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Analytical considerations in the investigation of mixed cryoglobulinemia.

To the Editor:

Schnabl et al. (1) recently presented a clinical case study of a patient with mixed cryoglobulinemia. It is important that such cases receive attention because cryoglobulinemia is often overlooked in diagnostic workups; however, several technical points regarding the analytical aspects of investigation require clarification.

In their protocol for the investigation of cryoglobulins, the authors advocate the use of the "cryocrit" as a means of quantifying and typing the cryoglobulin in question. Their comments about "cryocrit" are, in fact, confusing. In their Fig. 1, they indicate that the cryocrit should be performed on "washed precipitate," but in the Discussion they state that the cryocrit is the "(precipitate volume)/ (total volume of serum sample)." It is not clear whether they are recommending centrifugation of the washed precipitate or the original chilled serum sample. Cryocrit is at best a crude semiquantitative method to estimate the amount of cryoprotein present. Standardization and QC procedures are lacking.

Native cryoprecipitate contains both coprecipitants and contaminants, including other serum proteins, viral particles, and bacteria (2). Washing in a cold buffered saline solution may reduce the contamination, but it will also cause the loss of a variable amount of cryoglobulin. Given that most mixed cryoglobulins are present only in milligram concentrations, quantification by this method is unlikely to yield accurate results.

From a theoretical standpoint, the use of the cryocrit is rather dubious because it assumes that different cryoproteins pack and sediment with equal volumes. In particular, experience at this institution suggests that cryogels have very unpredictable sedimentation characteristics. The cryocrit is highly dependent on the speed and duration of centrifugation. Packing oft he gel after centrifugation is often irregular, with a variable volume of serum being retained around its surface, giving rise to difficulties in washing and necessitating careful manipulation.

Brouet et al. classified cryoglobulins into 3 main types, depending on their immunologic composition (3). Type I cryoglobulins are single monoclonal immunoglobulins (usually IgM or IgG), whereas type II and type III (mixed) cryoglobulins are immune complexes consisting of either monoclonal or polyclonal IgM, respectively, directed against polyclonal IgG.

The authors state that a "diffuse [gamma] region visible after protein electrophoresis ... suggested the presence of polyclonal [gamma]-globulins." The clonality of cryoglobulins can only be determined immunochemically. It is unclear, therefore, whether the patient's cryoglobulin was definitively "typed." The absence of an "M band" upon protein electrophoresis does not exclude the possibility of a monoclonal component being present. Furthermore, the inclusion of the cryocrit in Fig. 1 as a determination of the "type" is also misleading.

It is important to emphasize that the aim of typing is not solely to distinguish purely monoclonal from mixed cryoglobulinemia (i.e., type I from type II/III). Differentiating type II from type III has important prognostic implications, because a considerable number of patients with type II cryoglobulinemia may eventually develop non-Hodgkin lymphoma or other lymphatic/hepatic malignancies (4).

Also to be emphasized is that the cryoglobulin concentration is not an index of disease severity; low concentrations of mixed cryoglobulin may be associated with life-threatening presentations, whereas patients with relatively high concentrations can be asymptomatic. Other investigators have reported an apparent inverse relationship between the cryocrit and vasculitis (2). This situation is further complicated by the fact that many healthy individuals have detectable amounts of cryoglobulins; therefore, detection is not necessarily indicative of disease and needs to be interpreted in the specific clinical context of each case.

Overall, it is questionable whether quantification by such crude methods adds value to the diagnostic investigation, because there is no evidence that it informs clinical decision-making or patient management. Despite this fact, these crude quantitative measurements remain firmly entrenched in current practice (5).

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

References

(1.) Schnabl KL, Sibbald M, Gold WL, Chan PC, Adeli K. A patient with a leg rash, pedal edema, renal failure, and thrombocytopenia. Clin Chem 2009; 55:1419-24.

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

(3.) Brouet JC, Clauvel JP, Danon F, Klein M, Seligmann M. Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 1974;57:775-88.

(4.) Ferri C. Mixed cryoglobulinemia. Orphanet J Rare Dis 2008;3:25.

(5.) Vermeersch P, Gijbels K, Marien G, Lunn R, Egner W, White P, Bossuyt X. A critical appraisal of current practice in the detection, analysis, and reporting of cryoglobulins. Clin Chem 2008;54: 39-43.

Edward R. Smith

Brighton & Sussex University Hospitals NHS Trust Royal Sussex County Hospital Brighton, UK

Address correspondence to the author at: Department of Clinical Biochemistry Royal Sussex County Hospital Eastern Road Brighton BN2 5BE, UK Fax 01273-664828 E-mail edward.smith@bsuh.nhs.uk

Previously published online at DOI: 10.1373/clinchem.2009.134502

In Reply

E. Smith has pointed out that the use of cryocrit in the investigation of cryoglobulinemia has analytical and clinical limitations, an observation with which we concur in general. The emphasis of our case study, however, was not to make a judgment on which technique should be used, but rather to focus on the importance of including cryoglobulin analysis in the differential and to address the need for standardization between laboratories. We reported a schema of sample collection and analysis that merely reflects a practice that many laboratories still conduct today. Vermeersch et al. reported that up to 37% of the 140 surveyed laboratories included cryocrit or other estimates (e.g., total protein) in their patient reports (1). The lack of standardization of practice appears, at least in part, to be related to a difference in opinion on the usefulness of estimating cryoglobulin quantities. Although some investigators reported no relationship between cryoglobulin concentration and the severity of symptoms and disease activity (2), cryoglobulin concentrations have, nevertheless, been found to correlate with response to treatment with plasmapheresis, cytotoxic agents, and/or interferon a (3). Moreover, we did indicate in our report that cryocrit does not differentiate type 1 and type 2 cryoglobulinemia, and we recommended that serum protein electrophoresis and immunofixation should be conducted on resolubilized cryoprecipitate (4). Certainly, laboratories that do not have specialized equipment or a test in place to screen for cryoglobulins should consider at minimum estimating the cryocrit.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

References

(1.) Vermeersch P, Gijbels K, Marien G, Lunn R, Egner W, White P, Bossuyt X. A critical appraisal of current practice in the detection, analysis and reporting of cryoglobulins. Clin Chem 2008; 54:39-43.

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

(3.) Geltner D, Kohn R, Gorevic PD, Franklin EC. The effect of combination therapy on 5 mixed cryoglobulinemia patients with renal, neurological and vascular involvement. Arthritis Rheumat 1981;24:1121.

(4.) Schnabl KL, Sibbald M, Gold WL, Chan PC, Adeli K. A patient with a leg rash, pedal edema, renal failure and thrombocytopenia. Clin Chem 2009;55:1419-24.

Kareena L. Schnabl

P. Cheung Chan

Khosrow Adeli *

The Hospital for Sick Children and Sunnybrook Health Sciences Centre University of Toronto Toronto, Ontario, Canada

* Address correspondence to this author at: Hospital for Sick Children 555 University Avenue Toronto, Canada M5G1X8 Fax416-8136257 E-mail khosrow.adeli@sickkids.ca

Previously published online at DOI: 10.1373/clinchem.2009.137109
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Title Annotation:Letters to the Editor
Author:Smith, Edward R.
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Jan 1, 2010
Words:1393
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