Urine Hemosiderin: a Novel Marker to Assess the Severity of Chronic Venous Disease

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Urine hemosiderin: A novel marker to assess the severity of chronic venous disease

Paolo Zamboni, MD,a Marcello Izzo, MD,a Luisella Fogato, MD,a Sergio Carandina, MD,a and Vincenzo Lanzara, PhD,b Ferrara, Italy

Objective: Impaired venous drainage in severe chronic venous insufficiency (CVI) leads to microcirculatory overload, characterized by erythrocyte diapedesis and subsequent extravascular hemolysis, resulting in typical dermal hemosiderin deposition. We hypothesized that hemosiderin, normally absent, could be present in the urine in CVI. Methods: The three-phase study included 117 patients with CVI and 12 healthy control subjects, all of whom had undergone clinical examination and duplex scanning. In phase 1, current methods were used to test urine for hemosiderin in 61 persons: 12 healthy control subjects, 24 patients with mild CVI (clinical class C1 to C3), and 25 patients with severe CVI (clinical class C4 to C6). In phase 2, the concentration of urinary hemosiderin was determined in 45 consecutive patients with CVI, CEAP class 1 to 6. A score of 0 was assigned when typical hemosiderin granules were absent at microscopic examination, a score of 1 when one to three granules per field were detected; 2 when four to six granules were detected; and 3 when more than six granules were observed. Phase 3 included 23 patients with CVI (clinical class 2 to 6). Hemosiderin concentration was determined and a score assigned before patients underwent surgical procedures to correct primary CVI. Both hemosiderin testing and duplex scanning were repeated after 6 months. Results: Phase 1: Urine hemosiderin testing to determine presence or absence of CVI in patients with reflux detectable at duplex scanning yielded the following values: positive predictive value, 96% (95% confidence interval [CI], 86% to 100%); negative predictive value, 88% (CI, 68% to 97%); sensitivity, 94% (CI, 72% to 99%); specificity, 91% (CI, 83% to 99%); and diagnostic accuracy, 95% (CI, 86% to 99%). Phase 2: Hemosiderinuria score enabled classification of clinical severity of ؎ CVI. Mean scores, respectively, were clinical class 1, 0.18 ؎ 0.12; class 2, 0.75 ؎ 0.47; class 3, 1.67 ؎ 0.21; class 4, 1.86 class 5, 2.50 ؎ 0.28; and class 6, 1.92 ؎ 0.21 (P < .001). Phase 3: At 6-month follow-up, hemosiderin score was ;0.26 improved, from 2.48 ؎ 0.12 preoperatively to 0.78 ؎ 0.18 postoperatively (P < .0001). A score of 0 or 1 was associated with successful surgery, whereas a score of 2 or 3 reflected persistence of reflux. Conclusions: Determination of presence of hemosiderin in the urine is a new, sensitive, cost-effective, noninvasive, and repeatable test that enables detection of substantial microcirculatory overload in patients with CVI. (J Vasc Surg 2003; 37:132-6.)

Current tests used to differentiate normal from abnor- proposed a 10-component venous clinical severity score mal venous function, eg, duplex scanning, ambulatory ve- (VCS) to improve objective assessment of venous dis- nous pressure, foot volumetry, photoplethysmography, ease.4,5 and air plethysmography, do not enable classification of The ideal test for detecting this common chronic dis- clinical severity of chronic venous insufficiency (CVI), be- ease would be cost-effective, noninvasive, and easily repeat- cause sensitivity and specificity are unsatisfactory.1-3 Fur- able, and would detect a marker for CVI in urine or serum. thermore, clinical stage of disease as defined by section C of Impaired venous drainage in severe CVI (clinical class 4 the (CEAP) classification system, does not indicate severity to 6) leads to microcirculatory overload, characterized by of venous disease, because many of its components are proliferation of small blood vessels with trapping of white relatively static and others use detailed alphabetic designa- blood cells, and evidence of fibrin cuffs with ulceration.6-8 tions. A scoring scheme for disease severity must be quan- In addition, stasis favors erythrocyte diapedesis with subse- tifiable, with elements that can change in response to quent extravascular hemolysis and dermal hemosiderin treatment. Accordingly, the American Venous Forum has deposition. We hypothesized that hemosiderin, as in intravascular hemolytic disease, would be present in urine in such a From the Department of Surgery and Vascular Laboratory,a and 9-13 Department of Biochemistry and Molecular Biology,b University of Ferrara, model of extravascular hemolysis. Inasmuch as it is Italy. absent in normal urine, we sought to identify hemosiderin This work was supported by grants from the Italian Ministry of University as a new marker of chronic venous disease. and Scientific and Technological Research (MURST). Competition of interest: nil. Presented at the poster session of the Fourteenth Annual Meeting of the METHODS American Venous Forum, La Jolla, Calif, Feb 21-24, 2002. Reprint requests: Paolo Zamboni, MD, Department of Surgery, University Phase 1: Assessment of hemosiderinuria of Ferrara, 44100 Ferrara, Italy (e-mail: zmp@unife). Phase 1 included 61 subjects (38 women, 33 men; Copyright © 2003 by The Society for Vascular Surgery and The American mean age, 48), all of whom had undergone clinical exami- Association for Vascular Surgery. 1 0741-5214/2003/$30.00 ϩ 0 nation and duplex scanning. Twelve were healthy medical doi:10.1067/mva.2003.64 students without clinical or duplex scan evidence of CVI 132 JOURNAL OF VASCULAR SURGERY Volume 37, Number 1 Zamboni et al 133 who served as the control group. Twenty-four were patients with clinical or duplex scan evidence of mild CVI (clinical class C1 to C3); nine of these patients had C1 disease without duplex scan evidence of reflux. Twenty-five were patients with severe CVI (clinical class C4 to C6). The possibility of a simultaneous intravascular hemolytic condition was excluded by negative results of blood tests that included determination of hemochrome, haptoglobin, ferritin, transferrin, iron, and bilirubin concentrations; hemat- ocrit ratio; and antiglobulin testing (Coombs’ test).9 Cur- rent methods were used to test for hemosiderinuria in both patients and control subjects.13 Determination of urinary hemosiderin. Urinary hemosiderin was determined in a 10-mL sample of total daily urine centrifuged at 800g for 10 minutes, for microscopic examination of urinary sediment. After centrifugation, the Fig 1. A typical deeply blue-stained hemosiderin granule in the supernatant was poured off and the pellet resuspended in 5 urine of a patient with chronic venous insufficiency (magnification, ϫ mL of a hemosiderin staining reagent obtained by mixing 400). equal volumes of 2% potassium ferrocyanide (w/v) and 2% hydrochloric acid, obtained by diluting 17.5-fold the com- procedures for reflux elimination were carried out, sparing mercial analytical grade reagent. The sediment was thor- the saphenous vein14,15 Urine hemosiderin assessment and oughly mixed and allowed to stand for 10 minutes. Tubes duplex scanning were repeated at 6-month follow-up were centrifuged again, the supernatant was siphoned off, and the stained pellet was resuspended in a minimal volume Statistical analysis of liquid to wet the tube walls and transferred to a micros- Data are expressed as mean Ϯ SE. Statistical assessment copy slide. A cover slide was applied, and the specimen was of distribution of test results between clinical classes of studied at ϫ400 magnification. Typical deeply blue-stained disease was performed with the Kruskal-Wallis test. Differ- granules indicated a positive test result (Fig 1).9,13 ences in preoperative and postoperative urine hemosiderin scores were tested for significance with the Wilcoxon and Phase 2: Urine hemosiderin score Mann-Whitney U tests. P Ͻ .05 was considered significant. Phase 2 included 45 consecutive patients (13 men, 32 Positive predictive value (PPV), negative predictive value women; mean age, 58 years) with CVI. In all patients the (NPV), sensitivity, specificity, and diagnostic accuracy of diagnosis was confirmed with complete duplex scanning of the test were assessed, using the 95% confidence interval both legs. According to CEAP classification, CVI was (CI) when appropriate. scored as clinical class 1, with no evidence of reflux at duplex scanning, in 11 patients; class 2 in 4 patients; class 3 RESULTS in 6; class 4 in 7; class 5 in 4; and class 6 in 13. Phase 1. Urine hemosiderin test results were negative Urinary hemosiderin was detected according to the in all control subjects, but were positive in 7 of 24 (29%) methods described above. A score of 0 was assigned in the patients with mild CVI (clinical class 1 to 3) and 23 of 25 absence of typical granules; a score of 1 when one to three (92%) patients with severe CVI (clinical class 4 to 6) (P Ͻ granules per field were detected; 2 when four to six granules .0001, Kruskal-Wallis test) (Fig 2). were detected; and 3 when more than six granules were In assessing for the presence or absence of CVI in observed. Scores were then compared with clinical disease patients with reflux detected at duplex scanning, the test class. demonstrated PPV of 96% (CI, 86% to 100%); NPV, 88% The concentration of hemosiderin granules in the urine (CI, 68% to 97%); sensitivity, 94% (CI, 83% to 99%); sediment was not assessed. Hemosiderin score reflects the specificity, 91% (CI, 72% to 99%); and diagnostic accuracy, overall number of hemosiderin granules found in the uri- 95% (CI, 86% to 99%). nary sediment, which was not modified by changing the In contrast, according to CEAP clinical criteria, which volume in which the sediment was resuspended. include simple telangiectasia (class 1), PPV was 100%; NPV, 52% (CI, 31% to 73%); sensitivity, 71% (CI, 55% to Phase 3: Urinary hemosiderin at postoperative 84%); specificity, 100%; and diagnostic accuracy, 78% (CI, follow-up 64% to 88%). Phase 3 included 23 consecutive patients (11 men, 12 Phase 2. The proposed score based on urine hemo- women; mean age, 54 years) scheduled to undergo surgical siderin concentration enabled classification of CVI accord- correction of monolateral primary superficial CVI con- ing to clinical severity. Mean score in clinical class 1 was firmed at duplex scanning. Three patients had clinical class 0.18 Ϯ 0.12; in class 2, 0.75 Ϯ 0.47; in class 3, 1.67 Ϯ 2 disease; 4, class 3 disease; 6, class 4 disease; 3, class 5 0.21; in class 4, 1.86 Ϯ 0.26; in class 5, 2.50 Ϯ 0.28; and in disease; and 7, class 6 disease. Hemodynamic correction class 6, 1.92 Ϯ 0.21 (P Ͻ .001) (Fig 3). JOURNAL OF VASCULAR SURGERY 134 Zamboni et al January 2003

high molecular weight. Hemosiderin accumulates in the cells of the tubular epithelium and is only secondarily excreted when such cells are shed into the urine.12 This mechanism seems to apply to our findings. There is lack of a true standard between investigations used in patients with CVI. The CEAP classification, al- though somewhat rigid, is still the best method for defining the clinical severity of CVI.1 Recently the system was improved with the introduc- tion of the VCS score, which is quantifiable and gradable, with elements that can change in response to clinical vari- ations resulting from treatment or natural progression of disease.4,5 However, VCS scores also show small but significant intraobserver and interobserver variability, which could affect their reliability.5 We believe the urine hemosiderin test to be a valuable tool to be used in addition to clinical assessment, especially in differentiating between class 1 and 2 and class 3 and 4 CVI, in which both intraobserver and interobserver variation seems to occur more frequently.5 The main finding of our study is that positive results of the urine hemosiderin test enable differentiation of healthy subjects from patients with CVI and duplex scan–detectable reflux, with significant PPV, NPV, sensitivity, specificity, and accuracy levels. In addition, when the test is used in healthy subjects and patients with CVI, including those with class 1 disease without detectable reflux, it almost always demonstrates correct values for PPV and specificity but less accurate values for NPV, sensibility, and diagnostic accuracy, because of a higher number of false negative Fig 2. Urine hemosiderin test in healthy control subjects and results. patients with chronic venous insufficiency. Test results are always This raises the question of whether most patients with negative in healthy subjects and are more likely positive with class 1 disease should be considered to have CVI or a Ͻ increasing clinical severity of disease (P .001). different pathologic condition, often coexistent, mainly cosmetic, but with unrelated pathogenesis, pathophysio- Phase 3. Six-month follow-up demonstrated signifi- logic features, and prognosis. Usually in such patients ve- cant improvement in the hemosiderin score, from 2.48 Ϯ nous investigations do not demonstrate any venous reflux 0.12 preoperatively to 0.78 Ϯ 0.18 postoperatively (P Ͻ or obstruction, and the urine hemosiderin marker is not .0001). This corresponds to surgical outcome as assessed at positive. clinical examination and duplex scanning. Moreover, clinical classification of CVI as class 1 or 2 in The score worsened or did not change in only two patients with considerable dilatation of the venular system patients. In one, duplex scans revealed saphenofemoral is sometimes difficult. In such cases, it could be proposed recurrence of disease. In the other patient, superficial ve- that clinical class 2 be assigned only to disease in patients nous surgery was performed in the presence of coexisting with positive hemosiderin test results, drastically reducing deep reflux, which persisted at duplex scanning 6 months CEAP and VCS score interobserver and intraobserver vari- later. ability. Results of the proposed test appear to be consistently DISCUSSION positive in the more severe cases, and, when positive early Chronic venous stasis is indicative of erythrocyte diape- during clinical evolution of the disease, might precede by desis and dermal ferritin deposition as a result of extravas- years the dermal accumulation of visible hemosiderin de- cular hemoglobin catabolism. Subsequently, the increasing posits. This hypothesis remains to be proved in a rigorous overload of iron changes the structure of ferritin to hemo- longitudinal test. siderin.9-13,16-19 It is presumed that the hemosiderin stored In addition, results of the proposed hemosiderin test in the interstitial matrix partially drains through the lym- may lead to modification of the clinical management of phatic vessels and reaches the circulatory system through uncomplicated varicose veins. For example, in patients with the thoracic duct. varicose veins and negative urine hemosiderin test results The kidneys have a fundamental role in iron metabo- treatment could consist of use of elastic stockings along lism; however, hemosiderin cannot be filtered because of its with comprehensive follow-up. JOURNAL OF VASCULAR SURGERY Volume 37, Number 1 Zamboni et al 135

Fig 3. Distribution of urine hemosiderin scores among clinical classes of chronic venous insufﬁciency (P Ͻ .001).

The patient who prefers surgical treatment should be made aware that the main purpose of the procedure is cosmetic. Surgery could be reserved for patients with C2 disease with a positive test result, to prevent progression of the disease to class C4 to C6. Phase 2 of our study demonstrates how the proposed test scoring system enables rapid, albeit rough, quantifica- tion of the concentration of hemosiderin in the urinary sediment. In addition, the scoring system enables differentiation of the clinical classes of CVI (Fig 3). It is curious that the hemosiderin score was higher in clinical class 5 disease than in clinical class 6. Recently, nitric oxide was found to be highly concentrated in patients with healed ulcer- ations.20 This could be related to the natural drainage of Fig 4. Urine hemosiderin score obtained preoperatively and 6 Ͻ toxic agents through the ulcers, with consequent reduction months after superficial venous surgery (P .0001). in renal hemosiderin elimination. Finally, the proposed scoring system is useful for patient follow-up after surgery. Reduction of venous overload In conclusion, the hemosiderin test is a new, noninva- contributed to significant improvement in the hemosider- sive, repeatable, and cost-effective method for classifying inuria score within 6 months (Fig 4). This finding could chronic venous disease, and further studies are warranted. represent a possible future rapid, economic, and cost-effective tool for monitoring patient outcome after surgery, to REFERENCES complement the clinical CEAP or VCS score at office 1. Nicolaides AN, and the International Consensus Group. The investiga- evaluation. For example, in patients with preoperative clin- tion of chronic venous insufficiency: a consensus statement. Circulation ical class 4 or 5 disease, with irreversible skin damage, CVI 2000;102:126-63. is difficult to reclassify on the basis of physical examination 2. Belcaro G, Christopoulos D, Nicolaides AN. Basic data related to alone and, despite a successful surgical outcome, often normal and abnormal lower extremity hemodynamics. Ann Vasc Surg must be reevaluated with duplex scanning or other hemo- 1991;5:306-10. 3. Goldman MP, Weiss RA, Bergan JJ. Diagnosis and treatment of varicose dynamic tests. veins: a review. Dermatology 1994;31:393-413. According to our findings, instrumental investigations 4. Rutherford RB, Padberg FT Jr, Comerota AJ, Kistner R, Meissner MH, could be reserved for patients with urine hemosiderin Moneta GL. Venous severity scoring: an adjunct to venous outcome scores of 2 or 3. In our survey, when the score was 0 or 1 (ie, assessment. J Vasc Surg 2000;31:1307-12. 5. Meissner MH, Natiello C, Nicholls SC. Performance characteristics of trace amounts of hemosiderin), duplex scans were always the venous clinical severity score (VCS). J Vasc Surg 2002;36:889-95. negative for reflux. Moreover, in phases 1 and 2, a score of 6. Browse NL, Burnand KG. The cause of venous ulceration. Lancet 0 or trace amounts of hemosiderin in urine was always 1982;31:243-5. associated with normal venous function or mild disease. In 7. Coleridge Smith PD, Thomas P, Scurr JH, Dormandy JA. Causes of contrast, in the two patients with unchanged or worse venous ulceration: a new hypothesis. BMJ 1988;296:1726-7. 8. Wilkinson LS, Bunker C, Edwards JCW, Scurr JH, Coleridge Smith postoperative scores, duplex scans revealed recurrence of PD. Leukocytes: their role in the etiopathogenesis of skin damage in disease and concomitant deep reflux, respectively (Fig 4). venous disease. J Vasc Surg 1993;17:669-75. JOURNAL OF VASCULAR SURGERY 136 Zamboni et al January 2003

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