Research ArticleComparison between the Traditional and a Rapid Screening Testfor Cryoimmunoglobulins Detection

Federica Romitelli1 Leopoldo Paolo Pucillo2 Umberto Basile3 and Enrico Di Stasio1

1 Institute of Biochemistry and Clinical Biochemistry Catholic University of Sacred Heart Largo Francesco Vito 1 00168 Rome Italy2INMI ldquoL Spallanzanirdquo IRCCS 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 edistasiormunicattit

Received 8 November 2014 Accepted 3 January 2015

Academic Editor Norberto Ortego-Centeno

Copyright copy 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 proposedDesignandMethodsThe 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 timeand 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 ldquoreal timerdquo monitoring of cryoglobulinemia related diseases and for the evaluation of plasmapheresisefficacy

1 Introduction

Cryoglobulins are immunoglobulins that precipitate at tem-perature below 37∘C and redissolve on rewarming [1ndash4]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 5ndash10]

Immunochemical characterization of cryoglobulins hasled to their classification into three major groups [11] 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 infectiousautoimmune 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 12ndash15] 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 [18]

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 (4∘C) 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 [19]Howeverdifferent 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 [19ndash21]

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 pageshttpdxdoiorg1011552015783063

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 practiceMoreover 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

21 Samples Four hundred serum samples from patientswith clinical evidence of different disorders related to cryo-globulinemia were studied by both methods In particularstudieswere conducted in parallel on sera drawn for diagnos-tic purposes from 100 patients affected by type I cryoglob-ulinemia related diseases (monoclonal gammopathies andWaldenstromrsquos macroglobulinemia) 250 subjects suspectedof having cryoglobulinemia secondary toHCV infection and50 patients with autoimmune diseases (Sjogrenrsquos syndromerheumatoid 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

22 Blood Sampling Blood was drawn by venipuncture inVacutainer tubes prewarmed at 37∘C and left to clot for 2hours at the same temperature the serum was separated bycentrifugation (2500timesg for 10min) at 37∘C An aliquot ofeach sample (1800 120583L) was immediately tested with the rapidscreening assay and the remaining volume dispensed in agraduated Wintrobe tube for the traditional assay (time 0)

23 Cryoglobulins Determination by the Traditional MethodThe presence of precipitate in the samples was determinedby visual inspection before centrifugation after 1 3 7and 15 days of cold incubation (4∘C) 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 ldquominusrdquowhile cryoprecipitatelt1 ranging from 1 to 5 andgt5wasscored as ldquo+rdquo ldquo++rdquo and ldquo+++rdquo respectively On the 15th dayof cold incubation samples were centrifuged at 2500timesg for10min at 4∘C and the amount of cryoglobulins was estimatedas ldquocryocrit rdquo determined as the percentage of the volumeoccupied by the cryoprecipitate compared to the total volumeof serum after centrifugation occurred [19] According tothe cryocrit values obtained samples were divided into three

classes samples with cryocrit values higher than 10 withcryocrit values between 05 and 10 and with absence ofcryoglobulins

24 Cryoglobulins Determination by the Rapid Screening TestThe rapid method for cryoglobulins determination is basedon the detection of the scattered light by turbidimetry [22]Measurements were performed using a Cary3 dual-beamspectrophotometer (Varian Australia Pty Ltd MulgraveAustralia) on samples contained in a square cell (10mmpath length) whose temperature was maintained at 10∘CThis 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 [22] Briefly an aliquot (1800 120583L) 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) 119860max the maximumabsorbance value directly related to the cryoglobulin concen-tration [22] (2) the maximum slope (120575119860120575119905) of the aggrega-tion curve (3) the lag time (119905

0) obtained from extrapolation

of the maximum slope to the absorbance reading 1198600[22]

According to the 119860max 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 showing119860max values le01 absorbance units (94 of control sera) thesecond group assembled samples with 119860max values rangingfrom 01 to 02 absorbance units (5 of control sera) finallysamples showing 119860max values gt02 absorbance units wereassigned to the third group (1 of control sera) On the basisof a rigorous application of a 95 confidence interval 01 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 Results

31 Results of the Rapid and Traditional Test as a Functionof the Visual Inspection of Cryocrit at Different Days ofIncubation before Centrifugation In Table 1 results obtainedon cryoglobulinemic and controls (119899 = 500) samples bymeans of visual inspection at the 1st 3rd 7th and 15thdays of incubation and by the traditional (at the 15th day)and the rapid screening test (at time 0) are reported andcompared In addition the number of samples for eachcryoprecipitate score (see Section 2) is shown together withthe corresponding mean cryocrit value obtained after 15 daysof cold incubation and the rapid test results at time 0

At the first incubation day in most samples (286 572)no cryoprecipitate could be observed by visual inspection In

BioMed Research International 3

Table 1 Comparison of results obtainedwith the traditional and the rapid test For the traditional assay the number of samples (119899) at differentcryoprecipitate scores (see Section 2) by visual inspection at 1 3 7 and 15 days of cold incubation the mean cryocrit after centrifugationat the 15th day and the corresponding 119860max value from the rapid screening test at time 0 are reported The number of control samples isreported between brackets for the rapid and the traditional test at the 15th day of visual inspection

Visual inspection

Traditional test Rapid test (119860max)

Incubation days Cryocrit value atthe 15th day lt01

(119899)01-02

(119899)ge02(119899)

15 (119899) 7 (119899) 3 (119899) 1 (119899) mean plusmn SD[median range]

minus252(100) 257 261 286 0

mdash230(94)

21(5)

1(1)

+ 42 40 45 40 074 plusmn 002[075 05ndash1] 22 20 mdash

++ 105 102 93 88 484 plusmn 030[54 05ndash99] 11 75 19

+++ 101 101 101 86 1240 plusmn 120[89 05ndash565] mdash mdash 101

Table 2 Cross results from the performance of the traditional and the rapid cryoglobulin screening test Data are reported as number ofsamples and percentage (between brackets)

Cryoglobulinemic sera (119899 = 400) Control sera(119899 = 100)

Cryocrit gt 1(119899 = 185 46)

Cryocrit 05ndash1(119899 = 63 16)

Absence ofcryocrit

(119899 = 152 38)

Absence of cryocrit(119899 = 100 100)

119860max Patients () Patients () Patients () Subjects ()lt01 11 (6) 22 (34) 136 (89) 94 (94)01-02 56 (30) 39 (61) 16 (11) 5 (5)ge02 118 (64) 2 (3) mdash 1 (1)

forty (8) cases it was possible to detect a cryoprecipitate(+ score) whose cryocrit was lower than 1 at the 15th dayIn eighty-eight (176) cases scored as ++ the cryocrit at the15th day ranged from 1 to 5 and in 86 (172) (score +++)it was higher than 5 After fifteen days of cold incubationthe number of samples showing no cryoprecipitate decreased(252 versus 286) and together the number of samples withvisible cryoprecipitate with a cryocrit positioning between 1and 5 or higher than 5 increased (105 versus 88 and 101versus 86 samples resp)

The performance of the rapid screening test at time 0indicated that out of 252 samples negative for cryoglobulinswith the traditional test after 15 days of cold incubation 230showed absorbance values lt01 21 showed 01 lt 119860max lt02 and one sample was frankly positive for cryoglobulins(119860max gt 02) Out of 42 samples with cryocrit lower than1 with the rapid test 22 presented values of 119860max lt 01and 20 of 01 lt 119860max lt 02 Moreover out of 105 sampleswith a cryocrit ranging from 1 to 5 11 showed 119860max lt01 75 had absorbance values between 01 and 02 and 19were frankly positive (gt02) Finally all samples (101) showingby the traditional test cryocrit values gt5 displayed Absmaxlevels higher than 02 with the rapid test

32 Comparison of the Rapid and Traditional Test at the15th Day of Incubation after Centrifugation Table 2 shows

the overall results obtained from the comparative analysis ofthe rapid and the traditional screening tests According tothe classification reported in Section 2 samples distributionfor the traditional test showed that among the expectedcryoglobulinemic samples 46 had cryocrit values gt1 16had cryocrit values between 05 and 1 and no cryoprecip-itate was detectable in 38 of samples and in all controlsAbsorbance values ge01 by means of the rapid test werefound in 94 (174 out of 185) of samples with franklypositive cryocrit in 61 (41 out of 63) with low cryocrit (05ndash1) and in 9 of those with absence of cryoprecipitate (16patients plus 6 controls) The correlation between cryocritand turbidimetric readings was found statistically significant(Pearson coefficient = 0763 119875 lt 0001) However despitethis partial correspondence between the traditional and therapid screening test all of the patients with symptomaticcryoglobulinemic syndrome (119899 = 15) showed Absmax valueshigher than 05 (055ndash199 mean 117) and cryocrit rangingfrom 62 to 50 (mean 23)

Sensitivity and specificity of the rapid screening testcalculated in respect to the results of the traditional one were87 and 91 respectively Moreover diagnostic sensitivityand specificity of the rapid screening and the traditionaltest limited in respect to the presence of clinical symptomsof cryoglobulinemic syndrome were calculated by meansof ROC curves shown in Figure 1 AUCs and cutoff values

4 BioMed Research International

10

08

06

04

02

00

100806040200

1 minus specificity

Sens

itivi

ty

Figure 1 ROC curves of rapid (continuous line) and traditional(dotted line) tests in respect to the presence of clinical symptomsof cryoglobulinemic syndrome

determined at the 90 sensitivity for rapid and traditionaltest were 088 plusmn 030 (119875 lt 0001) and 023 (specificity 745)and 084 plusmn 041 (119875 lt 0001) and 26 (specificity 620)respectively

33 Cold-Induced Aggregation Curves of Type I and II Cryo-globulinemic Sera Shape and Parameters Figure 2 showsrepresentative cold-induced aggregation curves obtainedafter performing the rapid screening test on type I and typeII cryoglobulinemic serum samples and on a control serumsample normalized to the type II curve profile moreoverparameters derived from the phenomenological analysis ofthe process are reported These cryoglobulinemic samplesshared a comparable cryocrit value (sim5) obtained withthe traditional test The comparison between the two plotsshowed that despite a similar sigmoidal curve shape withcomparable maximum slopes and asymptotic absorbance(119860max) the 1199050 parameter observed was profoundly differentIn particular type I cryoglobulinemic sample showed a lagtime (119905

0) 10-fold higher than type II (119905

0ratio = 10 where

1199050ratio = (119905

0)type I(1199050)type II) Similar results were obtained

comparing 1199050values of all type I and type II cryoglobulins

achieving a mean 1199050ratio of 12 plusmn 4 The aggregation curves of

samples that have types II and III cryoglobulins did not showsignificantly different kinetic parameters (data not shown)

4 Discussion

Themethods used to date for the quantification of cryoglob-ulins have not been uniform between different laboratories[19ndash21 23] Traditional tests are based on cryoprecipitatequantification after prolonged (2ndash21 days) cold incubationof serum samples However the performance of this assay

00

02

04

06

08

10

0 10 20 30 40

Time (min)

Nor

mal

ized

abso

rban

ce

dAdt

Amax

t0

Figure 2 Typical aggregation curves obtained by the rapid screen-ing test performed on type II (∙) and type I (◻) cryoglobulinemicsera and on a sample of a control subject (998779) Curves are normalizedto the type II profiles original 119860max values were 132 140 and008 respectively When tested using the traditional method thecryoglobulinemic sample showed a cryocrit value of about 5Continuous and dotted lines refer to samples of type II and Icryoglobulinemic sera displaying the lowest and highest recordedlag time (119905

0

) values (1199050(min) = 02 and 29 and 119905

0

= 154 and 241resp)

in clinical laboratories poses considerable problems due tothe lack of standardization of preanalytical and analyticalprocedures Recently Vermeersch et al [19] evaluated thecurrent practice in the detection analysis and reportingof cryoglobulins by means of a questionnaire sent to 140laboratories participating in the UK National External Qual-ity Assessment Service quality control program The studyshowed that only 36 of laboratories respect the standardpreanalytical procedures to collect blood (tube preheatingtransport in container sedimentation andor centrifugationat 37∘C) and a wide variation at many steps of the analyticalprocedure (timing for cryoprecipitation at 4∘C washingandor resolubilization of cryoprecipitate etc) exists betweendifferent laboratories

Another important problem encountered in performingthe traditional test for cryoglobulin detection is due tothe false-negative results In fact since the definition ofcryoprecipitate presence depends on a visual inspection asmall amount of cryoglobulins in the sample could not bevisible andor cryoglobulins with peculiar physical aspectslike a cryogel could be missed

An alternative test to detect cryoglobulins in serumsamples was first proposed by Kalovidouris and Johnson [24]and recently reviewed by our group [22] Kalovidouris andJohnsonrsquos assay was based on the spectrophotometric detec-tion of a difference in optical density between two aliquotsof serum sample incubated at 4∘C and 37∘C respectively[24]The cryoglobulin screening assay defined here as ldquorapidscreening testrdquo is based on the same physical principle (lightscattering detection) although somemodifications have been

BioMed Research International 5

introduced In our approach a detailed phenomenologicalanalysis of the entire aggregation process with the identifi-cation of parameters describing its evolution over time foreach specimen was performed [22] Moreover the number ofpatients as well as the one of control subjects was notablyhigher than the number of cases reported by Kalovidourisand Johnson [24] and in addition sera from patients affectedby different disorders related to cryoglobulin typing (25type I 625 type II and 125 type III) were analyzed

We compared data from the performance of both classicaland rapid test for cryoglobulins screening on 500 serumsamples Only 1 (04) and 21 (83) out of 252 samplesnegative for cryoprecipitate showed Absmax ge 02 andbetween 01 and 02 respectively The major discrepanciesbetween the two screening tests were found within patientswith cryocrit gt1 Out of 185 patients frankly positive withthe traditional test 6 were negative with the rapid testand 30 reported absorbance values slightly altered (01-02) It is noteworthy to highlight the different phenomenonmonitored by the two assays the traditional test visuallydetects the amount of precipitated aggregates whereas theincrease in the scattered light on the basis of the rapid testis due to the formation of aggregates before precipitationoccurs Therefore results of samples positive to the rapid testand negative to the classical one could find their explanationin (1) a higher sensitivity of spectrophotometer signal inrespect to the macroscopic visual evaluation in detectinglow amounts of aggregatesprecipitate or (2) the possibilitythat cryoglobulins at low concentrations when undergoingthe aggregation process do not reach an adequate clusterdimension for overcoming the solubility limit thus notforming precipitate On the contrary the incongruence ofsamples positive to the traditional test and negative to therapid one could be reasoned by the following (1) the differentspan time of the two assays (ie days versus hours) whichdoes not exclude the possibility of a slow rate of aggregation(that could be undetectable in the first hour of observationand culminate in the precipitate formation only after daysof cold incubation) or (2) the occurrence of alternativeaggregation mechanisms (such as a gelnetwork formationor presence of macroscopic not precipitated aggregates)which interfere with the spectroscopic readings The lasthypothesis is supported by the observation of a high andexclusive frequency (45) of particular macroscopic aspects(gel consistency homogeneous turbidity) in such samples

A critical evaluation of such results has allowed us topropose a ldquofunctionalrdquo classification of cryoglobulins (1)ldquofastrdquo cryoglobulins positive results obtained with the rapidtest (showing an aggregation rate of minutes) as well aswith the traditional test ldquoslowrdquo cryoglobulins only thetraditional assay shows a positive result (precipitation rateof days) ldquoearlyrdquo cryoglobulins only the rapid assay showsa positive result The functional cryoglobulins classificationreported above might be helpful in the clinical approachand management of patients In fact ldquofastrdquo forms could beidentified as proteins able to rapidly aggregate and candidateto precipitate also in vivo ldquoslowrdquo forms could be identified asa laboratory finding of uncertain clinical relevance (probablyartefacts) or as consequence of an unspecific precipitation

originating from decrease of solubility of serum proteinsafter long cold incubation ldquoearlyrdquo cryoglobulins could beidentified as proteins found in samples at low concentrationsso that no visible precipitate could be detected with theclassical method

Finally the utility of the rapid test might be consid-ered in the therapeutic management of cryoglobulinemicpatients whose medical treatment includes plasmapheresisAs known plasmapheresis is a common procedure whichtransiently removes the circulating cryoglobulins reducingmorbidities associated with such disorder [25 26] Theefficacy of such treatment could be tested in a short timeand in a large number of patients by means of the rapid testenabling the patient to be treated by a different therapeuticapproach if poorly effectualThe utility of this approach needsfurther investigations in studies supported by specific clinicalprotocols

5 Conclusions

The present study proposes the rapid screening test as areliable and sensitive method to use in a complementaryway with the traditional test for the assessment of cryoglob-ulinemia Despite many advantages in using the rapid testin respect to the traditional one (high reproducibility noexaminer dependency and low cost results obtained within2 hours) and the possibility to discriminate monoclonal ormixed cryoglobulinemia by the 119905

0parameter cryocrit and its

immunological characterization by immunofixation remainfundamental for cryoglobulins typing whereas the rapid testcan be useful as a ldquoreal timerdquo monitoring of the diseasedevelopment and the evaluation of plasmapheresis efficacy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr M A Isgro for critical reading of thepaper Financial support by the Italian Ministry of Universityand Research (Linea D1 ldquoex-60rdquo 2012-2013 Universita Cat-tolica del Sacro Cuore) is gratefully acknowledged

References

[1] C H Hui and H H Lee ldquoCryoglobulinsrdquo Medical Journal ofAustralia vol 188 no 3 article 158 2008

[2] S Takada T Shimizu Y Hadano et al ldquoCryoglobulinemiardquoMolecular Medicine Reports vol 6 no 1 pp 3ndash8 2012

[3] E Morra ldquoCryoglobulinemiardquo Hematologythe Education Pro-gram of the American Society of Hematology pp 368ndash372 2005

[4] M Ramos-Casals J H Stone M C Cid and X Bosch ldquoThecryoglobulinaemiasrdquoTheLancet vol 379 no 9813 pp 348ndash3602012

[5] S Berentsen ldquoCold agglutinin-mediated autoimmune hemo-lytic anemia in Waldenstromrsquos macroglobulinemiardquo ClinicalLymphoma and Myeloma vol 9 no 1 pp 110ndash112 2009

6 BioMed Research International

[6] H Shirin Y Davidovitz Y Avni et al ldquoPrevalence of hepatitis Cvirus infection in patients with lymphoproliferative disordersrdquoIsrael Medical Association Journal vol 4 no 1 pp 24ndash27 2002

[7] M B Owlia R Sami M Akhondi and A Salimzadeh ldquoCryo-globulinaemia in hepatitis C-positive patients in Iranrdquo Singa-pore Medical Journal vol 48 no 12 pp 1136ndash1139 2007

[8] C Ferri L la Civita G Longombardo A L Zignego and GPasero ldquoMixed cryoglobulinaemia a cross-road between auto-immune and lymphoproliferative disordersrdquo Lupus vol 7 no 4pp 275ndash279 1998

[9] V Agnello ldquoThe etiology and pathophysiology of mixed cryo-globulinemia secondary to hepatitis C virus infectionrdquo SpringerSeminars in Immunopathology vol 19 no 1 pp 111ndash129 1997

[10] S de Vita L Quartuccio and M Fabris ldquoHepatitis C virusinfection mixed cryoglobulinemia and BLyS upregulationtargeting the infectious trigger the autoimmune response orbothrdquo Autoimmunity Reviews vol 8 no 2 pp 95ndash99 2008

[11] J C Brouet J P Clauvel F DanonM Klein andM SeligmannldquoBiologic and clinical significance of cryoglobulins A report of86 casesrdquo The American Journal of Medicine vol 57 no 5 pp775ndash788 1974

[12] M A Gertz ldquoCold agglutinin disease and cryoglobulinemiardquoClinical Lymphoma vol 5 no 4 pp 290ndash293 2005

[13] M D Wooten and H E Jasin ldquoMixed cryoglobulinemia andvasculitis a novel pathogenic mechanismrdquo Journal of Rheuma-tology vol 23 no 7 pp 1278ndash1281 1996

[14] J Damoiseaux ldquoThe diagnosis and classification of the cryo-globulinemic syndromerdquo Autoimmunity Reviews vol 13 no 4-5 pp 359ndash362 2014

[15] F Invernizzi F Saccardo M Galli G Monti and C ZanussildquoThe cryoglobulinemic syndromerdquo La Ricerca in Clinica e inLaboratorio vol 16 no 2 pp 269ndash274 1986

[16] M J Stone ldquoWaldenstromrsquosmacroglobulinemia hyperviscositysyndrome and cryoglobulinemiardquoClinical Lymphoma andMye-loma vol 9 no 1 pp 97ndash99 2009

[17] A Fornasieri S TazzariM Li et al ldquoElectronmicroscopy studyof genesis and dynamics of immunodeposition in IgMk-IgGcryoglobulin-induced glomerulonephritis in micerdquoThe Ameri-can Journal of Kidney Diseases vol 31 no 3 pp 435ndash442 1998

[18] C R Middaugh and G W Litman ldquoInvestigations of the mole-cular basis for the temperature-dependent insolubility of cryo-globulins VI Quenching by acrylamide of the intrinsic trypto-phan fluorescence of cryoglobulin and non-cryoglobulin IgMproteinsrdquo Biochimica et Biophysica Acta vol 535 no 1 pp 33ndash43 1978

[19] P Vermeersch K Gijbels G Marien et al ldquoA critical appraisalof current practice in the detection analysis and reportingof cryoglobulinsrdquo Clinical Chemistry vol 54 no 1 pp 39ndash432008

[20] GMotyckova andMMurali ldquoLaboratory testing for cryoglob-ulinsrdquo The American Journal of Hematology vol 86 no 6 pp500ndash502 2011

[21] R Sargur P White and W Egner ldquoCryoglobulin evaluationbest practicerdquo Annals of Clinical Biochemistry vol 47 no 1 pp8ndash16 2010

[22] E di Stasio P Bizzarri M Bove et al ldquoAnalysis of the dynamicsof cryoaggregation by light-scattering spectrometryrdquo ClinicalChemistry and Laboratory Medicine vol 41 no 2 pp 152ndash1582003

[23] Z K Shihabi ldquoCryoglobulins an important but neglected clin-ical testrdquo Annals of Clinical and Laboratory Science vol 36 no4 pp 395ndash408 2006

[24] A E Kalovidouris and R L Johnson ldquoRapid cryoglobulinscreening an aid to the clinicianrdquo Annals of the RheumaticDiseases vol 37 no 5 pp 444ndash448 1978

[25] A P Sanchez R Cunard and D M Ward ldquoThe selective ther-apeutic apheresis proceduresrdquo Journal of Clinical Apheresis vol28 no 1 pp 20ndash29 2013

[26] M A Rockx and W F Clark ldquoPlasma exchange for treat-ing cryoglobulinemia a descriptive analysisrdquo Transfusion andApheresis Science vol 42 no 3 pp 247ndash251 2010

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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 practiceMoreover 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

21 Samples Four hundred serum samples from patientswith clinical evidence of different disorders related to cryo-globulinemia were studied by both methods In particularstudieswere conducted in parallel on sera drawn for diagnos-tic purposes from 100 patients affected by type I cryoglob-ulinemia related diseases (monoclonal gammopathies andWaldenstromrsquos macroglobulinemia) 250 subjects suspectedof having cryoglobulinemia secondary toHCV infection and50 patients with autoimmune diseases (Sjogrenrsquos syndromerheumatoid 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

22 Blood Sampling Blood was drawn by venipuncture inVacutainer tubes prewarmed at 37∘C and left to clot for 2hours at the same temperature the serum was separated bycentrifugation (2500timesg for 10min) at 37∘C An aliquot ofeach sample (1800 120583L) was immediately tested with the rapidscreening assay and the remaining volume dispensed in agraduated Wintrobe tube for the traditional assay (time 0)

23 Cryoglobulins Determination by the Traditional MethodThe presence of precipitate in the samples was determinedby visual inspection before centrifugation after 1 3 7and 15 days of cold incubation (4∘C) 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 ldquominusrdquowhile cryoprecipitatelt1 ranging from 1 to 5 andgt5wasscored as ldquo+rdquo ldquo++rdquo and ldquo+++rdquo respectively On the 15th dayof cold incubation samples were centrifuged at 2500timesg for10min at 4∘C and the amount of cryoglobulins was estimatedas ldquocryocrit rdquo determined as the percentage of the volumeoccupied by the cryoprecipitate compared to the total volumeof serum after centrifugation occurred [19] According tothe cryocrit values obtained samples were divided into three

classes samples with cryocrit values higher than 10 withcryocrit values between 05 and 10 and with absence ofcryoglobulins

24 Cryoglobulins Determination by the Rapid Screening TestThe rapid method for cryoglobulins determination is basedon the detection of the scattered light by turbidimetry [22]Measurements were performed using a Cary3 dual-beamspectrophotometer (Varian Australia Pty Ltd MulgraveAustralia) on samples contained in a square cell (10mmpath length) whose temperature was maintained at 10∘CThis 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 [22] Briefly an aliquot (1800 120583L) 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) 119860max the maximumabsorbance value directly related to the cryoglobulin concen-tration [22] (2) the maximum slope (120575119860120575119905) of the aggrega-tion curve (3) the lag time (119905

0) obtained from extrapolation

of the maximum slope to the absorbance reading 1198600[22]

According to the 119860max 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 showing119860max values le01 absorbance units (94 of control sera) thesecond group assembled samples with 119860max values rangingfrom 01 to 02 absorbance units (5 of control sera) finallysamples showing 119860max values gt02 absorbance units wereassigned to the third group (1 of control sera) On the basisof a rigorous application of a 95 confidence interval 01 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 Results

31 Results of the Rapid and Traditional Test as a Functionof the Visual Inspection of Cryocrit at Different Days ofIncubation before Centrifugation In Table 1 results obtainedon cryoglobulinemic and controls (119899 = 500) samples bymeans of visual inspection at the 1st 3rd 7th and 15thdays of incubation and by the traditional (at the 15th day)and the rapid screening test (at time 0) are reported andcompared In addition the number of samples for eachcryoprecipitate score (see Section 2) is shown together withthe corresponding mean cryocrit value obtained after 15 daysof cold incubation and the rapid test results at time 0

At the first incubation day in most samples (286 572)no cryoprecipitate could be observed by visual inspection In

BioMed Research International 3

Table 1 Comparison of results obtainedwith the traditional and the rapid test For the traditional assay the number of samples (119899) at differentcryoprecipitate scores (see Section 2) by visual inspection at 1 3 7 and 15 days of cold incubation the mean cryocrit after centrifugationat the 15th day and the corresponding 119860max value from the rapid screening test at time 0 are reported The number of control samples isreported between brackets for the rapid and the traditional test at the 15th day of visual inspection

Visual inspection

Traditional test Rapid test (119860max)

Incubation days Cryocrit value atthe 15th day lt01

(119899)01-02

(119899)ge02(119899)

15 (119899) 7 (119899) 3 (119899) 1 (119899) mean plusmn SD[median range]

minus252(100) 257 261 286 0

mdash230(94)

21(5)

1(1)

+ 42 40 45 40 074 plusmn 002[075 05ndash1] 22 20 mdash

++ 105 102 93 88 484 plusmn 030[54 05ndash99] 11 75 19

+++ 101 101 101 86 1240 plusmn 120[89 05ndash565] mdash mdash 101

Table 2 Cross results from the performance of the traditional and the rapid cryoglobulin screening test Data are reported as number ofsamples and percentage (between brackets)

Cryoglobulinemic sera (119899 = 400) Control sera(119899 = 100)

Cryocrit gt 1(119899 = 185 46)

Cryocrit 05ndash1(119899 = 63 16)

Absence ofcryocrit

(119899 = 152 38)

Absence of cryocrit(119899 = 100 100)

119860max Patients () Patients () Patients () Subjects ()lt01 11 (6) 22 (34) 136 (89) 94 (94)01-02 56 (30) 39 (61) 16 (11) 5 (5)ge02 118 (64) 2 (3) mdash 1 (1)

forty (8) cases it was possible to detect a cryoprecipitate(+ score) whose cryocrit was lower than 1 at the 15th dayIn eighty-eight (176) cases scored as ++ the cryocrit at the15th day ranged from 1 to 5 and in 86 (172) (score +++)it was higher than 5 After fifteen days of cold incubationthe number of samples showing no cryoprecipitate decreased(252 versus 286) and together the number of samples withvisible cryoprecipitate with a cryocrit positioning between 1and 5 or higher than 5 increased (105 versus 88 and 101versus 86 samples resp)

The performance of the rapid screening test at time 0indicated that out of 252 samples negative for cryoglobulinswith the traditional test after 15 days of cold incubation 230showed absorbance values lt01 21 showed 01 lt 119860max lt02 and one sample was frankly positive for cryoglobulins(119860max gt 02) Out of 42 samples with cryocrit lower than1 with the rapid test 22 presented values of 119860max lt 01and 20 of 01 lt 119860max lt 02 Moreover out of 105 sampleswith a cryocrit ranging from 1 to 5 11 showed 119860max lt01 75 had absorbance values between 01 and 02 and 19were frankly positive (gt02) Finally all samples (101) showingby the traditional test cryocrit values gt5 displayed Absmaxlevels higher than 02 with the rapid test

32 Comparison of the Rapid and Traditional Test at the15th Day of Incubation after Centrifugation Table 2 shows

the overall results obtained from the comparative analysis ofthe rapid and the traditional screening tests According tothe classification reported in Section 2 samples distributionfor the traditional test showed that among the expectedcryoglobulinemic samples 46 had cryocrit values gt1 16had cryocrit values between 05 and 1 and no cryoprecip-itate was detectable in 38 of samples and in all controlsAbsorbance values ge01 by means of the rapid test werefound in 94 (174 out of 185) of samples with franklypositive cryocrit in 61 (41 out of 63) with low cryocrit (05ndash1) and in 9 of those with absence of cryoprecipitate (16patients plus 6 controls) The correlation between cryocritand turbidimetric readings was found statistically significant(Pearson coefficient = 0763 119875 lt 0001) However despitethis partial correspondence between the traditional and therapid screening test all of the patients with symptomaticcryoglobulinemic syndrome (119899 = 15) showed Absmax valueshigher than 05 (055ndash199 mean 117) and cryocrit rangingfrom 62 to 50 (mean 23)

Sensitivity and specificity of the rapid screening testcalculated in respect to the results of the traditional one were87 and 91 respectively Moreover diagnostic sensitivityand specificity of the rapid screening and the traditionaltest limited in respect to the presence of clinical symptomsof cryoglobulinemic syndrome were calculated by meansof ROC curves shown in Figure 1 AUCs and cutoff values

4 BioMed Research International

10

08

06

04

02

00

100806040200

1 minus specificity

Sens

itivi

ty

Figure 1 ROC curves of rapid (continuous line) and traditional(dotted line) tests in respect to the presence of clinical symptomsof cryoglobulinemic syndrome

determined at the 90 sensitivity for rapid and traditionaltest were 088 plusmn 030 (119875 lt 0001) and 023 (specificity 745)and 084 plusmn 041 (119875 lt 0001) and 26 (specificity 620)respectively

33 Cold-Induced Aggregation Curves of Type I and II Cryo-globulinemic Sera Shape and Parameters Figure 2 showsrepresentative cold-induced aggregation curves obtainedafter performing the rapid screening test on type I and typeII cryoglobulinemic serum samples and on a control serumsample normalized to the type II curve profile moreoverparameters derived from the phenomenological analysis ofthe process are reported These cryoglobulinemic samplesshared a comparable cryocrit value (sim5) obtained withthe traditional test The comparison between the two plotsshowed that despite a similar sigmoidal curve shape withcomparable maximum slopes and asymptotic absorbance(119860max) the 1199050 parameter observed was profoundly differentIn particular type I cryoglobulinemic sample showed a lagtime (119905

0) 10-fold higher than type II (119905

0ratio = 10 where

1199050ratio = (119905

0)type I(1199050)type II) Similar results were obtained

comparing 1199050values of all type I and type II cryoglobulins

achieving a mean 1199050ratio of 12 plusmn 4 The aggregation curves of

samples that have types II and III cryoglobulins did not showsignificantly different kinetic parameters (data not shown)

4 Discussion

Themethods used to date for the quantification of cryoglob-ulins have not been uniform between different laboratories[19ndash21 23] Traditional tests are based on cryoprecipitatequantification after prolonged (2ndash21 days) cold incubationof serum samples However the performance of this assay

00

02

04

06

08

10

0 10 20 30 40

Time (min)

Nor

mal

ized

abso

rban

ce

dAdt

Amax

t0

Figure 2 Typical aggregation curves obtained by the rapid screen-ing test performed on type II (∙) and type I (◻) cryoglobulinemicsera and on a sample of a control subject (998779) Curves are normalizedto the type II profiles original 119860max values were 132 140 and008 respectively When tested using the traditional method thecryoglobulinemic sample showed a cryocrit value of about 5Continuous and dotted lines refer to samples of type II and Icryoglobulinemic sera displaying the lowest and highest recordedlag time (119905

0

) values (1199050(min) = 02 and 29 and 119905

0

= 154 and 241resp)

in clinical laboratories poses considerable problems due tothe lack of standardization of preanalytical and analyticalprocedures Recently Vermeersch et al [19] evaluated thecurrent practice in the detection analysis and reportingof cryoglobulins by means of a questionnaire sent to 140laboratories participating in the UK National External Qual-ity Assessment Service quality control program The studyshowed that only 36 of laboratories respect the standardpreanalytical procedures to collect blood (tube preheatingtransport in container sedimentation andor centrifugationat 37∘C) and a wide variation at many steps of the analyticalprocedure (timing for cryoprecipitation at 4∘C washingandor resolubilization of cryoprecipitate etc) exists betweendifferent laboratories

Another important problem encountered in performingthe traditional test for cryoglobulin detection is due tothe false-negative results In fact since the definition ofcryoprecipitate presence depends on a visual inspection asmall amount of cryoglobulins in the sample could not bevisible andor cryoglobulins with peculiar physical aspectslike a cryogel could be missed

An alternative test to detect cryoglobulins in serumsamples was first proposed by Kalovidouris and Johnson [24]and recently reviewed by our group [22] Kalovidouris andJohnsonrsquos assay was based on the spectrophotometric detec-tion of a difference in optical density between two aliquotsof serum sample incubated at 4∘C and 37∘C respectively[24]The cryoglobulin screening assay defined here as ldquorapidscreening testrdquo is based on the same physical principle (lightscattering detection) although somemodifications have been

BioMed Research International 5

introduced In our approach a detailed phenomenologicalanalysis of the entire aggregation process with the identifi-cation of parameters describing its evolution over time foreach specimen was performed [22] Moreover the number ofpatients as well as the one of control subjects was notablyhigher than the number of cases reported by Kalovidourisand Johnson [24] and in addition sera from patients affectedby different disorders related to cryoglobulin typing (25type I 625 type II and 125 type III) were analyzed

We compared data from the performance of both classicaland rapid test for cryoglobulins screening on 500 serumsamples Only 1 (04) and 21 (83) out of 252 samplesnegative for cryoprecipitate showed Absmax ge 02 andbetween 01 and 02 respectively The major discrepanciesbetween the two screening tests were found within patientswith cryocrit gt1 Out of 185 patients frankly positive withthe traditional test 6 were negative with the rapid testand 30 reported absorbance values slightly altered (01-02) It is noteworthy to highlight the different phenomenonmonitored by the two assays the traditional test visuallydetects the amount of precipitated aggregates whereas theincrease in the scattered light on the basis of the rapid testis due to the formation of aggregates before precipitationoccurs Therefore results of samples positive to the rapid testand negative to the classical one could find their explanationin (1) a higher sensitivity of spectrophotometer signal inrespect to the macroscopic visual evaluation in detectinglow amounts of aggregatesprecipitate or (2) the possibilitythat cryoglobulins at low concentrations when undergoingthe aggregation process do not reach an adequate clusterdimension for overcoming the solubility limit thus notforming precipitate On the contrary the incongruence ofsamples positive to the traditional test and negative to therapid one could be reasoned by the following (1) the differentspan time of the two assays (ie days versus hours) whichdoes not exclude the possibility of a slow rate of aggregation(that could be undetectable in the first hour of observationand culminate in the precipitate formation only after daysof cold incubation) or (2) the occurrence of alternativeaggregation mechanisms (such as a gelnetwork formationor presence of macroscopic not precipitated aggregates)which interfere with the spectroscopic readings The lasthypothesis is supported by the observation of a high andexclusive frequency (45) of particular macroscopic aspects(gel consistency homogeneous turbidity) in such samples

A critical evaluation of such results has allowed us topropose a ldquofunctionalrdquo classification of cryoglobulins (1)ldquofastrdquo cryoglobulins positive results obtained with the rapidtest (showing an aggregation rate of minutes) as well aswith the traditional test ldquoslowrdquo cryoglobulins only thetraditional assay shows a positive result (precipitation rateof days) ldquoearlyrdquo cryoglobulins only the rapid assay showsa positive result The functional cryoglobulins classificationreported above might be helpful in the clinical approachand management of patients In fact ldquofastrdquo forms could beidentified as proteins able to rapidly aggregate and candidateto precipitate also in vivo ldquoslowrdquo forms could be identified asa laboratory finding of uncertain clinical relevance (probablyartefacts) or as consequence of an unspecific precipitation

originating from decrease of solubility of serum proteinsafter long cold incubation ldquoearlyrdquo cryoglobulins could beidentified as proteins found in samples at low concentrationsso that no visible precipitate could be detected with theclassical method

Finally the utility of the rapid test might be consid-ered in the therapeutic management of cryoglobulinemicpatients whose medical treatment includes plasmapheresisAs known plasmapheresis is a common procedure whichtransiently removes the circulating cryoglobulins reducingmorbidities associated with such disorder [25 26] Theefficacy of such treatment could be tested in a short timeand in a large number of patients by means of the rapid testenabling the patient to be treated by a different therapeuticapproach if poorly effectualThe utility of this approach needsfurther investigations in studies supported by specific clinicalprotocols

5 Conclusions

The present study proposes the rapid screening test as areliable and sensitive method to use in a complementaryway with the traditional test for the assessment of cryoglob-ulinemia Despite many advantages in using the rapid testin respect to the traditional one (high reproducibility noexaminer dependency and low cost results obtained within2 hours) and the possibility to discriminate monoclonal ormixed cryoglobulinemia by the 119905

0parameter cryocrit and its

immunological characterization by immunofixation remainfundamental for cryoglobulins typing whereas the rapid testcan be useful as a ldquoreal timerdquo monitoring of the diseasedevelopment and the evaluation of plasmapheresis efficacy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr M A Isgro for critical reading of thepaper Financial support by the Italian Ministry of Universityand Research (Linea D1 ldquoex-60rdquo 2012-2013 Universita Cat-tolica del Sacro Cuore) is gratefully acknowledged

References

[1] C H Hui and H H Lee ldquoCryoglobulinsrdquo Medical Journal ofAustralia vol 188 no 3 article 158 2008

[2] S Takada T Shimizu Y Hadano et al ldquoCryoglobulinemiardquoMolecular Medicine Reports vol 6 no 1 pp 3ndash8 2012

[3] E Morra ldquoCryoglobulinemiardquo Hematologythe Education Pro-gram of the American Society of Hematology pp 368ndash372 2005

[4] M Ramos-Casals J H Stone M C Cid and X Bosch ldquoThecryoglobulinaemiasrdquoTheLancet vol 379 no 9813 pp 348ndash3602012

[5] S Berentsen ldquoCold agglutinin-mediated autoimmune hemo-lytic anemia in Waldenstromrsquos macroglobulinemiardquo ClinicalLymphoma and Myeloma vol 9 no 1 pp 110ndash112 2009

6 BioMed Research International

[6] H Shirin Y Davidovitz Y Avni et al ldquoPrevalence of hepatitis Cvirus infection in patients with lymphoproliferative disordersrdquoIsrael Medical Association Journal vol 4 no 1 pp 24ndash27 2002

[7] M B Owlia R Sami M Akhondi and A Salimzadeh ldquoCryo-globulinaemia in hepatitis C-positive patients in Iranrdquo Singa-pore Medical Journal vol 48 no 12 pp 1136ndash1139 2007

[8] C Ferri L la Civita G Longombardo A L Zignego and GPasero ldquoMixed cryoglobulinaemia a cross-road between auto-immune and lymphoproliferative disordersrdquo Lupus vol 7 no 4pp 275ndash279 1998

[9] V Agnello ldquoThe etiology and pathophysiology of mixed cryo-globulinemia secondary to hepatitis C virus infectionrdquo SpringerSeminars in Immunopathology vol 19 no 1 pp 111ndash129 1997

[10] S de Vita L Quartuccio and M Fabris ldquoHepatitis C virusinfection mixed cryoglobulinemia and BLyS upregulationtargeting the infectious trigger the autoimmune response orbothrdquo Autoimmunity Reviews vol 8 no 2 pp 95ndash99 2008

[11] J C Brouet J P Clauvel F DanonM Klein andM SeligmannldquoBiologic and clinical significance of cryoglobulins A report of86 casesrdquo The American Journal of Medicine vol 57 no 5 pp775ndash788 1974

[12] M A Gertz ldquoCold agglutinin disease and cryoglobulinemiardquoClinical Lymphoma vol 5 no 4 pp 290ndash293 2005

[13] M D Wooten and H E Jasin ldquoMixed cryoglobulinemia andvasculitis a novel pathogenic mechanismrdquo Journal of Rheuma-tology vol 23 no 7 pp 1278ndash1281 1996

[14] J Damoiseaux ldquoThe diagnosis and classification of the cryo-globulinemic syndromerdquo Autoimmunity Reviews vol 13 no 4-5 pp 359ndash362 2014

[15] F Invernizzi F Saccardo M Galli G Monti and C ZanussildquoThe cryoglobulinemic syndromerdquo La Ricerca in Clinica e inLaboratorio vol 16 no 2 pp 269ndash274 1986

[16] M J Stone ldquoWaldenstromrsquosmacroglobulinemia hyperviscositysyndrome and cryoglobulinemiardquoClinical Lymphoma andMye-loma vol 9 no 1 pp 97ndash99 2009

[17] A Fornasieri S TazzariM Li et al ldquoElectronmicroscopy studyof genesis and dynamics of immunodeposition in IgMk-IgGcryoglobulin-induced glomerulonephritis in micerdquoThe Ameri-can Journal of Kidney Diseases vol 31 no 3 pp 435ndash442 1998

[18] C R Middaugh and G W Litman ldquoInvestigations of the mole-cular basis for the temperature-dependent insolubility of cryo-globulins VI Quenching by acrylamide of the intrinsic trypto-phan fluorescence of cryoglobulin and non-cryoglobulin IgMproteinsrdquo Biochimica et Biophysica Acta vol 535 no 1 pp 33ndash43 1978

[19] P Vermeersch K Gijbels G Marien et al ldquoA critical appraisalof current practice in the detection analysis and reportingof cryoglobulinsrdquo Clinical Chemistry vol 54 no 1 pp 39ndash432008

[20] GMotyckova andMMurali ldquoLaboratory testing for cryoglob-ulinsrdquo The American Journal of Hematology vol 86 no 6 pp500ndash502 2011

[21] R Sargur P White and W Egner ldquoCryoglobulin evaluationbest practicerdquo Annals of Clinical Biochemistry vol 47 no 1 pp8ndash16 2010

[22] E di Stasio P Bizzarri M Bove et al ldquoAnalysis of the dynamicsof cryoaggregation by light-scattering spectrometryrdquo ClinicalChemistry and Laboratory Medicine vol 41 no 2 pp 152ndash1582003

[23] Z K Shihabi ldquoCryoglobulins an important but neglected clin-ical testrdquo Annals of Clinical and Laboratory Science vol 36 no4 pp 395ndash408 2006

[24] A E Kalovidouris and R L Johnson ldquoRapid cryoglobulinscreening an aid to the clinicianrdquo Annals of the RheumaticDiseases vol 37 no 5 pp 444ndash448 1978

[25] A P Sanchez R Cunard and D M Ward ldquoThe selective ther-apeutic apheresis proceduresrdquo Journal of Clinical Apheresis vol28 no 1 pp 20ndash29 2013

[26] M A Rockx and W F Clark ldquoPlasma exchange for treat-ing cryoglobulinemia a descriptive analysisrdquo Transfusion andApheresis Science vol 42 no 3 pp 247ndash251 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

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International Journal of

Microbiology

BioMed Research International 3

Table 1 Comparison of results obtainedwith the traditional and the rapid test For the traditional assay the number of samples (119899) at differentcryoprecipitate scores (see Section 2) by visual inspection at 1 3 7 and 15 days of cold incubation the mean cryocrit after centrifugationat the 15th day and the corresponding 119860max value from the rapid screening test at time 0 are reported The number of control samples isreported between brackets for the rapid and the traditional test at the 15th day of visual inspection

Visual inspection

Traditional test Rapid test (119860max)

Incubation days Cryocrit value atthe 15th day lt01

(119899)01-02

(119899)ge02(119899)

15 (119899) 7 (119899) 3 (119899) 1 (119899) mean plusmn SD[median range]

minus252(100) 257 261 286 0

mdash230(94)

21(5)

1(1)

+ 42 40 45 40 074 plusmn 002[075 05ndash1] 22 20 mdash

++ 105 102 93 88 484 plusmn 030[54 05ndash99] 11 75 19

+++ 101 101 101 86 1240 plusmn 120[89 05ndash565] mdash mdash 101

Table 2 Cross results from the performance of the traditional and the rapid cryoglobulin screening test Data are reported as number ofsamples and percentage (between brackets)

Cryoglobulinemic sera (119899 = 400) Control sera(119899 = 100)

Cryocrit gt 1(119899 = 185 46)

Cryocrit 05ndash1(119899 = 63 16)

Absence ofcryocrit

(119899 = 152 38)

Absence of cryocrit(119899 = 100 100)

119860max Patients () Patients () Patients () Subjects ()lt01 11 (6) 22 (34) 136 (89) 94 (94)01-02 56 (30) 39 (61) 16 (11) 5 (5)ge02 118 (64) 2 (3) mdash 1 (1)

forty (8) cases it was possible to detect a cryoprecipitate(+ score) whose cryocrit was lower than 1 at the 15th dayIn eighty-eight (176) cases scored as ++ the cryocrit at the15th day ranged from 1 to 5 and in 86 (172) (score +++)it was higher than 5 After fifteen days of cold incubationthe number of samples showing no cryoprecipitate decreased(252 versus 286) and together the number of samples withvisible cryoprecipitate with a cryocrit positioning between 1and 5 or higher than 5 increased (105 versus 88 and 101versus 86 samples resp)

The performance of the rapid screening test at time 0indicated that out of 252 samples negative for cryoglobulinswith the traditional test after 15 days of cold incubation 230showed absorbance values lt01 21 showed 01 lt 119860max lt02 and one sample was frankly positive for cryoglobulins(119860max gt 02) Out of 42 samples with cryocrit lower than1 with the rapid test 22 presented values of 119860max lt 01and 20 of 01 lt 119860max lt 02 Moreover out of 105 sampleswith a cryocrit ranging from 1 to 5 11 showed 119860max lt01 75 had absorbance values between 01 and 02 and 19were frankly positive (gt02) Finally all samples (101) showingby the traditional test cryocrit values gt5 displayed Absmaxlevels higher than 02 with the rapid test

32 Comparison of the Rapid and Traditional Test at the15th Day of Incubation after Centrifugation Table 2 shows

the overall results obtained from the comparative analysis ofthe rapid and the traditional screening tests According tothe classification reported in Section 2 samples distributionfor the traditional test showed that among the expectedcryoglobulinemic samples 46 had cryocrit values gt1 16had cryocrit values between 05 and 1 and no cryoprecip-itate was detectable in 38 of samples and in all controlsAbsorbance values ge01 by means of the rapid test werefound in 94 (174 out of 185) of samples with franklypositive cryocrit in 61 (41 out of 63) with low cryocrit (05ndash1) and in 9 of those with absence of cryoprecipitate (16patients plus 6 controls) The correlation between cryocritand turbidimetric readings was found statistically significant(Pearson coefficient = 0763 119875 lt 0001) However despitethis partial correspondence between the traditional and therapid screening test all of the patients with symptomaticcryoglobulinemic syndrome (119899 = 15) showed Absmax valueshigher than 05 (055ndash199 mean 117) and cryocrit rangingfrom 62 to 50 (mean 23)

Sensitivity and specificity of the rapid screening testcalculated in respect to the results of the traditional one were87 and 91 respectively Moreover diagnostic sensitivityand specificity of the rapid screening and the traditionaltest limited in respect to the presence of clinical symptomsof cryoglobulinemic syndrome were calculated by meansof ROC curves shown in Figure 1 AUCs and cutoff values

4 BioMed Research International

10

08

06

04

02

00

100806040200

1 minus specificity

Sens

itivi

ty

Figure 1 ROC curves of rapid (continuous line) and traditional(dotted line) tests in respect to the presence of clinical symptomsof cryoglobulinemic syndrome

determined at the 90 sensitivity for rapid and traditionaltest were 088 plusmn 030 (119875 lt 0001) and 023 (specificity 745)and 084 plusmn 041 (119875 lt 0001) and 26 (specificity 620)respectively

33 Cold-Induced Aggregation Curves of Type I and II Cryo-globulinemic Sera Shape and Parameters Figure 2 showsrepresentative cold-induced aggregation curves obtainedafter performing the rapid screening test on type I and typeII cryoglobulinemic serum samples and on a control serumsample normalized to the type II curve profile moreoverparameters derived from the phenomenological analysis ofthe process are reported These cryoglobulinemic samplesshared a comparable cryocrit value (sim5) obtained withthe traditional test The comparison between the two plotsshowed that despite a similar sigmoidal curve shape withcomparable maximum slopes and asymptotic absorbance(119860max) the 1199050 parameter observed was profoundly differentIn particular type I cryoglobulinemic sample showed a lagtime (119905

0) 10-fold higher than type II (119905

0ratio = 10 where

1199050ratio = (119905

0)type I(1199050)type II) Similar results were obtained

comparing 1199050values of all type I and type II cryoglobulins

achieving a mean 1199050ratio of 12 plusmn 4 The aggregation curves of

samples that have types II and III cryoglobulins did not showsignificantly different kinetic parameters (data not shown)

4 Discussion

Themethods used to date for the quantification of cryoglob-ulins have not been uniform between different laboratories[19ndash21 23] Traditional tests are based on cryoprecipitatequantification after prolonged (2ndash21 days) cold incubationof serum samples However the performance of this assay

00

02

04

06

08

10

0 10 20 30 40

Time (min)

Nor

mal

ized

abso

rban

ce

dAdt

Amax

t0

Figure 2 Typical aggregation curves obtained by the rapid screen-ing test performed on type II (∙) and type I (◻) cryoglobulinemicsera and on a sample of a control subject (998779) Curves are normalizedto the type II profiles original 119860max values were 132 140 and008 respectively When tested using the traditional method thecryoglobulinemic sample showed a cryocrit value of about 5Continuous and dotted lines refer to samples of type II and Icryoglobulinemic sera displaying the lowest and highest recordedlag time (119905

0

) values (1199050(min) = 02 and 29 and 119905

0

= 154 and 241resp)

in clinical laboratories poses considerable problems due tothe lack of standardization of preanalytical and analyticalprocedures Recently Vermeersch et al [19] evaluated thecurrent practice in the detection analysis and reportingof cryoglobulins by means of a questionnaire sent to 140laboratories participating in the UK National External Qual-ity Assessment Service quality control program The studyshowed that only 36 of laboratories respect the standardpreanalytical procedures to collect blood (tube preheatingtransport in container sedimentation andor centrifugationat 37∘C) and a wide variation at many steps of the analyticalprocedure (timing for cryoprecipitation at 4∘C washingandor resolubilization of cryoprecipitate etc) exists betweendifferent laboratories

Another important problem encountered in performingthe traditional test for cryoglobulin detection is due tothe false-negative results In fact since the definition ofcryoprecipitate presence depends on a visual inspection asmall amount of cryoglobulins in the sample could not bevisible andor cryoglobulins with peculiar physical aspectslike a cryogel could be missed

An alternative test to detect cryoglobulins in serumsamples was first proposed by Kalovidouris and Johnson [24]and recently reviewed by our group [22] Kalovidouris andJohnsonrsquos assay was based on the spectrophotometric detec-tion of a difference in optical density between two aliquotsof serum sample incubated at 4∘C and 37∘C respectively[24]The cryoglobulin screening assay defined here as ldquorapidscreening testrdquo is based on the same physical principle (lightscattering detection) although somemodifications have been

BioMed Research International 5

introduced In our approach a detailed phenomenologicalanalysis of the entire aggregation process with the identifi-cation of parameters describing its evolution over time foreach specimen was performed [22] Moreover the number ofpatients as well as the one of control subjects was notablyhigher than the number of cases reported by Kalovidourisand Johnson [24] and in addition sera from patients affectedby different disorders related to cryoglobulin typing (25type I 625 type II and 125 type III) were analyzed

We compared data from the performance of both classicaland rapid test for cryoglobulins screening on 500 serumsamples Only 1 (04) and 21 (83) out of 252 samplesnegative for cryoprecipitate showed Absmax ge 02 andbetween 01 and 02 respectively The major discrepanciesbetween the two screening tests were found within patientswith cryocrit gt1 Out of 185 patients frankly positive withthe traditional test 6 were negative with the rapid testand 30 reported absorbance values slightly altered (01-02) It is noteworthy to highlight the different phenomenonmonitored by the two assays the traditional test visuallydetects the amount of precipitated aggregates whereas theincrease in the scattered light on the basis of the rapid testis due to the formation of aggregates before precipitationoccurs Therefore results of samples positive to the rapid testand negative to the classical one could find their explanationin (1) a higher sensitivity of spectrophotometer signal inrespect to the macroscopic visual evaluation in detectinglow amounts of aggregatesprecipitate or (2) the possibilitythat cryoglobulins at low concentrations when undergoingthe aggregation process do not reach an adequate clusterdimension for overcoming the solubility limit thus notforming precipitate On the contrary the incongruence ofsamples positive to the traditional test and negative to therapid one could be reasoned by the following (1) the differentspan time of the two assays (ie days versus hours) whichdoes not exclude the possibility of a slow rate of aggregation(that could be undetectable in the first hour of observationand culminate in the precipitate formation only after daysof cold incubation) or (2) the occurrence of alternativeaggregation mechanisms (such as a gelnetwork formationor presence of macroscopic not precipitated aggregates)which interfere with the spectroscopic readings The lasthypothesis is supported by the observation of a high andexclusive frequency (45) of particular macroscopic aspects(gel consistency homogeneous turbidity) in such samples

A critical evaluation of such results has allowed us topropose a ldquofunctionalrdquo classification of cryoglobulins (1)ldquofastrdquo cryoglobulins positive results obtained with the rapidtest (showing an aggregation rate of minutes) as well aswith the traditional test ldquoslowrdquo cryoglobulins only thetraditional assay shows a positive result (precipitation rateof days) ldquoearlyrdquo cryoglobulins only the rapid assay showsa positive result The functional cryoglobulins classificationreported above might be helpful in the clinical approachand management of patients In fact ldquofastrdquo forms could beidentified as proteins able to rapidly aggregate and candidateto precipitate also in vivo ldquoslowrdquo forms could be identified asa laboratory finding of uncertain clinical relevance (probablyartefacts) or as consequence of an unspecific precipitation

originating from decrease of solubility of serum proteinsafter long cold incubation ldquoearlyrdquo cryoglobulins could beidentified as proteins found in samples at low concentrationsso that no visible precipitate could be detected with theclassical method

Finally the utility of the rapid test might be consid-ered in the therapeutic management of cryoglobulinemicpatients whose medical treatment includes plasmapheresisAs known plasmapheresis is a common procedure whichtransiently removes the circulating cryoglobulins reducingmorbidities associated with such disorder [25 26] Theefficacy of such treatment could be tested in a short timeand in a large number of patients by means of the rapid testenabling the patient to be treated by a different therapeuticapproach if poorly effectualThe utility of this approach needsfurther investigations in studies supported by specific clinicalprotocols

5 Conclusions

The present study proposes the rapid screening test as areliable and sensitive method to use in a complementaryway with the traditional test for the assessment of cryoglob-ulinemia Despite many advantages in using the rapid testin respect to the traditional one (high reproducibility noexaminer dependency and low cost results obtained within2 hours) and the possibility to discriminate monoclonal ormixed cryoglobulinemia by the 119905

0parameter cryocrit and its

immunological characterization by immunofixation remainfundamental for cryoglobulins typing whereas the rapid testcan be useful as a ldquoreal timerdquo monitoring of the diseasedevelopment and the evaluation of plasmapheresis efficacy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr M A Isgro for critical reading of thepaper Financial support by the Italian Ministry of Universityand Research (Linea D1 ldquoex-60rdquo 2012-2013 Universita Cat-tolica del Sacro Cuore) is gratefully acknowledged

References

[1] C H Hui and H H Lee ldquoCryoglobulinsrdquo Medical Journal ofAustralia vol 188 no 3 article 158 2008

[2] S Takada T Shimizu Y Hadano et al ldquoCryoglobulinemiardquoMolecular Medicine Reports vol 6 no 1 pp 3ndash8 2012

[3] E Morra ldquoCryoglobulinemiardquo Hematologythe Education Pro-gram of the American Society of Hematology pp 368ndash372 2005

[4] M Ramos-Casals J H Stone M C Cid and X Bosch ldquoThecryoglobulinaemiasrdquoTheLancet vol 379 no 9813 pp 348ndash3602012

[5] S Berentsen ldquoCold agglutinin-mediated autoimmune hemo-lytic anemia in Waldenstromrsquos macroglobulinemiardquo ClinicalLymphoma and Myeloma vol 9 no 1 pp 110ndash112 2009

6 BioMed Research International

[6] H Shirin Y Davidovitz Y Avni et al ldquoPrevalence of hepatitis Cvirus infection in patients with lymphoproliferative disordersrdquoIsrael Medical Association Journal vol 4 no 1 pp 24ndash27 2002

[7] M B Owlia R Sami M Akhondi and A Salimzadeh ldquoCryo-globulinaemia in hepatitis C-positive patients in Iranrdquo Singa-pore Medical Journal vol 48 no 12 pp 1136ndash1139 2007

[8] C Ferri L la Civita G Longombardo A L Zignego and GPasero ldquoMixed cryoglobulinaemia a cross-road between auto-immune and lymphoproliferative disordersrdquo Lupus vol 7 no 4pp 275ndash279 1998

[9] V Agnello ldquoThe etiology and pathophysiology of mixed cryo-globulinemia secondary to hepatitis C virus infectionrdquo SpringerSeminars in Immunopathology vol 19 no 1 pp 111ndash129 1997

[10] S de Vita L Quartuccio and M Fabris ldquoHepatitis C virusinfection mixed cryoglobulinemia and BLyS upregulationtargeting the infectious trigger the autoimmune response orbothrdquo Autoimmunity Reviews vol 8 no 2 pp 95ndash99 2008

[11] J C Brouet J P Clauvel F DanonM Klein andM SeligmannldquoBiologic and clinical significance of cryoglobulins A report of86 casesrdquo The American Journal of Medicine vol 57 no 5 pp775ndash788 1974

[12] M A Gertz ldquoCold agglutinin disease and cryoglobulinemiardquoClinical Lymphoma vol 5 no 4 pp 290ndash293 2005

[13] M D Wooten and H E Jasin ldquoMixed cryoglobulinemia andvasculitis a novel pathogenic mechanismrdquo Journal of Rheuma-tology vol 23 no 7 pp 1278ndash1281 1996

[14] J Damoiseaux ldquoThe diagnosis and classification of the cryo-globulinemic syndromerdquo Autoimmunity Reviews vol 13 no 4-5 pp 359ndash362 2014

[15] F Invernizzi F Saccardo M Galli G Monti and C ZanussildquoThe cryoglobulinemic syndromerdquo La Ricerca in Clinica e inLaboratorio vol 16 no 2 pp 269ndash274 1986

[16] M J Stone ldquoWaldenstromrsquosmacroglobulinemia hyperviscositysyndrome and cryoglobulinemiardquoClinical Lymphoma andMye-loma vol 9 no 1 pp 97ndash99 2009

[17] A Fornasieri S TazzariM Li et al ldquoElectronmicroscopy studyof genesis and dynamics of immunodeposition in IgMk-IgGcryoglobulin-induced glomerulonephritis in micerdquoThe Ameri-can Journal of Kidney Diseases vol 31 no 3 pp 435ndash442 1998

[18] C R Middaugh and G W Litman ldquoInvestigations of the mole-cular basis for the temperature-dependent insolubility of cryo-globulins VI Quenching by acrylamide of the intrinsic trypto-phan fluorescence of cryoglobulin and non-cryoglobulin IgMproteinsrdquo Biochimica et Biophysica Acta vol 535 no 1 pp 33ndash43 1978

[19] P Vermeersch K Gijbels G Marien et al ldquoA critical appraisalof current practice in the detection analysis and reportingof cryoglobulinsrdquo Clinical Chemistry vol 54 no 1 pp 39ndash432008

[20] GMotyckova andMMurali ldquoLaboratory testing for cryoglob-ulinsrdquo The American Journal of Hematology vol 86 no 6 pp500ndash502 2011

[21] R Sargur P White and W Egner ldquoCryoglobulin evaluationbest practicerdquo Annals of Clinical Biochemistry vol 47 no 1 pp8ndash16 2010

[22] E di Stasio P Bizzarri M Bove et al ldquoAnalysis of the dynamicsof cryoaggregation by light-scattering spectrometryrdquo ClinicalChemistry and Laboratory Medicine vol 41 no 2 pp 152ndash1582003

[23] Z K Shihabi ldquoCryoglobulins an important but neglected clin-ical testrdquo Annals of Clinical and Laboratory Science vol 36 no4 pp 395ndash408 2006

[24] A E Kalovidouris and R L Johnson ldquoRapid cryoglobulinscreening an aid to the clinicianrdquo Annals of the RheumaticDiseases vol 37 no 5 pp 444ndash448 1978

[25] A P Sanchez R Cunard and D M Ward ldquoThe selective ther-apeutic apheresis proceduresrdquo Journal of Clinical Apheresis vol28 no 1 pp 20ndash29 2013

[26] M A Rockx and W F Clark ldquoPlasma exchange for treat-ing cryoglobulinemia a descriptive analysisrdquo Transfusion andApheresis Science vol 42 no 3 pp 247ndash251 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

10

08

06

04

02

00

100806040200

1 minus specificity

Sens

itivi

ty

Figure 1 ROC curves of rapid (continuous line) and traditional(dotted line) tests in respect to the presence of clinical symptomsof cryoglobulinemic syndrome

determined at the 90 sensitivity for rapid and traditionaltest were 088 plusmn 030 (119875 lt 0001) and 023 (specificity 745)and 084 plusmn 041 (119875 lt 0001) and 26 (specificity 620)respectively

33 Cold-Induced Aggregation Curves of Type I and II Cryo-globulinemic Sera Shape and Parameters Figure 2 showsrepresentative cold-induced aggregation curves obtainedafter performing the rapid screening test on type I and typeII cryoglobulinemic serum samples and on a control serumsample normalized to the type II curve profile moreoverparameters derived from the phenomenological analysis ofthe process are reported These cryoglobulinemic samplesshared a comparable cryocrit value (sim5) obtained withthe traditional test The comparison between the two plotsshowed that despite a similar sigmoidal curve shape withcomparable maximum slopes and asymptotic absorbance(119860max) the 1199050 parameter observed was profoundly differentIn particular type I cryoglobulinemic sample showed a lagtime (119905

0) 10-fold higher than type II (119905

0ratio = 10 where

1199050ratio = (119905

0)type I(1199050)type II) Similar results were obtained

comparing 1199050values of all type I and type II cryoglobulins

achieving a mean 1199050ratio of 12 plusmn 4 The aggregation curves of

samples that have types II and III cryoglobulins did not showsignificantly different kinetic parameters (data not shown)

4 Discussion

Themethods used to date for the quantification of cryoglob-ulins have not been uniform between different laboratories[19ndash21 23] Traditional tests are based on cryoprecipitatequantification after prolonged (2ndash21 days) cold incubationof serum samples However the performance of this assay

00

02

04

06

08

10

0 10 20 30 40

Time (min)

Nor

mal

ized

abso

rban

ce

dAdt

Amax

t0

Figure 2 Typical aggregation curves obtained by the rapid screen-ing test performed on type II (∙) and type I (◻) cryoglobulinemicsera and on a sample of a control subject (998779) Curves are normalizedto the type II profiles original 119860max values were 132 140 and008 respectively When tested using the traditional method thecryoglobulinemic sample showed a cryocrit value of about 5Continuous and dotted lines refer to samples of type II and Icryoglobulinemic sera displaying the lowest and highest recordedlag time (119905

0

) values (1199050(min) = 02 and 29 and 119905

0

= 154 and 241resp)

in clinical laboratories poses considerable problems due tothe lack of standardization of preanalytical and analyticalprocedures Recently Vermeersch et al [19] evaluated thecurrent practice in the detection analysis and reportingof cryoglobulins by means of a questionnaire sent to 140laboratories participating in the UK National External Qual-ity Assessment Service quality control program The studyshowed that only 36 of laboratories respect the standardpreanalytical procedures to collect blood (tube preheatingtransport in container sedimentation andor centrifugationat 37∘C) and a wide variation at many steps of the analyticalprocedure (timing for cryoprecipitation at 4∘C washingandor resolubilization of cryoprecipitate etc) exists betweendifferent laboratories

Another important problem encountered in performingthe traditional test for cryoglobulin detection is due tothe false-negative results In fact since the definition ofcryoprecipitate presence depends on a visual inspection asmall amount of cryoglobulins in the sample could not bevisible andor cryoglobulins with peculiar physical aspectslike a cryogel could be missed

An alternative test to detect cryoglobulins in serumsamples was first proposed by Kalovidouris and Johnson [24]and recently reviewed by our group [22] Kalovidouris andJohnsonrsquos assay was based on the spectrophotometric detec-tion of a difference in optical density between two aliquotsof serum sample incubated at 4∘C and 37∘C respectively[24]The cryoglobulin screening assay defined here as ldquorapidscreening testrdquo is based on the same physical principle (lightscattering detection) although somemodifications have been

BioMed Research International 5

introduced In our approach a detailed phenomenologicalanalysis of the entire aggregation process with the identifi-cation of parameters describing its evolution over time foreach specimen was performed [22] Moreover the number ofpatients as well as the one of control subjects was notablyhigher than the number of cases reported by Kalovidourisand Johnson [24] and in addition sera from patients affectedby different disorders related to cryoglobulin typing (25type I 625 type II and 125 type III) were analyzed

We compared data from the performance of both classicaland rapid test for cryoglobulins screening on 500 serumsamples Only 1 (04) and 21 (83) out of 252 samplesnegative for cryoprecipitate showed Absmax ge 02 andbetween 01 and 02 respectively The major discrepanciesbetween the two screening tests were found within patientswith cryocrit gt1 Out of 185 patients frankly positive withthe traditional test 6 were negative with the rapid testand 30 reported absorbance values slightly altered (01-02) It is noteworthy to highlight the different phenomenonmonitored by the two assays the traditional test visuallydetects the amount of precipitated aggregates whereas theincrease in the scattered light on the basis of the rapid testis due to the formation of aggregates before precipitationoccurs Therefore results of samples positive to the rapid testand negative to the classical one could find their explanationin (1) a higher sensitivity of spectrophotometer signal inrespect to the macroscopic visual evaluation in detectinglow amounts of aggregatesprecipitate or (2) the possibilitythat cryoglobulins at low concentrations when undergoingthe aggregation process do not reach an adequate clusterdimension for overcoming the solubility limit thus notforming precipitate On the contrary the incongruence ofsamples positive to the traditional test and negative to therapid one could be reasoned by the following (1) the differentspan time of the two assays (ie days versus hours) whichdoes not exclude the possibility of a slow rate of aggregation(that could be undetectable in the first hour of observationand culminate in the precipitate formation only after daysof cold incubation) or (2) the occurrence of alternativeaggregation mechanisms (such as a gelnetwork formationor presence of macroscopic not precipitated aggregates)which interfere with the spectroscopic readings The lasthypothesis is supported by the observation of a high andexclusive frequency (45) of particular macroscopic aspects(gel consistency homogeneous turbidity) in such samples

A critical evaluation of such results has allowed us topropose a ldquofunctionalrdquo classification of cryoglobulins (1)ldquofastrdquo cryoglobulins positive results obtained with the rapidtest (showing an aggregation rate of minutes) as well aswith the traditional test ldquoslowrdquo cryoglobulins only thetraditional assay shows a positive result (precipitation rateof days) ldquoearlyrdquo cryoglobulins only the rapid assay showsa positive result The functional cryoglobulins classificationreported above might be helpful in the clinical approachand management of patients In fact ldquofastrdquo forms could beidentified as proteins able to rapidly aggregate and candidateto precipitate also in vivo ldquoslowrdquo forms could be identified asa laboratory finding of uncertain clinical relevance (probablyartefacts) or as consequence of an unspecific precipitation

originating from decrease of solubility of serum proteinsafter long cold incubation ldquoearlyrdquo cryoglobulins could beidentified as proteins found in samples at low concentrationsso that no visible precipitate could be detected with theclassical method

Finally the utility of the rapid test might be consid-ered in the therapeutic management of cryoglobulinemicpatients whose medical treatment includes plasmapheresisAs known plasmapheresis is a common procedure whichtransiently removes the circulating cryoglobulins reducingmorbidities associated with such disorder [25 26] Theefficacy of such treatment could be tested in a short timeand in a large number of patients by means of the rapid testenabling the patient to be treated by a different therapeuticapproach if poorly effectualThe utility of this approach needsfurther investigations in studies supported by specific clinicalprotocols

5 Conclusions

The present study proposes the rapid screening test as areliable and sensitive method to use in a complementaryway with the traditional test for the assessment of cryoglob-ulinemia Despite many advantages in using the rapid testin respect to the traditional one (high reproducibility noexaminer dependency and low cost results obtained within2 hours) and the possibility to discriminate monoclonal ormixed cryoglobulinemia by the 119905

0parameter cryocrit and its

immunological characterization by immunofixation remainfundamental for cryoglobulins typing whereas the rapid testcan be useful as a ldquoreal timerdquo monitoring of the diseasedevelopment and the evaluation of plasmapheresis efficacy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr M A Isgro for critical reading of thepaper Financial support by the Italian Ministry of Universityand Research (Linea D1 ldquoex-60rdquo 2012-2013 Universita Cat-tolica del Sacro Cuore) is gratefully acknowledged

References

[1] C H Hui and H H Lee ldquoCryoglobulinsrdquo Medical Journal ofAustralia vol 188 no 3 article 158 2008

[2] S Takada T Shimizu Y Hadano et al ldquoCryoglobulinemiardquoMolecular Medicine Reports vol 6 no 1 pp 3ndash8 2012

[3] E Morra ldquoCryoglobulinemiardquo Hematologythe Education Pro-gram of the American Society of Hematology pp 368ndash372 2005

[4] M Ramos-Casals J H Stone M C Cid and X Bosch ldquoThecryoglobulinaemiasrdquoTheLancet vol 379 no 9813 pp 348ndash3602012

[5] S Berentsen ldquoCold agglutinin-mediated autoimmune hemo-lytic anemia in Waldenstromrsquos macroglobulinemiardquo ClinicalLymphoma and Myeloma vol 9 no 1 pp 110ndash112 2009

6 BioMed Research International

[6] H Shirin Y Davidovitz Y Avni et al ldquoPrevalence of hepatitis Cvirus infection in patients with lymphoproliferative disordersrdquoIsrael Medical Association Journal vol 4 no 1 pp 24ndash27 2002

[7] M B Owlia R Sami M Akhondi and A Salimzadeh ldquoCryo-globulinaemia in hepatitis C-positive patients in Iranrdquo Singa-pore Medical Journal vol 48 no 12 pp 1136ndash1139 2007

[8] C Ferri L la Civita G Longombardo A L Zignego and GPasero ldquoMixed cryoglobulinaemia a cross-road between auto-immune and lymphoproliferative disordersrdquo Lupus vol 7 no 4pp 275ndash279 1998

[9] V Agnello ldquoThe etiology and pathophysiology of mixed cryo-globulinemia secondary to hepatitis C virus infectionrdquo SpringerSeminars in Immunopathology vol 19 no 1 pp 111ndash129 1997

[10] S de Vita L Quartuccio and M Fabris ldquoHepatitis C virusinfection mixed cryoglobulinemia and BLyS upregulationtargeting the infectious trigger the autoimmune response orbothrdquo Autoimmunity Reviews vol 8 no 2 pp 95ndash99 2008

[11] J C Brouet J P Clauvel F DanonM Klein andM SeligmannldquoBiologic and clinical significance of cryoglobulins A report of86 casesrdquo The American Journal of Medicine vol 57 no 5 pp775ndash788 1974

[12] M A Gertz ldquoCold agglutinin disease and cryoglobulinemiardquoClinical Lymphoma vol 5 no 4 pp 290ndash293 2005

[13] M D Wooten and H E Jasin ldquoMixed cryoglobulinemia andvasculitis a novel pathogenic mechanismrdquo Journal of Rheuma-tology vol 23 no 7 pp 1278ndash1281 1996

[14] J Damoiseaux ldquoThe diagnosis and classification of the cryo-globulinemic syndromerdquo Autoimmunity Reviews vol 13 no 4-5 pp 359ndash362 2014

[15] F Invernizzi F Saccardo M Galli G Monti and C ZanussildquoThe cryoglobulinemic syndromerdquo La Ricerca in Clinica e inLaboratorio vol 16 no 2 pp 269ndash274 1986

[16] M J Stone ldquoWaldenstromrsquosmacroglobulinemia hyperviscositysyndrome and cryoglobulinemiardquoClinical Lymphoma andMye-loma vol 9 no 1 pp 97ndash99 2009

[17] A Fornasieri S TazzariM Li et al ldquoElectronmicroscopy studyof genesis and dynamics of immunodeposition in IgMk-IgGcryoglobulin-induced glomerulonephritis in micerdquoThe Ameri-can Journal of Kidney Diseases vol 31 no 3 pp 435ndash442 1998

[18] C R Middaugh and G W Litman ldquoInvestigations of the mole-cular basis for the temperature-dependent insolubility of cryo-globulins VI Quenching by acrylamide of the intrinsic trypto-phan fluorescence of cryoglobulin and non-cryoglobulin IgMproteinsrdquo Biochimica et Biophysica Acta vol 535 no 1 pp 33ndash43 1978

[19] P Vermeersch K Gijbels G Marien et al ldquoA critical appraisalof current practice in the detection analysis and reportingof cryoglobulinsrdquo Clinical Chemistry vol 54 no 1 pp 39ndash432008

[20] GMotyckova andMMurali ldquoLaboratory testing for cryoglob-ulinsrdquo The American Journal of Hematology vol 86 no 6 pp500ndash502 2011

[21] R Sargur P White and W Egner ldquoCryoglobulin evaluationbest practicerdquo Annals of Clinical Biochemistry vol 47 no 1 pp8ndash16 2010

[22] E di Stasio P Bizzarri M Bove et al ldquoAnalysis of the dynamicsof cryoaggregation by light-scattering spectrometryrdquo ClinicalChemistry and Laboratory Medicine vol 41 no 2 pp 152ndash1582003

[23] Z K Shihabi ldquoCryoglobulins an important but neglected clin-ical testrdquo Annals of Clinical and Laboratory Science vol 36 no4 pp 395ndash408 2006

[24] A E Kalovidouris and R L Johnson ldquoRapid cryoglobulinscreening an aid to the clinicianrdquo Annals of the RheumaticDiseases vol 37 no 5 pp 444ndash448 1978

[25] A P Sanchez R Cunard and D M Ward ldquoThe selective ther-apeutic apheresis proceduresrdquo Journal of Clinical Apheresis vol28 no 1 pp 20ndash29 2013

[26] M A Rockx and W F Clark ldquoPlasma exchange for treat-ing cryoglobulinemia a descriptive analysisrdquo Transfusion andApheresis Science vol 42 no 3 pp 247ndash251 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

introduced In our approach a detailed phenomenologicalanalysis of the entire aggregation process with the identifi-cation of parameters describing its evolution over time foreach specimen was performed [22] Moreover the number ofpatients as well as the one of control subjects was notablyhigher than the number of cases reported by Kalovidourisand Johnson [24] and in addition sera from patients affectedby different disorders related to cryoglobulin typing (25type I 625 type II and 125 type III) were analyzed

We compared data from the performance of both classicaland rapid test for cryoglobulins screening on 500 serumsamples Only 1 (04) and 21 (83) out of 252 samplesnegative for cryoprecipitate showed Absmax ge 02 andbetween 01 and 02 respectively The major discrepanciesbetween the two screening tests were found within patientswith cryocrit gt1 Out of 185 patients frankly positive withthe traditional test 6 were negative with the rapid testand 30 reported absorbance values slightly altered (01-02) It is noteworthy to highlight the different phenomenonmonitored by the two assays the traditional test visuallydetects the amount of precipitated aggregates whereas theincrease in the scattered light on the basis of the rapid testis due to the formation of aggregates before precipitationoccurs Therefore results of samples positive to the rapid testand negative to the classical one could find their explanationin (1) a higher sensitivity of spectrophotometer signal inrespect to the macroscopic visual evaluation in detectinglow amounts of aggregatesprecipitate or (2) the possibilitythat cryoglobulins at low concentrations when undergoingthe aggregation process do not reach an adequate clusterdimension for overcoming the solubility limit thus notforming precipitate On the contrary the incongruence ofsamples positive to the traditional test and negative to therapid one could be reasoned by the following (1) the differentspan time of the two assays (ie days versus hours) whichdoes not exclude the possibility of a slow rate of aggregation(that could be undetectable in the first hour of observationand culminate in the precipitate formation only after daysof cold incubation) or (2) the occurrence of alternativeaggregation mechanisms (such as a gelnetwork formationor presence of macroscopic not precipitated aggregates)which interfere with the spectroscopic readings The lasthypothesis is supported by the observation of a high andexclusive frequency (45) of particular macroscopic aspects(gel consistency homogeneous turbidity) in such samples

A critical evaluation of such results has allowed us topropose a ldquofunctionalrdquo classification of cryoglobulins (1)ldquofastrdquo cryoglobulins positive results obtained with the rapidtest (showing an aggregation rate of minutes) as well aswith the traditional test ldquoslowrdquo cryoglobulins only thetraditional assay shows a positive result (precipitation rateof days) ldquoearlyrdquo cryoglobulins only the rapid assay showsa positive result The functional cryoglobulins classificationreported above might be helpful in the clinical approachand management of patients In fact ldquofastrdquo forms could beidentified as proteins able to rapidly aggregate and candidateto precipitate also in vivo ldquoslowrdquo forms could be identified asa laboratory finding of uncertain clinical relevance (probablyartefacts) or as consequence of an unspecific precipitation

originating from decrease of solubility of serum proteinsafter long cold incubation ldquoearlyrdquo cryoglobulins could beidentified as proteins found in samples at low concentrationsso that no visible precipitate could be detected with theclassical method

Finally the utility of the rapid test might be consid-ered in the therapeutic management of cryoglobulinemicpatients whose medical treatment includes plasmapheresisAs known plasmapheresis is a common procedure whichtransiently removes the circulating cryoglobulins reducingmorbidities associated with such disorder [25 26] Theefficacy of such treatment could be tested in a short timeand in a large number of patients by means of the rapid testenabling the patient to be treated by a different therapeuticapproach if poorly effectualThe utility of this approach needsfurther investigations in studies supported by specific clinicalprotocols

5 Conclusions

The present study proposes the rapid screening test as areliable and sensitive method to use in a complementaryway with the traditional test for the assessment of cryoglob-ulinemia Despite many advantages in using the rapid testin respect to the traditional one (high reproducibility noexaminer dependency and low cost results obtained within2 hours) and the possibility to discriminate monoclonal ormixed cryoglobulinemia by the 119905

0parameter cryocrit and its

immunological characterization by immunofixation remainfundamental for cryoglobulins typing whereas the rapid testcan be useful as a ldquoreal timerdquo monitoring of the diseasedevelopment and the evaluation of plasmapheresis efficacy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr M A Isgro for critical reading of thepaper Financial support by the Italian Ministry of Universityand Research (Linea D1 ldquoex-60rdquo 2012-2013 Universita Cat-tolica del Sacro Cuore) is gratefully acknowledged

References

[1] C H Hui and H H Lee ldquoCryoglobulinsrdquo Medical Journal ofAustralia vol 188 no 3 article 158 2008

[2] S Takada T Shimizu Y Hadano et al ldquoCryoglobulinemiardquoMolecular Medicine Reports vol 6 no 1 pp 3ndash8 2012

[3] E Morra ldquoCryoglobulinemiardquo Hematologythe Education Pro-gram of the American Society of Hematology pp 368ndash372 2005

[4] M Ramos-Casals J H Stone M C Cid and X Bosch ldquoThecryoglobulinaemiasrdquoTheLancet vol 379 no 9813 pp 348ndash3602012

[5] S Berentsen ldquoCold agglutinin-mediated autoimmune hemo-lytic anemia in Waldenstromrsquos macroglobulinemiardquo ClinicalLymphoma and Myeloma vol 9 no 1 pp 110ndash112 2009

6 BioMed Research International

[6] H Shirin Y Davidovitz Y Avni et al ldquoPrevalence of hepatitis Cvirus infection in patients with lymphoproliferative disordersrdquoIsrael Medical Association Journal vol 4 no 1 pp 24ndash27 2002

[7] M B Owlia R Sami M Akhondi and A Salimzadeh ldquoCryo-globulinaemia in hepatitis C-positive patients in Iranrdquo Singa-pore Medical Journal vol 48 no 12 pp 1136ndash1139 2007

[8] C Ferri L la Civita G Longombardo A L Zignego and GPasero ldquoMixed cryoglobulinaemia a cross-road between auto-immune and lymphoproliferative disordersrdquo Lupus vol 7 no 4pp 275ndash279 1998

[9] V Agnello ldquoThe etiology and pathophysiology of mixed cryo-globulinemia secondary to hepatitis C virus infectionrdquo SpringerSeminars in Immunopathology vol 19 no 1 pp 111ndash129 1997

[10] S de Vita L Quartuccio and M Fabris ldquoHepatitis C virusinfection mixed cryoglobulinemia and BLyS upregulationtargeting the infectious trigger the autoimmune response orbothrdquo Autoimmunity Reviews vol 8 no 2 pp 95ndash99 2008

[11] J C Brouet J P Clauvel F DanonM Klein andM SeligmannldquoBiologic and clinical significance of cryoglobulins A report of86 casesrdquo The American Journal of Medicine vol 57 no 5 pp775ndash788 1974

[12] M A Gertz ldquoCold agglutinin disease and cryoglobulinemiardquoClinical Lymphoma vol 5 no 4 pp 290ndash293 2005

[13] M D Wooten and H E Jasin ldquoMixed cryoglobulinemia andvasculitis a novel pathogenic mechanismrdquo Journal of Rheuma-tology vol 23 no 7 pp 1278ndash1281 1996

[14] J Damoiseaux ldquoThe diagnosis and classification of the cryo-globulinemic syndromerdquo Autoimmunity Reviews vol 13 no 4-5 pp 359ndash362 2014

[15] F Invernizzi F Saccardo M Galli G Monti and C ZanussildquoThe cryoglobulinemic syndromerdquo La Ricerca in Clinica e inLaboratorio vol 16 no 2 pp 269ndash274 1986

[16] M J Stone ldquoWaldenstromrsquosmacroglobulinemia hyperviscositysyndrome and cryoglobulinemiardquoClinical Lymphoma andMye-loma vol 9 no 1 pp 97ndash99 2009

[17] A Fornasieri S TazzariM Li et al ldquoElectronmicroscopy studyof genesis and dynamics of immunodeposition in IgMk-IgGcryoglobulin-induced glomerulonephritis in micerdquoThe Ameri-can Journal of Kidney Diseases vol 31 no 3 pp 435ndash442 1998

[18] C R Middaugh and G W Litman ldquoInvestigations of the mole-cular basis for the temperature-dependent insolubility of cryo-globulins VI Quenching by acrylamide of the intrinsic trypto-phan fluorescence of cryoglobulin and non-cryoglobulin IgMproteinsrdquo Biochimica et Biophysica Acta vol 535 no 1 pp 33ndash43 1978

[19] P Vermeersch K Gijbels G Marien et al ldquoA critical appraisalof current practice in the detection analysis and reportingof cryoglobulinsrdquo Clinical Chemistry vol 54 no 1 pp 39ndash432008

[20] GMotyckova andMMurali ldquoLaboratory testing for cryoglob-ulinsrdquo The American Journal of Hematology vol 86 no 6 pp500ndash502 2011

[21] R Sargur P White and W Egner ldquoCryoglobulin evaluationbest practicerdquo Annals of Clinical Biochemistry vol 47 no 1 pp8ndash16 2010

[22] E di Stasio P Bizzarri M Bove et al ldquoAnalysis of the dynamicsof cryoaggregation by light-scattering spectrometryrdquo ClinicalChemistry and Laboratory Medicine vol 41 no 2 pp 152ndash1582003

[23] Z K Shihabi ldquoCryoglobulins an important but neglected clin-ical testrdquo Annals of Clinical and Laboratory Science vol 36 no4 pp 395ndash408 2006

[24] A E Kalovidouris and R L Johnson ldquoRapid cryoglobulinscreening an aid to the clinicianrdquo Annals of the RheumaticDiseases vol 37 no 5 pp 444ndash448 1978

[25] A P Sanchez R Cunard and D M Ward ldquoThe selective ther-apeutic apheresis proceduresrdquo Journal of Clinical Apheresis vol28 no 1 pp 20ndash29 2013

[26] M A Rockx and W F Clark ldquoPlasma exchange for treat-ing cryoglobulinemia a descriptive analysisrdquo Transfusion andApheresis Science vol 42 no 3 pp 247ndash251 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

[6] H Shirin Y Davidovitz Y Avni et al ldquoPrevalence of hepatitis Cvirus infection in patients with lymphoproliferative disordersrdquoIsrael Medical Association Journal vol 4 no 1 pp 24ndash27 2002

[7] M B Owlia R Sami M Akhondi and A Salimzadeh ldquoCryo-globulinaemia in hepatitis C-positive patients in Iranrdquo Singa-pore Medical Journal vol 48 no 12 pp 1136ndash1139 2007

[8] C Ferri L la Civita G Longombardo A L Zignego and GPasero ldquoMixed cryoglobulinaemia a cross-road between auto-immune and lymphoproliferative disordersrdquo Lupus vol 7 no 4pp 275ndash279 1998

[9] V Agnello ldquoThe etiology and pathophysiology of mixed cryo-globulinemia secondary to hepatitis C virus infectionrdquo SpringerSeminars in Immunopathology vol 19 no 1 pp 111ndash129 1997

[10] S de Vita L Quartuccio and M Fabris ldquoHepatitis C virusinfection mixed cryoglobulinemia and BLyS upregulationtargeting the infectious trigger the autoimmune response orbothrdquo Autoimmunity Reviews vol 8 no 2 pp 95ndash99 2008

[11] J C Brouet J P Clauvel F DanonM Klein andM SeligmannldquoBiologic and clinical significance of cryoglobulins A report of86 casesrdquo The American Journal of Medicine vol 57 no 5 pp775ndash788 1974

[12] M A Gertz ldquoCold agglutinin disease and cryoglobulinemiardquoClinical Lymphoma vol 5 no 4 pp 290ndash293 2005

[13] M D Wooten and H E Jasin ldquoMixed cryoglobulinemia andvasculitis a novel pathogenic mechanismrdquo Journal of Rheuma-tology vol 23 no 7 pp 1278ndash1281 1996

[14] J Damoiseaux ldquoThe diagnosis and classification of the cryo-globulinemic syndromerdquo Autoimmunity Reviews vol 13 no 4-5 pp 359ndash362 2014

[15] F Invernizzi F Saccardo M Galli G Monti and C ZanussildquoThe cryoglobulinemic syndromerdquo La Ricerca in Clinica e inLaboratorio vol 16 no 2 pp 269ndash274 1986

[16] M J Stone ldquoWaldenstromrsquosmacroglobulinemia hyperviscositysyndrome and cryoglobulinemiardquoClinical Lymphoma andMye-loma vol 9 no 1 pp 97ndash99 2009

[17] A Fornasieri S TazzariM Li et al ldquoElectronmicroscopy studyof genesis and dynamics of immunodeposition in IgMk-IgGcryoglobulin-induced glomerulonephritis in micerdquoThe Ameri-can Journal of Kidney Diseases vol 31 no 3 pp 435ndash442 1998

[18] C R Middaugh and G W Litman ldquoInvestigations of the mole-cular basis for the temperature-dependent insolubility of cryo-globulins VI Quenching by acrylamide of the intrinsic trypto-phan fluorescence of cryoglobulin and non-cryoglobulin IgMproteinsrdquo Biochimica et Biophysica Acta vol 535 no 1 pp 33ndash43 1978

[19] P Vermeersch K Gijbels G Marien et al ldquoA critical appraisalof current practice in the detection analysis and reportingof cryoglobulinsrdquo Clinical Chemistry vol 54 no 1 pp 39ndash432008

[20] GMotyckova andMMurali ldquoLaboratory testing for cryoglob-ulinsrdquo The American Journal of Hematology vol 86 no 6 pp500ndash502 2011

[21] R Sargur P White and W Egner ldquoCryoglobulin evaluationbest practicerdquo Annals of Clinical Biochemistry vol 47 no 1 pp8ndash16 2010

[22] E di Stasio P Bizzarri M Bove et al ldquoAnalysis of the dynamicsof cryoaggregation by light-scattering spectrometryrdquo ClinicalChemistry and Laboratory Medicine vol 41 no 2 pp 152ndash1582003

[23] Z K Shihabi ldquoCryoglobulins an important but neglected clin-ical testrdquo Annals of Clinical and Laboratory Science vol 36 no4 pp 395ndash408 2006

[24] A E Kalovidouris and R L Johnson ldquoRapid cryoglobulinscreening an aid to the clinicianrdquo Annals of the RheumaticDiseases vol 37 no 5 pp 444ndash448 1978

[25] A P Sanchez R Cunard and D M Ward ldquoThe selective ther-apeutic apheresis proceduresrdquo Journal of Clinical Apheresis vol28 no 1 pp 20ndash29 2013

[26] M A Rockx and W F Clark ldquoPlasma exchange for treat-ing cryoglobulinemia a descriptive analysisrdquo Transfusion andApheresis Science vol 42 no 3 pp 247ndash251 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology