Cryoproteins

Cryoglobulins do not have a consensus reference range. ^[1] Serum cryoglobulins in most individuals are in low concentrations (100-300 mg/L) among the high concentrations (60,000-80,000 mg/L) of normal serum proteins.

The cryocrit (percentage of packed cryoglobulins referred to total serum after centrifugation at 4°C ^[2] ) is the most practical and clinically useful parameter to predict a patient’s clinical outcome; 1% or more of cryocrit is abnormal.

Cryocrit is an important parameter used to quantify cryoglobulins. It can be determined using two methods. First, the degree of sedimentation in the Wintrobe tube can be measured, or, second, the specific immunoglobulin concentration can be determined with protein electrophoresis.

Low measured quantities of cryoglobulins may be pathogenic. Therefore, it is important to always correlate the clinical findings in the patient with test results obtained.

Determining the immunophenotype of the cryoglobulins is essential to offer clues about the possible underlying diagnoses. This is done by analyzing cryoprecipitate solubilized at 37°C through the processes of immunodiffusion and immunofixation, which used antibodies that bind to immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) (kappa or lambda), C3, and C4. ^[1]

Cryoglobulinemia is classified based on cryoglobulin composition according to the Brouet classification, shown in the table below.

Table 1. Brouet Classification of Cryoglobulinemia. (Open Table in a new window)

Specimen: Blood

Container: Red-top tube, Wintrobe tube, 2 other tubes

Collection Method: Routine venipuncture

Refrigeration, heparin therapy, hemolysis, and lipemia can invalidate the specimen.

Technique

The authors start by collecting two 10-mL red-top tubes of blood (without an anticoagulant, since the sample is then allowed to clot during immersion in warm water). These samples are immersed in warm water and maintained at a temperature of 37°C during transportation to the laboratory. In the laboratory, the clotted sample will be separated from the side of the tube with a Pasteur pipette. The remaining serum is separated into 3 different tubes and maintained at 37°C.

The Wintrobe tube is used for the quantification of the cryoprecipitate. The second tube, which contains a larger quantity of serum, is useful for cryoprecipitate observation. Finally, the third tube is important for determining the solubility of the cryoprecipitate on rewarming to 37°C.

The 3 serum-containing tubes are then kept at 4°C and analyzed after 72 hours. ^[1] Type I cryoprecipitate may appear as early as 24 hours (at concentrations >5 mg/mL). Type III cryoglobulins may require 7 days to precipitate in a small sample (< 1 mg/mL). ^[1]

Description

Cryoglobulins are single or mixed immunoglobulins that undergo reversible precipitation at low temperatures and that arise from B-cell lymphoproliferative disorders. An immunoglobulin monomer is a "Y"-shaped molecule that consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds. ^[5]

Following precipitation after exposure to lower temperatures, cryoglobulin-containing immune complexes form and trigger a systemic inflammatory response.

Cryoglobulins preferentially target the kidney and skin. The reason why the kidney is a desired organ was explained by a study that showed that the most important electrolyte variable that controls the size and the rate of cryoprecipitate formation at both low temperature and 37°C is the chloride anion. The kidney is a vital organ in maintaining chloride homeostasis.

Indications/Applications

Mixed cryoglobulinemia is the most common documented extrahepatic manifestation of hepatitis C virus (HCV) infection. In patients infected with HCV, abnormal immune response to HCV leads to the formation of immune complexes of the mixed cryoglobulinemia variant. ^[4] However, clinicians should be aware that only 5%-10% of individuals with chronic HCV infection have clinically evident mixed cryoglobulinemia syndrome, so negative cryoglobulin results in these patients does not exclude the disease. ^{[6, 7]}

Patients with symptoms of hyperviscosity and thrombosis may require workup for type I cryoglobulinemia to evaluate for secondary causes of cryoglobulinemia. These have been listed in Table 1.

Patients with manifestations of renal immune-complex disease, commonly membranoproliferative glomerulonephritis, joint involvement, cutaneous vasculitis, and peripheral neuropathy, require workup for types II and III cryoglobulinemia. The Meltzer triad (purpura, arthralgia, weakness) ^[8] is seen in up to 25%-30% of patients with types II and III cryoglobulinemia. These patients may develop rapidly progressive renal failure if not treated.

Secondary causes of cryoglobulinemia and their association with specific types of cryoglobulinemia are listed in Table 1. ^[4]

Considerations

Some reasons for false-negative results include accident refrigeration of a sample or failing to transport a sample at 37°C.(2) Lipemia causes serum turbidity secondary to lipids, potentially interfering with correct cryoglobulin identification. ^[1]

False-positive results are common in patients on heparin therapy, so cryoglobulinemia should not be investigated during heparin treatment. ^[9] The reason for this is that there is a complex formation between heparin and fibronectin. Fibronectin is a cold insoluble protein. Therefore, the heparin-fibronectin complexes are cold precipitable and can be mistaken for cryoprecipitate formation during laboratory analysis. ^[9]

Interestingly, some healthy individuals can have detectable levels of cryoglobulins with an unknown significance. ^[1] When quantifying abnormal values for cryoglobulins, a positive result of 1% or more of cryocrit is abnormal. However, in some cases, even a minute amount of cryoglobulin may be pathogenic and be associated with severe symptoms. Therefore, clinicians must correlate the clinical findings with cryoglobulin test results. In the event that clinical suspicion is still very high despite undetectable cryoglobulin levels, measuring for the presence of rheumatoid factor and hypocomplementemia is predictive of the disease. ^[4]

There is a high incidence of detectable cryoglobulins in widely prevalent diseases such as HCV infection. Many other clinical conditions, as mentioned above, also require workup for cryoglobulinemia. It is clear that testing for cryoglobulins is underused in clinical practice ^[10] because of lack of standardized reference values. It may be very useful to work on developing more reliable testing methods and references as a new frontier in the near future. ^[10]

A study by Chang et al suggested that in HCV infection, mixed cryoglobulinemia may manifest as an attempt by the immune system to expel HCV. For example, the investigators noted that in HCV patients with baseline mixed cryoglobulinemia, HCV RNA positivity rates were higher but the HCV RNA levels themselves were lower. ^[11]