti-α chain Ab, M: anti-μ chain Ab, K: anti-κ chain Ab, L: anti-λ chain Ab).
monoclonal Igs, favoring weak intermolecular bonds and their aggregation at low temperature. Physicochemical properties of cryoprecipitates (variation of solubility in function of temperature, ionic strength or pH)
confirm the involvement of intermolecular non-covalent, weak interactions such as hydrogen bonds, hydrophobic and electrostatic interactions in the aggregation of monoclonal cryoglobulins [13,20,21,39–41].
Fig. 4. Flowchart for detection, characterization and quantification of cryoglobulins and their activity.
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Table 1 Diagnosis approach of cryoglobulinemia and biological interferences. Clinical context
Cutaneous vasculitis, Raynaud's phenomenon, peripheral neuropathy, arthralgia-weakness-purpura Important: healthy individuals can have cryoglobulin at low concentration Cryoglobulin detection Good pre-analytic conditions: Blood collected and transported at ~37 °C ⇨ If Negative: to be confirmed by 2 other detections in blood ⇨ If Positive: see ↓ → In case of positive cryoglobulin In the cryoprecipitate • Typing: to find the associated disease detection after purification • Quantification: to evaluate the risk of vasculitis, to follow evolution and response to treatment • RF activity: to confirm the risk of cold precipitation in mixed cryoglobulin In the serum • Complement exploration:
Cutaneous or renal biopsy Diagnosis of underlying pathology
- Complement profile with decreased C4 frequently associated with active cryoglobulin • RF activity of serum collected in warm condition Leukocytoclastic vasculitis, … Type 1 membranoproliferative glomerulonephritis, …. • Type I: - Lymphoproliferative diseases (MGUS, myeloma, Waldenström macroglobulinemia, chronic lymphoid leukemia, lymphoma, …) • Type II or III mixed cryoglobulins:
Biological interferences associated with the presence of cryoglobulin
- Infectious diseases: frequent with HCV, or HBV, HIV, bacterial or parasitic diseases autoimmune diseases: Sicca syndrome, systemic erythematosus lupus, rheumatoid arthritis - Possibility of lymphoproliferative disease if monoclonal IgM cryoglobulin has RF activity (often in Waldenström macroglobulinemia) • If no underlying disease: essential/primitive cryoglobulin • Interferences with complete blood count: leukocytosis, thrombocytosis or thrombocytopenia • Serum gel formation: prevents serum/plasma sampling by automated systems • Nephelometry or turbidimetry analysis: immune complexes due to RF activity increase nonspecific turbidity • Ig cold precipitation: - False hypogammaglobulinemia and hypoproteinemia, - Decreased monoclonal peak on electrophoresis - Risk of non-detection of specific antibodies, especially with ELISA techniques
Van der Waals interactions are also involved for IgM molecules where large surface area and pentameric structure favor these interactions [21]. Different structures of cryoprecipitates were shown in vitro for IgG [25– 27,42] and IgM [26,43] monoclonal cryoglobulins: amorphous, cryogel or crystals. These different precipitation morphologies depend on the isotype, Ig concentration and precipitating temperature and are associated with disease severity [42]. The most frequent aggregate structure is amorphous and the most severe is a crystal responsible for occlusion of small and mediumsize vessels, leading to tissue necrosis [26,42]. All these properties have clinical consequences. The higher precipitation temperature for high concentrated cryoglobulins could explain the renal involvement of type I cryoglobulin in part due to the high protein concentration in the glomeruli. The slow type I cryoglobulin crystallization due to a nucleation phenomenon could explain the delayed apparition of cutaneous signs after an extended time in the cold. The surface property of monoclonal cryoglobulins is also altered at physiological temperature suggesting that cryoglobulins may represent the outer edge of solubility distribution of total serum Ig [44]. Pathologic signs may develop when these abnormal Ig are produced at high concentration during various disease states. This could explain why a low concentration of cryoglobulin is commonly detected in the serum of healthy subjects and that all monoclonal Ig are not cryoprecipitable, even at high concentration. 3.2. Type II and III mixed cryoglobulins The mechanism of cold precipitation of mixed cryoglobulins combines immune complex formation and precipitation in small and medium-size vessels. These immune complexes are formed by the association of Ig with RF activity, more often monoclonal IgM and polyclonal IgG. For mixed cryoglobulins, the size of immune complexes is temperature dependent with more IgG bound to IgM below 15 °C and increase of immune complex size [45]. As for the monoclonal cryoglobulin
aggregates, immune complex precipitation is inhibited by ionic strength increase and extreme pH [13]. This suggests the formation of one or few electrostatic contacts between IgM and IgG, allowing the size increase of the immune complexes. Cold insolubility of mixed cryoglobulins is due to the insolubility of RF complexes rather than extensive lattice formation [45]. Immune complex formation is not sufficient to explain cold precipitability of mixed cryoglobulins. As for type I cryoglobulins, Igs involved in mixed cryoglobulin immune complexes must have cold precipitation properties. This is linked to RF IgM rather than associated IgG [13,46]. These IgM preferentially bind with IgG1, less with IgG2 and IgG4, and not with IgG3 subclasses [13,47]. The predominance of IgM Kappa is described particularly for Ig with RF activity [48,49]. The preferential use of an uncommon V region subgroup VkIII and sub-subgroup VkIIIb genes among the light chain of human monoclonal IgM kappa was documented through sequence analyses of cryoglobulins and RF of different origin (rheumatoid arthritis, Sjogren's syndrome) [47,50,51]. The RF specificity of monoclonal cryoglobulins is acquired by somatic hypermutation of malignant or non-malignant B-cell clones [48,52] and the major cross idiotype WA on RF IgM is described in 60% of mixed cryoglobulins [53]. These IgM, synthetized by WA B cell clones, are clearly involved in IgM cryoprecipitability and cryoglobulin pathogenicity. In HCV chronic infection, clonal WA B cells are found preferentially in patients with cryoglobulinemic vasculitis and associated with higher risk of B cell malignancies [50,54]. 4. Clinical aspects 4.1. Diagnosis approach of cryoglobulinemia and biological follow-up In Table 1, we propose an approach of cryoglobulinemia diagnosis and the risks of biological error associated with the presence of
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Fig. 5. A: Proposed pathological mechanisms of type I cryoglobulins. Monoclonal cryoglobulins form aggregates that can increase in size in cold temperature. Obstruction of medium and small sized vessels by these aggregates leads to vasculitis responsible for purpura, livedo reticularis or necrosis. B: Proposed pathological mechanisms of mixed cryoglobulins Mixed cryoglobulins (type II cryoglobulin) form immune complexes. When the temperature decreases, the complexes' size increases and may precipitate in small and medium sized vessels. Complement is activated, and this leads to leukocytoclastic vasculitis responsible for purpura, livedo reticularis or necrosis.
cryoglobulin in the serum. The presence of a cryoglobulin should be monitored regularly, especially in mixed cryoglobulinemia containing monoclonal Igs. Even in small quantities, their presence indicates a monoclonal clone of B cells that lead to a lymphoma, particularly in patients with HCV infection and Sjögren's syndrome [50,55–57].
4.2. Clinical manifestations of cryoglobulinemia The presence of cryoglobulin may be asymptomatic or be associated with severe clinical manifestations such as kidney failure. Vasculitis is the main symptom of cryoglobulinemia due to obstruction of small or medium-size vessels, by cryoglobulin aggregate or immune complex precipitation (Fig. 5A and B). The skin temperature depending on the environment (air and humidity) is about 28–30 °C vs. 37 °C for the body, especially in the extremities, explaining the frequency of cutaneous vasculitis responsible for purpura, livedo and ulceration. General symptoms of cryoglobulinemia were first described as the Meltzer's triad associating arthralgia–purpura–weakness [10]. Cryoglobulin most frequent clinical manifestations are intermittent purpuric lesions affecting the lower limbs. The lesions regress spontaneously leaving scar patches on the skin (hemosiderin deposition) and may lead to livedo reticularis. The purpura may be accompanied by Raynaud's phenomenon [58–60]. Ulcers may occur, usually around malleoli, as well as necrosis especially at the extremities, and are more frequent in type I cryoglobulinemia [61–65]. Arthralgia affects the hands, knees and hips. Peripheral neuropathies either sensory, motor or mixed, are seen in 30–50% of patients with cryoglobulins, they result in paresthesia and pain usually of the lower limbs [60]. Renal involvement such as type 1 membranoproliferative glomerulonephritis is seen in 10–30% of patients, which can progress to acute or chronic renal failure after several years [62,66–68]. Other organs may be affected such as the lungs (hemorrhagic alveolitis), heart or intestines.
An association between mixed cryoglobulinemia and HCV infection was first described in 1989 [50,55]. In these patients, vasculitis, kidney failure, neurologic impairment and non-Hodgkin B-cell lymphoma are more frequent [55,65]. Immunosuppressive therapy added to their treatment by interferon and ribavirin, often did not lead to total disappearance of cryoglobulins [69,70]. New antiviral drugs have shown remission of cryoglobulinemic vasculitis associated with viral clearance, confirming the link between anti-HCV chronic response and cryoglobulinemia [71,72]. 5. Conclusion Cryoglobulins can lead to vasculitis of small and medium-size vessels. Precipitation of aggregates of monoclonal cryoglobulin or immune complexes is favored by cold temperature and by environmental chemical conditions. For their detection, good pre-analytical conditions are critical for transport and coagulation at 37 °C. Cryoglobulin typing and quantification are important to confirm the role of cryoglobulins in disease and response to treatment. References [1] Wintrobe M, Buell M. Hyperproteinemia associated with multiple myeloma. Bull Johns Hopkins Hosp 1933;52:156–65. [2] Lerner AB, Watson CJ. Studies of cryoglobulins; unusual purpura associated with the presence of a high concentration of cryoglobulin (cold precipitable serum globulin). Am J Med Sci 1947;214:410–5. [3] Lospalluto J, Dorward B, Jr Miller W, Ziff M. Cryoglobulinemia based on interaction between a gamma macroglobulin and 7S gamma globulin. Am J Med 1962;32: 142–7. [4] Costanzi JJ, Coltman CA, Clark DA, Tennenbaum JI, Criscuolo D. Cryoglobulinemia associated with a macroglobulin; studies of a 17.5S cryoprecipitating factor. Am J Med 1965;39:163–72. [5] Meltzer M, Franklin EC. Cryoglobulinemia–a study of twenty-nine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med 1966;40: 828–36.
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