Damage assessment in adult IgA vasculitis. Cross-sectional results of a multicentre cohort.

OBJECTIVES: Immunoglobulin (Ig) A vasculitis affects children more commonly than adults and previous literature lacks any formal damage assessment. Our aim in this study is to investigate the disease course, relapse rates and prognostic factors in adult patients with IgA vasculitis and to evaluate the disease-related damage. METHODS: We assembled a retrospective cohort of adult IgA vasculitis from six tertiary Rheumatology Centres in Turkey. The demographics, clinical characteristics, treatment and outcomes of patients were abstracted from medical records. RESULTS: The study included 130 (male/female: 85/45) patients and the mean age was 42.2±17 years. Cutaneous manifestations and arthritis/arthralgia were the most common clinical manifestations. One hundred thirteen patients (86.9%) were treated with oral glucocorticoids (GC). As additional immunosuppressive (IS) agents, azathioprine was given to 44 (34.9%) and pulse cyclophosphamide to 18 (12.6%) patients. Seventy-nine patients (60%) had follow-up of median 15 (IQR 7-40) months. Twelve (15%) patients relapsed during follow-up. The mean VDI score was 0.4 in the last visit. Nineteen (24.7%) patients had at least one damage item at the end of follow-up. Most frequent damage items were renal 11 (42%), ocular 4 (15%) and cardiovascular 4 (15%). CONCLUSIONS: In this cohort the most frequent damage item was renal and was related to the disease itself. Damage score was higher in patients with more severe disease and treated more aggressively. Our results suggest that more effective treatment options are needed in a subgroup of patients with IgA vasculitis to prevent the damage related with the vasculitis, especially with more severe disease.

An integrative approach for the assessment of peritubular capillaritis extent and score in low-grade microvascular inflammation-associations with transplant glomerulopathy and graft loss.

Background: Peritubular capillaritis (ptc), reported by the ptc score, is a major feature of kidney allograft rejection and microvascular inflammation (MVI). MVI sum scores (ptc + glomerulitis score ≥2) are accepted diagnostic surrogates of human leucocyte antigen (HLA)-antibody interaction. However, low-grade inflammation is common and ptc scores (number of leucocytes/capillary) may not mirror all aspects of ptc morphology. Recently we observed a relationship of the diffuse extent of ptc (inflammation of >50% of the renal cortex) with graft loss and significantly higher donor-specific antibody levels, suggesting potential inclusion of diffuse ptc as an additional surrogate of antibody-antigen interaction. Methods: We sought to assess how a combination of ptc score and extent in low-grade inflammation (ptc1) affects transplant glomerulopathy (TG) and graft loss risk. Patients (n = 616) were assessed for MVI in first indication biopsies. Cases with a ptc score of 1 but diffuse extent (ptc1diffuse, g-score = 0, n = 26) were considered additional surrogates of HLA-antibody interaction and compared with MVI ≥2 and MVI <2. Results: The ptc1diffuse and MVI score ≥2 subjects had worse graft survival (42% and 59%) compared with an MVI score <2 (70%) (P = 0.002). The incorporation of ptc1diffuse in the MVI score ≥2 increased the receiver operating characteristics curve for TG [area under the curve (AUC) 0.602; P = 0.008] compared with a Banff MVI score ≥2 (AUC 0.56; P = 0.12); cases with baseline TG were excluded. In multivariate analysis, ptc1diffuse remained independently related to TG (odds ratio 3.89; P = 0.008) and graft loss (hazard ratio 2.64; P = 0.001) even after inclusion of all rejection episodes. Conclusion: An integrated view of ptc morphology including diffuse ptc in MVI is superior for TG and graft loss risk assessment.

Long-term damage assessment in patients with microscopic polyangiitis and renal-limited vasculitis using the Vasculitis Damage Index.

OBJECTIVES: The Vasculitis Damage Index (VDI) is used to define the degree of damage occurring in patients with systemic vasculitis. We conducted a retrospective study of 30 patients with microscopic polyangiitis (MPA) and renal-limited vasculitis (RLV). METHODS: The clinical data and VDI of the 30 patients enrolled in the study were collected and assessed for a period of 5 years. RESULT: The VDI score, which was 2.5 at 1 year after the initial diagnosis, increased gradually to 4.3 at 5 years post-diagnosis. The degrees of musculoskeletal and ocular damage significantly increased during the 5-year period (p = 0.001 and p = 0.002, respectively). The most frequent damage items in the VDI were cataract (13 %), hypertension (12 %), diabetes mellitus (9 %), and osteoporosis (6 %). The VDI score was significantly higher in the groups of patients who showed relapse or MPA than in the groups of patients who did not show relapse or RLV at 5 years (p = 0.02 and p = 0.03, respectively). In addition, a significant correlation was found between the VDI score at 5 years and the Birmingham Vasculitis Activity Score at diagnosis (p = 0.04, r = 0.4). CONCLUSION: The VDI was found to be a useful tool for determining the severity of damage caused by disease and the effects of treatment. The individual contributions of the VDI items may also be applied to treatment decisions.

Optical coherence tomography and histopathological assessment of delayed arterial healing after drug-eluting stent implant in a pig coronary model.

BACKGROUND: Delayed healing, such as persistent inflammation and fibrin deposition, and vascular dysfunction after drug-eluting stent has been reported. Histological validation of coronary optical coherence tomography (OCT) morphology has not yet been done. METHODS: Sirolimus eluting stents (SES, n=8) and bare metal stents (BMS n=8) were implanted in pig coronary arteries. One month after implantation, an acetylcholine challenge test and OCT were performed. The OCT texture pattern of the neointima was classified into one of the three categories; Layered type, Homo type, and Hetero type. Hearts were harvested for histopathological scoring of inflammation and intramural thrombus. RESULTS: Inflammation and intramural thrombus scores were higher in the Hetero type than in the Layered type and Homo type. OCT intensity of the Homo type was higher than that of the Layered type and Hetero type. Most SES were of the Hetero type. Conversely, most BMS were of the Homo type. SES exhibited higher inflammation and intramural thrombus than BMS (1.72 ± 0.89 vs 1.00 ± 0.00, P=0.0003, 2.39 ± 0.70 vs 0.92 ± 0.28, P<0.001 respectively). After acetylcholine injection, the diameter change was 4.31 ± 4.80% for SES versus -3.68 ± 6.81% for BMS (P=0.024). CONCLUSIONS: The Hetero type texture pattern in OCT images was associated with histological inflammation and intramural thrombus predominantly found in SES, and is related to endothelial dysfunction.

The histological assessment of cutaneous vasculitis.

Vasculitis is defined as inflammation directed at vessels, which compromises or destroys the vessel wall leading to haemorrhagic and/or ischaemic events. Skin biopsy is the gold standard for the diagnosis of cutaneous vasculitis, whose manifestations include urticaria, infiltrative erythema, petechiae, purpura, purpuric papules, haemorrhagic vesicles and bullae, nodules, livedo racemosa, deep (punched out) ulcers and digital gangrene. These varied morphologies are a direct reflection of size of the vessels and extent of the vascular bed affected, ranging from a vasculitis affecting few superficial, small vessels in petechial eruptions to extensive pan-dermal small vessel vasculitis in haemorrhagic bullae to muscular vessel vasculitis in lower extremity nodules with livedo racemosa. Skin biopsy, extending to subcutis and taken from the earliest, most symptomatic, reddish or purpuric lesion is crucial for obtaining a high-yielding diagnostic sample. Based on histology, vasculitis can be classified on the size of vessels affected and the dominant immune cell mediating the inflammation (e.g. neutrophilic, granulomatous, lymphocytic, or eosinophilic). Disruption of small vessels by inflammatory cells, deposition of fibrin within the lumen and/or vessel wall coupled with nuclear debris allows for the confident recognition of small vessel, mostly neutrophilic vasculitis (also known as leukocytoclastic vasculitis). In contrast, muscular vessel vasculitis can be identified solely by infiltration of its wall by inflammatory cells. Extravasation of red blood cells (purpura) and necrosis are supportive, but not diagnostic of vasculitis as they are also seen in haemorrhagic and/or vaso-occlusive disorders (pseudovasculitis). Vasculitic foci associated with extravascular granulomas (palisaded neutrophilic and granulomatous dermatitis), tissue eosinophilia, or tissue neutrophilia signal the risk for, or co-existence of systemic disease. This essential histological information coupled with direct immunofluorescence and anti-neutrophil cytoplasmic data and clinical findings enables more precise and accurate diagnosis of localized and systemic vasculitis syndromes.

Assessment of damage in vasculitis: expert ratings of damage.

OBJECTIVES: Current measures of damage in vasculitis do not account for the possibility that some forms of damage may exert greater impact than others. As part of an international effort to revise how damage is quantified in vasculitis clinical research, an exercise was performed to measure expert ratings of damage items. METHODS: Members of the Vasculitis Clinical Research Consortium and European Vasculitis Study Group were given a list of 129 items of damage related to WG and microscopic polyangiitis (MPA). Participants were asked to rate each item of damage on an integer scale from 0 to 10, where 10 represented the most severe form of damage and 0 indicated 'no impact'. RESULTS: A multidisciplinary panel of 50 investigators from North America, Europe and Australia-New Zealand participated. The highest median ratings (8-10) were assigned to items of damage associated with malignancy, tissue ischaemia, the central nervous system and cardiopulmonary manifestations. The mean scores ranged from 1.3 to 9.5. The highest s.d.s (>or=2.5) were associated with forms of damage that may benefit from surgical intervention or may not be causally associated with WG or MPA. Lower scores were assigned by nephrologists in comparison with rheumatologists and by Americans in comparison to Europeans, although the difference in median ranks used by these groups was not statistically significant (P > 0.05 for the comparisons). CONCLUSIONS: This exercise represents an important step in the development of a weighting system that may increase the utility of damage index scores for the assessment of patients with vasculitis.

Damage assessment in systemic vasculitis.

Systemic vasculitides were initially reported as acute, progressive, severe, and life-threatening diseases. The introduction of glucocorticoids and cyclophosphamide for the treatment of vasculitis improved survival dramatically, but morbidity has remained high. Damage develops as a consequence of recurrent or persistent active vasculitis or its treatment. It is defined as the accumulation of nonhealing scars that are unlikely to respond to immunosuppressive therapy. Damage assessment is essential in systemic vasculitis because it may facilitate patient stratification in clinical trials and possibly in clinical practice. Moreover, it may avoid unnecessary use of immunosuppressive therapy. The Vasculitis Damage Index, developed and validated in 1997, has been very useful in solving many matters in systemic vasculitis and is currently the only validated damage-assessment tool available. However, the vasculitis community has recognized that there is a growing need to improve the evaluation of damage in vasculitis. The development of a Combined Damage Assessment index, which would permit a more appropriate and standardized approach to disease assessment applicable to systemic vasculitis, has been proposed.

The use of C3d and C4d immunohistochemistry on formalin-fixed tissue as a diagnostic adjunct in the assessment of inflammatory skin disease.

BACKGROUND: Direct immunofluorescent (DIF) testing defines an important diagnostic adjunct in the classification of various inflammatory skin conditions; it requires fresh tissue, a laboratory equipped to perform the procedure, and a pathologist skilled in its interpretation. Although advances have been made in the development of antibodies that can be applied to paraffin-embedded tissue, there has been no reported success on the application of paraffin tissue-based immunohistochemistry as a potential substitute for DIF testing on skin biopsy material. OBJECTIVE: We applied C3d and C4d immunohistochemistry on paraffin-embedded, formalin-fixed tissue to define a potential application of these two antibodies as a diagnostic adjunct in the evaluation of various inflammatory skin diseases. DESIGN: A natural language search identified cases submitted for both light microscopic and DIF studies from July 2006 to August 2007. We prospectively included similar cases encountered from August 2007 to March 2008. We correlated the C3d and C4d staining pattern with the DIF and light microscopic findings. RESULTS: All cases of scarring discoid lupus erythematosus (LE) (20/20) and systemic LE (5/5) showed prominent granular C3d along the dermoepidermal junction (DEJ) and a positive lupus band test result in the latter by DIF. All systemic LE cases demonstrated granular DEJ C4d with C3d or C4d in blood vessels (BV). There was a negative lupus band test result without DEJ C3d or C4d in all cases of subacute cutaneous lupus erythematosus (SCLE) (15/15). There were, however, deposits of C4d within epidermal keratinocytes (7/7), corresponding to IgG decoration of keratinocytes by DIF and the presence of anti-Ro antibodies. Dermatomyositis cases showed prominent mural C3d and C4d in BV corresponding to C5b-9 by DIF (12/12) and one case of hydroxyurea-induced dermatomyositis lacked this staining. Although by DIF all dermatomyositis cases had a negative lupus band test result, 25% of cases showed staining for C3d along the DEJ (3/9). Bullous pemphigoid cases demonstrated homogenous DEJ C3d (17/17) whereas C4d was characteristically negative; there was 100% concordance with linear IgG and C3d by DIF. Eighty two percent of pemphigus cases demonstrated prominent intercellular C3d and C4d, roughly mirroring the intercellular pattern for IgG and complement seen by DIF (9/11). Porphyria cases showed homogeneous and granular C3d (11/11) and C4d (7/11), mirroring the vascular immunoglobulin and C5b-9 by DIF. All cases of urticarial (5), leukocytoclastic (6), and lymphocytic (1) vasculitis exhibited prominent mural C3d and C4d in BV, whereas Henoch-Schönlein purpura (10/10) showed primarily mural BV C3d without C4d, with IgA by DIF. Three cases of relapsing polychondritis showed C3d and C4d within chondrocyte nuclei (3/3), in contrast to negative staining in chondrodermatitis nodularis helicis (0/2). Hypersensitivity reactions were negative for C3d and C4d. LIMITATIONS: The small sample size in each category is a limitation. The lack of literature precedent with regard to immunohistochemical assessment of extracellular antigens on paraffin-embedded tissue in skin samples is another limitation of this study. CONCLUSIONS: When correlated with the light microscopic and clinical findings, the C3d and C4d assay has significant application in the assessment of select inflammatory skin diseases including vasculopathic conditions, collagen vascular disease, and autoimmune vesiculobullous disorder. It may prompt further DIF testing or, in some instances, may even define a reasonable substitute for DIF and/or add to the morphologic assessment of a biopsy specimen submitted for routine light microscopic assessment primarily in the setting of autoimmune vesiculobullous disease and collagen vascular disease.

The future of damage assessment in vasculitis.

Damage denotes the aspects of chronic disease that do not reverse with therapy. This concept is particularly important for the primary systemic vasculitides, since the careful differentiation between activity and damage may help avoid unnecessary exposure to cytotoxic medications. Damage significantly influences both longterm prognosis and quality of life. Because the primary systemic vasculitides have diverse manifestations, the use of a damage assessment instrument is crucial to ensure reproducibility. The Vasculitis Damage Index (VDI) is the only validated measure for damage assessment in vasculitis. Use of the VDI in recent clinical trials has shown that it may not adequately determine the full spectrum of damage experienced by patients with vasculitis of small- and medium-size vessels. We propose reexamining the way in which damage is assessed, focusing on vasculitides of small- and medium-size vessels, and outline an initiative to create a substantially revised and improved damage assessment instrument using data-driven approaches. This initiative is part of a larger international effort to create a unified approach to disease assessment for the primary systemic vasculitides.

Semiquantitative assessment of surgical lung biopsy: predictive value and impact on survival of patients with diffuse pulmonary infiltrate.

PURPOSE: Surgical lung biopsy has been studied in distinct populations, mostly going beyond clinical issues to impinge upon routine histopathological diagnostic information in diffuse infiltrates; however, detailed tissue analyses have rarely been performed. The present study was designed to investigate the prognostic contribution provided by detailed tissue analysis in diffuse infiltrates. METHODS: Medical records and surgical lung biopsies from the period of 1982 to 2003 of 63 patients older than 18 years with diffuse infiltrates were retrospectively examined. Lung parenchyma was histologically divided into 4 anatomical compartments: interstitium, airways, vessels, and alveolar spaces. Histological changes throughout these anatomical compartments were then evaluated according to their acute or chronic evolutional character. A semiquantitative scoring system was applied to histologic findings to evaluate the intensity and extent of the pathological process. We applied logistic regression to predict the risk of death associated with acute and chronic histological changes and to estimate the odds ratios for each of the independent variables in the model. RESULTS: Impact on survival was found for male gender (P = 0.03), presence of diffuse alveolar damage (P = 0.001), and chronic histological changes (P = 0.0004) on biopsy. Thus, being male was associated with a slightly lower risk (O.R. = 0.18; P=0.03) of dying than being female. Death risk was increased 17 times in the presence of acute histological changes such as diffuse alveolar damage and 2.5 times in the presence of chronic histological changes. CONCLUSION: Detailed analysis of histological specimens can provide more than a nosological diagnosis: this approach can provide valuable information concerning prognosis.

Semiquantitative assessment of surgical lung biopsy: predictive value and impact on survival of patients with diffuse pulmonary infiltrate

PURPOSE: Surgical lung biopsy has been studied in distinct populations, mostly going beyond clinical issues to impinge upon routine histopathological diagnostic information in diffuse infiltrates; however, detailed tissue analyses have rarely been performed. The present study was designed to investigate the prognostic contribution provided by detailed tissue analysis in diffuse infiltrates. METHODS: Medical records and surgical lung biopsies from the period of 1982 to 2003 of 63 patients older than 18 years with diffuse infiltrates were retrospectively examined. Lung parenchyma was histologically divided into 4 anatomical compartments: interstitium, airways, vessels, and alveolar spaces. Histological changes throughout these anatomical compartments were then evaluated according to their acute or chronic evolutional character. A semiquantitative scoring system was applied to histologic findings to evaluate the intensity and extent of the pathological process. We applied logistic regression to predict the risk of death associated with acute and chronic histological changes and to estimate the odds ratios for each of the independent variables in the model. RESULTS: Impact on survival was found for male gender (P = 0.03), presence of diffuse alveolar damage (P = 0.001), and chronic histological changes (P = 0.0004) on biopsy. Thus, being male was associated with a slightly lower risk (O.R. = 0.18; P=0.03) of dying than being female. Death risk was increased 17 times in the presence of acute histological changes such as diffuse alveolar damage and 2.5 times in the presence of chronic histological changes. CONCLUSION: Detailed analysis of histological specimens can provide more than a nosological diagnosis: this approach can provide valuable information concerning prognosis.

PROPOSIÇÃO: A biópsia pulmonar cirúrgica tem sido estudada em populações distintas, geralmente abordando aspectos histopatológicos puramente diagnósticos em infiltrados pulmonares difusos, além de dados clínicos. Contudo, análises teciduais detalhadas em tais casos têm sido pouco exploradas. O presente estudo foi delineado com o intuito de se investigar a contribuição prognóstica fornecida pela análise histológica detalhada em infiltrados difusos. MÉTODOS: Foram examinados retrospectivamente os prontuários e biópsias pulmonares cirúrgicas de 63 pacientes maiores de 18 anos, com infiltrados difusos, de 1982 a 2003. O parênquima pulmonar foi dividido em 4 compartimentos histológicos: interstício, vias aéreas, vasos e espaços alveolares. Alterações histológicas de cada compartimento histológico foram então avaliadas de acordo com seu caráter evolutivo agudo ou crônico. Um escore semiquantitativo foi aplicado a achados histopatológicos com o intuito de se avaliar a intensidade e a extensão do processo patológico. Aplicamos regressão logística para predizer o risco de morte para alterações histológicas agudas e crônicas e para estimar a razão de probabilidades para cada uma das variáveis independentes do modelo. RESULTADOS: O impacto sobre a sobrevida foi observado para o gênero masculino (p=0.03), para a presença de dano alveolar difuso (p=0.001) e para alterações histológicas crônicas (p=0.0004) em biópsias. Assim, homens apresentariam menor chance (O.R. = 0.18; P=0.03) de morrer do que mulheres. O risco de morte foi 17 vezes maior na presença de alterações histológicas agudas como dano alveolar difuso e 2,5 vezes na presença de alterações histológicas crônicas. CONCLUSÃO: A análise detalhada de espécimes histológicos pode proporcionar maiores e mais valiosas informações de valor prognóstico do que o simples diagnóstico nosológico.

Clinical and biological assessment in systemic necrotizing vasculitides.

The systemic vasculitides are multi-system disorders with significant mortality and morbidity and frequent relapses. Treatment is usually effective but fraught with potentially serious effects. Disease Assessment is important to ensure that patients receive the appropriate treatment. Disease Assessment should comprise measurement of disease activity, chronic irreversible damage and impairment of function. Serological markers can be helpful in assessing disease activity but lack sufficient sensitivity and specificity to be used on their own. Radiological techniques such as Magnetic Resonance Imaging, Ultrasound and Positron Emission Tomography show promise in the large vessel vasculitides but require validation in large studies. Clinical Assessment tools are the current gold standard for the assessment of disease activity, damage and function.

[Update on the pathomorphological assessment of vasculopathies in pulmonary arterial hypertension]. / Vaskulopathien bei pulmonal-arterieller Hypertonie Update zur pathomorphologischen Beurteilung.

Pulmonary arterial hypertension (PAH) is a term which has recently been redefined and includes idiopathic pulmonary arterial hypertension, familial pulmonary arterial hypertension PAH related to specific pathological conditions (e.g. connective tissue diseases), as well as PAH caused by veno-occlusive disease or capillary hemangiomatosis. The clinical manifestation seems to be related to a peculiar pathological anatomy involving small, muscular pulmonary arteries, capillaries and veins. In addition to common hypertrophy of the tunica media, other vascular compartments may also be affected by intimal thickening or adventitial fibrosis. Moreover, complex lesions, such as so called plexiform lesions and arteritis can be present in certain forms of the disease. While the recent identification of responsible gene mutations in subgroups of patients have shed some light on disease evolution, therapeutic strategies must currently rely on vasodilative and antimitogenic drugs acting on the intimal and medial level of the affected pulmonary vessels. The clinical outcome of patients suffering from PAH remains poor, underlining our need for a better comprehension of disease pathophysiology, and thus for the characterization of specific histomorphological patterns.

[Clinical assessment of microscopic polyangiitis in elderly patients].

OBJECTIVE: To evaluate the influence of the age at disease onset on the clinical symptoms, laboratory findings, treatment, and complications of microscopic polyangiitis (MPA). PATIENTS: From 1999 to 2001, we encountered 4 MPA patients with disease onset at age 65 or older (average 77.3, all were female: the elderly group). For comparison, 4 MPA patients with disease onset a 64 years or younger (average 44.7, two were male: the non-elderly group) were used. RESULTS: There was no statistically significant difference in clinical features between the two groups. All patients in the elderly group were referred to our hospital, because of fever of unknown origin or suspicion of connective tissue disease. The elderly group had a longer duration from the first admission to the start of treatment. Renal biopsies were done in all of the non-elderly group and one of the elderly group. The diagnosis of the other 3 patients of the elderly group was based on muscle or nerve biopsy, showing necrotizing vasculitis. At the time of diagnosis, antibodies to myeloperoidase (MPO-ANCA) were positive in 7 of 8 patients (87.5%). 2 patients of the non-elderly group were died of heart failure and hepatic failure by cyclophosphamide (CYC). The other 6 patients achieved substantial improvement. CONCLUSIONS: Muscle or nerve biopsy helped clinical management of elderly patients when renal biopsies could not be done. IVCY was relatively safe and effective treatment for MPA in elderly as well as non-elderly patients.

Clinical and pathological assessment of acute vascular rejection in the transplant kidney.

Acute vascular rejection (AVR) in kidney transplantation is the most important factor influencing graft prognosis. We focus on patients whose grafts were lost because of AVR, and assessed their clinical characteristics and histological findings of biopsied renal grafts. Biopsied specimens exhibited AVR in 43 patients who underwent kidney transplantation in the Kidney Center of Tokyo Women's Medical University from 1995 to 1999. In the follow-up from 1 to 5 yr (median: 2.5 yr) we classified these patients into three groups: favourable prognosis group (FPG), relatively poor prognosis group (RPPG) and poor prognosis group (PPG). Light microscopic study for histological grading of acute rejection according to the Banff scheme and detection of the C4d complement deposition on peritubular capillaries by the immunofluorescence method were performed. Based on the results, the donors of RPPG and PPG were significantly older than those of FPG, and all factors of acute rejection according to the Banff scheme were not statistically significantly different among the three groups. However, an acute tubular injury mimicking acute tubular necrosis (ATN) was observed in the biopsy specimens from PPG. In conclusion, an older donor is a risk factor of poor prognosis of the graft with AVR, and acute tubular injury mimicking ATN is one of the important features that enables the prediction of graft failure originating from AVR in kidney transplantation.

Diagnostic strategies for the histological examination of muscle biopsy specimens for the assessment of vasculitis in rheumatoid arthritis.

For the diagnosis of rheumatoid vasculitis (RV), histological examination of a blindly-taken muscle biopsy is advocated. If fibrinoid necrosis (FN) is observed in one or more tissue sections of the biopsy the diagnosis of RV is confirmed. The diagnostic value of such histological investigation depends on the prevalence of FN in biopsies of RV patients, the number of tissue sections that are investigated, and the sampling design with which the tissue sections are obtained from the biopsy. In this paper we determine the mathematical relation between the sensitivity of the RV diagnosis and these three factors for four different models for the FN distribution in RV biopsies. The goodness-of-fit of these models was assessed by analysing 18 829 tissue sections of the biopsies of 56 patients, among which were 24 (otherwise histologically proven) RV patients. It appeared that the sensitivity was moderate: FN was observed in only 14 out of 24 (58 per cent) muscle biopsies of the RV patients. The prevalence of FN in the tissue sections of those 14 biopsies was low, about 17 per cent according to the best fitting model. We proved that the sampling design, maximizing the minimum distance between tissue sections (equidistant sampling) was optimal, and that with such optimal sampling, examination of about 20 tissue sections was sufficient. With practical sampling designs, however, considerably more tissue sections had to be inspected. FN appeared to cluster in the RV biopsies, and a first-order Markov model satisfactorily described the (auto)correlation between adjacent tissue sections in RV biopsies.