Test of the Month
Laboratory testing for cryoglobulins†
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Cryoglobulins are immunoglobulins that precipitate below 37°C and can cause multiorgan damage. There are three types of cryoglobulins: Type I (also called simple), which is mostly associated with monoclonal gammopathy and/or other hematologic disorders and Type II and Type III (known as mixed cryoglobulins), which are associated with infectious and systemic diseases. Testing for cryoglobulins is complicated by lack of reference range, standards, and stringency in maintaining testing temperature conditions. Identification of cryoprecipitate can be critical for patient care; therefore, correct testing conditions are crucial for reliable cryoglobulin testing. The patient's blood sample should be kept at 37°C initially to avoid premature precipitation of cryoglobulins and thereby decreasing the yield for subsequent identification. This could cause a false negative result. After warm centrifugation or warm cell precipitation, the clear serum is observed at 4°C for formation of cryoprecipitate. The cryoprecipitate is then washed in cold buffer, and the resulting precipitate is warmed to 37°C and subjected to further analysis by immunodiffusion and immunofixation. In addition to Meltzer's triad of purpura, weakness and arthralgias, cryoglobulinemias have protean manifestations involving skin, joints, kidney, nervous system, as well as the hematopoietic system. The management of cryoglobulinemia especially in patients with organ damage remains difficult. Treatment of cryoglobulinemia focuses on management of the underlying lymphoproliferative disorder or infectious or systemic causes. Medical management may also include corticosteroids and other immunosuppressive agents and plasmapheresis. Rituximab therapy seems to abrogate the aberrant B cell response. Am. J.Hematol. 86:500–502, 2011. © 2011 Wiley-Liss, Inc.
Cryoglobulins are immunoglobulins which precipitate below 37°C from patient's serum and redissolve when rewarmed in vitro [1, 2]. They were first observed in 1933 by Wintrobe and Buell  in a patient with Raynaud's phenomenon. They were called cryoglobulins (cold precipitable serum globulin) in 1947 . In the 1960s, a clinical profile of palpable purpura, asthenia, arthralgia, and renal disease was recognized, caused by immune complex deposition and cryoglobulinemia . There are three types of cryoglobulins depending on their characteristics and interactions with other proteins (Table I) .
Table I. Cryoglobulin Classification
|Type I (simple)||Monoclonal immunoglobulins (IgG, IgM, or IgA) or Bence Jones protein/monoclonal free light chains||Waldenstrom's macroglobulinemia|
|Monoclonal gammopathy associated with lymphoproliferative disorder|
|Light chain disease|
|Type II (mixed)||Monoclonal immunoglobulins (IgM, IgG, or IgA) and polyclonal immunoglobulin (mostly IgG)||Hepatitis C|
|Chronic lymphocytic leukemia|
|Type III (mixed)||Polyclonal immunoglobulins of all isotypes||Essential cryoglobulinemia|
|Systemic lupus erythematosus|
|Viral infections (Hepatitis B and C, CMV, EBV, HIV)|
|Endocarditis, other bacterial infections|
The laboratory workup of suspected cryoglobulin should be performed in patients with unexplained multiorgan disease involvement such as skin, liver, renal, peripheral nerve manifestations, Raynaud's syndrome, and in patients with the typical triad (first described by Meltzer and hence known as Meltzer's triad) of purpura, weakness, and arthralgias .
Monoclonal cryoglobulinemia (Type I) is often seen in patients with hematologic malignancies and can cause hyperviscosity syndrome. Mixed cryoglobulinemia (Types II and III) often presents as a systemic vasculitis due to immune complex deposition in the small vessels and may be the presenting feature in patients with infectious and systemic disorders . Patients may present with palpable purpura, weakness, arthralgia as well as liver and renal involvement, peripheral neuropathy, sicca syndrome, Raynaud's phenomenon, and B cell non-Hodgkin lymphoma . Around 92% of patients (range 70–100% depending on patient population) with mixed cryoglobulinemia have Hepatitis C infection (HCV) versus 1.8% having Hepatitis B [8, 9]. Conversely, about 40–66% of patients with HCV have elevated circulating monoclonal and/or polyclonal immunoglobulins. The clinical syndrome of cryoglobulinemia is manifested in only about 5% of HCV patients . Among the Type III cryoglobulins, the mixed cryoglobulinemia can be associated with organ involvement and small and medium size vessel vasculitis and is again seen in infectious and rheumatologic diseases . Table II summarizes salient clinical and laboratory features of the cryoglobulinemia syndromes.
Table II. Clinical and Laboratory Features of Cryoglobulinemia
|Arthralgia ≫ arthritis||+||++||+++|
|Purpura||+, usually nonpalpable||+++, palpable||+++, palpable|
|Cryocrit||>5%||<5% (1–2)||<5% (1–2)|
|Autoantibodies (ANA, ENA, AMA, etc.)||−||++||++|
Laboratory Testing for Cryoglobulins
For cryoglobulin identification, two 10 mL red top tubes of sample (without an anticoagulant) are drawn from a patient and transported to the laboratory at 37°C (commonly, this is achieved by submerging the tubes in warm water during transportation to the laboratory). The sample is allowed to clot at 37°C for 1 hr. The clot is separated from the sides of the tube with a Pasteur pipette, and the serum is separated by warm (37°C) centrifugation and aliquoted into three tubes at 37°C. One tube is the Wintrobe tube for quantification of the cryoprecipitate (a Wintrobe tube has markings that are used to quantify). The second tube contains a larger quantity of serum, which is used for cryoprecipitate observation and subsequent analysis. The third tube is used to determine the solubility of the cryoprecipitate on rewarming to 37°C, a feature of cryoglobulin. If a warm centrifuge is not available, serum should be separated in a 37°C water bath after allowing cells to clot and settle at 37°C for an hour without centrifugation, being careful to ensure that the red cells and fibrin clot are not aspirated with the serum. It is important for the sample temperature not to drop below 37°C until serum separation is complete to avoid premature precipitation. Improper sample handling may result in a missed diagnosis of cryoglobulinemia . Increasing concentration of cryoglobulins increases the temperature at which the precipitate forms. The three serum containing tubes are then kept at 4°C and analyzed at 72 hr. Type I cryoprecipitate may appear as early as 24 hr, but mixed cryoglobulins may precipitate after several days; therefore, some laboratories wait until 7 days if there is no precipitate formed initially.
After visual observation, the precipitate formed at 4°C can be reported in one of three ways: (1) cryocrit (percent of total volume), (2) protein content, or (3) immunoglobulin content . There is no widely accepted reference range for cryoglobulins. Using protein content for reference values is complicated by wide variation of total protein content in cryoprecipitate possibly due to coprecipitation of other proteins. Reference values for individual immunoglobulins in the cryoprecipitate have been described , but cryoprecipitate protein and immunoglobulin contents are rarely reported by clinical laboratories. The most practical and clinically useful are the cryocrit values in a longitudinal analysis of patient's clinical outcome.
If there is a precipitate formed at 72 hr at 4°C, the precipitate is washed and recovered in cold phosphate-buffered saline. Washing of the sample at 4°C facilitates removal of the contaminating normal serum proteins, thus allowing analysis of the cryoglobulin exclusively. The cryoprecipitate is solubilized at 37°C and analyzed by immunodiffusion and immunofixation to identify the cryoglobulin type. Many laboratories use antibodies to IgG, IgA, IgM, kappa or lambda as well as to C3 and C4 to identify the immunoreactants. The clonality is determined by immunofixation electrophoresis. Incorporating anti-albumin antibodies in the analysis serves to ensure quality of the washing of the cryoglobulins and confirmation of removal of contaminating serum proteins.
False negative results can occur due to the following reasons. (1) Improper handling of the patient's sample: If the sample is not transported at 37°C before processing, cryoprecipitate may form in transit during clotting of the sample, thereby decreasing the chance of cryoglobulin recovery. Thus, proper collection, optimum clotting, and centrifugation are essential to avoid false negative results. (2) Storage of the sample at a temperature higher than 4°C may impede or abrogate cryoprecipitate formation and contribute to false negative results. (3) Lipemia in a patient's sample causes serum turbidity due to lipids and may also interfere with correct cryoglobulin identification. Positive results of 1% or more of cryocrit are abnormal but in some cases even a minute amount of cryoglobulins may be pathogenic, therefore the immunophenotype as well as the clinical context are important in deciding the clinical relevance of cryoglobulin test results.
Difficulties with the current testing technique for cryoglobulins include the requirement of large sample volume (10–15 cm3) and the time to analysis of 3 or more days. Moreover, there are no standards and controls available for the cryoglobulin test, including no widely used reference ranges for cryocrit protein content or immunoglobulin content values [7, 10]. Visual inspection of cryoprecipitate is less accurate at low concentrations of cryoglobulins, therefore improving sensitivity and specificity of cryoglobulin testing is an important topic as in most cases the concentration of cryoglobulins do not have to directly correlate with intensity of symptoms . Standardized testing technique is important to isolate cryoglobulins which are present in low concentrations compared to normal serum protein . Interestingly, some healthy individuals can have detectable levels of cryoglobulins with an unknown significance. Given the variability of testing conditions used in different laboratories and the lack of test standards and reference values, further investigation into standardization of the cryoglobulin testing should be performed in the future. The biological importance and activity of cryoglobulins such as its ability to activate proinflammatory complement proteins needs to be defined as well.
Treatment of Cryoglobulinemia
Treatment of Type I cryoglobulinemia focuses on decreasing the formation of the cryoprecipitable monoclonal proteins. Prewarming blood transfusions, steroids, less commonly splenectomy (for idiopathic cases) and treatment of the underlying lymphoproliferative disorder are some of the treatment options. In patients with hyperviscosity, plasmapheresis and cytotoxic agents are useful. Treatment of Types II and III cryoglobulinemia includes treatment of infections such as HCV, steroids, other immunosuppressant agents, plasmapheresis (especially for patients with hyperviscosity syndrome), cytotoxic chemotherapy, and rituximab [12–17]. Plasmapheresis has a limited role but can be useful in the acute phase. Low antigen diet (avoidance of macromolecular antigen containing foods such as diary products, meats and eggs, and gluten) has been used in some patients, improving the function of the reticuloendothelial system thereby possibly improving clearance of immune complexes. Symptoms of polyarthritis may respond to steroids, hydroxychloroquine, or cyclosporine for more severe conditions . The treatment course for cryoglobulinemia is usually guided by improvement in symptoms, decrease in cryocrit, restoration of complement activity, and decrease in rheumatoid factor activity.