A Report of 86 Cases - UW Hematology Protocolswilliams.medicine.wisc.edu/cryoglobulins.pdf · 2005. 4. 26. · REVIEWS A Report of 86 Cases JEAN-CLAUDE BROUET, M.D. JEAN-PIERRE CLAUVEL,

REVIEWS

A Report of 86 Cases

JEAN-CLAUDE BROUET, M.D.JEAN-PIERRE CLAUVEL, M.D.

FRAN<;:OISE DANON, M.D.

MICHEL KLEIN, M.D.

MAXIME SELIGMANN, M.D.

Paris, France

From the Research Institute on Blood Diseases,Hopital Saint-Louis, Paris, France. This workwas supported in part by the French National In-stitute for Health and Medical Research(INSERM U. 108) and by the Centre National dela Recherche Scientifique (ERA 239). Requestsfor reprints should be addressed to Dr. J. C.Brouet, Laboratory of Immunochemistry, Re-search Institute on Blood Diseases, HopitalSaint-Louis, 75475 Parix Cedex 10, France.Manuscript accepted February 21, 1974.

Eighty-six patients with cryoglobulinemia repeatedly underwentcomplete immuno.chemical and clinical evaluation during thecourse of their disease. Immunochemical analysis of the purifiedcryoglobulins allowed us to classify them into three groups. TypeI cryoglobulins are made of isolated monoclonal immunoglobulin:IgM (11 cases), IgG (7 cases), IgA (2 cases) or Bence Jonesprotein (1 case). Type II cryoglobulins are mixed cryoglobulin~with a monoclonal component possessing antibody activitytowards polyclonallgG. These cryoglobulins were mainly IgM-lgG(19 cases), sometimes IgG-lgG (2 cases) or IgA-lgG (1 case).Type III cryoglobulins (43 cases) are mixed poly clonal cryoglobu-lins, i.e., composed of one or more classes of poly clonal immuno-globulins and sometimes nonimmunoglobulin molecules such asbeta1C or lipoprotein. Most of these type III cryoglobulins are alsoimmunoglobulin-anti-immunoglobulin immune complexes. Thisclassification enabled us to establish correlations between the bi-ologic findings and the clinical features as well as the underlyingdiseases.

Cutaneous and vasomotor symptoms were most severe in pa-tients with type I and II cryoglobulins. The usual clinical picture inpatients with type II or III cryoglobulins consisted of chronic vas-cular purpura and mild Raynaud's phenomenon. Renal and neuro-logic involvement were more frequent in patients with type II andIII cryoglobulins, and were of major prognostic signilicance. In ourseries, immunoproliferative and autoimmune disorders were themost frequent diseases associated with cryoglobulinemia. Theformer were associated with type I or II cryoglobulins and the lat-ter mainly with type III cryoglobulins. Of note is that idiopathiccryoglobulinemia accounted" for nearly 30 per cent of 1he casesdespite repeated careful clinical evaluation and a mean follow upof 9 years.

In 10 per cent of the cases, acute and severe symptoms ne-cessitated emergency treatment with plasmapheresis and che-motherapy which allowed a satisfactory initial remission in all butone patient. Conversely, no treatment was definitively effective inpatients with chronic symptoms such as vascular purpura.

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t

I

Most proteins involved in cryoprecipitation are immu-noglobulins. Cryoglobulins with a single monoclonalimmunoglobulin were recognized first. the most fre-quent being immunoglobulin G (lgG) or M (lgM). Fol-lowing LoSpalluto's report [1]. cryoglobulins com-posed of two or more immunoglobulins belonging todifferent classes were described and called mixedcryoglobulins, most of which are in fact immunecomplexes [2-4]. IgM-lgG cryoglobulins are the mostfrequent type of mixed cryoglobulins. and the IgMantibody can be either monoclonal or polyclonal[4.5], whereas the antigen is polyclonal IgG. Mono-clonal cryoglobulins with or without antibody activityare found mostly in immunoproliferative diseases.Mixed cryoglobulins are frequent in autoimmune dis-orders. A number of microbial, viral or parasitic in-fections are often associated with asymptomatic and

~ent cryoQlobulins~We describe 86 patients with cryoglobulinemia

who were studied and followed in a department ofhematology and immunopathology. Our chief aimwas to establish in individual patients possible corre-lations between the immunochemical characteristicsof the cryoglobulin and the clinical symptoms or as-sociated diseases.

.;,....

IMMUNOCHEMICAL STUDIES

Isolation and Purification of the Cryoglobulin. Bloodwas obtained from the fasting patient in a room keptat a temperature of 37°C, and the blood, collectedwithout anticoagulant, was allowed to clot at 37°C.The serum, after centrifugation at 37°C, was trans-ferred into narrow tubes which allowed easy detec-tion of minute amounts of cryoprecipitate. The serumsamples were checked for cryoglobulin until the 8thday. The cryoprecipitate was purified by four centrif-ugations and washings at 4°C, the two first being fol-lowed by redissolution of the cryoprecipitate at 37°C.The cryoglobulin level" was evaluated by measuringultraviolet absorption at 280 m,u of an acid dissolvedaliquot of the cryoprecipitate. When the amount ofcryoglobulin was appreciable, the cryoglobulin levelwas evaluated by centrifugation of the serum in a

.~i

hematocrit tube at +4°C or by comparing the serumprotein level before and after cryoprecipitation. Theinitial temperature of cryoprecipitation was definedas the higher temperature at which the serum be-came opalescent. This parameter showed great vari-ability (+10oC to +36°C) from patient to patient.Immunochemlcal Analysis. The purified cryoprecip-itate was characterized by electrophoresis (at a con-centration of 8 to 15 mg/ml) and by immunoelectro-phoretic study of the purified cryoglobulin against an-tiserum to whole human serum and monospecific an-tiserums to gamma, alpha and mu chains (Figure 1).The monoclonal or polyclonal nature of the immuno-globulin(s) found in the cryoprecipitate was mainlyassessed by typing of light chains. In mixed cryo-globulins, the depolymerization by thiol reagentsoften enabled the immunoelectrophoretic determina-tion of the light chain type(s) of the IgM component.However, when the amount of cryoglobulin was low,purification of its immunoglobulin components by pre-parative ultracentrifugation in a sucrose density gra-dient at acid pH was necessary prior to light chaintyping by double diffusion analysis in gel (Figure 2).

Following this analytical study, we classified thecryoglobulins into three categories: (1) monoclonalcryoglobulins made of immunoglobulins with only oneclass or subclass of heavy and/or light chain; (2)mixed cryoglobulins with a monoclonal componentmade of immunoglobulins belonging to two differentclasses, one of which is monoclonal (we have sepa-rated this category of cryoglobulins because of theoutstanding significance of the presence of a mono-clonal immunoglobulin within a mixed cryoglobulin);and (3) polyclonal mixed cryoglobulins which aremade of heterogeneous immunoglobulin moleculesbelonging usually to two or more different classes -

and sometimes of additional serum proteins. This'"classification is arbitrary, but it has been found usefulfor the study of clinicobiologic correlations in pa-tients. '"

Monoclonal Cryoglobulins (Type I Cryoglobullns):-As shown in Table I, 25 per cent of the 86 cryoglobu-lins were purely monoclonal. The IgM cryoglobulins

++ - -I

Figure 1. Immunoelectrophoretic pat-tern of various types of cryoglobulins. 1,monoclonallgG. 2, monoclonal IgM. 3,mixed IgM-lgG cryoglobulin with mono-clonallgM. 4, mixed polyclonallgM-lgA-IgG cryoglobulin. A = polyvalent horseantiserum to normal human serum pre-viously absorbed by IgG globulins. B =rabbit antiserum monospecific forgamma heavy chains.

+

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SIGNIFICANCE OF CRYOG.OBULINS-BROUET ET AL.

D.O. 280 m}J

19s

Figure 2. ~entification of the heavy Iand light chains of the two components lof a mixed IgM lambda-lgG cryoglobulin.Upper, 195 and 75 fractions separatedby preparative ultracentrifugation in asucrose gradient. Lower, immunodiffu-sion studies of the 198 and 75 fractionswith antiserums against mu chains (A);gamma chains (8); kappa chains (C);lambda chains (0).

2 3 4 5 6 7 8 9 10 11 12 13 14 fraction number

rameters. Increasing the concentration of the purifiedcryoglobulin resulted in an increase of the tempera-ture at which precipitation occurred. Acid and alka-line pH, high ionic strength and low concentrations ofurea (0.5 M), guanidine hydrochloride (0.2 M) or sodi-um dodecyl sulphate (0.1 per cent) all resulted in adecrease in the amount of cold precipitate. These re-sults and those from previous reports [7-9J stronglysuggest that weak electrostatic and hydrophobicforces occur during cold-induced denaturation. -

Our results pointed to the major importance of theover-all structure of the immunoglobulin molecule forcold precipitation to occur. Fab, F(ab')2 and Fc frag-ments as well as gamma chains from a IgG 1 kappacryoglobulin did not precipitate in the cold. HoweverSaha et al. [10 J observed a slight cryoprecipitationof isolated .gamma chains and of the F(ab')2 frag-ment from an IgG cryoglobulin. Dimerization of Fab

are the most frequent within this group. We havefound two IgA kappa and one Bence Jones lambdacryoglobulins. The serum level of these monoclonalcryoglobulins is usually high (1 to 30 mg/ml), and theimmunoglobulin readily precipitates in the cold. Usu-ally, a flocculent precipitate is observed; in other in-stances, such as our two IgA cryoglobulins, a gelati-nous precipitate occurred. A single cryoglobulin (lgG2kappa) gave a crystalline precipitate. Six IgG cryo-globulins belonged to the IgG 1 subgroup, four to theIgG2 and one to the IgG3. Thus the frequency of IgG2class is unusually high as in another report [6]. Therelative frequence of kappa or lambda light chaintypes of IgG or IgM cryoglobulins was similar to thatobserved among noncold precipitable monoclonal

immunoglobulin.The cryoprecipitability of the monoclonal immuno-

globulin was affected by various physicochemical pa-

Cryoglobulin Level

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SIGNIFICANCE OF CRYOGLOBULINS-BROUET ET AL.

or F(ab')2 fragments of two IgG cryoproteins wasshown to occur in ultracentrifugal studies [10,11].Reduced and alkylated IgM subunits and mu heavychains obtained from eight of our cryomacroglobulinswere not able to precipitate in the cold. In a single re-port the reduction to monomeric subunits did notabolish cold precipitability [9]. Hybrid IgM moleculesmade of subunits of cryo- and noncryomacroglobu-lins still retained some degree of cryoprecipitability.The Bence Jones lambda cryoprotein was a mixtureof a covalent dimer (80 per cent) and of a native mo-nomer (20 per cent). The isolated covalent dimerprecipitated at +4°C above a molar concentration of4.10-5 M. The isolated monomer or the reduced al-kylated covalent dimer was unable to cryoprecipitateat 4.10 -4 M although ultracent~if~gal analysis at

37°C showed that it was in th~~orm of a non-covalent dimer, demonstrating the importance of theinterchain disulfide bridge in the cryophenomenon.Amino acid analysis revealed a high content of tyro-sine residues (10 per monomer). Difference spec-trum studies showed that tryptophan exposure oc-curred when the temperature decreased. Circular di-chroism analysis showed that the secondary orderedbeta structure was unaffected by temperature de-crease and that only the near ultraviolet spectrumwas modified.

In accordance with previous data [12], the aminoacid composition of light and heavy chains of an IgG1kappa cryoglobulin was similar to that of a noncryo-IgG1 molecule. Carbohydrate analysis revealed astriking lack of sialic acid. However the carbohydratecomposition of several other cryoglobulins showedno remarkable abnormality. Cryoglobulins cannot bepresently considered as abnormal molecules with aunique size, shape or charge characteristics. More-over it is noteworthy that one monoclonallgG 1 kappacryoglobulin of our series exhibited an antistreptolysinantibody activity [13]. X-ray studies of crystalline im-munoglobulin may provide a suitable model for a bet-ter understanding of the cryoprecipitation phenome-non [14].Mixed Cryoglobulins with Monoclonal Component(Type II Cryoglobulins). These cryoglobulins aremade of two immunoglobulin components, one ofwhich is monoclonal. This variety accounts for 26 percent of our cryoglobulins and IgM-lgG cryoglobulinsare by far the most frequent within this group (TableI). The serum level of these cryoglobulins is usuallyhigh. However the serum level of the monoclonalcomponent is sometimes too low to give a character-istic spike, or the interaction between the two com-ponents of the cryoglobulin can mask the electropho-retic homogeneity of the monoclonal protein.

IgM represents the monoclonal component of themixed IgM-lgG cryoglobulins. Isolation of the mono-clonal IgM was performed by gel filtration in pH 4.5,

i

0.2 M acetate buffer. The two separate componentsisolated from the cryoprecipitate were each solublein the cold. The cryoprecipitability was restored bythe addition of purified IgG or of IgG Fc fragments tothe monoclonallgM but not by the addition of unrelat-ed IgM to the polyclonal IgG of the cryoprecipitate.F(ab")2 mu fragments of the IgM were able to com-bine with IgG. Metzger and Stone provided most re-vealing data upon the thermodynamic properties ofthe reaction between such monoclonal IgM and IgG[2,3,15]. The IgM has an homogeneous binding con-stant (6.9 104 M-1). Each of the 10 Fab fragments ofIgM combines with a single antigenic determinant ofhuman IgG although the pentameric molecule bindsonly five IgG molecules. The solubility characteristicsof the small IgM-lgG complexes are determined bythe salt concentration, the pH or temperature of theenvironment, but these parameters do not modify theequilibrium constant between the monoclonal IgMand IgG.

In our series, the monoclonal IgM had kappa lightchains except in two cases in which lambda chainswere found. This is of interest since almost all re-ported monoclonal IgM with anti-lgG activity hadkappa light chains. This situation is most probably rel-evant to the antibody combining site specificity. Intwo cases, a mixed IgM kappa-lgG cryoglobulinshowed an anti-I antibody-like activity. Further studiesof these peculiar cryoglobulins are obviously needed.

In two cases IgG-lgG cryoglobulins were found.auchterlony double diffusion experiments demon-strated that these two purified cryoprecipitates eachcontained two IgG subclasses (respectively, IgG3-IgG1 and IgG3-lgG2) and both types of light chains. Inone of these cases, no monoclonal spike was appar-ent on electrophoresis of the serum or even of theisolated cryoglobulin. The immunoelectrophoreticstudy disclosed the presence of both kappa andlambda molecules in the IgG bow. However myelomaplasma cells of this patient synthesized in vitro onlyIgG3 molecules with lambda light chains. IgG3 is theprevalent subclass among the monoclonal IgG anti-bodies with an anti-lgG activity [16]. Complex forma-tion between Fab or F(ab')2 fragments of monoclonalIgG rheumatoid factors and normal IgG or their F.cfragments was first shown by Grey et al. [16] in ul-tracentrifugal experiments. The binding constant isweak (3 104 M-1) [17]. The detection of the:;e mixedIgG-lgG cryoglobulins is sometimes very difficult sincethe monoclonal immunoglobulin may account for al-most all the protein in the cryoprecipitate and F IIlatex test is seldom positive. Since we have not per-formed systematically such ultra centrifugal experi-ments, we may have underestimated the real fre-quency of these mixed IgG cryoglobulins in our se-ries.

The monoclonal component of the IgA-lgG cryo-


S~IFICANCE OF CRYOGLOBUUNS-BROUET ET AL.

globulin was an IgA 1 kappa protein which gave ahighly viscous cryogel with polyclonal IgG at 35°C.Such monoclonal IgA with anti-lgG antibody activityhave seldom been reported [18,19], and the thermo-dynamic parameters of the reaction have not beenthoroughly worked out.

A peculiar pattern of specificity for these IgM, IgGor IgA monoclonal rheumatoid factors has not yetemerged. The monoclonal IgM of our mixed IgM-lgGcryoglobulins often reacted only with some subclass-es of aggregated monoclonal IgG. Such restrictedspecificity has been described in several laboratories

:[16,20,21]. A monoclonal IgM was shown tocryoprecipitate with IgG molecules which were de-void of sialic acid [22]. More than two-thirds ofmonoclonal rheumatoid factors react only with pri-mate IgG, and the remainder react both with humanand rabbit IgG [23,24]. Cross idiotypic specificitiesbetween various monoclonal IgM rheumatoid factorswere demonstrated in two reports [25,26]. Similaramino acid sequences in the hypervariable regions ofthe heavy or light chains may account for some ofthese cross reactions [26].Mixed Polyclonal Cryoglobulins (Type III Cryoglobu-lins). This group accounts for half of our cryoglobu-lins. IgM-lgG cryoglobulins were the most frequent.The level of these cryoglobulins was usually very low(0.1 to 1 mg/ml) (Table 1).NomonoclonaLimmuno-globulin could be detected by electrophoretic or im-munoelectrophoretic studies of the purified cryo-globulin. In only two-thirds of these cases was theamount of cryoglobulin sufficient to allow furtherstudies. Both kappa and lambda chains were found inthe various immunoglobulin components of thecryoprecipitate isolated by preparative ultracentrifu-gation in acidic medium. Rheumatoid activity, asshown by positive F II latex test, was usually demon-strated either at 37°C with the isolated cryoglobulinor at +4°C with its purified IgM component. Waaler-Rose sheep red blood cell reaction was occasionallypositive in low titer. Chavin and Franklin [27] demon-strated the anti-lgG antibody activity of the polyclonalIgM in such a mixed cryoglobulin. Although IgA-con-taining cryoglobulins are not unusual in Wager et al.'sexperience [28], we have in several cases detectedonly minute amounts of IgA by double diffusion analy-sis of the dissolved precipitate at high concentration.However, in four mixed polyclonal IgM-lgG-lgA cryo-globulins, each of the three major classes of immu-noglobulin was present in appreciable amounts (Fig-ure 1), and the F II latex test was positive. Two poly-clonal IgM cryoprecipitates were described in pa-tients with trypanosomiasis [5]. Various componentsof the complement system may be found in polyclo-nal IgG or IgM-lgG cryoprecipitates [29-31]. In somesuch cases, C 1q presumably plays a major role in thecryoprecipitability since IgG and C1q isolated from the

IgM-IgG-C1q cryoglobulins studied by Stastny and liff[29] precipitated in the cold when mixed in the ab-sence of IgM. Cryoglobulins made of beta lipoproteinand monoclonal IgM were described [32]. The trueimmunologic nature of this interaction was not dem-onstrated. The presence of small amounts of deoxy-ribonucleic acid (DNA) in some cryoprecipitates hasbeen reported [33], but the significance of this find-ing is still uncertain.

Apart from rheumatoid factor activity, antibody ac-tivity at high titers was very seldom reported in isolat-ed polyclonaJ cryoglobulins. Antinuclear activity dis-appeared after cryoprecipitation in the serum of pa-tients with infectious mononucleosis [34]. We foundsuch a decrease in antinuclear factor activity in only1 of 10 cryoprecipitating serums with very high levelsof antinuclear factors. However nuclear antigenswere not found in this cryoprecipitate. In other stud-ies, antibacterial, antiviral, anticardiolipid, antinuclearor red cell antibody activities found in the serum werenot significantly decreased in the supernatant afterprecipitation in the cold, and the corresponding anti-body activities were usually not concentrated in thecryoprecipitate [29,35-37].

One can visualize the significance of these POIy-clonal cryoglobulins in at least two ways: (1) thecryoglobulin may be made of different classes of an-tibodies directed against a same unidentified antigen;and (2) alternatively the cryoglobulin could be due tothe interaction of polyclonal rheumatoid factors withIgG molecules bound to an unknown antigen. Thesecond hypothesis seems more appealing; it couldaccount for most of the findings in human subjectsand is in accordance with recent experimental data[38].

CLINICAL STUDY

Full clinical investigation included, if not contraindicat-ed, extensive hematologic study with Iymphangio-gram and bone marrow biopsy; hepatic, renal andneurologic evaluation; and search for clinical or bio-logic autoimmune disorders. Cutaneous, muscularand renal biopsies were performed if clinically indi-cated. These studies were repeated in many patientsduring the follow up.Presenting Symptoms. In two thirds of our patients.skin lesions or vasomotor attacks were the majorpresenting symptoms. Cold sensitivity was apparentin less than half of these patients. In rare instances,the initial symptoms consisted of renal failure, muco-sal bleeding, visual disturbance or abdominal pains. Infour cases, the diagnosis of cryoglobulinemia wassuggested by unusual routine laboratory findings suchasan increased sedimentation rate at 37°C contrast-ing with a normal rate at room temperature, rouleauxformation and false results in blood counts, especiallyin electronic procedures. In 25 per cent of the cases,

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iS~K;AfoK:-E OF CRYOGLOBULINS-BROUET ET AL.

Figure 3. Left, typical cutaneous le-sions in a patient with type III cryoglobu-lin: extensive vascular purpura with live-do and leg ulcer. Right, orange plaqueson hand and forearm (which wereshown to be caused by vascular andextravascular deposition of IgM kappacryoglobulin) and nail lesions in a patientwith IgM cryoglobulin.

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leading, after some years, to brownish pigmentationof the skin. The rashes were often preceeded byitching or a burning sensation localized to the purpu-ric areas. Painful swelling of the ankles was frequent-ly observed. In two patients, a single rash occurredalthough the cryoglobulin did not disappear. In twoother patients with infrequent purpura for manyyears, sudden and repeated rashes were followed bythe onset of a polyneuropathy or a glomerulonephri-tis.

Skin necrosis and/or supra malleolar ulcers werefound in 5 per cent of our cases. Necrosis affectedthe tip of the nose, the ears, fingers, toes or legs. Inall instances the limited area of skin necrosis ap-peared after exposure to severe cold.

Urticaria and livedo were constantly induced bythe cold and usually associated with vascular purpu-ra. In one patient with IgM kappa cryoglobulin theskin lesions were most unusual. Orange skin infiltra-tion was present on the hands, arms, face, scalp andnose. Involvement of the nails was also observed(Figure 3, right). Ultrastructural studies showed wide-spread proteinaceous deposits inside and outside thevessels of the dermis which stained by anti-mu andanti-kappa antiserums on immunofluorescence.

Mild or severe Raynaud's phenomenon was thepresenting symptom in 15 per cent of our cases andwas most often associated with vascular purpura. In25 per cent of the patients these symptoms werevery severe. The syncopal attacks often worsenedwith time, and necrosis of the fingertips was ob-served in three cases. Cyanosis or erythrocyanosisinduced by cold was found in 9 per cent of our cases.In some patients acrocyanosis was associated withcircumscribed painless white or purple areas dissem-

the cryoglobulin was detected through a systematicsurvey for cryoproteins. More than half of those withhigh level type I cryoglobulins and only 15 per cent ofthose with mixed cryoglobulins were asymptomatic.Cutaneous and Vasomotor Symptoms. Vascularpurpura was the most frequent symptom, usually be-ginning in the lower limbs. In 25 per cent of thecases, the purpura gradually extended to the thighsand the lower part of the abdomen, or to the but-tocks. In a few cases, the arms were involved. Theface and the trunk were always spared. Palatal orgastric purpura was found in two cases. Petechiaand/or papula were the most common lesions (Fig-ure 3, left). Necrotic purpura was found in four pa-tients. Bullous or vesicular lesions were infrequent.Ecchymoses, erythematous spots and dermal nod-ules were associated with the purpura rashes in 20patients. and in 10 patients the clinical picture wasthat of the so-called Gougerot trisymptom. Skin biop-sy was performed in 30 patients. The usual picturewas an acute angiitis characterized by fibrinoid ne-crosis of the vascular walls and perivascular infiltra-tion of pyknotic polymorphonuclear leukocytes. Thevessels of the upper dermis were most often af-fected. In addition, intravascular hyalin deposits cor-responding most likely to the cryoprotein were foundin four cases.

Cold enhanced the purpura rashes in only 30 percent of the cases. On the other hand, crops of pur-pura were often triggered by standing or sustainedefforts. In eight cases, the purpura was eventuallytriggered by drugs (penicillin, acetyl salicylic acid,vaccins) or upper respiratory tract infections. Mostpatients had successive purpuric rashes, usuallyonce or twice a month, lasting for 1 or 2 weeks and


SIGNIFICANCE OF CRVoc.-OBULINS-BROUET ET AL.

TABLE II Incidence of Symptoms Observed in 86 Patients-

Incidence (%) Accordingto Cryoglobulin Type

II

GeneralIncidence

(%) III

15401518

40155

25152005

6020000

401520355500

700

10144

602

581225050

55141085

509

35211772

Symptoms

Cutaneous symptomsVascular purpuraDistal necrosisUrticariaLivedoLeg ulcers

Raynaud's phenomenonAcrocyanosisArticular manifestationsRenal symptomsNeurologic symptomsHemorrhagesAbdominal painsArterial thrombosis

cells. Four other patients (including the two with sud-den anuria) showed large endomembranous depositswithout any cell proliferation. Focal glomerulitis withendomembranous or subepithelial deposits wasfound in four patients. In the last case, the picturewas that of a membranous glomerulonephritis. In 14cases, immunofluorescent studies of the biopsyspecimens were performed and showed positivestaining with anti-ig and anti-beta,C antiserums withgranular deposition along the basement membrane.The immunoglobulin class(es) present in these glo-merular deposits were identical to those found in the

cryoglobulin.In five patients, renal failure was the major cause

of the death. Four patients, still living, have hadchronic renal insufficiency for 2 to 12 years.

Meltzer et al. [4] stressed the frequency of glo-merulonephritis in patients with mixed cryoglobulinand showed the occurrence of voluminous endotheli-al deposits and cell proliferation. Subsequent reportsconfirmed these findings [55-60]. Isolated depositsnearly occluding the capillary lumen were reported ina few patients with monoclonal cryoglobulins[52,60,61]. The present data show that the inci-dence of renal injury is highest in those with type IIcryoglobulins (Table II), but no clear-cut correlationemerged from the comparison between the immuno-chemical type of the cryoglobulin and the histologicpattern. Diffuse glomerulonephritis with endomem-branous deposits was observed in patients with allthree types whereas isolated massive deposits werefound only in those with type I or II, i.e., with mono-clonal components. The biopsy specimen disclosedno histologic abnormalities in three additional patientswith type III mixed polyclonal cryoglobulins and nor-mal urinary findings. Conversely, voluminous en-domembranous deposits were found in two patients

inated all over the body occurring dramatically, for in-stance, during sea baths.

As shown in Table II, vascular purpura or mild va-somotor symptoms were often found in patients withmixed cryoglobulins (type II or 1/1). On the other hand,skin necrosis, necrotic purpura and severe Ray-naud's phenomenon were hallmarks of patients withtype I or II cryoglobulins. In patients with mixed cryo-globulin, cutaneous or vasomotor symptoms usuallyran a chronic course with no dramatic changes in theclinical symptoms over the years. Conversely, inthose with monoclonal cryoglobulins, mild cutaneoussymptoms m.ay precede for years major vascular or

visceral injuries.These findings are in keeping with previous re-

ports, although correlations between clinical and im-munochemical data have rarely been worked out[39-44]. Meltzer et al. [4] first stressed the impor-tance of purpura as well as arthralgias in the clinicalpattern of mixed cryoglobuJins. Immunofluorescentstudies in patients with acute vasculitis showed thedeposition of IgG, IgM and beta1C in the vessel walls[42,45]. A monoclonal or mixed cryoglobulin is foundin about 20 per cent of the patients with cold urticaria[ 46 f. In a case reported by Costanzi and Coltman[47], the monoclonallgG cryoglobulin presumably in-teracted with C1 esterase. Urticaria may be trans-ferred passively to normal recipients with isolatedcryoglobulin [47]. Distal necrosis, extensive gan-grene and recurrent necrosis of the fingers, leadingto successive amputations, have been observed inpatients- with type I cryoglobulins [48-51]. Wide-spread intravascular deposition of proteinaceous ma-terial-has been noted in these patients [52-54].Renal Symptoms. Glomerular injury was found in 21per cent of our patients, disregarding those with sys-temic lupus erythematosus. Anuria and glomerulone-phritis were the presenting symptoms in only two pa-tients. In eight patients, renal damage was found dur-ing the first evaluation. In the other eight patients,clinical or laboratory evidence of glomerulonephritisdeveloped 6 months to 10 years after the initial eval-uation. Subacute or chronic glomerulonephritis wasthe usual clinical pattern (14 patients).

A renal biopsy specimen was obtained from 17patients. Diffuse glomerulonephritis with cell prolifer-ation and endomembranous deposits was found ineight patients, with scattered polymorphonuclearneutrophils and/or acute vasculitis of small vessels insome. Endomembraneous deposits were usuallymoderate and discrete (sometimes with wire loop as-pects), whereas in three patients the deposits almostoccluded the capillary lumen (Figure 4). In two in-stances, ultrastructural studies showed intermembra-noendothelial localization of the deposits. In onecase, crystalline inclusions were found in endothelial


-)t

S~IFK;ANCE OF CRYOGLOBULINS-BROUET ET AL

Figure 4. Voluminous endomembra-nous deposits in a patient with type 1/cryoglobulinemia and proliferative glo-merulonephritis.

,~cable in six of our patients who were affected withperiarteritis nodosa, multiple myeloma or Walden-strom's macroglobulinemia.Miscellaneous. Painful abdominal crisis was ob-served in three patients, one without satisfactory ex-planation, one with hematemesis due to gastric pur-pura and one with intestinal infarction due to mesen-teric arterial occlusion by hyaline thrombi.

Hemorrhage were observed only in six patientswith multiple myeloma or macroglobulinemia. This isin keeping with other reports [52,53,64], and it is dif-ficult to decide if the cryophenomenon plays a crucialrole in the bleeding tendency. However in two pa-tients, the Ivy test was markedly abnormal at 20°Cand returned to normal values at 37°C. In one ofthese, the level of factor V was less than 5 per centat 20°C and was normal at 37°C.

Granular sludge in conjunctival and retinal vesselswas very frequent, mainly in those with monoclonalcryoglobulins. Retinal vein thrombosis was found inthe first reported case of cryoglobulinemia [64].Comments: Two main mechanisms may be responsi-ble for the clinical findings we describe in patientswith cryoglobulinemia.

Intravascular deposits of cryoglobulin were firstsuggested on clinical (cold-induced symptoms of vas-cular insufficiency) as well as histologic (occlusion ofvarious sized vessels) grounds. It was assumed thatcryoprecipitation occurred in vivo or that locai hyper-viscosity enhanced sludging in small vessels and/ormajor vascular insufficiency. However similar intra-vascular deposits may be demonstrated in visceralvessels which are protected from thermic variations.Attention should thus be paid to abnormalities in thelocal microcirculation which are likely to occur in thekidney since protein concentration and hematocritare increased at the end of the capillary loops, lead-

with severe cold-induced symptoms and high levelsof type I or II cryoglobulin despite normal laboratorytests.Neurologic Symptoms. Neurologic involvement wasfound in 17 per cent of our patients and was morefrequent in those with type III cryoglobulinemia than inthose with type I or II cryoglobulinemia (Table II). Innine patients. paresthesia and numbness were themain features. The motor defect appeared somemonths or years later. and progressed in a distal andsymmetrical fashion. It was ascertained in each caseby electromyogram and neuromuscular biopsy. Thelatter was of no help in elucidating the mechanism ofthe neurologic injury. The sensory motor neuropathyusually had a protracted course, and the neurologicdefect stabilized 2 to 4 years after its onset. How-ever, in two patients, the neurologic symptoms disap-peared after several years of follow-up. In four otherpatients. the neurologic symptoms appeared abruptlyand progressed in an asymmetric course. and inthree of these. muscle biopsy showed a picture ofperiarteritis nodosa. Central neurologic involvementwas found in only two patients: one with cryomacro-globulinemia had a transient hemiplegia and the otherwith type III cryoglobulinemia had a temporary blind-ness.

Logothetis et al. [98] reviewed the neurologic in-volvement in patients with cryoglobulinemia. Althoughthey reported an incidence of only 7 per cent. theover-all clinical presentation agreed with our findingsand those of others [4.62]. Central neurologic in-volvement was seldom reported [4,48.63]. Cerebralhyaline thrombi and purpura were found in a patientwith myeloma [63]. The distinctive features of cryo-globulinemic neuropathy are difficult to delineate.Whether the cryoglobulin of the underlying illness isresponsible for the neurologic symptoms was debat-


SIGNIFICANCE OF CRYOGlOBULINS-BROUET ET Al

ing to a decrease in the initial temperature of coldprecipitation and to a sharp increase in viscosity.

The demonstration of the immune complex natureof most mixed cryoglobulins led to postulate thatsuch complexes may be responsible of in vivo acutevasculitis. This hypothesis is heightened by severalfacts: (1) histologic similarities between the cuta-neous or renal lesions found in patients with mixedcryoglobulinemia and in those with acute serum sick-ness; (2) immunofluorescence demonstration of spe-cific deposition in the vascular walls of the immuno-globulin found in the cryoprecipitate; (3) detection ofcirculating immune complexes at 37°C by ultracen-trifugal studies of some serums with mixed cryo-globulins [4,56,57,65,66]; and (4) decrease of theserum complement lev~1 at the onset of visceral in-juries in mixed cryoglobulinemia. However, we foundlow total complement .evel in monoclonal as well asin mixed cryoglobulins and were unable to establishcorrelations between complement levels and renallesions. In vitro experiments showed anticomplemen-tary activity of isolated cryoglobulins [67,68]. Rotheret al. [69] demonstrated a two stage complementactivation: complement was bound to the cryoglobu-lin at +4°C but the reaction went to completion onlyat +37°C.

It is clear from Table II that the symptoms relatedto the mechanical vascular insufficiency are mainlyfound in patients with type I or II cryoglobulins, thelevels of which are usually high. On the other hand,acute cutaneous vasculitis and glomerulonephritisare most frequent in patients with type II and III mixedcryoglobulins. Patients with type I( cryoglobulins mayexhibit features of both varieties of injuries, i.e., vas-culitis in the skin and glomerular obstruction withoutcell proliferation in the kidney. However, exceptionsto these schematic rules were found: some patientswith minute amounts of type III cryoglobulins hadprominent symptoms of vascular insufficiency; twopatients with monoclonal IgG cryoglobulins exhibitedan immune complex type of nephritis with granulardeposition of beta1C and IgG endomembranousthrombi, although the possibility of mixed IgG-lgGcryoglobulin had been ruled out. The pathogeneticimportance of cryoglobulins is sometimes difficult toassess since some signs and symptoms, usually con-sidered as directly related to the cryoglobulin, may bedue to the associated diseases or may be found inpatients with noncryoprecipitating monoclonal immu-noglobulin or rheumatoid factors

multiple myeloma. Four had symptoms directly relat-ed to the cryoglobulin. Extreme cold sensitivity withsevere vasomotor symptoms and/or cutaneous ne-crotic lesions were constant findings in these pa-tients. One patient with mixed IgG-lgG cryoglobulinalso exhibited extensive vascular purpura with acutevasculitis. Polyneuropathy was a dramatic event inanother patient in whom transient clinical improve-ment was obtained twice with extensive plasmapher-esis and chemotherapy. No correlation could be es-tablished between the severity of the cold-inducedsymptoms and the level of the cryoglobulin. Our pa-tients with the most dramatic symptoms had cryo-globulins which precipitated above 33°C. It is note-worthy that cold-induced symptoms were often pres-ent several years before the diagnosis of multiplemyeloma was made and increased in severity withtime. This finding outlines the need for repeated eval-uation of these patients. Such symptoms shouldallow a relatively early diagnosis of myeloma. andtreatment at this stage may prevent the occurenceof life-threatening symptoms, such as extensivegangrene, which were reported in early cases[48.50.51).

Waldenstrom's macroglobulinemia: Nearly 10 percent of monoclonal IgM were cryog/obulins in our ex-perience. Fifteen patients in this series had Walden-strom's macroglobulinemia. Seven had a type I cryo-globulin and eight a type II cryoglobulin with a mono-clonal IgM. The strikingly high incidence of anti-lgGantibody activity of monoclonal IgM in serum as well

ASSOCIATED DISEASES (TABLE III)

Hematologic Diseases. Multiple myeloma: Six percent of the myeloma proteins studied in our laborato-ry were cryoglobulins. Eight patients in this series had

783November 1974 The American Journal of Medicine Volume 57

S~IFK;ANCE OF CRYOGlOBULINS-BROUET ET Al

j

,

nephritis. In one patient, a cryoglobulin was found 15years after the onset of the disease and somemonths before a relapse.

Various type III cryogtobulins (lgM-lgG, IgM-lgG-C1q,IgG-C1q) are found in active systemic lupus erythe-matosus [29,30,56,71] and are most frequent whena glomerulitis and/or a low level of complement arepresent [29]. The titer of antinuclear antibodies wasnot higher in the cryoprecipitate than in whole serum[29]. Koffler et al. [72] showed that rheumatoid fac-tor activity was present in the renal capillary depositsof a patient with systemic lupus erythematosus andmixed cryoglobulin.Sjogren's syndrome: In eight patients, Sjogren's syn-drome was associated with a type II or type III cryo-globulin. In accordance with previous findings[73,74], all these patients exhibited, besides thesicca syndrome, extrasalivary symptoms such asvascular purpura, Raynaud's phenomenon, polyneu-ropathy and necrotizing angiitis. The occurrence ofglomerulitis in three of our patients is striking sinceglomerular injury is exceptional in Sjogren's syn-drome without cryoglobulinemia [73] and empha-sizes the pathogenetic role of the cryoglobulin. Thecourse of the disease was complicated in three pa-tients by the occurrence of a lymphoma.Other autoimmune disorders: Four patients had defi-nite rheumatoid arthritis. No evidence of malignantarthritis was documented in these patients despite along-standing history. One patient with probable rheu-matoid arthritis had a crystallizable IgG2 cryoglobulinwith necrotic purpura. We did not study the synovialfluid in which different varieties of cryoproteins havebeen described [75]; their significance is still uncer-tain.

We found a cryoglobulin in two patients with idio-pathic thrombopenic purpura and in seven patientswith autoimmune hemolytic anemia. Four of the latterwere affected with cold agglutinin disease and theIgM kappa cryoglobulin exhibited strong anti-I activity.

Four patients with type III cryoglobulinemia had theclinical and histologic features of classic periarteritisnodosa. In two, the vascular disease was associatedwith Sjogren's syndrome. The occurrence of cryo-globulins in periarteritis nodosa has seldom been re-ported [76-78]. In view of the possible role of circu-lating immune complexes involving Australia antigenin the pathogenesis of vascular injuries [79], it is ofinterest that one of our patients had acute hepatitis 6years before the onset of periarteritis nodosa. Aus-tralia antigen was present at high concentration inthis serum.Essential Cryoglobu!inemla. The cryoglobulinemiaappeared idiopathic in 29 patients despite carefulevaluation during follow-up. Mixed polyclonal cryo-globulins were the most frequent. Five of these pa-

as on the surface of leukemic lymphocytes [70]suggests that autoantibody-synthesizing clones aremore likely to undergo neoplastic transformation.

Presenting symptoms related to the cryoglobu-linemia were found in 80 per cent of these patientsand were present from 1 to 10 years before evalua-tion leading to diagnosis. Raynaud's phenomenonwas present in 10 patients, vascular purpura in 7 (6of whom had mixed cryoglobulins), glomerular injuryin 5 and neurologic symptoms in 3. Kidney biopsiesshowed massive intracapillary deposits in three pa-tients (two with IgM and one with IgM-lgG cryoglobu-lin) and proliferative nephritis in two patients withmixed cryoglobulinemia.

The presence of 3 cryoglobulin may modify theusual symptoms of Waldenstrom's macroglobulin-emia [53,65]. Vascular purpura or Raynaud's phe-nomenon are not seen in patients without cryoglobu-linemia. Glomerular lesions are far in excess of those:>bserved in macroglobulinemia without cryoglobulinn our experience and have a major prognostic signif-cance. An extreme decrease in IgG levels has been.eported in patients with mixed IgM-lgG cryoglobu-inemia [57,66] and was present in three of our pa-ients. Waldmann et al. [66] demonstrated in such a)atient a decreased synthesis of IgG but also an in-:reased catabolism of those subclasses of IgG which:ombined with the monoclonallgM.)ther hematologic diseases: A diagnosis of chronic,mphocytic leukemia was made in three patientsnd that of well differentiated lymphocytic lymphoma1 six. In two patients, the lymphoma was disclosedeveral years after the diagnosis of cryoglobulinemiand negative hematologic evaluation. All these pa-ents had a type II or III mixed cryoglobulin, usuallyresent in small amounts. A systematic surveylowed us that the incidence of cryoglobulinemia inlese diseases is lower than 2 per cent. Major symp-Ims could be assigned to the cryoglobulin in six ofJr patients, and two died of renal failure. Of note isat, in addition to the Iymphoproliferative disease,'0 patients had autoimmune hemolytic anemia and1e had Sjogren's syndrome.Cryoglobulinemia was also found in two patientsth reticulum cell sarcoma and in individual patientsith Hodgkin's disease, myelofibrosis, reticuloen-Itheliosis or polycythemia vera.Itolmmune Diseases. Mixed cryoglobulin is a fre-ent finding in diseases with a proved or presumedtoimmune mechanism, and it belonged to type III in0 thirds of our cases. A symptomatic cryoglobulin~ceded for many years the onset of overt autoim-Ine diseases in five patients.stemlc lupus erythematosus: Four patients withtive systemic lupus erythematosus had a transient'e III cryoglobulin and three of them had gldmerular


SIGNIFICANCE OF CRYOGLOBULINS-BROUET ET Al.

tients had a monoclonal IgG. The first symptoms hadappeared from 3 to 20 years before evaluation. Twopatients had severe proliferative glomerulonephritis,two had necrotic purpura and one died from mesen-teric arterial obstruction. Although no patient had evi-dence of myeloma or of other malignancy, chemo-therapy was prompted by the severity of the symp-toms. Only two patients had apparently essential type1/ cryoglobulinemia, one with IgM kappa-lgG cryo-globulin, the other with a high level of IgA kappa-lgGcryoglobulin. Because of extreme cold sensitivity withpermanent erythrocyanosis, the latter patient neededrepeated plasmapheresis and chemotherapy. Dra-matic improvement ensued, and the patient is stil/free of symptoms 5 years later.

Among the 22 patients with essential type II/ cryo-globulin, a female prevalence was observed. Patientswere 35 to 70 years old at the onset of symptoms.The mean duration of the disease averages 9 yearsat the present time. Vascular purpura and Raynaud'sphenomenon were the main symptoms. Arthralgiaswere found in half of these patients. Mild leukopeniawas a common finding. Moderate polyclonal hyper.;.gammaglobulinem.ia was frequent (mean 1.7 g/ 1 00m/). Transient positive Coombs' test without hemolyt-icanemia or a significant titer of speckled antinuclearantibodies was found in nine patients. The courseWc8s benign in 11 patients. In four others the myal-gias, paresthesias or vasomotor symptoms weremost disabling. In the last seven patients, renal orneurologic injuries appeared some months or yearsafter the onset of the cutaneous symptoms. In someof these patients, fever and weight loss were sugges-tive of diffuse vasculitis.

It is worth emphasizing that the diagnosis of es-sential cryoglobulinemia should be considered onlyafter extensive evaluation and fol/ow-up. In 10 of ourpatients the initial tentative diagnosis of essentialcryoglobulinemia had to be dismissed 2 to 10 yearslater when autoimmune diseases or hematologicmalignancies became apparent.Comments: The incidence of the various associateddiseases in our patients differs from that of otherlarge series [4,7,28,80] and reflects the fact that ourown cases were col/ected in a department devotedto hematology and clinical immunology. In our series,it appears clearly from Table 1/1 that patients with im-munoproliferative diseases mainly have type I or 1/cryoglobulins, whereas in most patients with autoim-mune disorders, the cryoglobulin belongs to type 1/ ortype II/. It is not astonishing that both groups of asso-ciated diseases are found in patients with type 1/cryoglobulinemia. The autoantibody nature of themonoclonallgM in this variety of cryoglobulins and itshigh incidence among Waldenstr6m's macroglobulinspoints to the possible relationship between autoim-

munity and malignancy. Indeed in our patients, 10 (12per cent) had both Iymphoproliferative and autoim-mune diseases. Since essential cryoglobulinemia ac-counts for 33 per cent of the cases included in thisstudy, it is noteworthy that we have found no cryo-globulin in 100 normal control subjects under 60years old and 4 per cent in older healthy subjects.Using different isolation procedures, Cream [81]found a much higher incidence of minute amounts ofcryoglobulin in normal control subjects.

Cryoglobulinemia in infectious diseases is not in-cluded in this series. Mixed cryoglobulins are encoun-tered in the acute phase of viral illnesses such as in-fectious mononucleosis or cytomegalovirus infection[34,82-84], in subacute bacterial endocarditis [39,85], leprosy [86,87], primary and secondary syphilis[88] and in the course of parasitic infections such astrypanosomiasis [5,39]. A high frequency of mixedcryoglobulins has been found in acute poststrepto-coccal nephritis or in various types of subacute ne-phritis [31,89]. Chronic hepatic diseases may be as-sociated with cryoglobulinemia [4,90-92]. A 4 percent incidence of mixed cryoblobulin was found in asystematic survey of 100 patients with alcoholic cir-rhosis [93]. A direct role of the cryoglobulin in thegenesis of the various visceral injuries found in pa-tients with infectious, renal or hepatic diseases hasnot been established. These cryoglobulins belongusually to type III, sometimes to type II and are oftentransient; their level is usually very low. In contrast toour findings in hematologic and autoimmune disor-ders, symptoms directly related to the presence ofa cryoglobulin are rarely reported in these diseases.However the occurrence of cryoglobulins in infec-tious diseases may have an important pathogenicsignificance since cryoglobulins were found in thecourse of active experimental immunization with pro-tein or nonprotein antigens [94,95], recalling currentetiologic concepts of autoimmunity. In this respectthe finding of IgM cryoglobulins in 25 per cent of NZBmice aged 4 months or older is of interest [96].

TREATMENT

The treatment of the underlying diseases and of thesymptoms linked to the cryoglobulin should be evalu-ated separately. There is a sharp contrast betweenthe dramatic effectiveness of symptomatic treatmentin many patients with severe manifestations and thelittle benefit obtained in most patients with chronicsymptoms such as vascular purpura. In the formerpatients, plasmapheresis is the main emergencytreatment [97]. When indicated, plasmapheresisshould be performed with withdrawal of 400 to 600ml of plasma each day until sustained clinical im-provement is attained, which is usually after 2 to 4weeks. Plasmapheresis should then be performed



usually twice a week until a stable reduction of thecryoglobulin level is maintained. When the initial tem-perature of precipitation is high, plasmapheresisshould be carried out at 37°C, and the separation ofplasma from blood cells should be performed withcare in order to avoid hemolysis. Such long-termplasmapheresis therapy was applied to eight patientsin this series. They had type I or II cryoglobulinemiawith high levels. The clinical diagnosis was Walden-strom's macroglobulinemia in five patients, idiopathicmonoclonal gammopathy in two and multiple myelo-ma in one. The indication for plasmapheresis restedupon the presence of one or more of the followingsevere symptoms: vascular insufficiency (Raynaud'sphenomenon or distal necrosis), renal and/or neuro-logic involvement. A satisfactory clinical improve-ment was obtained in all but one patient and was onlytransient in two. In five patients the initial improve-ment was followed by a satisfactory remission underchemotherapy, and they are doing well without plas-mapheresis after 2 to 5 years of follow up. Otheremergency therapeutic procedures, such as support-ive therapy of renal failure, may be rewarding. In twopatients three episodes of anuria were successfullytreated by hemodialysis, whereas in two others acuterenal faiiur-e led to death despite therapeutic efforts.

In cryoglobulinemia without severe symptoms andin the absence of an underlying disease requiring ac-tive treatment, the decision as to the need of treat-ment and its choice are difficult. Beneficial resultscan be obtained by minimizing cold exposure. Avoid-ance of prolonged standing is most important in pa-tients with purpura. Hydroxychloroquine has occa-sionally been of some benefit in our experience.When manifestations with a relatively poor prognosis,such as renal failure or neurologic involvement, arepresent, or when the patient experiences disabling

paresthesias or myalgias, more active therapy is le-gitimate. Prednisone and/or so-called immunosup-pressive agents have met with limited success[45,97] but have been effective in a few of our pa-tients. Improvement appeared related to the anti-in-flammatory effects of these drugs rather than to anyreduction in the concentration of the cryoglobulin. It isnoteworthy that cytotoxic drugs may modify the anti-gen:antibody ratio and therefore precipitate immunecomplex vasculitis. In our limited experience, penicil-lamine, which has been used for treatment of IgMmonoclonal or mixed cryoglobulinemia, has been in-effective.

The choice and effectiveness of cytotoxic agentsin cryoglobulinemia associated with multiple myelo-ma, Waldenstrom's macroglobulinemia or malignantlymphoma are the same as in random patients withthese diagnoses. In patients with monoclonal cryo-globulinemia and without overt myeloma or macro-globulinemia, the hazard of severe manifestations re-lated to the cryoglobulinemia justifies active treat-ment. In this whole group of patients, the reduction ofthe level of cryoglobulin usually leads to diminution inthe symptoms related to the cryoglobulinemia. In pa-tients with autoimmune disease, the presence of acryoglobulin does not usually modify the therapeuticattitude. Controlled therapeutic trials, which have notbeen performed in the present series, are certainlyneeded in order to evaluate the effectiveness of vari-ous treatments in mixed polyclonal cryoglobulinemia.

";7

ACKNOWLEDGMENT

We are grateful to Dr. L. Morel-Maroger for interpret-ing the kidney biopsies, to Miss Y. Signoret for excel-lent technical assistance and to Mrs. Miglierina forher help in preparing this manuscript.

.;

',:C'

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A Report of 86 Cases - UW Hematology Protocolswilliams.medicine.wisc.edu/cryoglobulins.pdf · 2005. 4. 26. · REVIEWS A Report of 86 Cases JEAN-CLAUDE BROUET, M.D. JEAN-PIERRE CLAUVEL,