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Heat-insoluble cryoglobulin in a patient with essential type II cryoglobulinemia and cryoglobulin-occlusive membranoproliferative glomerulonephritis: Case report and literature review
Clinica Chimica Acta 406 (2009) 170–173
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Case report
Heat-insoluble cryoglobulin in a patient with essential type II cryoglobulinemia and cryoglobulin-occlusive membranoproliferative glomerulonephritis: Case report and literature review Qing H. Meng a,⁎, Rajni Chibbar a, Derek Pearson b, Joanne Kappel b, John Krahn a a b
Department of Pathology and Laboratory Medicine, Royal University Hospital, University of Saskatchewan, 103 Hospital Drive, Saskatoon, SK, Canada S7N 0W8 Department of Medicine, Royal University Hospital, University of Saskatchewan, 103 Hospital Drive, Saskatoon, SK, Canada S7N 0W8
a r t i c l e
i n f o
Article history: Received 28 April 2009 Received in revised form 11 May 2009 Accepted 12 May 2009 Available online 20 May 2009 Keywords: Cryoglobulin Heat-insoluble cryoglobulin Membranoproliferative glomerulonephritis Renal failure Monoclonal gammopathy of undetermined significance
a b s t r a c t Background: A type of heat-insoluble cryoglobulin has been rarely reported and poorly understood. We report the case of a 79 y-old female who was admitted to hospital due to edema and renal failure. Methods: Serial biochemical, immunological, and histological investigations were conducted. Results: This patient had elevated serum urea and creatinine with positive rheumatoid factor and low serum C3 and C4. Her serum was positive for cryoglobulin at 4 °C. The precipitate did not dissolve at 37 °C until it was heated to 56 °C. Electrophoresis of the cryoglobulin demonstrated a monoclonal spike in the gamma region characterized as IgG-κ and polyclonal IgM by immunofixation. Bone marrow aspiration showed presence of 5% plasma cells. Histological examination of renal biopsy revealed a diffuse increase in mesangial matrix, cellularity and endocapillary proliferation. Numerous monocyte/macrophages were present within mesangium and capillary lumina. Focal double contouring of glomerular basement membrane with subendothelial deposits and “hyaline thrombi” were noted. Accordingly, a type II heat-insoluble cryoglobulinemia associated with membranoproliferative glomerulonephritis and monoclonal gammopathy of undetermined significance was made. Conclusions: The unusual heat-insoluble cryoglobulins may indicate severe clinical consequence. Proper laboratory procedure and careful examination of cryoglobulin will assure early recognition and detection of heat-insoluble cryoglobulins. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Cryoglobulins are predominantly immunoglobulin complexes that precipitate when cooled and redissolve when heated [1,2]. Because these proteins precipitate at low temperature, cryoglobulins may be associated with a variety of diseases including lymphoproliferative disorders, autoimmune diseases, and viral infections when patients are exposed to the cold [2,3]. Common clinical findings in patients with cryoglobulinemia include purpura, vasculitis, glomerulonephritis, synovitis with joint swelling and pain, and serositis–pleural effusion and pain [3]. The detection of cryoglobulin has become a routine practice using well established techniques and procedures in clinical laboratories. Yet another type of so-called heat-insoluble cryoglobulin is rarely reported and poorly understood. Rare cases of type I heat-insoluble cryoglobulin associated with glomerulonephritis and multiple myeloma have been described [4,5]. In addition, a patient with type II heat-insoluble cryoglobulin associated with Sjögren's syndrome was reported by
Hent et al. [6]. Different from the previous reports, we recently encountered a case with heat-insoluble type II cryoglobulin associated with membranoproliferative glomerulonephritis and monoclonal gammopathy of undetermined significance (MGUS). 2. Case reports and laboratory investigations 2.1. Case presentation A 79 y old female had been recently admitted to the hospital and diagnosed as having membranoproliferative glomerulonephritis with chronic renal failure. She had been in good health until recently she started to have hematuria and edema. 3. Methods Comprehensive biochemical, immunological, and histological investigations were conducted to establish the diagnosis and pathological cause for the case with renal failure. The results were shown below with reference ranges in bracket. 3.1. Laboratory findings
⁎ Corresponding author. Fax: +1 306 655 2193. E-mail address: [email protected] (Q.H. Meng). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.05.013
She was anemic with RBC 3.15 × 1012/l (3.20–5.40× 1012), hemoglobin 93 g/l (110– 160), hematocrit 0.280 l/l (0.330–0.480), MCV 88.7 fl (79–99), MCH 29.8 pg (27.0–32.0),
Q.H. Meng et al. / Clinica Chimica Acta 406 (2009) 170–173 MCHC 332 g/l (320–360), RDW 16.7% (11.5–15.0%), reticulocytes 171 × 109/l (25–75 × 109) and immature reticulocyte fraction 0.43 (0.10–0.30 for adults). Serum iron was 7 μmol/l (10–29), total iron binding capacity (TIBC) 40 μmol/l (45–72), transferrin saturation 18% (14–51%), and ferritin 380 μg/l (20–120). Overall, the electrolytes (following hemodialysis), lipid profile, and liver function testing were all normal. Significant laboratory findings included: elevated serum urea 21.5 mmol/l (3.7–7.0), creatinine 294 μmol/l (45– 110), positive rheumatoid factor 336 IU/ml (0.0–30.0), low serum C3, 0.56 g/l (0.90–2.00) and marginally low serum C4, 0.15 g/l (0.15–0.40) on several occasions. Serological autoantibody testing including anticardiolipin G, anticardiolipin M, anti-gangliosidemonosialic acid 1, antinuclear antibodies, anti-extractable nuclear antigen, anti-doublestranded DNA, and anti-neutrophilic cytoplasmic antibodies were negative. Serological tests for hepatitis B, hepatitis C, and HIV were all negative. Serum total protein was 45 g/l (60–80) and albumin 25 g/l (35.0–52.0). Serum protein electrophoresis revealed that there was a monoclonal band in gamma region (1.1 g/l), which was characterized as IgG-κ and polyclonal IgM by immunofixation electrophoresis. Serum immunoglobulin levels were quantified as IgG 5.60 g/l (6.94–16.20), IgM 0.99 g/l (0.60–2.65), and IgA 0.85 g/l (0.70–3.80). Serum free κ light chains were 101.20 mg/l (3.30–19.40), free λ light chains 26.70 mg/l (5.70–26.30), and free κ/λ ratio 3.79 (0.26– 1.65). Urine protein was 2.35 g/l and albumin 1.98 g/l. There was a faint band in gamma region on electrophoresis but was too low to be quantitated. Cryoglobulin investigation was conducted following the standard protocol [7]. Her serum was positive for cryoglobulin showing moderate amount of white and jelly-like precipitate at 4 °C. The precipitate did not dissolve at 37 °C but dissolved when it was heated to 56 °C for 10 min (Fig. 1). Electrophoresis of the cryoglobulin demonstrated a monoclonal spike in the gamma region, which was characterized as monoclonal IgG-κ and polyclonal IgM-κ by immunofixation (Fig. 2). The redissolved precipitate pellet was quantified as IgG 7.87 g/l and IgM 3.54 g/l. Bone marrow aspiration showed plasma cells accounting for 5% of all nucleated cells identified by CD138 immunostaining of the biopsy. Combined with serum protein electrophoresis, immunofixation electrophoresis, and clinical data, the patient was identified as MGUS [8]. Histological examination of renal biopsy revealed a membranoproliferative, type I glomerulonephritis characterized by diffuse increase in mesangial matrix, cellularity and endocapillary proliferation (Fig. 3). Numerous CD 68 positive monocyte/macrophages were present within mesangium and capillary lumina. Focal double contouring of glomerular basement membrane with subendothelial deposits and “hyaline thrombi” were noted. The “hyaline thrombi” were weakly eosinophilic stained with PAS and slightly red stained with Masson trichrome stain. Immunofluorescence staining revealed strongly positive, diffuse granular capillary wall and focal mesangial staining for IgG, C3 and κ light chain. There was also positive staining for IgM whereas IgA staining was negative. Significant findings on electron microscopic examination revealed mesangial interposition and scattered subendothelial and mesangial, electron dense deposits (Fig. 4). There were numerous macrophages with phagolysosomes within glomerular capillary lumina and mesangium. Based on the above findings, the essential type II heat-insoluble cryoglobulinemia with the membranoproliferative glomerulonephritis and MGUS was made.
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Fig. 2. Immunofixation electrophoresis of the cryoglobulin. The cryoglobulin in this case was characterized by immunofixation electrophoresis as monoclonal IgG-κ and polyclonal IgM-κ.
Cryoglobulins are immunoglobulins mainly made of IgM or IgG and are classified into 3 types of cryoglobulins [1,2]. Type I cryoglobulin consists of a monoclonal single homogenous immunoglobulin usually IgM or
IgG. Type I cryoglobulinemia is associated with MGUS, Waldenstrom's macroglobulinemia, multiple myeloma, idiopathic nonmalignant monoclonal cryoglobulinemia, or paroxysmal cold hemoglobinuria. Clinical findings include Raynaud's phenomenon, purpura, cutaneous vascularitis, and discoloration of extremities. Type II cryoglobulins are classified as mixed cryoglobulins composed of a monoclonal component usually IgM which has rheumatoid factor activity and a polyclonal component IgG. Type II cryoglobulinemia is associated with autoimmune disorders with multiple organ involvement such as vasculitis, glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. It may also be seen in infections such as hepatitis B or C infection, infectious mononucleosis, cytomegalovirus, and toxoplasmosis. Type II cryoglobulinemia is slightly more common in females than males. In some cases it is difficult to distinguish between the type I and type II cryoglobulinemia, since in many instances a monoclonal immunoglobulin is the major component present in the mixed type and the diffused polyclonal component may be missed if it is not carefully checked [9]. Type III cryoglobulins are mixed cryoglobulins which consist of two or more polyclonal immunoglobulins. Type III cryoglobulinemia is usually associated with the same disease spectrum as Type II cryoglobulinemia. The patient we described here possesses many laboratory and clinical features of mixed cryoglobulinemia including low serum C3 and C4 levels, positive rheumatoid factor, essential type II cryoglobulinemia, MGUS, glomerulonephritis etc.
Fig. 1. Cryoglobulin at 4 °C and 37 °C. Cryoglobulin was formed at 4 °C (middle) and 37 °C (right) gel visible at the bottom of the tube. The left tube with no cryoglobulin formation was a negative control.
Fig. 3. Histological examination of renal biopsy. Light microscopy showed a membranoproliferative pattern with a diffuse increase in mesangial matrix and cellularity with endocapillary proliferation. Numerous inflammatory cells were present within mesangium and intracapillary lumina with “hyaline thrombi” (original magnification × 400).
4. Discussion
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Fig. 4. Electron microscopy of renal biopsy. Electron microscopy revealed mesangial interposition, intraluminal capillary “hyaline thrombi”, and macrophages with phagolysosomes (original magnification × 6000).
In the laboratory setting, cryoglobulins usually precipitate at 4 °C and redissolve at 37 °C. However, temperatures for cryoprecipitation may vary and in some cases, cryoprecipitation may occur close to body temperature. The mechanisms of cryoglobulin formation remain unclear although several hypotheses have been proposed [2]. Several factors may facilitate the precipitation including temperature, protein concentration, pH and ionic strength. Under physiologic conditions of pH and ionic strength, protein concentration is the most important factor in determining the temperature at which a particular cryoglobulin will precipitate. In general, the higher the protein concentration, the higher the temperature at which precipitation occurs. The higher the temperature at which the cryoglobulins precipitate, the more dramatic are the symptoms [10]. The physical characteristics of the cryoprecipitate vary from a gelatinous precipitate to flocculent or crystalline precipitate. The presence of other factors such as hepatitis B, hepatitis C, fibrinogen, and complement components could also affect the temperature of precipitation [2]. Cryoglobulin-induced diseases are associated with abnormal precipitation in the cold. Normally a small amount of cryoglobulin is produced in some people and removed by the liver through a specific hepatocellular receptor [11]. Even deposited in glomeruli, the cryoglobulin can still be removed through phagocytosis by monocytes/macrophages [12]. Increased production and/or decreased removal of cryoglobulin leads to cryoglobulin accumulation and precipitation in tissues and organs, resulting in cryoglobulin-induced diseases [13,14]. The clinical consequence of cryoglobulinemia is the intravascular precipitation of immunoglobulins and immunoglobulin mediated vasculitis, particularly in the skin, peripheral nerves, and kidneys [14]. Clinical manifestations of cryoglobulinemia depend on the tissues or organs involved. The deposition of cryoglobulins in the solid organs such as kidney can cause renal failure [15]. Although type II and III cryoglobulinemia associated membranoproliferative glomerulonephritis are seen in clinical practice, a type II heatinsoluble cryoglobulinemia associated with membranoproliferative glomerulonephritis and MGUS has not been reported [4,6,16]. Ishimura et al. reported a patient with heat-insoluble type I cryoglobulinemia and glomerulonephritis [4]. Hent et al. reported a case of heat-insoluble type II cryoglobulinemia associated with Sjögren syndrome and glomerulo-
nephritis [6]. The immunoglobulins of heat-insoluble type II cryoglobulin reported by Hent et al. [6] are monoclonal IgM-κ and polyclonal IgG-κ whereas in our case, the immunoglobulins of heat-insoluble type II cryoglobulin are monoclonal IgG-κ and polyclonal IgM-κ in addition to other different laboratory and clinical features. The massive deposition of immunocryoglobulins in kidneys is proposed to be responsible for the pathogenesis of chronic glomerular injury and eventually leading to renal failure. The underlying mechanisms of cryoglobulin associated membranoproliferative glomerulonephritis have been studied. It is generally accepted that immunocryoglobulin complex-induced complement activation and inflammatory cell infiltration is responsible for the development of glomerolonephritis [16]. Increased cryoglobulin complex deposition in the basement membrane, capillary lumina, and mesangium of the glomeruli promotes inflammation via complement activation and mononuclear leukocyte recruitment, resulting in kidney injury and the development of membranoproliferative glomerulonephritis [17,18]. Cryoglobulin-induced up-regulation and expression of protease nexin-1, tissue plasminogen activator, and TGF-β1 are responsible for infiltration of monocyte/macrophage, marked expansion of extracellular matrix, and mesangial deposits of cryoglobulins [19]. Overexpression of thymic stromal lymphopoietin, a cytokine that promotes B-cell development, leads to the development of mixed cryoglobulinaemia with renal disease closely resembling human cryoglobulin-associated membranoproliferative glomerulonephritis [20,21]. On the other hand, suppression of cryoglobulin production and deposition effectively prevented renal injury and the development of membranoproliferative glomerulonephritis [22]. In theory, heat-insoluble cryoglobulin, which precipitate at higher temperature, would readily precipitate and deposit in kidney and cause kidney injury, leading to the development of membranoproliferative glomerulonephritis through mechanisms discussed above. Previous study indicated that human monoclonal immunoglobulins from myeloma and cryoglobulinemia share common idiotype, suggesting that monoclonal immunoglobulins could arise from proliferation of a clone that normally produces a natural antibody [23]. In our case, the immunoglobulin phenotypes are the same in both serum and cryoglobulin. There has been no specific treatment for cryoglobulinemia. The primary goal of treatment is to treat the underlying disease including anti-virus, steroid hormones, and immunosuppresants etc. The most effective therapy for cryoglobulinemia is to remove cryoglobulins by plasmapheresis [13]. Our patient was treated with oral dexamethasone 40 mg daily and received hemodialysis 3 times a week. After 2 week treatment, symptoms dramatically resolved and patient's condition dramatically improved. Cryoglobulin is a common laboratory finding and the detection of cryoglobulin provides useful information for patient care. The unusual heat-insoluble cryoglobulins may indicate severe clinical consequence as reported by us and others. Proper laboratory procedure and careful examination of cryoglobulin will assure early recognition and detection of heat-insoluble cryoglobulins. Early detection of heatinsoluble cryoglobulins would certainly provide important information relevant to clinical diagnosis, treatment, and prognosis. Acknowledgement The authors thank Ms. Karen Nogier for technical assistance.
References [1] Brouet JC, Clauvel JP, Danon F, Klein M, Seligmann M. Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 1974;57:775–88. [2] Ferri C, Zignego AL, Pileri SA. Cryoglobulins. J Clin Pathol 2002;55:4–13. [3] Gorevic PD, Kassab HJ, Levo Y, et al. Mixed cryoglobulinemia: clinical aspects and long-term follow-up of 40 patients. Am J Med 1980;69:287–308. [4] Ishimura E, Nishizawa Y, Shoji S, et al. Heat-insoluble cryoglobulin in a patient with essential type I cryoglobulinemia and massive cryoglobulin-occlusive glomerulonephritis. Am J Kidney Dis 1995;26:654–7.
Q.H. Meng et al. / Clinica Chimica Acta 406 (2009) 170–173 [5] Modarressi A, Kuriyan M, Harvey G, Strair R. Heat insoluble cryoglobulin associated with gangrene in multiple myeloma. J Clin Apher 2003;18:190–3. [6] Hent RC, Bergkamp FJ, Weening JJ, Schut NH. A patient with heat insoluble cryoglobulin. Nephrol Dial Transplant 2000;15:917–8. [7] Vermeersch P, Gijbels K, Mariën G, et al. A critical appraisal of current practice in the detection, analysis, and reporting of cryoglobulins. Clin Chem 2008;54:39–43. [8] Durie BG, Kyle RA, Belch A, et al. Myeloma management guidelines: a consensus report from the Scientific Advisors of the International Myeloma Foundation. Hematol J 2003;4:379–98. [9] Bloch KJ. Cryoglobulinemia and hepatitis C virus. N Engl J Med 1992;327:1521–2. [10] Letendre L, Kyle RA. Monoclonal cryoglobulinemia with high thermal insolubility. Mayo Clin Proc 1982;57:629–33. [11] Levo Y. Nature of cryoglobulinaemia. Lancet 1980;1:285–7. [12] Fornasieri A, D Amico G. Type II mixed cryoglobulinaemia, hepatitis C virus infection, and glomerulonephritis. Nephrol Dial Transplant 1996;11(Suppl 4):25–30. [13] Siami GA, Siami FS. Plasmapheresis and paraproteinemia: cryoprotein-induced diseases, monoclonal gammopathy, Waldenström's macroglobulinemia, hyperviscosity syndrome, multiple myeloma, light chain disease, and amyloidosis. Ther Apher 1999;3:8–19. [14] Shihabi ZK. Cryoglobulins: an important but neglected clinical test. Ann Clin Lab Sci 2006;36:395–408. [15] Siami GA, Siami FS. Cryofiltration apheresis for treatment of cryoglobulin-induced glomerulonephritis in renal transplant. Transplant Proc 1995;27:2583–4.
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[16] Alpers CE, Smith KD. Cryoglobulinemia and renal disease. Curr Opin Nephrol Hypertens 2008;17:243–9. [17] Cohen RA, Bayliss G, Crispin JC, et al. T cells and in situ cryoglobulin deposition in the pathogenesis of lupus nephritis. Clin Immunol 2008;128:1–7. [18] Trendelenburg M, Fossati-Jimack L, Cortes-Hernandez J, et al. The role of complement in cryoglobulin-induced immune complex glomerulonephritis. J Immunol 2005;175:6909–14. [19] Taneda S, Hudkins KL, Mühlfeld AS, et al. Protease nexin-1, tPA, and PAI-1 are upregulated in cryoglobulinemic membranoproliferative glomerulonephritis. J Am Soc Nephrol 2008;19:243–51. [20] Taneda S, Segerer S, Hudkins KL, et al. Cryoglobulinemic glomerulonephritis in thymic stromal lymphopoietin transgenic mice. Am J Pathol 2001;159:2355–69. [21] Iyoda M, Hudkins KL, Wietecha TA, et al. All-trans-retinoic acid aggravates cryoglobulin-associated membranoproliferative glomerulonephritis in mice. Nephrol Dial Transplant 2007;22:3451–61. [22] Iyoda M, Hudkins KL, Becker-Herman S, et al. Imatinib suppresses cryoglobulinemia and secondary membranoproliferative glomerulonephritis. J Am Soc Nephrol 2009;20:68–77. [23] Barbouche MR, Guilbert B, Makni S, Gorgi Y, Ayed K, Avrameas S. Common idiotypes expressed on human, monoclonal, abnormal immunoglobulins and cryoglobulins with polyreactive autoantibody activities. Clin Exp Immunol 1993;91:196–201.
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Case report
Heat-insoluble cryoglobulin in a patient with essential type II cryoglobulinemia and cryoglobulin-occlusive membranoproliferative glomerulonephritis: Case report and literature review Qing H. Meng a,⁎, Rajni Chibbar a, Derek Pearson b, Joanne Kappel b, John Krahn a a b
Department of Pathology and Laboratory Medicine, Royal University Hospital, University of Saskatchewan, 103 Hospital Drive, Saskatoon, SK, Canada S7N 0W8 Department of Medicine, Royal University Hospital, University of Saskatchewan, 103 Hospital Drive, Saskatoon, SK, Canada S7N 0W8
a r t i c l e
i n f o
Article history: Received 28 April 2009 Received in revised form 11 May 2009 Accepted 12 May 2009 Available online 20 May 2009 Keywords: Cryoglobulin Heat-insoluble cryoglobulin Membranoproliferative glomerulonephritis Renal failure Monoclonal gammopathy of undetermined significance
a b s t r a c t Background: A type of heat-insoluble cryoglobulin has been rarely reported and poorly understood. We report the case of a 79 y-old female who was admitted to hospital due to edema and renal failure. Methods: Serial biochemical, immunological, and histological investigations were conducted. Results: This patient had elevated serum urea and creatinine with positive rheumatoid factor and low serum C3 and C4. Her serum was positive for cryoglobulin at 4 °C. The precipitate did not dissolve at 37 °C until it was heated to 56 °C. Electrophoresis of the cryoglobulin demonstrated a monoclonal spike in the gamma region characterized as IgG-κ and polyclonal IgM by immunofixation. Bone marrow aspiration showed presence of 5% plasma cells. Histological examination of renal biopsy revealed a diffuse increase in mesangial matrix, cellularity and endocapillary proliferation. Numerous monocyte/macrophages were present within mesangium and capillary lumina. Focal double contouring of glomerular basement membrane with subendothelial deposits and “hyaline thrombi” were noted. Accordingly, a type II heat-insoluble cryoglobulinemia associated with membranoproliferative glomerulonephritis and monoclonal gammopathy of undetermined significance was made. Conclusions: The unusual heat-insoluble cryoglobulins may indicate severe clinical consequence. Proper laboratory procedure and careful examination of cryoglobulin will assure early recognition and detection of heat-insoluble cryoglobulins. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Cryoglobulins are predominantly immunoglobulin complexes that precipitate when cooled and redissolve when heated [1,2]. Because these proteins precipitate at low temperature, cryoglobulins may be associated with a variety of diseases including lymphoproliferative disorders, autoimmune diseases, and viral infections when patients are exposed to the cold [2,3]. Common clinical findings in patients with cryoglobulinemia include purpura, vasculitis, glomerulonephritis, synovitis with joint swelling and pain, and serositis–pleural effusion and pain [3]. The detection of cryoglobulin has become a routine practice using well established techniques and procedures in clinical laboratories. Yet another type of so-called heat-insoluble cryoglobulin is rarely reported and poorly understood. Rare cases of type I heat-insoluble cryoglobulin associated with glomerulonephritis and multiple myeloma have been described [4,5]. In addition, a patient with type II heat-insoluble cryoglobulin associated with Sjögren's syndrome was reported by
Hent et al. [6]. Different from the previous reports, we recently encountered a case with heat-insoluble type II cryoglobulin associated with membranoproliferative glomerulonephritis and monoclonal gammopathy of undetermined significance (MGUS). 2. Case reports and laboratory investigations 2.1. Case presentation A 79 y old female had been recently admitted to the hospital and diagnosed as having membranoproliferative glomerulonephritis with chronic renal failure. She had been in good health until recently she started to have hematuria and edema. 3. Methods Comprehensive biochemical, immunological, and histological investigations were conducted to establish the diagnosis and pathological cause for the case with renal failure. The results were shown below with reference ranges in bracket. 3.1. Laboratory findings
⁎ Corresponding author. Fax: +1 306 655 2193. E-mail address: [email protected] (Q.H. Meng). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.05.013
She was anemic with RBC 3.15 × 1012/l (3.20–5.40× 1012), hemoglobin 93 g/l (110– 160), hematocrit 0.280 l/l (0.330–0.480), MCV 88.7 fl (79–99), MCH 29.8 pg (27.0–32.0),
Q.H. Meng et al. / Clinica Chimica Acta 406 (2009) 170–173 MCHC 332 g/l (320–360), RDW 16.7% (11.5–15.0%), reticulocytes 171 × 109/l (25–75 × 109) and immature reticulocyte fraction 0.43 (0.10–0.30 for adults). Serum iron was 7 μmol/l (10–29), total iron binding capacity (TIBC) 40 μmol/l (45–72), transferrin saturation 18% (14–51%), and ferritin 380 μg/l (20–120). Overall, the electrolytes (following hemodialysis), lipid profile, and liver function testing were all normal. Significant laboratory findings included: elevated serum urea 21.5 mmol/l (3.7–7.0), creatinine 294 μmol/l (45– 110), positive rheumatoid factor 336 IU/ml (0.0–30.0), low serum C3, 0.56 g/l (0.90–2.00) and marginally low serum C4, 0.15 g/l (0.15–0.40) on several occasions. Serological autoantibody testing including anticardiolipin G, anticardiolipin M, anti-gangliosidemonosialic acid 1, antinuclear antibodies, anti-extractable nuclear antigen, anti-doublestranded DNA, and anti-neutrophilic cytoplasmic antibodies were negative. Serological tests for hepatitis B, hepatitis C, and HIV were all negative. Serum total protein was 45 g/l (60–80) and albumin 25 g/l (35.0–52.0). Serum protein electrophoresis revealed that there was a monoclonal band in gamma region (1.1 g/l), which was characterized as IgG-κ and polyclonal IgM by immunofixation electrophoresis. Serum immunoglobulin levels were quantified as IgG 5.60 g/l (6.94–16.20), IgM 0.99 g/l (0.60–2.65), and IgA 0.85 g/l (0.70–3.80). Serum free κ light chains were 101.20 mg/l (3.30–19.40), free λ light chains 26.70 mg/l (5.70–26.30), and free κ/λ ratio 3.79 (0.26– 1.65). Urine protein was 2.35 g/l and albumin 1.98 g/l. There was a faint band in gamma region on electrophoresis but was too low to be quantitated. Cryoglobulin investigation was conducted following the standard protocol [7]. Her serum was positive for cryoglobulin showing moderate amount of white and jelly-like precipitate at 4 °C. The precipitate did not dissolve at 37 °C but dissolved when it was heated to 56 °C for 10 min (Fig. 1). Electrophoresis of the cryoglobulin demonstrated a monoclonal spike in the gamma region, which was characterized as monoclonal IgG-κ and polyclonal IgM-κ by immunofixation (Fig. 2). The redissolved precipitate pellet was quantified as IgG 7.87 g/l and IgM 3.54 g/l. Bone marrow aspiration showed plasma cells accounting for 5% of all nucleated cells identified by CD138 immunostaining of the biopsy. Combined with serum protein electrophoresis, immunofixation electrophoresis, and clinical data, the patient was identified as MGUS [8]. Histological examination of renal biopsy revealed a membranoproliferative, type I glomerulonephritis characterized by diffuse increase in mesangial matrix, cellularity and endocapillary proliferation (Fig. 3). Numerous CD 68 positive monocyte/macrophages were present within mesangium and capillary lumina. Focal double contouring of glomerular basement membrane with subendothelial deposits and “hyaline thrombi” were noted. The “hyaline thrombi” were weakly eosinophilic stained with PAS and slightly red stained with Masson trichrome stain. Immunofluorescence staining revealed strongly positive, diffuse granular capillary wall and focal mesangial staining for IgG, C3 and κ light chain. There was also positive staining for IgM whereas IgA staining was negative. Significant findings on electron microscopic examination revealed mesangial interposition and scattered subendothelial and mesangial, electron dense deposits (Fig. 4). There were numerous macrophages with phagolysosomes within glomerular capillary lumina and mesangium. Based on the above findings, the essential type II heat-insoluble cryoglobulinemia with the membranoproliferative glomerulonephritis and MGUS was made.
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Fig. 2. Immunofixation electrophoresis of the cryoglobulin. The cryoglobulin in this case was characterized by immunofixation electrophoresis as monoclonal IgG-κ and polyclonal IgM-κ.
Cryoglobulins are immunoglobulins mainly made of IgM or IgG and are classified into 3 types of cryoglobulins [1,2]. Type I cryoglobulin consists of a monoclonal single homogenous immunoglobulin usually IgM or
IgG. Type I cryoglobulinemia is associated with MGUS, Waldenstrom's macroglobulinemia, multiple myeloma, idiopathic nonmalignant monoclonal cryoglobulinemia, or paroxysmal cold hemoglobinuria. Clinical findings include Raynaud's phenomenon, purpura, cutaneous vascularitis, and discoloration of extremities. Type II cryoglobulins are classified as mixed cryoglobulins composed of a monoclonal component usually IgM which has rheumatoid factor activity and a polyclonal component IgG. Type II cryoglobulinemia is associated with autoimmune disorders with multiple organ involvement such as vasculitis, glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. It may also be seen in infections such as hepatitis B or C infection, infectious mononucleosis, cytomegalovirus, and toxoplasmosis. Type II cryoglobulinemia is slightly more common in females than males. In some cases it is difficult to distinguish between the type I and type II cryoglobulinemia, since in many instances a monoclonal immunoglobulin is the major component present in the mixed type and the diffused polyclonal component may be missed if it is not carefully checked [9]. Type III cryoglobulins are mixed cryoglobulins which consist of two or more polyclonal immunoglobulins. Type III cryoglobulinemia is usually associated with the same disease spectrum as Type II cryoglobulinemia. The patient we described here possesses many laboratory and clinical features of mixed cryoglobulinemia including low serum C3 and C4 levels, positive rheumatoid factor, essential type II cryoglobulinemia, MGUS, glomerulonephritis etc.
Fig. 1. Cryoglobulin at 4 °C and 37 °C. Cryoglobulin was formed at 4 °C (middle) and 37 °C (right) gel visible at the bottom of the tube. The left tube with no cryoglobulin formation was a negative control.
Fig. 3. Histological examination of renal biopsy. Light microscopy showed a membranoproliferative pattern with a diffuse increase in mesangial matrix and cellularity with endocapillary proliferation. Numerous inflammatory cells were present within mesangium and intracapillary lumina with “hyaline thrombi” (original magnification × 400).
4. Discussion
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Fig. 4. Electron microscopy of renal biopsy. Electron microscopy revealed mesangial interposition, intraluminal capillary “hyaline thrombi”, and macrophages with phagolysosomes (original magnification × 6000).
In the laboratory setting, cryoglobulins usually precipitate at 4 °C and redissolve at 37 °C. However, temperatures for cryoprecipitation may vary and in some cases, cryoprecipitation may occur close to body temperature. The mechanisms of cryoglobulin formation remain unclear although several hypotheses have been proposed [2]. Several factors may facilitate the precipitation including temperature, protein concentration, pH and ionic strength. Under physiologic conditions of pH and ionic strength, protein concentration is the most important factor in determining the temperature at which a particular cryoglobulin will precipitate. In general, the higher the protein concentration, the higher the temperature at which precipitation occurs. The higher the temperature at which the cryoglobulins precipitate, the more dramatic are the symptoms [10]. The physical characteristics of the cryoprecipitate vary from a gelatinous precipitate to flocculent or crystalline precipitate. The presence of other factors such as hepatitis B, hepatitis C, fibrinogen, and complement components could also affect the temperature of precipitation [2]. Cryoglobulin-induced diseases are associated with abnormal precipitation in the cold. Normally a small amount of cryoglobulin is produced in some people and removed by the liver through a specific hepatocellular receptor [11]. Even deposited in glomeruli, the cryoglobulin can still be removed through phagocytosis by monocytes/macrophages [12]. Increased production and/or decreased removal of cryoglobulin leads to cryoglobulin accumulation and precipitation in tissues and organs, resulting in cryoglobulin-induced diseases [13,14]. The clinical consequence of cryoglobulinemia is the intravascular precipitation of immunoglobulins and immunoglobulin mediated vasculitis, particularly in the skin, peripheral nerves, and kidneys [14]. Clinical manifestations of cryoglobulinemia depend on the tissues or organs involved. The deposition of cryoglobulins in the solid organs such as kidney can cause renal failure [15]. Although type II and III cryoglobulinemia associated membranoproliferative glomerulonephritis are seen in clinical practice, a type II heatinsoluble cryoglobulinemia associated with membranoproliferative glomerulonephritis and MGUS has not been reported [4,6,16]. Ishimura et al. reported a patient with heat-insoluble type I cryoglobulinemia and glomerulonephritis [4]. Hent et al. reported a case of heat-insoluble type II cryoglobulinemia associated with Sjögren syndrome and glomerulo-
nephritis [6]. The immunoglobulins of heat-insoluble type II cryoglobulin reported by Hent et al. [6] are monoclonal IgM-κ and polyclonal IgG-κ whereas in our case, the immunoglobulins of heat-insoluble type II cryoglobulin are monoclonal IgG-κ and polyclonal IgM-κ in addition to other different laboratory and clinical features. The massive deposition of immunocryoglobulins in kidneys is proposed to be responsible for the pathogenesis of chronic glomerular injury and eventually leading to renal failure. The underlying mechanisms of cryoglobulin associated membranoproliferative glomerulonephritis have been studied. It is generally accepted that immunocryoglobulin complex-induced complement activation and inflammatory cell infiltration is responsible for the development of glomerolonephritis [16]. Increased cryoglobulin complex deposition in the basement membrane, capillary lumina, and mesangium of the glomeruli promotes inflammation via complement activation and mononuclear leukocyte recruitment, resulting in kidney injury and the development of membranoproliferative glomerulonephritis [17,18]. Cryoglobulin-induced up-regulation and expression of protease nexin-1, tissue plasminogen activator, and TGF-β1 are responsible for infiltration of monocyte/macrophage, marked expansion of extracellular matrix, and mesangial deposits of cryoglobulins [19]. Overexpression of thymic stromal lymphopoietin, a cytokine that promotes B-cell development, leads to the development of mixed cryoglobulinaemia with renal disease closely resembling human cryoglobulin-associated membranoproliferative glomerulonephritis [20,21]. On the other hand, suppression of cryoglobulin production and deposition effectively prevented renal injury and the development of membranoproliferative glomerulonephritis [22]. In theory, heat-insoluble cryoglobulin, which precipitate at higher temperature, would readily precipitate and deposit in kidney and cause kidney injury, leading to the development of membranoproliferative glomerulonephritis through mechanisms discussed above. Previous study indicated that human monoclonal immunoglobulins from myeloma and cryoglobulinemia share common idiotype, suggesting that monoclonal immunoglobulins could arise from proliferation of a clone that normally produces a natural antibody [23]. In our case, the immunoglobulin phenotypes are the same in both serum and cryoglobulin. There has been no specific treatment for cryoglobulinemia. The primary goal of treatment is to treat the underlying disease including anti-virus, steroid hormones, and immunosuppresants etc. The most effective therapy for cryoglobulinemia is to remove cryoglobulins by plasmapheresis [13]. Our patient was treated with oral dexamethasone 40 mg daily and received hemodialysis 3 times a week. After 2 week treatment, symptoms dramatically resolved and patient's condition dramatically improved. Cryoglobulin is a common laboratory finding and the detection of cryoglobulin provides useful information for patient care. The unusual heat-insoluble cryoglobulins may indicate severe clinical consequence as reported by us and others. Proper laboratory procedure and careful examination of cryoglobulin will assure early recognition and detection of heat-insoluble cryoglobulins. Early detection of heatinsoluble cryoglobulins would certainly provide important information relevant to clinical diagnosis, treatment, and prognosis. Acknowledgement The authors thank Ms. Karen Nogier for technical assistance.
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