Cryoglobulins are immunoglobulins that precipitate at temperatures lower than 37°C. These cryoglobulins deposit in medium to small blood vessels, causing injury to the endothelium and leading to end-organ damage. The pre-analytical phase is important for optimal cryoglobulin testing. However, laboratories often are challenged to maintain 37°C during key preanalytical phases such as blood collection, centrifugation, and specimen transport1, 2. Literature suggests various strategies to maintain the temperature of 37°C during these phases, including heel warmers applied to the collection site, using water or sand baths, and dry incubators to maintain specimen temperature at 37 °C1, 2.
However, there is no practical guideline among the lab medicine community on handling/implementation of the cryoglobulin testing process. This scientific short will provide suggestions on cryoglobulin testing based on our experience.
- Collection/transportation/processing of serum samples: Serum sample is preferred to be collected in plain red top tubes (no additives) rather than serum separator tubes (SST) containing gel. Immunoglobulins may adsorb to the gel in SST, leading to false negative results.
- Our institution adopted the strategy of maintaining the temperature of red top tubes and other collection supplies with gel packs and thermos flasks that are kept at 37°C at all times. This is achieved by storing the gel and thermos flasks in a 37°C incubator.
- Clinical labs often face challenges to maintain robust pre-analytical processing of cryoglobulin samples collected apart from the receiving laboratory due to the need for heated centrifuges that can maintain temperature at 37°C, as well as maintaining temperature during transport. Indeed, blood samples must be maintained at 37°C until the serum is removed from cells to reduce the presence of false negatives. We validated our transportation process using gel packs and thermos flasks by determining the rate of temperature decline per hour. This validation included different scenarios such as opening of the thermos by lab staff/courier and immediately closing the lid, opening of thermos and failure to close the lid properly, and accidental dropping of thermos flask during transportation. In general, the flask and gel maintained temperature for 4 hours when gel packs stored at 37 °C were transported in thermos flasks. Processing: Blood samples should be centrifuged at 37°C at 2000-2500 g for 10 minutes3. Provided the serum is removed while maintaining the 37°C during processing the samples are ready for cryoglobulin analysis.
- Reporting elements:
- Positive/negative: Previous studies report that the presence of cryoglobulin should be checked up to 7 days4 due to smaller cryoprecipitate formation in Type II and Type III cryoglobulinemia (based on the Brouet’s classification). Prior to starting cryoglobulin testing the specimen must be at 37°C for 24 hours followed by splitting the serum. The performing laboratory needs to check each sample daily to look for presence of precipitate in the specimen at 4°C relative to normal patient matched serum everyday for precipitate formation. On day 7, one of the red top tubes should be kept in the incubator at 37°C and another at 4°C for final reporting.
- Cryocrit %: We report cryocrit% threshold ≥1% by centrifuging the serum sample at 4°C and performing the calculation using formula: volume of precipitate/volume of serum x100.
- Immunotyping: the cryoprecipitate is washed 3 times with phosphate buffer saline (PBS) and is re-suspended in PBS. Samples should be kept at 37°C until the time of loading in gel for running immunofixation. Type I cryoglobulins show monoclonal proteins while type II and type III cryoglobulins have polyclonal immunoglobulins5. Utilization: Our discussion with stakeholders indicated that typing cryoglobulins is crucial at the time of diagnosis but does not require frequent clone typing for monitoring. Knowing the type of cryoglobulin early on can reduce the time for treatment decision while waiting for the final confirmation by biopsy. Hence, one of the strategies that could be employed by clinical labs is to build two tests (diagnostic and monitoring) for cryoglobulins testing in laboratory information system with only diagnostic offering immunofixation. Alternatively, delta checks could be performed to check for previous positive specimens. Cryoglobulin test interpretation should be performed by well-trained staff and signed out similar to serum protein electrophoresis at your institution.
References
- Sargur R, White P, Egner W. Cryoglobulin evaluation: best practice? Ann Clin Biochem: Int J Lab Med. 2010; 47 (1): 8-16.
- Motyckova G, Murali M. Laboratory Testing for Cryoglobulins. Am J Hematology. 2011; 86: 500-502.
- Romitelli F, Pucillo LP, Basile U, Stasio ED. Comparison between the Traditional and a Rapid Screening Test for Cryoimmunoglobulins Detection. Biomed Res Int. 2015; 26; 1-6.
- Kolopp-Sarda MN, Nombel A, Miossec P. Cryoglobulins Today: Detection and Immunologic Characteristics of 1,675 Positive Samples From 13,439 Patients Obtained Over Six Years. Arthritis Rheumatol. 2019 Nov;71(11):1904-1912.
- Napodano C, Gulli F, Rapaccini GL, Marino M, Basile U. Cryoglobulins: Identification, Classification, and novel biomarkers of mysterious proteins. Adv Clin Chem. 2020 31; 104-299.
