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The complement system is part of the innate immune system activated by three distinct pathways: classical, lectin and alternative. It is also involved in retinal development and homoeostasis. Dense deposit disease is a rare renal disease associated with mutations in Complement factor H and overactivity of the alternative complement pathway. As well as glomerulonephritis, many affected individuals have retinal drusen and may be at risk of vision loss due to macular atrophy or choroidal neovascularisation. We discuss the reclassification of dense deposit disease as a type of C3 glomerulonephropathy, and hypothesise on the mechanisms of retinal abnormalities. Drusen have also been described in individuals with other types of glomerulonephritis involving abnormalities of the classical (membranoproliferative glomerulonephritis type 1) or lectin (IgA nephropathy, lupus nephritis) complement pathways. Although drusen are found in abnormalities of all three complement pathways, the age at onset, aetiology, and the threat to vision differs. This review describes drusen and other retinal abnormalities associated with the glomerulonephritides due to abnormal activation in each of the three complement activation pathways, and provides the first report of drusen occurring in a patient with the recently reclassified C3 glomerulonephritis with homozygous variant V62I in complement factor H . Optometric management of young patients presenting with retinal drusen is discussed, and complement-based therapies for visual loss are reviewed.

This document follows up on a 2017 revised international consensus on anti-neutrophil cytoplasm antibodies (ANCA) testing in granulomatosis with polyangiitis and microscopic polyangiitis and focuses on the clinical and diagnostic value of ANCA detection in patients with connective tissue diseases, idiopathic interstitial pneumonia, autoimmune liver diseases, inflammatory bowel diseases, anti-glomerular basement membrane (GBM) disease, infections, malignancy, and during drug treatment. Current evidence suggests that in certain settings beyond systemic vasculitis, ANCA may have clinical, pathogenic and/or diagnostic relevance. Antigen-specific ANCA targeting proteinase-3 and myeloperoxidase should be tested by solid phase immunoassays in any patient with clinical features suggesting ANCA-associated vasculitis and in all patients with anti-GBM disease, idiopathic interstitial pneumonia, and infective endocarditis associated with nephritis, whereas in patients with other aforementioned disorders routine ANCA testing is not recommended. Among patients with autoimmune liver diseases or inflammatory bowel diseases, ANCA testing may be justified in patients with suspected autoimmune hepatitis type 1 who do not have conventional autoantibodies or in case of diagnostic uncertainty to discriminate ulcerative colitis from Crohn's disease. In these cases, ANCA should be tested by indirect immunofluorescence as the target antigens are not yet well characterized. Many questions concerning the optimal use of ANCA testing in patients without ANCA-associated vasculitis remain to be answered., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anti-neutrophil cytoplasmic antibodies (ANCAs) are valuable laboratory markers used for the diagnosis of well-defined types of small-vessel vasculitis, including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). According to the 1999 international consensus on ANCA testing, indirect immunofluorescence (IIF) should be used to screen for ANCAs, and samples containing ANCAs should then be tested by immunoassays for proteinase 3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs. The distinction between PR3-ANCAs and MPO-ANCAs has important clinical and pathogenic implications. As dependable immunoassays for PR3-ANCAs and MPO-ANCAs have become broadly available, there is increasing international agreement that high-quality immunoassays are the preferred screening method for the diagnosis of ANCA-associated vasculitis. The present Consensus Statement proposes that high-quality immunoassays can be used as the primary screening method for patients suspected of having the ANCA-associated vaculitides GPA and MPA without the categorical need for IIF, and presents and discusses evidence to support this recommendation.

Objective: To determine if a programme of progressive resistance exercise, mobilisation and orientation, in addition to usual care, was superior to usual care alone in the prevention of incident delirium in older hospitalised patients., Design: A randomised controlled trial., Setting: The study was performed at a secondary referral hospital in Melbourne, Australia between May 2005 and December 2007., Participants: 648 consecutive medical inpatients aged 65 years or older who had been in hospital for less than 48 h and who did not have delirium., Intervention: Participants were randomly allocated to a twice-daily programme of progressive resistance exercise tailored to individual ability, mobilisation and orientation in addition to usual care or to usual care alone., Measurements: Delirium was measured using the Confusion Assessment Method at baseline and every 48 h until discharge. Secondary outcome measures were severity and duration of delirium, discharge destination and length of stay., Results: Delirium occurred in 4.9% (95% CI 2.3% to 7.3%) of the intervention group (15/305) and in 5.9% (20/339; 95% CI 3.8% to 9.2%) of the group receiving usual care. No difference was observed between groups (χ(2); p=0.5). The intervention had no effect on delirium duration, severity, discharge destination or length of stay., Conclusion: A programme of progressive resistance exercise and orientation was not effective in reducing incident delirium in hospitalised elderly patients.

Increasing number of older patients are admitted to hospital with hip fractures. This review evaluates the common medical problems that arise as a consequence of having a hip fracture. Older patients with fractures commonly have co-morbidities that require evaluation prior to and after surgery. Joint acute orthopaedic-geriatric units have been established to provide comprehensive orthopaedic and medical care with some studies showing a reduction in postoperative complications and mortality. Recommendations surrounding the care of the older orthopaedic patient include early surgical fixation, the use of prophylactic antibiotics and thromboembolic prophylaxis, good perioperative pain control to improve ambulation, delirium detection and management to decrease the risk complications, such as institutionalisation, the avoidance of malnutrition, urinary tract management, osteoporosis management and the promotion of early mobilisation to improve functional recovery. Physicians are well placed to manage these patients with orthopaedic surgeons during the perioperative period. Sufficient evidence exists for most recommendations for fracture patients, but further research is needed in most areas.

Background: The "classical" antineutrophil cytoplasmic antibody (C-ANCA) pattern seen on indirect immunofluorescence (IIF) is characterised by granular cytoplasmic staining showing central or interlobular accentuation, and is strongly associated with antiproteinase-3 antibodies (PR3-ANCA) and Wegener's granulomatosis. However, many laboratories report C-ANCA in the presence of any cytoplasmic IIF staining, regardless of pattern, which risks reducing the diagnostic value of this pattern., Aims: To classify different cytoplasmic ANCA patterns and thus determine whether stringent application of the classical criteria for C-ANCA would produce better correlation between C-ANCA and (1) PR3-ANCA enzyme linked immunosorbent assay (ELISA) results; (2) a diagnosis of systemic vasculitis (including Wegener's granulomatosis)., Methods: 72 sera with cytoplasmic IIF collected over a two year period were analysed by IIF and a commercial PR3-ANCA ELISA kit., Results: Three IIF patterns were defined: "classical/true" C-ANCA as described above (n = 27 (37.5%)); "flat" ANCA with homogeneous cytoplasmic staining (n = 21 (29%)); and "atypical" ANCA which included all other cytoplasmic patterns (n = 24 (33.5%)). Twenty five of the 27 true C-ANCA sera (92.5%) contained PR3-ANCA (p < 0.0001), but none of the 21 with flat ANCA and only one of the 24 with atypical ANCA. From clinical data on 23 of the 27 true C-ANCA positive patients, 20 (87%) had evidence of Wegener's granulomatosis or systemic vasculitis (p < 0.0001 v the other two patterns). However, none of 19 sera with flat ANCA and clinical data had evidence of systemic vasculitis., Conclusions: Restricting the term "c-ANCA" to the "classical" description of central/interlobular accentuation on IIF, will improve its correlation with PR3-ANCA positivity and a diagnosis of systemic vasculitis.

Aim: To describe the neutrophil fluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) with different antigen specificities, and by other auto- and alloantibodies., Background: Most sera from patients with active generalised Wegener's granulomatosis result in diffusely granular cytoplasmic neutrophil fluorescence with internuclear accentuation (cANCA) and proteinase 3 (PR3) specificity. About 80% of the sera from patients with microscopic polyangiitis result in perinuclear neutrophil fluorescence with nuclear extension (pANCA) and myeloperoxidase (MPO) specificity, or a cANCA pattern with PR3 specificity. However, many different neutrophil fluorescence patterns are noted on testing for ANCA in routine immunodiagnostic laboratories., Methods: Sera sent for ANCA testing, or containing a variety of auto- and alloantibodies, were studied. They were examined by indirect immunofluorescence according to the recommendations of the first international ANCA workshop, and for PR3 and MPO specificity in commercial and in-house enzyme linked immunosorbent assays (ELISA)., Results: Sera with typical cANCA accounted for only half of all neutrophil cytoplasmic fluorescence. Other sera had "flatter" fluorescence without internuclear accentuation, and the corresponding antigens included MPO and bactericidal/permeability increasing protein (BPI), but were usually unknown. Peripheral nuclear fluorescence without nuclear extension occurred typically when the antigens were BPI, lactoferrin, lysozyme, elastase, or cathepsin G. Most types of ANA were evident on ethanol fixed neutrophil nuclei. AntidsDNA, antiRo, and antilamin antibodies resembled pANCA. Antimicrobial and antiribosomal antibodies produced cytoplasmic fluorescence, and antiGolgi antibodies, a pANCA. Sera from patients with anti-smooth muscle antibodies were associated with cytoplasmic fluorescence. There was no neutrophil fluorescence with anti-skeletal muscle and anti-heart muscle antibodies, anti-liver/kidney microsomal, antithyroid microsomal, or antiadrenal antibodies. Alloantibodies such as antiNB1 typically resulted in cytoplasmic fluorescence of only a subpopulation of the neutrophils., Conclusions: The ability to distinguish between different neutrophil fluorescence patterns, and the patterns seen with other auto- and alloantibodies is helpful diagnostically. However, the demonstration of MPO or PR3 specificity by ELISA will indicate that the neutrophil fluorescence is probably clinically significant, and that the diagnosis is likely to be Wegener's granulomatosis or microscopic polyangiitis.

The basement membranes of the glomerulus, thyroid and adrenal all contain the Goodpasture antigen, the target of autoantibodies in antiglomerular basement membrane (GBM) disease. Antithyroid antibodies can be associated with antiGBM disease, and there have been occasional reports of antithyroid antibodies in Alport syndrome, an inherited kidney disease where the GBM lacks the Goodpasture antigen. The aim of this study was to determine how often antithyroid and antiadrenal autoantibodies occurred in antiGBM disease, Alport syndrome and a related condition, thin basement membrane disease (TBMD). Sera from patients with antiGBM disease (n = 19), Alport syndrome (n = 5) or TBMD (n = 13) were tested for antithyroglobulin, antithyroid microsomal and antiadrenal antibodies. Five of the patients with antiGBM disease (5/19, 26%, P NS) had antimicrosomal, and one had antithyroglobulin, antibodies (1/19, 5%, P NS). No patient with Alport syndrome had antithyroid antibodies. One with TBMD (1/13, 8%, P NS) had antithyroglobulin and antimicrosomal antibodies at titres of 1/400 and 1/25,600, respectively. Both patients with antithyroglobulin antibodies had previously been diagnosed with hypothyroidism. No one with antiGBM disease, Alport syndrome or TBMD had antiadrenal antibodies. Antithyroid microsomal antibodies do not occur significantly more often in patients with antiGBM disease than in normals, and antithyroid and antiadrenal antibodies are not associated with Alport syndrome or TBMD.

Bacterial and viral infections may be associated with the onset of a number of autoimmune diseases and relapses of these conditions. We describe a patient in whom there was a close temporal relationship between a suppurative wound infection and the onset of microscopic polyarteritis. The clinical features of this disease responded to treatment with high dose prednisolone and cyclophosphamide. The patient had several further infective episodes while being treated, but there were no disease exacerbations or relapses related to these. Anti-neutrophil cytoplasmic antibodies (ANCA) were never demonstrated in this patient. Thus while it is likely that the infection precipitated the onset of the systemic vasculitic illness, this occurred independently of ANCA.

In this study of antineutrophil cytoplasmic antibody (ANCA)-associated diseases, we determined the prevalence of other autoantibodies and the antigen specificities of ANCA. ANA were common, occurring in 7 of 36 (19%) patients with Wegener's granulomatosis, in 16 of 34 (47%) patients with microscopic polyarteritis, in 6 of 11 (55%) patients with segmental necrotising glomerulonephritis and in 8 of 18 (44%) of those with ANCA-associated systemic vasculitis without renal involvement. ANA were associated more often with pANCA and microscopic polyarteritis than with cANCA (P < 0.05). Patterns were speckled (n = 23), homogeneous (n = 10) or nucleolar (n = 4). Anticardiolipin antibodies were also common, occurring in 10 of 25 (40%) patients with Wegener's granulomatosis, in 8 of 14 (57%) patients with microscopic polyarteritis and in 6 of 18 (33%) of those with a systemic vasculitis. However, anticardiolipin antibodies did not correlate with the presence of ANCA in any of the disease groups. Anti-GBM antibodies were demonstrated in only 2 of 25 (8%) patients with Wegener's granulomatosis, in 1 patient with microscopic polyarteritis (1/14, 7%) and in 1 with segmental necrotising glomerulonephritis (1/11, 9%). All four patients with anti-GBM antibodies had either cANCA or pANCA. In the second part of the study, the target antigens of ANCA were determined in Wegener's granulomatosis, microscopic polyarteritis, systemic vasculitis, inflammatory bowel disease, rheumatoid arthritis and systemic lupus erythematosus (SLE). Of the 19 sera with cANCA, 13 (68%) were directed against proteinase 3; other antigens were myeloperoxidase (1/19, 5%), elastase and lactoferrin together (1/19, 5%), lysozyme (1/19, 5%) or unknown (3/19, 16%). Of the 12 (58%) sera from patients with Wegener's granulomatosis who had cANCA, 7 bound to proteinase 3. Antimyeloperoxidase antibodies were present in 14 of 45 (31%) sera with pANCA; other antigens were proteinase 3 (5/45, 11%), elastase (3/45, 78%), lactoferrin (1/45, 2%), cathepsin G (5/45, 11%) or unknown (17/45, 38%). Antimyeloperoxidase antibodies were common in microscopic polyarteritis (6/14, 43%) and systemic vasculitis (5/16, 31%). However, the majority of target antigens in systemic vasculitis and rheumatoid arthritis with pANCA were not determined. "Atypical" ANCA were present in four patients, one with inflammatory bowel disease (1/8, 13%) and three with SLE (3/15, 20%). The specificities were cathepsin G, cathepsin G plus lactoferrin, or unknown in two sera. A recent report has suggested that bactericidal/permeability-increasing protein may be the target in patients with inflammatory bowel disease.

alpha 1-antitrypsin (alpha 1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of alpha 1-AT phenotypes associated with dysfunctional alpha 1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of alpha 1-AT. Phenotypes usually associated with a moderate or severe reduction in alpha 1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n = 2), S (n = 1) or Z (n = 1) (P < 0.05). However, the corresponding alpha 1-AT serum levels were normal (n = 3) or elevated (n = 1). None of the 31 sera with anti-PR3 antibodies had low levels of alpha 1-AT. No abnormal alpha 1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of alpha 1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum alpha 1-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal alpha 1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal alpha 1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have alpha 1-AT phenotypes that are usually associated with low serum levels of alpha 1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal alpha 1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.

Background: Anti-neutrophil cytoplasmic antibodies (ANCA) are typically associated with small vessel vasculitides. They are also found in situations where other autoantibodies are common, sometimes after infections and possibly in individuals who have received multiple blood transfusions., Aims: The aim of this study was to determine the incidence of ANCA in a variety of haematological disorders, where these predisposing factors may be at work., Methods: Sera from patients with myelodysplasia (n = 26), acute myeloid leukaemia (AML) (n = 3), and myeloproliferative (n = 25) or lymphoproliferative syndromes (n = 16) were screened for ANCA using a crude neutrophil cytoplasmic extract ELISA and indirect immunofluorescent examination of normal peripheral blood neutrophils. Positive results were confirmed by ELISAs for anti-proteinase 3, anti-myeloperoxidase or anti-elastase antibodies., Results: ANCA were demonstrated in two patients with myelodysplasia, both with chronic myelomonocytic leukaemia and greater than 5% blasts in the bone marrow. Both of these individuals were infected at the time that ANCA were demonstrated and other autoantibodies were present. One of these individuals had never had evidence of any vasculitis; the other probably developed myelodysplasia after treatment with cyclophosphamide for Wegener's granulomatosis. ANCA were demonstrated in one individual with AML secondary to myelodysplasia. ANCA were also found in a patient with lymphoma in whom autoantibodies against red cells and platelets were already noted. ANCA were demonstrated in one further individual with lymphomatoid granulomatosis, a condition that resembles Wegener's granulomatosis clinically and histologically, but which is treated as a lymphoma. No ANCA were present in any of the patients with myeloproliferative syndromes., Discussion: ANCA probably occur secondary to immune dysregulation in myelodysplasia and the lymphoproliferative conditions and they are not necessarily associated with the presence of a vasculitis.

Anti-proteinase 3 antibodies are a subgroup of anti-neutrophil cytoplasmic antibodies (ANCA), and we have established an ELISA for their detection using high performance liquid chromatography (HPLC)-purified protein. This assay is sensitive and specific: inhibition studies have shown that despite the homology between proteinase 3 and elastase there is no cross-reactivity between the corresponding antibodies for their targets. Anti-proteinase 3 antibodies were associated most often with cytoplasmic fluorescence (17/22, 77%), but occasionally with a perinuclear (3/22, 14%) or atypical pattern (1/2). These antibodies were found in 23 out of 76 sera (30%) that were positive in an ELISA based on a crude neutrophil cytoplasmic extract, and they were associated with both 29 and 55 kD bands on Western blots. Anti-proteinase 3 antibodies were found in most individuals with active Wegener's granulomatosis (10/13, 77%), but less often in individuals with microscopic polyarteritis (2/10, 20%) or segmental necrotizing glomerulonephritis (3/6, 50%). However, anti-proteinase 3 antibodies were not detected in any of 32 sera from individuals with rheumatoid arthritis or systemic lupus erythematosus (SLE). Occasionally anti-proteinase 3 antibodies were associated with anti-glomerular basement membrane antibodies (1/11, 9%) or with anti-myeloperoxidase antibodies (1/11, 9%). IgM anti-proteinase 3 antibodies were uncommon (2/22 sera, 9%), and no IgA antibodies were demonstrated in any of 22 sera from patients with active systemic vasculitis. Significantly more individuals presented with anti-proteinase 3 antibodies in April-May-June, suggesting that an infective agent prevalent in Autumn might have a causative role in the associated diseases. Anti-proteinase 3 antibodies are the most common target antigen associated with Wegener's granulomatosis and cytoplasmic fluorescence.

Many autoantibodies have been described in HIV-infected individuals. We have examined the incidence, associations and prognostic significance of anti-nuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and anti-glomerular basement membrane (GBM) antibodies in individuals with HIV infections. One hundred and five patients, with asymptomatic infections (n = 37), AIDS-related complex (n = 32) or AIDS (n = 36) were studied. Plasma from 24 of these (23%) were positive for ANA: most demonstrated speckled fluorescence (n = 21) and were of low titre (1+ in 18). ANCA were demonstrated by IIF in 18 individuals (17%) and all fluorescent patterns were seen; 6 of these plasma were also positive in the ELISAs for antibodies to proteinase 3, myeloperoxidase or elastase. Thirteen plasma were positive for ANCA in the neutrophil cytoplasm ELISA; 10 of these were also positive in the specific ELISAs. A total of 30 plasma bound to proteinase 3, myeloperoxidase or elastase in specific ELISAs, in 6 cases with 2 specificities. Finally, 18 plasma (17%) contained anti-GBM antibodies by ELISA, but none of 4 plasma tested in inhibition assays was specific. ANA, ANCA and anti-GBM antibodies were not uncommon in HIV-infected individuals but the presence of these antibodies was not associated with the clinical manifestations of the corresponding autoimmune diseases. In addition, there was no correlation between the demonstration of these antibodies and the immunological status of the individual (apart from a correlation between CD4 counts less than 400/microliters with anti-GBM antibodies), the presence of an opportunistic infection, the development of malignancy or reduced survival. Some of these antibodies may arise from polyclonal activation, or be due to "sticky" serum since we have shown that the presence of anti-GBM antibodies correlated with the demonstration of ANCA by ELISA. These antibodies are not more common in hypergammaglobulinemic plasma but some may be due to heat-treatment of the plasma. The clinician caring for HIV-infected individuals needs to be aware of these "false-positive" antibody results.

Sera from 105 HIV-infected patients were tested for anti-neutrophil cytoplasm antibodies (ANCA) by indirect immunofluorescence (IIF), by specific ELISAs using target proteins of ANCA and by a neutrophil cytoplasm extract ELISA. Forty-four sera were positive. These included 18 positive by IIF, 7 with anti-neutrophil proteinase 3 antibodies, 26 with anti-myeloperoxidase antibodies and 2 with anti-elastase antibodies. Four sera were positive in the neutrophil cytoplasm extract ELISA but not in the specific ELISAs. None of these patients had clinical evidence of a cutaneous or systemic vasculitis. In addition there was no correlation between the presence of ANCA and the stage of disease, intercurrent bacterial or viral infection, anti-nuclear antibodies (ANA) and positive hepatitis B or syphilis serology.

A cutaneous or systemic vasculitis occurs in myelodysplasia as well as in myeloproliferative and lymphoproliferative disorders. The most common lesion is a leucocytoclastic vasculitis, with neurological or joint involvement occurring less often. The vasculitis may appear contemporaneously with or precede the clinical onset of the blood dyscrasia. Occasionally the lesions respond dramatically to the use of steroids but in general, patients with vasculitis have a worse prognosis than those with uncomplicated myelodysplasia. Vasculitis and myelodysplasia appear together too often for the association to be coincidental and the vasculitis in most cases cannot be attributed to intercurrent infections, therapeutic agents or a pre-existing rheumatological disorder. While autoantibodies are frequently present in myelodysplasia, and ANA and anti-neutrophil cytoplasm antibodies (ANCA) are found in other vasculitides, neither of these antibodies is associated with the vasculitis of myelodysplasia. There has however been one report of ANCA in Sweet's syndrome a non-vasculitic skin condition that also occurs in the myelodysplastic syndromes.

The demonstration of circulating anti-glomerular basement membrane (GBM) antibodies is almost diagnostic for anti-GBM disease and Goodpasture's syndrome. These antibodies are, however, occasionally present in SLE and diabetes, in association with IgA disease and membranous nephropathy and after transplantation in Alport's syndrome. In addition, we describe circulating anti-GBM antibodies in a research worker who handled GBM and in whom coeliec disease later developed, and in an individual with epidermolysis bullosa acquisita. Neither patient had impaired renal function nor an abnormal urinary sediment, suggesting either that these antibodies were of low affinity, or that additional factors are required for the pathogenesis of an aggressive glomerular lesion when circulating anti-GBM antibodies are present. In at least one of these individuals anti-GBM antibodies may have developed after the exposure of basement membrane collagen type IV to activated immunological mediators and cells.

The gene causing adult polycystic kidney disease (APKD) in most northern European families has been localised to the short arm of chromosome 16, close to the alpha globin gene (PKD1). A DNA probe 3' to the alpha globin locus (3'HVR) has been used to test such families but a second genetic locus (PKD2) was recently proposed when two families from Italy failed to show linkage to that locus. The presence of two or more loci could significantly reduce the value of linked probe analysis as a tool for the diagnosis of APKD and we have therefore examined five Mediterranean families using the 3'HVR probe. In these families we have not demonstrated any mutations at the second locus. The 3'HVR gene remains the most useful probe in making the diagnosis of APKD disease in an Australian Caucasian population, and the certainty with which the diagnosis is made may be enhanced by the additional use of other nearby probes when the defect involves the PKD1 locus.

Anti-neutrophil cytoplasm antibodies (ANCA) occur occasionally in rheumatoid arthritis (RA), but their incidence and clinical significance have been unclear. In this study we have investigated 58 patients with RA. In 22 patients the disease was inactive and the remaining 36 with active disease were further subdivided into those without clinical evidence of vasculitis (26), those with cutaneous vasculitis (8) and those with systemic vasculitis (2). ANCA were demonstrated by indirect immunofluorescence in 10 of the 58 patients (17%). While both perinuclear (pANCA) and cytoplasmic (cANCA) staining were detected, pANCA were more common (70%). Neutrophil-specific anti-nuclear antibodies (ANNA) were demonstrated in a further eight sera (14%) and ANA were detected on Hep-2 cells in 30 of the 58 sera (52%). ELISAs for the detection of anti-myeloperoxidase and anti-elastase antibodies were then established. Five sera with pANCA and five that contained ANNA were negative for both anti-myeloperoxidase and anti-elastase antibodies, suggesting other as yet unidentified cytoplasmic antigens as the target molecules. However, anti-myeloperoxidase or anti-elastase antibodies were found in four sera that had homogeneous or speckled ANA on both Hep-2 cells and neutrophils. One serum contained both antibodies. The presence of ANCA detected by indirect immunofluorescence or of anti-myeloperoxidase or anti-elastase antibodies in these patients with RA was not associated with disease activity nor with the demonstration of cutaneous vasculitis or renal disease (P NS). A possible association with systemic vasculitis remains to be confirmed. There is an incomplete correlation between indirect immunofluorescence patterns and antibody specificity in ELISA systems.

Alport's syndrome is a heterogeneous group of inherited abnormalities of basement membranes that may result in progressive renal failure, defective hearing and lens abnormalities. The glomerular basement membrane (GBM) characteristically has areas of reduplication, lamellation and attenuation on electron microscopic examination. In the majority of affected males and some females, there is reduced or variable binding of serum from patients with anti-GBM disease (Goodpasture's syndrome) to these basement membranes. These sera contain antibodies directed against the Goodpasture antigen which has been thought to be located in the non-collagenous domain of the alpha3 chain of type IV collagen and is presumed to be important in cross-linking of the collagen molecules. The reduced staining for the Goodpasture antigen suggests that this structure is either absent or masked in Alport's syndrome. We have tested DNA from six unrelated individuals with Alport's syndrome. All had been transplanted for renal failure. The diagnosis of Alport's syndrome was made on the characteristic electron microscopic appearance of the renal basement membranes (n = 4), the presence of sensori-neural deafness (n = 4), a family history of Alport's syndrome (n = 5) and the presence of circulating inhibitable anti-GBM antibody activity post-transplant (n = 2). Oligonucleotides (20mers) corresponding to the 5' and 3' ends of the known 25 amino acid sequence for the putative Goodpasture antigen were used as primers for amplification of genomic DNA. The products were then blotted and probed with an intermediate 19-mer DNA. All Alport's patients contained a 75-bp fragment corresponding to the published peptide sequence for the non-collagenous domain of the alpha 3 chain of type IV collagen, suggesting that a large deletion of this region, the putative Goodpasture antigen, is unlikely to account for the defect in Alport's syndrome.

We have studied 495 sera that were referred to us from patients suspected on clinical and/or histological grounds to have a small vessel vasculitis. These sera were tested for antibodies against neutrophil cytoplasm antigens (anti-neutrophil cytoplasm antibodies, ANCA) using assays based on neutrophil acid extract, myeloperoxidase and elastase. Such antibodies are commonly found in Wegener's granulomatosis (WG) and microscopic polyarteritis (MPA), and sometimes in other small vessel vasculitides. One hundred and twenty-six of these sera (25%) were positive in the acid extract ELISA, 68 (14%) in the assay for anti-myeloperoxidase antibodies and 35 (16%) in the assay for anti-elastase antibodies. A total of 166 sera (34%) were positive for antibodies against neutrophil cytoplasm constituents. No ANCA, anti-myeloperoxidase or anti-elastase antibodies were detected in 26 convalescent sera from patients either with WG or MPA, or who had previously been positive. The mean time between positive and negative sera was eight weeks (range three weeks to six months) and three out of three who relapsed again developed ANCA of the same specificity as the original sera. Of the 228 sera also tested for anti-GBM antibodies, 13 (5.7%) were positive. All these contained antibodies against neutrophil cytoplasm constituents (three against the acid extract, eight against myeloperoxidase and two against elastase). Forty-nine of the 74 sera (66%) tested for ANA were positive. Twenty-nine (39%) had a speckled and 20 (27%) had a homogeneous pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

The Goodpasture antigen is the target recognized by anti-glomerular basement membrane (GBM) antibodies in anti-GBM disease or Goodpasture's syndrome. This structure is present in all normal GBM, but when serum containing anti-GBM antibodies is used to examine renal tissue from most males with classical Alport's syndrome, the Goodpasture antigen appears to be missing. The nature of the Goodpasture antigen is uncertain although it has been putatively and controversially localized to the non-collagenous domain of a novel type IV collagen chain (alpha 3) by one group, and a short peptide sequence has been published (M2). We have performed several experiments to determine whether M2 represents the Goodpasture antigen and we have also studied the corresponding sequence of the alpha 4 chain of type IV collagen (M3). Firstly, we demonstrated by polymerase chain reaction (PCR) amplification using specific priming oligonucleotides that mRNAs corresponding to M2 and M3 were found within the kidney and that the published sequences were correct. When heterologous antibodies were raised against M2 and M3 these bound specifically to GBM in an ELISA based on collagenase-digested basement membrane and this binding could be inhibited by incubation with collagenase-digested GBM but not with ovalbumin. On further examination of the target molecules using Western blots, the anti-M2 antibody bound to a single high molecular weight band of collagen-digested GBM in contrast to the anti-M3 antibody that bound to the same bands as Goodpasture serum. We then established ELISAs for anti-M2 and anti-M3 activity using the peptides M2 and M3. While rabbit anti-M2 and M3 antibodies bound specifically to their respective peptides in these ELISAs, there was no binding of three high titre Goodpasture's syndrome sera or two sera from Alport's syndrome patients with inhibitable anti-GBM antibody post-renal transplant. We have shown that the sequences of M2 and M3 correspond to proteins present within the collagenase-resistant part of the GBM, suggesting that these do represent parts of novel type IV collagen chains. However, sera containing anti-GBM antibodies did not bind to either peptide in solid-phase ELISAs, and these antibodies may recognize a different peptide sequence, features of the tertiary structure of these peptides or interactions between collagen chains.

Autoantibodies that produce a perinuclear pattern on indirect immunofluorescent examination of ethanol-fixed neutrophils (pANCA) are found in about half of all cases of microscopic polyarteritis. These antibodies are often directed against myeloperoxidase or elastase and we have developed sensitive reproducible ELISAs for their detection and study. Seven sera from 19 patients with microscopic polyarteritis or segmental necrotizing glomerulonephritis contained anti-myeloperoxidase or anti-elastase antibodies or both. In contrast, only one of 18 sera from patients with Wegener's granulomatosis, where the pattern of immunofluorescence is predominantly cytoplasmic, had anti-myeloperoxidase antibodies and no anti-elastase antibodies were detected. Using sera from patients with microscopic polyarteritis, both anti-myeloperoxidase and anti-elastase antibodies were demonstrated to be of high affinity. There was no immunoglobulin class, subclass or light chain restriction noted. Anti-myeloperoxidase and anti-elastase antibodies were also found occasionally in anti-glomerular basement membrane disease, mixed connective tissue disease, systemic lupus erythematosus, post-streptococcal glomerulonephritis and atypical pneumonia. In further studies these antibodies were not associated with other lung infections, although anti-elastase antibodies were noted in one of 14 sera positive for ASOT that were tested. Anti-myeloperoxidase antibodies were found more frequently than anti-elastase antibodies and these antibodies were occasionally present together. In addition some sera with pANCA had neither anti-myeloperoxidase nor anti-elastase antibodies. The target molecules in these cases remain unclear.

Anti-idiotypic antibodies are antibodies that are directed against the variable region of the corresponding antibody molecule and have been postulated to have a regulatory role in the immune response. Circulating anti-idiotypic antibodies have been reported in several antibody-mediated autoimmune diseases. We have established the following detection system for anti-idiotypic antibodies in human anti-glomerular basement membrane (GBM) disease. Spleen cells harvested from BALB/c mice immunised with human GBM were incubated with GBM-coated sheep red blood cells in the presence of guinea pig complement. Each spleen cell that produced anti-GBM antibodies resulted in a surrounding plaque where antibodies lysed the GBM-coated RBC. The number of plaques could be inhibited by the addition of heterologous anti-idiotypic antibodies to the plaquing mixture. Anti-idiotypic antibodies were then sought in both acute and convalescent phase sera from patients with anti-GBM disease and in laboratory staff who repeatedly handled GBM and sera containing anti-GBM antibodies. Anti-GBM antibodies were removed from all material before testing and affinity-purified preparations contained concentrations of IgG corresponding to the range that resulted in inhibition when affinity-purified rabbit anti-idiotypic antibody was examined. No anti-idiotypic antibodies could be demonstrated in any of the sera or IgG preparations examined. We conclude that if circulating anti-idiotypic antibodies are present in patients with anti-GBM disease, they must be infrequent, or at concentrations below the limits of detection of this assay (less than 1 mcg/mL). The observations of anti-idiotypic antibodies in other antibody-mediated diseases need to be confirmed.

Antibodies that are directed against cytoplasmic constituents of neutrophils and monocytes (anti-neutrophil cytoplasm antibodies, ANCA) have been described in Wegener's granulomatosis, microscopic polyarteritis (MPA) and some cases of segmental necrotizing glomerulonephritis (SNGN). Other antibodies occasionally described in Wegener's granulomatosis and MPA include anti-nuclear antibodies (ANA) and anti-glomerular basement membrane (GBM) antibodies. We have studied the diversity of the corresponding antigens in ANCA-associated renal diseases. Sera from 46 patients with active histologically proven Wegener's granulomatosis, MPA and SNGN were tested for ANCA by indirect immunofluorescent examination of normal peripheral blood neutrophils. Thirty-four sera (74%) were positive; 16 were associated with diffuse cytoplasmic staining (cANCA) and 18 with perinuclear staining (pANCA). In addition, five demonstrated antineutrophil-specific nuclear staining (ANNA). On Western blotting of the neutrophil extract, five sera recognized a 29-kD molecule recently identified as neutrophil proteinase 3. Two sera with typical cANCA bound to molecules of 36, 38 and 116 kD and another to a molecule of 22 kD. The final serum associated with pANCA bound to a molecule of about 12 kD. Thirteen sera out of 46 (28%) tested in an ELISA contained anti-myeloperoxidase antibodies; 10 of these were associated with pANCA and two others with ANNA. Three sera of 17 (18%) tested contained anti-elastase antibodies; these also contained anti-myeloperoxidase antibodies and were associated with pANCA. However, eight sera with pANCA were negative for anti-myeloperoxidase antibodies and three of these were also negative for anti-elastase antibodies, suggesting further unidentified target antigen or antigens associated with the pANCA. Fifteen of the 34 sera positive for ANCA also demonstrated anti-nuclear staining on Hep-2 cells (53%) in a speckled, homogeneous, or nucleolar pattern. ANA were significantly associated with the presence of pANCA (P less than 0.01), and levels of ANA and ANCA fell in parallel after treatment. One serum with a pANCA was also positive for anti-GBM antibodies. Inhibition studies using ELISAs for anti-GBM antibodies indicated that there was no cross-reactivity between target molecules recognized by these antibodies. The diversity of target molecules recognized by ANCA suggests that cross-reactivity with bacterial structures is less likely as the primary aetiological event in the development of these antibodies than tissue destruction; and that cross-reactivity with vascular endothelium is also unlikely as the pathogenetic basis of vessel disease.

Circulating antibodies directed against cytoplasmic constituents of granulocytes and monocytes (anti-neutrophil cytoplasm antibodies, ANCA) are found in about 80% of active cases of Wegener's granulomatosis and microscopic polyarteritis. Antibodies are detected by indirect immunofluorescence on normal peripheral blood leucocytes, or by ELISA or radio-immunoassay using a neutrophil cytoplasm extract. There are 2 patterns of staining seen on indirect immunofluorescent examination of leucocytes and a number of different molecules recognised on Western blots. In Wegener's granulomatosis diffusely granular cytoplasmic staining (cANCA) is associated with a 29KD molecule that has recently been identified as neutrophil proteinase 3. A finely granular pattern may also be seen in microscopic polyarteritis. A perinuclear pattern (pANCA) is present in some other cases of segmental necrotising glomerulonephritis and occasionally in rheumatoid arthritis but almost never in Wegener's granulomatosis. This pattern is often associated with the presence of anti-myeloperoxidase and anti-elastase antibodies. Levels of anti-neutrophil cytoplasm antibodies usually reflect disease activity but a pathogenetic role for these antibodies in the development of small vessel vasculitides is, as yet, unproven.

Phase-contrast microscopy of the urine of a young Greek woman with macroscopic haematuria showed sickling of the red blood cells. The diagnosis of sickle-cell trait was confirmed with haemoglobin electrophoresis, and an intravenous pyelogram demonstrated the typical medullary cavities seen in this disease. Urine collected from the left ureter, from which the haematuria originated, grew 10(6) Ureaplasma urealyticum/ml. With Doxycycline therapy the macroscopic haematuria and the sickled cells in the urine resolved, but red cell casts and an excessive number of glomerular red blood cells persisted in the urine, confirming the presence of glomerulonephritis. Ureaplasma urealyticum has not previously been described in association with a sickling episode nor with the scarring of a sickle cell kidney. The possible role of this infection is discussed.

We examined 40 female patients for the presence of fastidious bacteria in the bladder by culture of mid catheter and suprapubic aspiration urine specimens. Gardnerella vaginalis and Ureaplasma urealyticum were the most frequently isolated fastidious species. The suprapubic aspirate was positive in 3 patients with more than 10(5) colony-forming units per ml. Gardnerella vaginalis and 7 with more than 10(2) colony-forming units per ml. Ureaplasma urealyticum in the mid catheter urine specimen, while it was negative for bacteria in 6 patients with less than 10(4) colony-forming units per ml. coryneform bacilli or lactobacilli in the mid catheter urine specimen. The results show that culture of mid catheter urine samples provides a reasonable guide to the presence of bacteriuria owing to fastidious micro-organisms and can be used when a suprapubic aspiration specimen cannot be obtained easily.

Reconstitution experiments in the heterologous phase of nephrotoxic nephritis (HNTN) have shown that tissue injury is directly related to the number of polymorphonuclear neutrophils (PMN) infiltrating the glomeruli. Recent studies have indicated that endotoxin pretreatment in this model enhances tissue injury under certain conditions. The aim of this project was to investigate the relationship between glomerular PMN infiltration and tissue injury after endotoxin in HNTN. A dose of nephrotoxic globulin (NTG) that resulted in low levels of albuminuria was chosen. The administration of E. coli lipopolysaccharide (LPS) to Sprague-Dawley rats 24 h before the induction of HNTN resulted in the earlier appearance of larger numbers of glomerular PMN than in animals injected with nephrotoxic globulin alone (P less than 0.05 at 1/2 and 1 h). However there was no significant increase in albuminuria nor in renal histological damage. There was no difference in the amount or rate of glomerular deposition of the NTG or rat complement between the two groups of rats. The more rapid accumulation of glomerular PMN can, however, be accounted for by the LPS-induced activation of endothelium, since endotoxin was detected in the kidneys 24 h after the injection of LPS. No endotoxin was present in the serum at the time of injection of NTG. Thus we have demonstrated in a model of PMN-mediated tissue injury, that an increase in PMN influx is not necessarily associated with exaggerated injury.

Anti-neutrophil cytoplasm antibodies (ANCA) are present in the serum of patients with Wegener's granulomatosis, microscopic polyarteritis, and some other small vessel vasculitides. There are at least 2 different anti-neutrophil cytoplasm antibodies identified by their distinctive cytoplasmic staining patterns on indirect immunofluorescence examination. The only antigen identified to date is myeloperoxidase which has a perinuclear distribution on alcohol-fixed neutrophils and monocytes. We have established an ELISA that detects all anti-neutrophil cytoplasm antibodies and one specific for anti-myeloperoxidase antibodies. In the ELISA for anti-neutrophil cytoplasm antibodies, all sera with diffuse cytoplasmic or perinuclear neutrophil staining on indirect immunofluorescence examination bound at levels greater than the normal range (34%, m + 4SD). Three convalescent sera that were negative by indirect immunofluorescence examination were also negative in the assay. Positive sera could be detected at a dilution of 2 and inhibition studies showed that the binding was specific for the neutrophil extract. However, the presence of anti-neutrophil antibodies (ANA), anti-mitochondrial antibodies or immune complexes resulted occasionally in binding in the positive range. Where positive binding was noted in non-vasculitic segmental necrotizing glomerulonephritis, the binding could not be inhibited by pre-incubation with the neutrophil extract. The ELISA for ANCA is a sensitive, objective screening technique that can be performed in parallel with the assay for anti-glomerular basement antibodies to exclude the presence of anti-neutrophil cytoplasm antibodies in patients presenting with rapidly progressive glomerulonephritis. The ELISA for anti-myeloperoxidase antibodies may identify a subset of patients with distinct clinical or prognostic features.

A functional comparison between elicited peripheral blood neutrophils has been made in vivo and in vitro. Preliminary experiments showed that separation of peripheral blood cells on a metrizamide gradient yielded too few neutrophils for efficient radiolabelling with indium (In): hence a mixed cell preparation comprising 80% neutrophils was elicited in the peripheral blood of adult male rats by the administration of endotoxin (0.25 mg i.a.) and cobra venom factor (200 microliter i.p.) 20 h before. Peritoneal neutrophils were collected 4 h after the i.p. injection of 6 ml thioglycollate. Both populations differed markedly from normal peripheral neutrophils on the in vitro testing of random locomotion, chemotaxis and phagocytosis of Candida. After labelling with IIIIn-tropolonate, a greater proportion (mean = 8%) of peripheral blood cells localized to an E. coli/Freund's complete adjuvant-induced abscess compared with peritoneal neutrophils (mean = 3%). The abscess could be visualized externally by scanning with both cell preparations, but the distribution of activity differed markedly. The greater hepatic sequestration of peritoneal neutrophils suggested cell damage or activation. To overcome the difficulty of harvesting normal peripheral blood neutrophils in the rat, either of these populations can be used to follow the kinetics of inflammation. However, elicited peripheral blood cells yield a higher proportion of responding cells.

Certain fastidious organisms such as U urealyticum and G vaginalis can be isolated from the aspirated bladder urine of pregnant women more frequently than conventional urinary pathogens such as Escherichia coli [1]. They can be isolated even more often from the aspirated bladder urine of patients with renal disease, but rarely from that of healthy men or nonpregnant women [2]. We investigated the incidence of bacteriuria due to these two organisms--particularly U urealyticum--in patients with preeclampsia. U urealyticum was isolated more frequently (rate, 20%), and usually in higher colony counts, from the urine of patients with preeclampsia than from that of healthy pregnant women (rate, 7%). G vaginalis was isolated with approximately the same frequency as U urealyticum from specimens of bladder urine; both organisms were isolated from the urine of 11 patients (eight healthy women and three with preeclampsia). High colony counts of G vaginalis were also found more frequently in patients with preeclampsia. In both groups other fastidious organisms were isolated in a total of only five patients, and in four of these five cases U urealyticum and/or G vaginalis were also isolated from the same specimen. Urine cultures were more frequently positive in patients with moderately severe hypertension (blood pressure, greater than 160/100 mm Hg) than in those with mild hypertension (blood pressure, 140/90-160/100 mm Hg, occurring in nine (53%) of 17 patients and in nine (26.5%) of 34 patients, respectively. This difference was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

The administration of the polyclonal activator HgCl2 (1 mg/kg i.p.) to Brown Norway (BN) rats on days 0, 2, 4 and 7 resulted in the cyclical production of anti-glomerular basement membrane (GBM) antibodies, the first peak of which occurred at day 14 with a smaller peak at day 26. Glomerular anti-GBM antibody levels were also raised at day 14. Renal injury as measured by urinary loss of albumin and complement (C3) was also cyclical, being maximal on days 15 and 23-26. However, urinary protein excretion was significantly diminished on the days corresponding to the first peak of circulating antibody levels if peripheral monocyte counts were reduced by the repeated injection of anti-monocyte antiserum. Protein excretion was also reduced after the administration of anti-polymorphonuclear neutrophil (PMN) antiserum. Finally, glomerular protein excretion was independent of depletion of serum C3 levels to less than 10% of pooled normal sera by the repeated administration of Cobra Venom Factor (CVF) on days 9 and 11. These findings demonstrate that in the absence of progressive tissue injury in this model, glomerular protein excretion fluctuates according to circulating levels of anti-GBM antibody and that, despite being independent of complement, tissue injury may be increased in the presence of complement activation.

A 77-year-old man presented with macroscopic haematuria and oligo-anuric renal failure. A renal biopsy showed moderate linear glomerular basement membrane (GBM) staining with lambda light chains alone, but both circulating IgG and lambda light chain activity were present. There was no evidence of a serum or urinary paraprotein and a bone marrow biopsy was normal. Serum lambda light chains did not bind to renal GBM when examined by indirect immunofluorescence. This case suggests that light chains are capable of binding directly to epitopes in the GBM and may initiate injury although by a different mechanism from the IgG anti-GBM molecule.

The mechanisms by which intercurrent bacterial infections are associated with increased tissue injury in some forms of glomerulonephritis may include complement activation by bacteria and subsequent increased glomerular neutrophil (PMN) infiltration. We have studied the effect of complement activation after cobra venom factor (CVF) in anti-glomerular basement membrane (GBM) antibody-mediated disease. A single injection of CVF 24 h before the administration of heterologous nephrotoxic globulin (NTG) to Sprague-Dawley rats resulted in greatly increased albuminuria in some animals on the second day of this model. This phenomenon was reproducible and depended on the presence of circulating PMN and complement. We have previously shown that the administration of CVF on days 9 and 11 of the HgCl2 model in inbred Brown Norway rats, resulted in increased albuminuria in all animals at day 17 (p less than 0.05). The administration of small amounts of CVF with consequent complement activation in antibody-mediated disease represents a model for the increased injury seen after infection in human disease.

The association of anti-glomerular basement membrane (GBM) antibody-mediated glomerulonephritis and glomerular immune complexes is common and probably arises from a number of mechanisms. In the series, glomerular immune complexes were identified in 6 of 17 patients who initially presented with anti-GBM disease. In four cases, glomerular immune complexes were noted in renal biopsies obtained at clinical presentation; in the other two, they were first demonstrated seven and 28 months after presentation, when circulating anti-GBM antibody levels were undetectable. Circulating immune complexes were detected in only two of six patients, either 28 months before or 17 months after the demonstration of the glomerular membranous lesion. The association of glomerular immune complexes and anti-GBM disease may be coincidental with immunologically-unrelated immune complexes localizing in the GBM for physico-chemical reasons; or the presence of glomerular-bound anti-GBM antibodies may predispose to the deposition of molecules with particular affinity for these antibodies. One patient with glomerular immune complexes used heroin, which may be associated with immune complex formation and the development of glomerulonephritis; and one patient was subsequently thought to have systemic lupus erythematosus (SLE). An antecedent infection was found in two of the four patients who had glomerular complexes at presentation, but in only three of 13 with uncomplicated anti-GBM disease. Three of 6 patients with superimposed glomerular complexes had a history of exposure to organic solvents before the onset of disease, while none in the group with anti-GBM disease alone had.

Adult polycystic kidney disease (APKD) is one of the most common inherited diseases in man. A diagnosis based on the demonstration of renal cysts with ultrasonography or computerised tomography may be inconclusive in early adulthood, the crucial years before child-bearing is complete. Here we describe the improved diagnostic probability that is possible using genetic linkage studies. A 24-year-old woman, whose father and younger sister were affected by APKD, was demonstrated to have a single cyst in each kidney. These findings were insufficient for a diagnosis of APKD and for this reason genetic linkage studies were undertaken. DNA was extracted from peripheral blood leukocytes from the presenting individual, and her immediate and extended family; the DNA was cut with the restriction enzyme PvuII, electrophoresed in a 0.7% agarose gel and blotted onto nitrocellulose before probing with a 32P-labelled 4 kb fragment. This contained DNA from the hypervariable region (3' hypervariable region, 3'HVR) that is linked to the gene for APKD on the short arm of chromosome 16 and has been used in other family studies by Reeders et al. We correlated the findings on Southern blotting with ultrasound evidence of APKD and found that the disease segregated with a 7.0 kb fragment in the presenting individual's father and sister. She was shown to have inherited this allele also; the use of this technique thus increased the probability of her having APKD from 50% to 96.5%.