Research ArticleComparison between the Traditional and a Rapid Screening Testfor Cryoimmunoglobulins Detection
Federica Romitelli,1 Leopoldo Paolo Pucillo,2 Umberto Basile,3 and Enrico Di Stasio1
1 Institute of Biochemistry and Clinical Biochemistry, Catholic University of Sacred Heart, Largo Francesco Vito 1, 00168 Rome, Italy2I.N.M.I. L. Spallanzani I.R.C.C.S. Laboratorio di Biochimica Clinica e Farmacologia, Via Portuense 292, 00149 Rome, Italy3Department of Laboratory Medicine, Catholic University of Sacred Heart, Largo Francesco Vito 1, 00168 Rome, Italy
Correspondence should be addressed to Enrico Di Stasio; firstname.lastname@example.org
Received 8 November 2014; Accepted 3 January 2015
Academic Editor: Norberto Ortego-Centeno
Copyright 2015 Federica Romitelli et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
Objectives. A new rapid, automatic, and sensitive screening test useful to detect cryoglobulins in serum samples is proposed.DesignandMethods.The increase of turbidity during the cryoglobulin aggregationwasmonitored spectrophotometrically in sera from 400patients with clinical evidence of cryoglobulinemia related disorders and 100 controls. Results were correlated to those obtained bythe traditional method. Results. Kinetics of the aggregation curves were described by their maximum turbidity increase, lag time,and slope. Despite a partial correspondence between the traditional and the rapid test, patients with symptomatic cryoglobulinemiashowed turbidity values significantly higher than the determined cutoff. Moreover, a functional classification of cryoglobulins isproposed. Conclusions. Due to its high reproducibility, operator independence, low cost, and results obtained within 2 hours, therapid test can be used as a real time monitoring of cryoglobulinemia related diseases and for the evaluation of plasmapheresisefficacy.
Cryoglobulins are immunoglobulins that precipitate at tem-perature below 37C and redissolve on rewarming .The interest in these proteins has been growing becauseof their association with a number of diseases, includinglymphoproliferative and autoimmune disorders and hepatitisC virus (HCV) infection [1, 510].
Immunochemical characterization of cryoglobulins hasled to their classification into three major groups . TypeI represents individual monoclonal immunoglobulins, gen-erally associated with lymphoproliferative diseases; type IIconsists of mixed immunoglobulins with a monoclonal com-ponent and is strongly associated with HCV infection; typeIII is constituted by amixture of polyclonal immunoglobulinsand is associated mainly with a wide range of infectious,autoimmune, and liver diseases.
Multiple but still poorly understood physiopathologicalmechanisms have been hypothesized for the comprehensionof the cellular and molecular events leading to the clinical
expression of cryoglobulinemic syndrome [9, 1215]. Inparticular, it has been proposed that cryoprecipitation occursbecause of the rapid formation of cold-insoluble IgM-IgGimmune complexes [16, 17] or simply by a decreasing solubil-ity phenomenon, resulting from an unfavorable interactionbetween cryoglobulins and solvent at low temperatures .
Stating that blood collection and transport proceduresfor cryoglobulin screening test remain a critical issue, alsothe methods used in clinical practice for the quantificationof cryoglobulins are not standardized. Traditionally, cryopre-cipitation is detected in vitro by incubating serum samplesat low temperatures (4C) for 2 to 15 days and the result ofthe assay is reported as a cryocrit, which is the percent of theprecipitate in respect to the total serumvolume .However,different span time for sample inspection and poor sensitivityin appreciating low levels of cryoglobulins constitutes a severelimitation to the standardization and clinical interpretation ofquantitative results in clinical practice .
In this paper we propose an alternative fast and eas-ily reliable method for cryoglobulins detection in serum
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 783063, 6 pageshttp://dx.doi.org/10.1155/2015/783063
2 BioMed Research International
samples based on light scattering (turbidity). In respect tothe traditional, time-consuming method, the performanceof this rapid assay allows following the full kinetic processof cryoglobulin aggregation, before precipitation occurs. Acomparative analysis between the traditional and the alter-native screening test was performed on suspected cryoglobu-linemic and noncryoglobulinemic sera, in order to evaluatethe diagnostic sensitivity and specificity, as well as the advan-tages and the limitations of the two assays in clinical practice.Moreover, the kinetic analysis of the cryoaggregation processmay provide a first attempt to a functional characterization ofcryoglobulins, based on the comprehension of physiopatho-logical mechanisms leading to cryoprecipitation.
2. Materials and Methods
2.1. Samples. Four hundred serum samples from patientswith clinical evidence of different disorders related to cryo-globulinemia were studied by both methods. In particular,studieswere conducted in parallel on sera, drawn for diagnos-tic purposes, from 100 patients affected by type I cryoglob-ulinemia related diseases (monoclonal gammopathies andWaldenstroms macroglobulinemia), 250 subjects suspectedof having cryoglobulinemia secondary toHCV infection, and50 patients with autoimmune diseases (Sjogrens syndrome,rheumatoid arthritis). All patients were asymptomatic, withthe exception of 15 subjects who had clinical signs of acutecryoglobulinemic syndrome. Moreover one hundred serafrom healthy volunteers served as control group. The studywas conducted according to the principles of the Helsinkideclaration after approval by the local institution reviewboard and written informed consent was obtained from allsubjects.
2.2. Blood Sampling. Blood was drawn by venipuncture inVacutainer tubes prewarmed at 37C and left to clot for 2hours at the same temperature; the serum was separated bycentrifugation (2500g for 10min) at 37C. An aliquot ofeach sample (1800 L) was immediately tested with the rapidscreening assay and the remaining volume dispensed in agraduated Wintrobe tube for the traditional assay (time 0).
2.3. Cryoglobulins Determination by the Traditional Method.The presence of precipitate in the samples was determinedby visual inspection, before centrifugation, after 1, 3, 7,and 15 days of cold incubation (4C) and was expressedas percentage of the volume occupied by the precipitatedproteins compared to the total volume of serum. Before thefinal quantification, a subjective scoring of cryoprecipitatewas adopted: absence of cryoprecipitate was scored as while cryoprecipitate5%,wasscored as +, ++, and +++, respectively. On the 15th dayof cold incubation, samples were centrifuged at 2500g for10min at 4C and the amount of cryoglobulins was estimatedas cryocrit %, determined as the percentage of the volumeoccupied by the cryoprecipitate compared to the total volumeof serum, after centrifugation occurred . According tothe cryocrit values obtained, samples were divided into three
classes: samples with cryocrit values higher than 1.0%, withcryocrit values between 0.5 and 1.0%, and with absence ofcryoglobulins.
2.4. Cryoglobulins Determination by the Rapid Screening Test.The rapid method for cryoglobulins determination is basedon the detection of the scattered light by turbidimetry .Measurements were performed using a Cary3 dual-beamspectrophotometer (Varian Australia Pty. Ltd., Mulgrave,Australia) on samples contained in a square cell (10mmpath length), whose temperature was maintained at 10C.This temperature was chosen to prevent the misting of thecuvette walls in instruments without nitrogen flow and thetemperature dependence of the aggregation curve is reportedin a previous paper . Briefly, an aliquot (1800 L) ofserum samples, collected as described inBlood Sampling, wasdispensed in a cuvette in a spectrophotometer, the systemwasblanked, and cryoglobulins aggregationwas recorded readingthe absorbance values at 350 nm, until precipitation occurred.
Three parameters were used to describe the evolution ofthe aggregation process over time: (1) max, the maximumabsorbance value, directly related to the cryoglobulin concen-tration ; (2) the maximum slope (/) of the aggrega-tion curve; (3) the lag time (
0) obtained from extrapolation
of the maximum slope to the absorbance reading 0.
According to the max values, indicative of the quanti-tative measurement of cryoglobulins, samples were dividedinto three groups on the basis of percentile distribution in thecontrol sera: the first group comprehended samples showingmax values 0.1 absorbance units (94% of control sera); thesecond group assembled samples with max values rangingfrom 0.1 to 0.2 absorbance units (5% of control sera); finally,samples showing max values >0.2 absorbance units wereassigned to the third group (1% of control sera). On the basisof a rigorous application of a 95% confidence interval, 0.1 isthe cutoff for positivity of the test.
In order to determine the diagnostic accuracy of rapidand traditional test for the presence of clinical symptomsof cryoglobulinemic syndrome we analyzed the ROC curvesand calculated area under curves (AUCs). Cutoff values weredetermined at 90% sensitivity criterion derived directly fromthe ROC curves.
3.1. Results of the Rapid and Traditional Test as a Functionof the Visual Inspection of Cryocrit at Different Days ofIncubation before Cen