HLA Class I typing of volunteers for a bone marrow registry: QC analysis by DNA-based methodology identifies serological typing discrepancies in the assignment of HLA-A and B antigens.

Until recently, the majority of newly recruited volunteer donors were typed for HLA-A and -B by serology onto the National Marrow Donor Program Registry. Quality control of this serological typing performed by contracted laboratories was carried out by retesting approximately 1% of each laboratory's test volume utilizing DNA-based techniques (SSOP). The criteria used for selection included samples presumed to be homozygotes, samples with split antigen specificities and samples with antigens considered to be difficult to define. Out of 1983 samples analyzed, 156 HLA-A (3.9%) and 265 HLA-B (6.7%) locus discrepancies were identified. Review of these discrepancies by both the serological and QC laboratory revealed that the majority of discrepancies were due to errors in serological typing. Serological discrepancies were categorized as follows: blank antigens identified (36.8%) and misassignments (63.2%). Misassignments were defined as either the incorrect assignment of antigens within a group ("wrong split"), or a complete misassignment. Antigens reported as blanks most frequently belonged to the A19 and A28 groups and to the B70, 46 and 40 groups. The most frequent misassignments within groups were the A19 and A10 groups, and the B40 and B15 groups. Other HLA-A misassignments included A2 vs A28 or A2 vs A69, while other HLA-B misassignments included B35 and B70. This QC analysis showed that serological typing of class I antigens for the purposes of NMDP registry typing is prone to a significant error rate. Careful evaluation and selection of contracted laboratories by the NMDP suggests methodological limitations rather than poor performance as the main cause of these observations.

Novel HLA-B alleles identified in potential marrow donors: B*3917, B*1405 and B*3528.

Sequence-specific oligonucleotide probe hybridization and sequence-specific primer polymerase chain reaction (PCR) typing of volunteer bone marrow donors suggested the presence of variants of known HLA-B alleles in five individuals. PCR products encompassing HLA-B locus exons 1 through 3 were prepared and subcloned. Three African-American individuals had a novel HLA-B*39 allele (B*3917), and another African-American was found to have a novel HLA-B*14 allele (B*1405). In a third individual of Hispanic origin, a novel HLA-B*35 allele (B*3528) was identified. These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations.

Novel HLA-B*15 alleles identified in potential marrow donors.

Several methods for low-resolution class I typing of potential bone marrow donors are available. The National Marrow Donor Program (NMDP) has initiated pilot projects for large-scale DNA-based class I typing to initially characterize donors. Sequence-specific oligonucleotide probe hybridization and sequence-specific primer polymerase chain reaction (PCR) screening of 3,500 NMDP potential donors suggested the presence of variants of known HLA-B*15 variants in 3 donors. PCR products encompassing HLA-B locus exons 1 through 3 were prepared and subcloned. Sequencing revealed 3 alleles differing from known HLA-B*15 alleles by nucleotide substitutions resulting in predicted novel HLA-B antigens. The new alleles occur in distinct ethnic groups. These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations.

A novel HLA-B*40 allele and novel exon 1 sequences of two B*40 alleles identified in potential marrow donors.

Sequence-specific oligonucleotide probe hybridization and sequence-specific primer polymerase chain reaction (PCR) typing suggested the presence of variants of HLA-B*40 in three individuals. Two were part of 3,500 potential marrow donors being screened for the National Marrow Donor Program, while the third was a clinical specimen. PCR products encompassing HLA-B locus exons 1 through 3 were prepared and subcloned. In one individual, a native of the Pacific Islands, sequencing revealed a novel HLA-B*40 allele (B*4023). In two other individuals, a previously unknown exon 1 sequence was determined for HLA-B*4016 (ethnicity unknown) and B*4020 (Hispanic). These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations.

Familial antiphospholipid antibody syndrome: criteria for disease and evidence for autosomal dominant inheritance.

OBJECTIVE: To develop diagnostic criteria for a familial form of antiphospholipid antibody syndrome (APS), identify families with >1 affected member, examine possible modes of inheritance, and determine linkage to potential candidate genes. METHODS: Family members of probands with primary APS were analyzed for clinical and laboratory abnormalities associated with APS. Families with > or =2 affected members were analyzed by segregation analysis and typed for candidate genetic markers. RESULTS: Seven families were identified. Thirty of 101 family members met diagnostic criteria for APS. Segregation studies rejected both environmental and autosomal recessive models, and the data were best fit by either a dominant or codominant model. Linkage analysis showed independent segregation of APS and several candidate genes. CONCLUSION: Clinical and laboratory criteria are essential to identify the spectrum of disease associated with APS. We believe a set of criteria was developed that can precisely define affected family members with APS. Modeling studies utilizing these criteria strongly support a genetic basis for disease in families with APS and suggest that a susceptibility gene is inherited in an autosomal dominant pattern. However, in these families, APS was not linked with HLA, Fas, or other candidate genes, including beta2-glycoprotein 1, HLA, T cell receptor beta chain, Ig heavy chain, antithrombin III, Fas ligand, factor V, complement factor H, IgK, and Fas.

Fine mapping of a genetic locus for Peutz-Jeghers syndrome on chromosome 19p.

Peutz-Jeghers syndrome (PJS) was recently mapped in a single report to the telomeric region of chromosome 19p (A. Hemminki et al., Nat. Genet., 15: 87-90, 1997). Our studies confirm this location and provide further localization of the PJS locus. In the five families examined, there were no recombinants with the marker D19S886. The multipoint log odds score at D19S886 is 7.52, and we found no evidence for genetic heterogeneity. We also found that all carriers expressed the PJS phenotype and no noncarriers displayed PJS sequellae, indicating complete penetrance with no sporadic cases.

The prevalence of systemic lupus erythematosus in Northern Ireland.

Using six sources of patient ascertainment, 467 patients were initially identified as having a diagnosis of systemic lupus erythematosus (SLE) in Northern Ireland. The diagnosis was subsequently altered in 45 of these, either by the patient, their family, or their physician. The corrected source data were then reanalysed by the 'capture-recapture' technique, which suggested a further 71 patients missed during the initial identification process. Eighty-eight patients underwent physical examination, and 14 were found to have a diagnosis other than SLE. A final estimated figure of 415 patients was obtained, giving a point prevalence on August 1, 1993 of 25.4 per 10,000 for the Province (95% confidence interval 22.1-28.7 per 100,000).

Clinical features associated with a positive anticardiolipin antibody in Irish patients with systemic lupus erythematosus.

Anticardiolipin antibodies (ACA) are one of a number of autoantibodies found in patients with systemic lupus erythematosus (SLE) and their presence has been associated with clinical features of the antiphospholipid syndrome (APS). The aim of this study was to determine which clinical features are associated with a positive anticardiolipin antibody in Irish patients with SLE. Ninety-five Irish patients with SLE were studied. All were examined thoroughly, had their full history taken, and had case records reviewed. The presence of any clinical feature associated with the APS was noted. Sera from these patients were tested for IgG and IgM ACA. The only significant association found was between a history of venous thrombosis and the presence of ACA, although several other features were more common in ACA positive patients. There were no significant associations with one or other isotype. This study serves as a reminder to consider the possibility of venous thrombosis, and other clinical features, if an SLE patient is positive for ACA.

A clinical and serological comparison of familial and non-familial systemic lupus erythematosus in Ireland.

Seventeen families, in which the diagnosis of SLE could be verified in two relatives, were included in the study. The diagnosis was made according to the revised 1982 ARA criteria. We compared the 34 cases of familial SLE in these 17 families with 34 non-familial SLE controls matched for age, sex, ethnicity and duration of disease. Comparisons were made for the presence of 26 clinical and 11 serological features. The frequency of clinical features was similar between the groups. The frequency of anti-Ro antibody was higher in the non-familial group (15 out of 34 compared to 6 out of 34, P = 0.036, McNemar's test), although this was not significant after application of Bonferoni's correction for the number of comparisons. No cases of familial IgA or familial complement deficiency were identified. It was noted that 10 of the 34 non-familial patients and only one of the familial patients had the combination of anti-Ro antibody and photosensitivity. The findings of this study support the hypothesis that familial SLE and non-familial SLE are the same clinical entity, although there are differences in the subtypes of disease.

Kikuchi's disease as a presenting feature of mixed connective tissue disease.

Necrotising histiocytic lymphadenitis (Kikuchi's disease) is a recognised cause of benign lymphadenopathy. It has been reported in association with both adult Still's disease, systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). We report a case of mixed connective tissue disease (MCTD), in which biopsy of enlarged lymph nodes occurring as a presenting feature showed the characteristic features of Kikuchi's disease. This finding supports the belief that Kikuchi's disease and the autoimmune rheumatic disorders may share a common aetiology.

Oxalate nephrosis and renal sclerosis after renal transplantation in a cat.

A 10-year-old castrated domestic shorthair cat received two renal allografts, 14 days apart, for the treatment of chronic renal failure. Oxalate nephrosis developed in both allografts, and they became nonfunctional. During the transplantation period, the cat was not exposed to exogenous sources of oxalate, and there was no evidence of primary type 2 hyperoxaluria before surgery. Urologic surgery, in particular renal transplantation, has been identified as a factor that can precipitate renal failure in human patients with decompensated renal function and hyperoxaluria. If hyperoxaluria was present before surgery in this cat, it was most likely caused by increased absorption or decreased metabolism of dietary oxalate.

Evaluation of a method of ureteroneocystostomy in cats.

Renal transplantation is a successful treatment for terminal renal failure in cats. Ureteral obstruction at the bladder wall or stoma has been a technical complication encountered in approximately 50% of clinical transplants. The small (0.4 mm) diameter of the feline ureter makes standard techniques described for ureteroneocystostomy unsatisfactory for cats. In this study, we used microsurgical techniques to oppose ureteral mucosa to intact bladder mucosa in an attempt to form a stricture-free stoma. In each of 5 anatomically normal cats, 1 ureter was microsurgically implanted in the bladder. Ultrasonographic examination of the kidneys, ureters, and bladder was performed twice weekly for 8 weeks. Excretory urography was performed at postoperative weeks 1, 2, 4, 8, and 12. Biopsy specimens were taken from the kidney on the surgically treated side 4 weeks after surgery. At 12 weeks, the kidney, ureter, and ureterovesical junction from the treated side were removed and submitted for histologic evaluation. At 1 week, all cats had enlargement of the kidney, renal pelvis, and ureteral lumen on the treated side. This enlargement gradually decreased, and by week 8, there was no difference in comparison with the control side. Ureteroneocystostomy that requires tunneling of the ureter through the bladder wall may result in hydroureter and hydronephrosis, which may resolve. Recognition of these changes may prevent unwarranted surgical intervention in cases of suspected obstruction. The technique described in this study has been used in 15 cats receiving renal allografts. None required surgical correction of ureteral obstruction.

Renal transplantation for treatment of end-stage renal failure in cats.

Renal transplantation was performed as treatment of end-stage renal failure in 23 cats. Twenty-two cats had chronic renal disease and 1 cat had acute renal disease associated with ethylene glycol-induced toxicosis. Sixteen cats were discharged from the hospital. Nine survived a mean of 8.4 +/- 6.5 months, and 7 cats continue to survive at the time of this report (mean 12.6 months). Seven cats died within 2 weeks of surgery. All renal allografts were obtained from unrelated blood-crossmatch-compatible donors. No deaths were attributable to acute renal allograft rejection, demonstrating the successful maintenance of renal allografts by use of cyclosporine and prednisolone immunosuppression in cats.

Definition of the gracilis musculocutaneous flap for distant transfer in cats.

Dissection, injection, and surgical studies in feline cadavers and in anesthetized cats were conducted to determine the feasibility of using the gracilis muscle as the basis for a free musculocutaneous flap. The vascular pedicle of the flap consisted of the femoral artery and vein. Mean length (1.6 +/- 0.2 cm) of the vascular pedicle and mean artery (1.33 +/- 0.19 mm) and vein (2.55 +/- 0.38 mm) diameters were satisfactory for microvascular transfer. Fluorometry revealed overlying cutaneous perfusion in the flaps on the basis of their muscle vascular pedicles. To ensure survival of the flap, the muscular branches of the femoral artery and vein supplying the gracilis muscle had to be carefully preserved during surgical elevation of the flap.

Pathologic studies of acute rejection of mismatched feline musculocutaneous flaps. Effect of cyclosporine and prednisolone.

The gracilis musculocutaneous flap was developed as an allograft model to study acute rejection and immunosuppression in the cat. Twelve adult cats received a MLC incompatible flap. Six of the cats received cyclosporine oral solution and prednisolone (0.5 mg/kg/24 hr) for 100 days and six cats were not treated. Trough whole-blood levels of cyclosporine in the treatment group were maintained at approximately 750 ng/ml for 70 days, then 500 ng/ml for the remaining 30 days. Three flaps failed due to technical problems; 5 flaps were studied in the treatment group and 4 in the untreated group. All 5 flaps in the treatment group survived the 100 day treatment period and were rejected 30 +/- 26 days following cessation of treatment. Prior to discontinuation of treatment, with the exception of one cat, inflammatory changes associated with rejection were not observed in biopsy specimen. The flaps in the untreated group survived 13 +/- 1.5 days. Histopathologic examination of the flaps revealed little difference in the appearance of acute rejection and rejection after cessation of therapy. The most prominent lesion was a vasculitis with extensive perivascular lymphohistocytic inflammation. The lymphoid infiltrates consisted predominantly of T cells of both major classes (CD4 and CD8). Full-thickness epidermal necrosis and subsequent bacterial invasion followed vascular compromise.

Mizoribine serum levels associated with enterotoxicity in the dog.

To prevent or minimize mizoribine enterotoxicity in organ transplant recipients and to differentiate mizoribine enterotoxicity from other causes of enteritis, serum levels of mizoribine that produced subclinical and clinical signs of enterotoxicity were determined in the dog. When mizoribine was administered orally at 12-hr intervals, half the dogs studied showed clinical evidence of gastrointestinal disturbances (vomiting, diarrhea, and anorexia) without histopathologic signs of enterotoxicity. Using a 24-hr oral-dose schedule, clinical signs of gastrointestinal disturbances and histopathologic evidence (mucosal degeneration, crypt degeneration, and necrosis) of enterotoxicity were encountered when the mean 12-hr mizoribine serum level was 0.97 +/- 0.4 microgram/ml or greater. Histopathologic signs of enterotoxicity with repeated positive fecal occult blood assays and without clinical signs of gastrointestinal disturbances occurred when the mean 12-hr serum level was 0.53 +/- 0.17 microgram/ml or greater. Oral administration of cyclosporine did not exacerbate mizoribine enterotoxicity in the dog when administered with mizoribine at a dose that produced histopathologic signs of enterotoxicity.

Identification of muscle flaps in small animals.

Muscle flaps in small animals have been used quite frequently in experimental research. Their use in clinical veterinary surgery, however, has not yet been extensively explored. This paper describes the anatomy and clinical use of some of the muscle flaps that have been investigated and used in small animals.

Use of flaps and/or grafts for repair of skin defects of the distal limb of the dog and cat.

Skin flaps and/or skin grafts can be used to cover wounds of the distal limb of the dog and cat. Flaps are more suitable for poorly vascularized areas, radiation injuries, areas of motion, and pressure points. Reconstruction should not be limited to slowly healing or nonhealing wounds. Skin flaps and grafts can also be used to cover fresh surgical wounds created by excision of large neoplastic or granulomatous lesions.

Preputial reconstruction in a dog.

A 2-year-old dog was examined after a 22-month history of intermittent drying and chafing of the distal portion of the penis after traumatic loss of the cranial portion of the prepuce and fracture of the os penis at 8 weeks of age. A multiple-staged surgical procedure was performed to reconstruct the prepuce, including free buccal mucosal grafting and transfer of a peripreputial bipedicle subdermal plexus flap. Preputial reconstruction provided mucosal and cutaneous coverage of the penis.