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By using the Coulter Counter S-Plus IV erroneous measurements of leukocytes, platelets and red cells were experienced in blood from two patients with cryoglobulinaemia. Leukocyte levels were estimated too high owing to contamination of the leukocyte counting chamber by unlysed erythrocytes. One of the patients had leukopenia, but the Coulter Counter reported white blood cell counts within normal limits. Rewarming of the sample to 37 degrees C eliminated the problem.

An impaired function of splenic macrophages, measured as the clearance rate of erythrocytes coated with IgG (E-IgG), was observed in 7 out of 8 cryoglobulinemic patients with severe urinary abnormalities and systemic symptoms, and in 0 out of 6 patients without urinary symptoms and only mild systemic signs of disease. The E-IgG clearance rate was not related to HLA or Rh phenotype, patients' age or disease duration. Moreover, longitudinal studies showed this parameter to be strictly related to disease activity. To investigate the nature of the defect, five series of analyses were planned using peripheral blood phagocytes (PBP) from 8 patients: a) detection of cell-bound immune material by using the antibody CE59 directed to the Fc fragment of IgG modified by the antibody-antigen reaction; b) cytofluorometric and/or radiometric analyses of the cell surface expression of HLA II, CR1 and FcR structures by means of specific monoclonal antibodies (MoAbs); c) electron microscopy (EM) examination of diverse combinations of cryoglobulins incubated with PBP from patients and normals; d) analysis of cryoglobulin-induced inhibition of E-IgG phagocytosis; e) measurement of the generation of chemiluminescence (CL) in response to Zymosan, Phorbol Myristate Acetate and n-Formyl-Methyonine-Leucine-Phenilalanine (n-FMLP). Patients' PBP were found to have a higher amount of cell-bound immune material as compared to normals (p less than 0.01). CR1 and FcR expression was not different from controls, whereas a slight increase in percentage of monocytes bearing HLA II structures was found in patients (p less than 0.05). Upon EM examination no obvious differences were found in the internalization capacity of cryoglobulins between patients and controls. The CL production was lower than normal (p less than 0.02), whatever stimulus used, with a maximal impairment for n-FLMP (p less than 0.005), the most specific test probe for cytoskeleton integrity. Finally, a remarkable cryoglobulin-induced inhibition of E-IgG phagocytosis was shown. A combination of saturation mechanisms and intracellular abnormalities could underlie the apparent discrepancy between E-IgG clearance defect and preserved potential of cryoglobulin internalization in cryoglobulinemia.

Efficiency of albumin (Alb) and immunoglobulin G (IgG) separation from cryoprecipitable proteins (CPP) and IgM was compared between a membrane plasma fractionator with a mean pore diameter (MPD) of 0.1 micron, surface area of 0.65 m2, and a new large pore diameter filter with a MPD of 4.3 microns, and surface area of 0.14 m2. In five in vitro tests at 4 degrees C using polyclonal cryoglobulinemic (CG) plasma from a patient treated by plasma exchange (PE), the large pore filter retained only 22% of the mass of Alb, and a comparable amount of CPP and IgM, as compared to the plasma fractionator, at a fraction of its total retention capacity. The new filter can replace PE in treatment of polyclonal CG.

Our aim was to assess whether the amount of complement C3b/C4b receptors (CR1) on erythrocytes shows a correlation to disease activity in various connective tissue diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and essential mixed cryoglobulinemia (EMC). Using an anti-CR1 monoclonal antibody, 26 patients with SLE, 34 with RA and 22 patients with EMC were investigated for erythrocyte CR1 expression. The control group consisted of 30 healthy individuals. The mean number of CR1/erythrocyte in the control group was 568 +/- 197 (range 174-1060), significantly higher than studied (EMC:379 +/- 248; p = 0.0005;SLE 147 +/- 56, p less than 0.0001; RA 298 +/- 177, p less than 0.0001). In patients with RA and in SLE, but not in patients with EMC, the number of CR1 numbers and anticardiolipin antibody (aCl) titers (r2 = 0.493; p = 0.034). A statistically significant correlation between CR1 numbers and CH50 values was found in patients with SLE, while in 3 patients with RA 4 months of therapy with cyclosporine A led to a further 30% reduction in CR1 number. Our conclusions are that (a) the decreased expression of erythrocyte CR1 is apparently a common feature of patients with various connective tissue diseases; (b) several acquired factors such as disease activity, complement activation, aCl and drugs may contribute to the loss of CR1 from erythrocytes; (c) in patients with RA and SLE, but not in patients with EMC, CR1 enumeration on erythrocytes may serve as a variable for clinical monitoring.

Cryoglobulin was isolated from a horse which had glomerulo-nephritis and a history of swelling and skin ulcers of the limbs in the winter. The isolated cryoglobulin showed a single peak on a gel permeation chromatography column with an apparent molecular mass (Mr) of 180,000 which could be divided into two gamma bands by cellulose acetate electrophoresis. Immunoelectrophoretic analysis revealed that the cryoglobulin formed two precipitation lines with anti-horse IgG. Spur formation was observed when the cryoglobulin and the IgG purified from a normal healthy horse were cross-reacted with anti-horse IgG on a double diffusion gel. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the reduced conditions showed that the isolated cryoglobulin consisted of two doublets of polypeptides with Mr values of 52,000 and 50,000, and 31,000, and 30,000, corresponding to the heavy chain and the light chain of the horse IgG molecules, respectively. These results suggest that the isolated cryoglobulin might consist of two different IgG molecules, and that the manifestations such as foot swelling with skin ulcers and renal failures were all induced by the cryoglobulin in the serum.

A 93-year-old woman presented with profound anemia (hematocrit 23% [0.23]); there was clumping of her red cells in test tubes and on peripheral blood smears. There was also a marked decrease in erythroid precursors in the bone marrow and reticulocytopenia in the peripheral blood. An IgM kappa monoclonal gammopathy was found in low concentration (approximately 1%) in her serum, and the cold agglutinins had a titer of 2560. However, the cold agglutinin titer of the supernatant after cryoglobulin precipitation was 40. Redissolving the cryoglobulin in the supernatant resulted in a cold agglutinin titer of 1280. Moreover, the addition of the patient's whole serum inhibited erythroid colony formation in culture. The inhibition was removed by cryoprecipitation of the cryoglobulin. The patient was given steroid therapy, to which she responded with reticulocytosis and an elevation of hematocrit. By 3 months, the cold agglutinin titer had fallen to 10. She remained well 4 years later. Whereas reports of cryoprecipitable cold agglutinins are rare, this case is unique because there have been no previous reports that these cold active proteins also have erythroid stem cell-suppressant properties.

The mixed cryoglobulinemia is considered to be a nonmalignant human B-cell proliferation that frequently produces a monoclonal IgM with anti-IgG activity (rheumatoid factor). Using murine monoclonal anti-idiotypic antibodies specific for private or minor idiotopes on monoclonal IgM from three patients suffering from nonmalignant mixed cryoglobulinemia, we investigated the presence of the CD5 antigen on the monoclonal IgM producing cells in these patients. It is shown by two-color cytofluorometric analysis that the majority of the peripheral blood monoclonal IgM rheumatoid factor secreting cells is CD5 negative in these three patients. One of the monoclonal rheumatoid factor K variable regions was sequenced at the protein level and belongs to the human VK III group, as a high proportion of monoclonal rheumatoid factors and some B-cell chronic lymphocytic leukemia (CLL) membrane bound Igs. Thus, despite the preferential use of similar VK genes and the absence of somatic mutation affecting these variable regions in both malignant B-cell CLL and nonmalignant mixed cryoglobulinemia, these proliferating B cells differ in the CD5 membrane expression.

Cryofibrinogenemia, a rare disease, is diagnosed by the presence of reversible cryoprecipitate in plasma, which may cause various clinical manifestations of thromboembolic or hemorrhagic disorders. A 66-year-old female patient suffered from recurrent reticulate erythema, purpura and gangrene on the distal portion of the extremities and face during the winter for 5 years. Cryofibrinogenemia was proven with the following evidences: (1) Histopathologic findings revealed many eosinophilic cylinders, about 6-9 microns in diameter, deposited within the lumen of the blood vessels in the dermis and subcutaneous tissue. There was no evidence of vasculitis. These cylinders were fibrinogen in nature, as proven by direct immunofluorescence. (2) Cryofibrinogen was detected in the citrated plasma, which was confirmed by double immunodiffusion, the presence of an IgG-kappa M protein was also revealed by immunofixation. Debridement and skin grafts were performed, and prednisolone combined with cyclophosphamide were given. Skin lesions improved without recurrence, and the "cryocrit" decreased from 13.6% to less than 2% during the follow up. We conclude that if there is any case with the similar cutaneous manifestations, cryofibrinogenemia should be considered.

We investigated the influence of carbohydrate on the binding of human rheumatoid factors (RF) to the Fc fragment of IgG. The monoclonal RF studied were derived from the serum of patients with mixed cryoglobulinemia or from hybridomas generated from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. Polyclonal RF were derived from patients with RA. The carbohydrate located on the Fc fragment, regardless of whether it contained different amounts of mannose or reduced amounts of galactose, or was removed, did not affect the binding of the RF. In contrast, the isotype of the Fc was found to be critical. Two groups of hybridoma RF could be delineated. One group bound preferentially to IgG1 and/or IgG2, and a second group (primarily from patients with RA) bound preferentially to IgG3 and/or IgG4. Our results indicate that the isotype of the Fc fragment, and not the extent of galactosylation, influences the binding of the RF.

Blood, plasma and serum viscosity and blood filtration were investigated in 43 unselected mixed cryoglobulinemia patients. A hyperviscosity syndrome was present in only one patient, and rapidly improved after plasma exchange and cyclophosphamide therapy. A clear-cut increase in blood viscosity was infrequently observed in mixed cryoglobulinemia, although significant differences were present in the plasma and serum viscosity of patients and controls. In contrast, blood filtration was severely impaired in a high percentage of cases (51 and 72% of the values recorded at 37 degrees and 25 degrees C, respectively), and was on the average significantly higher in patients than in controls. Indirect evidence suggests that blood viscosity is at least in part related to cryoglobulins. In 19 patients studied before and after cryoglobulin removal, serum viscosity significantly decreased when the serum was deprived of cryoglobulins. In addition, the cryocrit correlated with all the hemorheological parameters with the exception of blood filtration. The hemorheological findings were compared with multisystemic features of the disease, i.e. liver, renal, lung, neurologic, vascular and funduscopic alterations. The potential clinical relevance of the hemorheological parameters was stressed by the close correlation between blood filtration parameters and serum creatinine. Furthermore, by discriminant analysis, viscosity and blood filtration changes were the serological parameters most significantly associated with the presence of renal, liver and neurological involvement. Thus, hemorheological parameters are frequently abnormal in mixed cryoglobulinemia patients, and seem to play a significant clinical role; they should therefore be included in the work-up of these patients.

As many as 130 patients with Sjögren's disease (SD) were examined for blood cryoglobulins during 1977-1982. Cryoglobulinemia was discovered in 25 (19.2%) patients with SD. The clinical manifestations such as severe xerostomia, appreciable increase of the parotid salivary glands, hepatosplenomegaly, purpura, polyneuropathy, lesions of the lungs and kidneys were mostly detectable in SD patients with cryoglobulinemia. Ten cryoprecipitates of SD patients with cryoglobulinemia showed the monoclonal immunoglobulins IgMk-9 and IgA-1. All the patients had high titers of antinuclear antibodies and 90% manifested antinuclear Ro/La antibodies. Over the 5-year period. SD patients with cryoglobulinemia manifested the growth of hepatosplenomegaly, ulcerous-necrotic vasculitis, polyneuropathy, polyneuritis, cerebral vasculitis, lesions of the lungs and kidneys. The development of the grave systemic manifestations of the disease was attended by a decrease of immunological activity and the rise of inflammatory activity. The 5-year survival of SD patients with cryoglobulinemia was 64% against 98% in SD patients without cryoglobulinemia (p less than 0.001).

A new type of interference of cryoglobulins on hemocytometric tests is described. The precipitation of temperature-dependent proteins produced a pseudolymphocytosis on a three-part differential leukocyte count of Coulter S-Plus VI, whereas unaffected results, identical to the microscopical count, were obtained using the cytometer Coulter VCS. The laboratory detection of cryoglobulin interference on hematological data is very important in patients with underlying diseases, where the accuracy of absolute and differential leukocyte counts is critical for follow-up. Histograms from the Coulter S-Plus VI can help detect these cases.

Plasma fibronectin (FN) was measured in 17 patients with essential mixed cryoglobulinaemia (EMC) and in 17 normal subjects by single radial immunodiffusion (RID) and enzyme-linked immunosorbent assay (ELISA). In 9 patients the presence of FN in the cryoprecipitates was also assessed by immunoblotting. In the EMC group, plasma FN levels were significantly lower than in control subjects, using both methods, and FN was constantly demonstrated in EMC cryoprecipitates. Capillaroscopic observation of the capillary bed in skin and bulbar conjunctiva, performed in all cases, showed severe alterations of microcirculation in EMC patients. A negative correlation between plasma FN and capillaroscopic abnormalities of skin capillaries was observed. These data support the hypothesis that plasma fibronectin plays a role in the pathogenesis of systemic vasculitis in EMC.

C4-binding protein (C4BP) concentration was measured in sera of patients with systemic lupus erythematosus (SLE) (59) and mixed essential cryoglobulinemia (MEC) (6). The mean concentration of C4BP was not significantly different from the normal controls in both groups of patients; 1 patient with MEC and 11 patients with SLE had concentrations below the normal range. In addition there was no significant correlation between C4BP and C3, C4 or factor B concentrations in patients with SLE. These results suggest that C4BP is not consumed in these two diseases where strong activation of the classical pathway is known to occur in vivo. In addition, the significantly increased C4BP/C4 ratio, evident in both groups of patients, may provide a protective mechanism against C3 conversion by the classical pathway.

Fourteen cases of Essential Mixed Cryoglobulinemia (EMC) are described in this report. Clinical and laboratory manifestations in our patients were similar to those previously reported in literature, although involvement of the peripheral nervous system was much more prevalent in our series. We suggest that peripheral neuropathy should be systematically searched in EMC patients.

In order to gain insight into the role of IgG Fc receptors in essential mixed cryoglobulinaemia (EMC), we analyzed quantitatively the expression of such molecules on polymorphonuclear leukocytes (PMN) and assessed them, functionally, on PMN, tissue macrophages and circulating monocytes in 6 HLA-B8-, DR3-negative patients with EMC and glomerulonephritis. The Fc receptors expressed on PMN surface, as estimated by using AB 8.28 monoclonal antibody to PMN Fc receptor and 125I-labelled rabbit F(ab')2 fragments to mouse IgG as detecting system, were found to be quantitatively normal in EMC patients, whereas PMN Fc- receptor-mediated phagocytosis was reduced. Moreover, the patients were found to have prolonged macrophage clearance of IgG-sensitized autologous erythrocytes and reduced kinetics of ingestion of IgG-coated material by circulating monocytes. This impairment of Fc receptor function may play a remarkable role in the tissue localization of immune material in this disease.

An 82-year-old woman with essential mixed cryoglobulinemia type II (IgM K IgG) presented with moderate renal failure and nephritic syndrome. Mesangiocapillary glomerulonephritis with mesangial and subendothelial granular deposits containing IgG, IgM, and C3 in conjunction with small-vessel vasculitis was seen on renal biopsy. Renal symptomatology preceded by a period of 10 months the development of leg ulcers and purpura. The onset of the skin lesions was accompanied by an acute decline of renal function and an increase in liver alkaline phosphatase. Plasmapheresis with a 50% plasma exchange each week over 12 weeks led to improvement in renal function, healing of leg ulcerations, disappearance of purpura, and a return to the baseline of alkaline phosphatase in association with the disappearance of circulating cryoglobulins.

Cryoglobulinemias in connective tissue diseases (CTD) represent, according to various authors, 12-30% of all cryoglobulinemia cases. Among CTD, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Sjögren's syndrome are the diseases most frequently involved in the presence of cryoglobulins (8-48%). The cryoglobulinemias are mostly of the third type and sometimes of the second type. When clinical symptoms are present, usually they are represented by Raynaud's phenomenon, arthralgias, urticaria, purpura and liver involvement. However, the presence of cryoglobulins in a patient with CTD often does not correlate with clinical picture and other laboratory findings. Eight of our 28 cases (15 SLE and 13 RA) showed third type cryoglobulinemias (7 IgM-IgG and one IgM-IgG-Clq) with a remarkable decrease of serum C4 levels.

Procoagulant activity (PCA) of peripheral blood mononuclear cells was studied in vitro in 14 consecutive patients with essential mixed cryoglobulinemia (EMC). Mononuclear cells tested immediately after isolation expressed significantly higher PCA than cells from a matched control group (P less than 0.01). PCA generated by patients' cells after incubation at 37 degrees C for four hours without any stimulant was significantly higher than that produced by control cells (P less than 0.001). Lower mononuclear cell PCA was observed in the subgroup of patients treated with low doses of prednisone than in untreated patients. In two patients given high-dosage prednisone, cell PCA was markedly reduced. These findings suggest that mononuclear cells may be activated for PCA production in vivo by cryoglobulins or other unknown stimuli. Mononuclear phagocytes, by producing PCA in vivo, might be directly implicated in the local fibrin deposition in tissue lesions of EMC.

In 3 patients with cryoglobulinemia (case 1: type I: cases 2 and 3: type II) spurious leukocyte and platelet counts were seen using automatic particle counters, with up to 2.5X and 3X counts for leukocytes and platelets respectively, with peculiar volume histograms. All these anomalies were absent with manual counting and were still evident in warmed blood samples from 1 of the 3 patients. The corresponding blood smears showed numerous deposits, rectangular in the type I cryoglobulinemia and flake-like in the other two (type II) patients, responsible for the erroneous automatic counting. Ultrastructural study showed in cases 2 and 3 they consisted of dense amorphous protein clusters; and in case 1, a periodic disposition of hollow rods (each with 21-24 nm external diameter). Comparison with ultrastructure descriptions of isolated cryoglobulins from the literature showed that crystals made of hollow rods might be preferentially associated with monoclonal GK immunoglobulin. Close scrutiny of histograms from automatic cell counters and stained blood films is necessary to detect falsely elevated counts in patients with cryoglobulinemia, and in the management of suppressive therapy.

Isolated and washed cryoglobulins obtained from 30 patients with different diseases were studied for the presence of fibronectin by immunoelectrophoresis. Two of the isolated cryoglobulins were of the type I, 3 were of the type II, while the other 25 were of type III. Despite extensive washing, fibronectin was detected in all the cryoprecipitates. Our results indicate the constant presence of fibronectin in the cryoprecipitates from patients with different diseases and types of cryoglobulin. The possible mechanisms by which fibronectin could act in the precipitation of immunoglobulins are discussed.

Isolated and washed cryoimmunoglobulins from 15 patients were studied for the presence of fibronectin by immunodiffusion tests. Two of the isolated cryoglobulins proved to the pure monoclonal immunoglobulins (IgGl kappa and IgGl lambda), nine contained both monoclonal IgM kappa and polyclonal IgG and four were composed of polyclonal IgG, IgM and IgA by immunoelectrophoresis. Double immunodiffusion analysis detected the presence of fibronectin in each of the separated cryoimmunoglobulins. In a solid phase radioimmunoassay, I125 labelled purified fibronectin proved to bind to isolated human monoclonal myeloma proteins of IgGl kappa and IgG3 lambda subclasses. Fibronectin seems to be present not only in mixed polyclonal but also in mixed monoclonal-polyclonal and in monocomponent cryoimmunoglobulins, and it may be bound to one of the immunoglobulin components of the cryoglobulins.

Three patients with cutaneous vasculitis and one patient with digital gangrene had a negative or equivocal test for cryoglobulins but a positive result in a modified assay for cryoproteins ( hypocryoglobulins [ HGs ]) in which serum is rendered hypotonic by dilution with an equal volume of distilled water before incubation in the cold. Each cryoprecipitate contained a mixture of immunoglobulins, and in two instances, a monoclonal component was demonstrated. Rheumatoid factor activity was found in two precipitates. All four patients improved with plasmapheresis, and two subsequently responded to alkylating agents. Seven patients with conventional cryoglobulins had precipitation from diluted serum as well, but none had a substantial increase in precipitation in the HG assay. Only two of five patients with seropositive rheumatoid arthritis had abnormal levels of HG. Hypocryoglobulins are a new category of abnormally insoluble serum proteins, probably closely related to conventional cryoglobulins, which are readily detected in a simple precipitation assay.

The acquired form of cold induced urticarial syndrome can be found associated with serum cryoproteins, in idiopathic form (generally IgE mediated) and transitory forms associated with other factors. The viral infections, specially infectious mononucleosis and hepatitis B can cause urticaria, mostly chronic, although infrequently produces cold urticaria. We present a case of a 13 year old patient with history suggestive of cold urticaria wherein we have found the existence of a mixed polyclonal cryoglobulinemia, IgG-IgA (exceptionally associated) and serologic markers of hepatitis B, HBsAb and HBsAb (the last being suggestive of a recent infection) 3 months from the urticaria, without recent or past history of hepatitis B infection. We also observed an elevated total serum IgE and peripheral blood eosinophilia. The provocation test presented an evolution similar to the cryoglobulinemia and markers of hepatitis B (after 18 months were negative) but serum IgE and eosinophilia remain elevated until the present time. All of this make us think that the patient could have suffered a subclinical form of hepatitis B which triggered off a cryoglobulinemia, presenting as cold urticaria.

Two patients with cryoglobulinemia were treated with plasmapheresis in combination with immunosuppressive therapy. One had Waldenstrom's macroglobulinemia, and the other suffered from systemic lupus erythematosus. Membrane plasmapheresis, using membrane plasma separators, was performed, and the initial plasmapheresis treatments were unsatisfactory because of clotting of hollow fibers by cryoglobulin. The plasma filtration rate and the clearance of immunoglobulins fell rapidly with the clotting of the hollow fibers. This technical difficulty was overcome by prediluting the blood with normal saline before passing it through the membrane plasma separator. The mechanism of preventing clotting of hollow fiber by saline predilution is likely to be the reduction of serum viscosity and the lowering of cryoglobulin concentration. This modification is simple and efficient and enables membrane plasmapheresis to be performed effectively in patients with cryoglobulinemia without complicated and expensive machines.

Fibronectin is a dimeric glycoprotein with a molecular weight of 440,000. It is a soluble constituent of plasma and other body fluids and a fibrillar matrix protein of connective tissue. The two components are structurally similar and convertible. The possibility of multiple molecular interactions gives rise to a variety of biological functions. The regulation of cell growth and the reduced shedding of fibronectin from malignant cells raises the question as to whether fibronectin is valid as a tumour marker. In wound healing and chronic inflammation fibronectin serves as a scaffold for the formation of collagen. As opsonic protein it maintains reticuloendothelial function. Especially in shock, fibronectin may become the limiting factor of unspecific host defence mechanisms. The value of a substitution therapy will be discussed.

We studied the effects of cryoapheresis combined with different immunosuppressive treatments on the course of the glomerulonephritis of essential mixed cryoglobulinemia. The study was carried out on 11 patients. The effects of immunosuppressive treatments on cryoglobulin rebound after cryoapheresis varied widely. In those responding with sustained reduction in serum cryoglobulin levels, creatinine clearance increased, an effect that lasted several years in 4 patients. In one patient cryoglobulin disappeared, with almost fully recovery of renal function and normalization of blood pressure. One patient died of acute liver failure shortly after the first observation and another entered regular dialysis treatment. All the other patients are still alive after follow-up of 2-9 years. These results compare favourably with those reported by other investigators and suggest that cryoapheresis and cytostatic drugs are beneficial for glomerulonephritis associated with essential mixed cryoglobulinemia.

Fourteen patients with essential mixed cryoglobulinemia were studied. The relevance of fibronectin (FN) in cryoprecipitation seems to be of relative importance since only two thirds of the cryoprecipitates have a higher cryocrit level when FN is present. On the other hand, the other samples show a lower precipitating activity after plasma heating at 60 degrees C. This reduction may reflect either a structural FN modification by heating or the denaturation of other cryoprecipitating molecules. It is possible, even if not necessary, that at least in vitro FN plays a role in the cryoprecipitate formation.

There is a close connection between molecular changes of the Ig molecule with sinking temperature and the syndrome of hyperviscosity. The cryoglobulins are usually macroglobulins or complexes between IgM and other immunoglobulin classes. Many of these molecules can also be characterized as euglobulins. The connection between precipitation in the cold and in solutions with low electrolyte content is studied. Data on these connections are presented. The dependence of relative viscosity on the temperature has been investigated in a large number of sera from patients with different diseases. It is clear that most of these cold-sensitive globulins are macroglobulins (IgM). A number of viscosity curves at different temperatures in such sera are given. Also a few sera from patients with IgG myeloma have similar curves. The change in viscosity with lower temperatures implies that relative viscosity has to be determined at 37 degrees C in order to correspond to the condition in the living body. In practice, however, it is usually correct to state that IgM levels above 40 g/l are an indication that plasmapheresis should be considered. The best clinical sign is obtained by ophthalmoscopy. Difficulties with the counting of leukocytes and platelets in electronic counters may be caused by the presence of macroglobulins, usually but not always, cryoglobulins.

Four men and 2 women with Essential Mixed Cryoglobulinemia and a membrano-proliferative glomerulonephritis were treated with prolonged Plasma Exchange without the addition of cytotoxic agents. All patients had Nephrotic Syndrome and Renal Insufficiency. Three of them presented a rapid deterioration of renal function just prior to Plasma Exchange treatment. Total number of procedures varied for each patients from 24 to 105. Serum creatinine decreased significantly in those patients with rapid deterioration of renal function, while it was not modified in the 3 with stable chronic renal failure. In no instance major side effects were recorded, and relapses of the disease did not occur, after gradually tapering of Plasma Exchange sessions. These data suggest that Plasma Exchange alone, if early instituted, may be an effective and safe treatment of Essential Mixed Cryoglobulinemia Glomerulonephritis.

The investigated membranes having a pore size of 3 micron or less can retain over 95% of cryoprecipitable proteins and immune complexes with nearly complete passage of other plasma proteins at 4 degrees C. Based on this result, the G-3 module consisting of V-3000 membrane was chosen for further study. The G-3 module (0.148 m2 of surface area) processed a 10 times higher plasma volume per unit surface area with higher selectivity than the Asahi Plasmaflo AP 06M. Separation efficiency of cryoprecipitable proteins and immune complexes in cryoglobulinemic plasmas by cryofiltration at 4 degrees C is related to the composition of the cryoprecipitable protein; three of four plasmas contained cryoprecipitable proteins that were removable by cryofiltration. Cryoprecipitation was too slow in one of four plasmas. For this plasma SDS PAGE showed its light chain to be of higher molecular weight than other plasmas. Two of four plasmas had elevated amounts of IC that were removed by cryofiltration.

A 43-year-old monk had generalized purpura, arthritis of both shoulders, erythrocyanosis of lip and oral mucous membrane, Raynaud's phenomenon and uveitis. Platelets were normal. Cryoglobulin and cryofibrinogen were positive. Biopsy revealed vasculitis. No underlying infection, collagen vascular disease, lymphoproliferative, myeloproliferative and malignancy were found. He was diagnosed as having essential cryoglobulinemia and cryofibrinogenemia.