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A critical appraisal of current practice in the detection, analysis, and reporting of cryoglobulins.

Cryoproteins are serum proteins that reversibly precipitate at temperatures below 37 [degrees]C. There are 2 types of cryoproteins, cryoglobulins and cryofibrinogens. Cryoglobulins are immunoglobulins that precipitate both from serum and plasma, whereas cryofibrinogens, which consist of fibrinogen-fibrin complexes, precipitate only from plasma (1).

Cryoglobulinemia was 1st described by Wintrobe and Buell in 1933 and has been associated with the Meltzer triad of palpable purpura, arthralgia, and asthenia. The clinical significance of cryoglobulinemia is the consequence of intravascular precipitation of immunoglobulins, which can produce (reversible) mechanical obstruction of small vessels leading to the Raynaud phenomenon and immune complex-mediated vasculitis, particularly in the skin, peripheral nerves, and kidneys (2).

There are 2 distinct types of cryoglobulins. Type I cryoglobulins are monoclonal immunoglobulins associated with immunoproliferative disorders. Type I cryoglobulins account for about 10% of cryoglobulins, usually precipitate within 24 h, and can produce symptoms of hyperviscocity and thrombosis (2). Mixed cryoglobulins, in contrast, are composed of immune complexes containing polyclonal immunoglobulins with (type II) or without (type III) a monoclonal component and can take up to 1 week to become apparent by precipitation. Mixed cryoglobulins account for approximately 90% of cryoglobulins and are associated with chronic inflammatory diseases and infection. Mixed cryoglobulinemia is characterized by immune complex-mediated vasculitis and multiple organ involvement (2).

Detection of cryoglobulinemia can aid in the diagnosis of certain syndromes and is part of the diagnostic work-up for other conditions (e.g., monoclonal gammopathies) (3). The Chapel Hill nomenclature for systemic vasculitis is the only disease classification system in which the presence of cryoglobulins is mentioned, i.e., for essential cryoglobulinemic vasculitis (4). Quantitiation of cryoglobulins can be used to make therapeutic decisions (plasmapheresis) and to assess the efficacy of therapeutic interventions.

Although serum and plasma are necessary for detecting cryofibrinogenemia, cryoglobulin analysis is traditionally performed only on serum (1). Several authors have described different analytical approaches for cryoglobulin detection. Unfortunately, there is no internationally accepted standard (2, 5). Some laboratories determine cryocrit, and others quantify total protein content. Furthermore, no formal reference values exist (6). This lack of standardization and of reference values hampers comparison between studies and interpretation by clinicians. Reports in the literature about missed diagnosis of cryoglobulinemia further highlight the need for standardization (7, 8).


To establish the current practice in the detection, analysis, and reporting of cryoglobulins, we sent a questionnaire to 140 laboratories participating in the UK National External Quality Assessment Service quality control program. A total of 137 laboratories that search for cryoglobulins were included for further analysis (Fig. 1). The number of samples analyzed per year varied between 1 and 1500 (median 40). The 25th and 75th percentiles were 15 and 85, respectively. (The questionnaire is available in the online data supplement at


It is well recognized to be crucial that blood should be collected, transported, and clotted at 37 [degrees]C, and most authors stress the need for warm centrifugation to avoid false-negative results (6, 9, 10). Temperature control is particularly important for type I cryoglobulins, which are often present at concentrations >5 g/L (1, 11). High concentrations of monoclonal cryoglobulins tend to precipitate at higher temperatures, and it has been suggested that the higher the temperature at which cryoglobulins precipitate, the more dramatic the symptoms (1, 11). There are also reports of missed diagnosis of type II cryoglobulins due to inadequate temperature control (7, 8). For type III cryoglobulins temperature control is not crucial because they slowly precipitate over a period of days (1, 2).

In our survey, 48 of the 137 laboratories (35%) took all the standard precautions: preheating of tubes, transport at 37 [degrees]C, and sedimentation/centrifugation at 37 [degrees]C (Table 1). Insufficient temperature control is recognized as the single most important cause of missed diagnosis of cryoglobulinemia (1). Inadequate temperature control during blood collection and transport before the sample arrives in the laboratory are a common cause of concern for laboratories. Often non-preheated tubes were used (40%) and samples were brought to the laboratory by hand-held transport (19%). When adequate temperature control cannot be ensured during sample collection and transport, then patients should be required to come to the laboratory for sample collection to avoid false-negative results. In many laboratories (47%) the sample was centrifuged without maintaining the temperature at 37 [degrees]C, most likely because of a lack of a heated centrifuge. When no preheated centrifuge is available, cells should be allowed to sediment at 37 [degrees]C, and serum should be separated at 37 [degrees]C without centrifugation.


After serum separation, most laboratories (66%) allowed the sample to cryoprecipitate at different temperatures. Although most authors report performing cryoprecipitation only at 4 [degrees]C, some authors find that cryoprecipitation at different temperatures has merit (10). The duration of cryoprecipitation at 4 [degrees]C varied from 12 h to 9 days (median 3 days). Although monoclonal immunoglobulin cryoprecipitates (type I) usually appear within 24 h, mixed cryoglobulins may appear only after several days (1, 9). A minimum incubation period of at least 3 days (9) to preferably 7 days (1, 6, 12) is required to avoid missing a diagnosis of mixed cryoglobulinemia.

All laboratories performed visual observation of the cryoprecipitate. Most laboratories (72%) compared the same sample at different temperatures and approximately 1 in 3 laboratories (32%) compared serum and plasma. After cryoprecipitation, 60% of laboratories washed the sample, 81% resolubilized the cryoprecipitate at 37 [degrees]C, and 19% concentrated the sample before further analysis. The number of washes ranged from 1 to 5, with a median of 3. Washing the cryoprecipitate and checking the resolubility at 37 [degrees]C is recommended to avoid contamination from precipitation of normal serum proteins (6).

Seven laboratories that refer positive samples for confirmation were excluded from further analysis (Fig. 1).


There was wide variation in the type of results reported, ranging from a simple qualitative negative/positive result to a quantitative report including cryoglobulin type and total protein content and/or immunoglobulin concentration in the cryoprecipitate. Of the 127 laboratories included for the postidentification phase, 50 (39%) reported the cryocrit, the total protein content, and/or the immunoglobulin concentration in the cryoprecipitate (quantitative); 48 (38%) answered negative/ positive only; and 29 (23%) provided additional information without reporting the cryoglobulin concentration in the cryoprecipitate (nonquantitative).

Forty-six of the 50 laboratories that provided a quantitative report and 55 of the 77 laboratories that provided a nonquantitative report performed immunofixation analysis on the cryoprecipitate. Of the 101 laboratories (57%) that further immunotyped the cryoprecipitate, 98 performed immunofixation or immunoelectrophoresis, and 3 used protein electrophoresis only.


For type I cryoglobulins, which are often present at concentrations >5 g/L, (1), reporting the presence or absence of an important visual cryoprecipitate that can be resolubilized is acceptable. For mixed cryoglobulins, however, which constitute >90% of the total cryoglobulins and are typically observed in chronic inflammatory conditions (6), further analysis and quantification is important to improve sensitivity and specificity, because visual inspection of mixed cryoglobulins at low concentrations lacks accuracy for detection and quantification. Diagnosis of low concentrations of mixed cryoglobulins is important because there is, in general, no relationship between the cryoglobulin concentration and the severity of the symptoms (2, 13).


Of the 11 laboratories that provided reference values, only 3 (2%) applied cryoprecipitate-specific reference values, whereas the other 8 used reference values for serum proteins and/or immunoglobulins, which are not valid for the cryoprecipitate. Three laboratories that had cryoprecipitate-specific reference values provided different reference values using total protein content: 0-10 mg/L serum (no reference provided), 0-50 mg/L serum (no reference provided), and 0-80 mg/L (12). None of the participating laboratories provided cryoprecipitate-specific reference values as immunoglobulin concentrations.

The lack of formal cryoprecipitate-specific reference values hampers the direct quantification of cryoglobulins, because many healthy individuals also have detectable amounts of cryoglobulins (2, 6). Although several authors have looked at total protein content in the cryoprecipitate to establish reference values, (12, 14, 15), only 3 laboratories (2%) provided cryoprecipitate-specific reference values for total protein content. This result could be related to the lack of standardization of cryoglobulins detection, variation in the suggested reference values, methodological problems (e.g., young healthy controls instead of diseased controls), or the lack of hepatitis C (HCV) serology. There is an association between essential cryoglobulinemia and HCV infection (5).

Total protein content and cryocrit are only indirect measures of the cryoglobulin concentration because they also detect the presence of other proteins such as albumin and fibrinogen. When cryocrit is determined without washing and resolubilizing the precipitate, particular care has to be taken when interpreting the results (6). Direct quantification of immunoglobulins in the cryoprecipitate could overcome this problem, but no cryoprecipitatespecific reference values exist for immunoglobulins.


The results of our questionnaire underline the need for standardization of the detection, analysis, and reporting of cryoglobulins. To avoid missing a diagnosis of monoclonal cryoglobulins, laboratories should ensure that the sample temperature does not drop below 37 [degrees]C during the preanalytical phase. For the diagnosis of mixed cryoglobulins, serum should be allowed to cryoprecipitate at 4 [degrees]C for at least 3 to preferably 7 days, the cryoprecipitate should be washed and resolubilized at 37 [degrees]C, and the cryoglobulin should be quantified to improve accuracy and allow comparison of results between laboratories. Because cryocrit and total protein are only indirect measures of the cryo-immunoglobulin concentration, further research is warranted to provide reference values for cryo-immunoglobulins.

Grant/funding Support: None declared.

Financial Disclosures: None declared.

Received April 11,2007; accepted October 10, 2007.

Previously published online at DOI: 10.1373/clinchem.2007.090134


(1.) Gorevic PD, Galanakis D. Cryoglobulins, cryofibrinogenemia and pyroglobulins. In: Detrick B, Hamilton RG, Folds JD, eds. Manual of molecular and clinical laboratory immunology. 7th ed: ASM Press, 2006:101-11.

(2.) Shihabi ZK. Cryoglobulins: an important but neglected clinical test. Ann Clin Lab Sci 2006; 36:395-408.

(3.) Keren DF, Alexanian R, Goeken JA, Gorevic PD, Kyle RA, Tomar RH. Guidelines for clinical and laboratory evaluation patients with monoclonal gammopathies. Arch Pathol Lab Med 1999; 123:106-7.

(4.) Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, et al. Nomenclature of systemic vasculitides: proposal of an international consensus conference. Arthritis Rheum 1994;37:187-92.

(5.) Ferri C, Zignego AL, Pileri SA. Cryoglobulins. J Clin Pathol 2002;55:4 -13.

(6.) Kallemuchikkal U, Gorevic PD. Evaluation of cryoglobulins. Arch Pathol Lab Med 1999;123: 119-25.

(7.) Kay J, McCluskey RT. Case records of the Massachusetts General Hospital. Case 31-2005: a 60-year-old man with skin lesions and renal insufficiency. N Engl J Med 2005;353:1605-13.

(8.) Bakker AJ, Slomp J, de Vries T, Boymans DA, Veldhuis B, Halma K, Joosten P. Adequate sampling in cryoglobulinaemia: recommended warmly. Clin Chem Lab Med 2003;41:85-9.

(9.) Foerster J. Cryoglobulins and cryoglobulinemia. Wintrobe's clinical hematology. 10th ed. 2000; 2725-37.

(10.) Dammacco F, Sansonno D, Piccoli C, Tucci FA, Racanelli V. The cryoglobulins: an overview. Eur J Clin Invest 2001;31:628 -38.

(11.) Letendre L, Kyle RA. Monoclonal cryoglobulinemia with high thermal insolubility. Mayo Clin Proc 1982;57:629-33.

(12.) Cream JJ. Cryoglobulins in vasculitis. Clin Exp Immunol 1972;10:117-26.

(13.) Gorevic PD, Kassab HJ, Levo Y, Kohn R, Meltzer M, Prose P, Franklin EC. Mixed cryoglobulinemia: clinical aspects and long-term follow-up of 40 patients. Am J Med 1980;69:287-308.

(14.) Romaszko JP, Tridon A, Jouanel P, Subtil E, Dibet P, Betail G. Detection et analyse des cryoglobulines: etude comparative d'une population de malades et une population de temoins sains. Pathol Biol (Paris) 1993;41:525-9.

(15.) Mola EM, Gharavi AE, Joshi VR, Patel BM, Colaco CB, Hughes GR. Enhancement of cryoprecipitation from hypotonic serum: a comparative study in normals and in patients with connective tissue diseases. Clin Exp Rheumatol 1983;1:35-40.

Pieter Vermeersch, [1] Koenraad Gijbels, [1,2] Godelieve Marien, [1] Rod Lunn, [3] William Egner, [3] Peter White, [3] and Xavier Bossuyt [1] *

[1] Department of Laboratory Medicine, Immunology, University Hospitals Leuven, Catholic University of Leuven, Belgium; [2] Current address: Department of Laboratory Medicine, Ziekenhuis Oost-Malle, Zoersel, Belgium; [3] United Kingdom National External Quality Assessment Service (UKNEQAS), Department of Immunology, Sheffield, United Kingdom.

* Address correspondence to this author at: Laboratory Medicine, Immunology, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium. Fax 0032-16-34-79-31; e-mail
Table 1. Temperature precautions in the preanalytical phase
(137 laboratories).

 Preheating and
 Transport at transport at 37
 Preheating 37 [degrees]C [degrees]C
 (n = 84) (n = 108) (n = 73)

Sedimentation 37 12 12 11
 [degrees]C (n = 15)
Centrifugation 37 40 49 37
 [degrees]C (n = 56)
Centrifugation at room 32 46 25
 temperature (n = 65)
Heparin tube only 0 1 0
 (n = 1)
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Article Details
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Title Annotation:Opinion
Author:Vermeersch, Pieter; Gijbels, Koenraad; Marien, Godelieve; Lunn, Rod; Egner, William; White, Peter; B
Publication:Clinical Chemistry
Article Type:Survey
Date:Jan 1, 2008
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