Viral infections post-renal transplant (Tx) impact on outcome. Increased rejection rates and decreased renal function secondary to acute CMV, EBV and HHV-6 infections are well described. However, the clinical significance of a mere presence of these viruses on kidney tissue biopsy remains questionable. Thirty-six kidney biopsies obtained from 17 renal transplants (five females) and two combined liver-kidney recipients (one female) were retrospectively evaluated. Age at Tx ranged from 1.7 to 17.2 yr (median = 7.4). Biopsies were performed as protocol biopsies or when renal function deteriorated, between 6 weeks and 11 yr post-Tx (median = 1.2 yr). Immunosuppression included steroids and combination of tacrolimus/cyclosporin, mycophenolate mofetil/azathioprin and induction therapy. Fourteen patients received antiviral prophylaxis (ganciclovir/valganciclovir/acyclovir). Renal tissue was classified according to Banff '97 criteria. Tissue CMV, EBV, HHV-6 and HHV-7 was analyzed by PCR. We used an estimation of GFR from average plasma Cystatin C (CysC) and slopes of 1/CysC to assess renal function. The 16/36 biopsies were positive for one virus; 5/36 biopsies were positive for two viruses. In the infected group, Banff '97 scores for interstitial fibrosis (ci) and tubular degeneration/atrophy (ct) were significantly higher (p < 0.03 vs. the non-infected group for both). The slope of 1/CysC, or the proportion of patients on antiviral prophylaxis, did not differ significantly between both groups. In conclusion, a significant number of kidney biopsies showed PCR positivity for CMV, EBV, HHV-6 and HHV-7. This was associated with a significantly higher Banff score for ci and ct; while renal function was not affected. Further controlled studies are required.

Background: Infections caused by herpes virus, in particular, Epstein-Barr virus (EBV), remain a major challenge in solid organ transplantation. Little is known about the significance of tissue EBV load. Methods: Twenty-three tissue biopsy specimens (19 kidney, 3 gastrointestinal, and 1 tonsil specimen) and 2 bronchoalveolar lavage specimens from 14 pediatric transplant recipients (10 kidney, 3 liver, 1 combined transplant) were subject to tissue EBV polymerase chain reaction (PCR) semiquantitative analysis and enzyme-linked immunosorbent assay (ELISA) methods. Results of biopsies were correlated with clinical data. Results: Five of 14 patients had clinically diagnosed EBV disease: 2 patients presented with a septic picture with multiorgan failure and pneumonitis; 1 patient had mononucleosis; 1 patient had an increase in serum creatinine level, lymphadenopathy, and chronic fatigue; and 1 patient had EBV nephritis. These 5 patients underwent 12 biopsies at the time of clinically active infection; 8 biopsies had positive results (up to 111 copies/10 μL of extracted DNA). Conversely, 1 of the remaining 13 tissue biopsy specimens from asymptomatic patients had positive results on ELISA, but undetectable viral load, whereas 8 patients had a positive EBV immunoglobulin G titer with historic evidence of EBV replication in the blood. No patient without evidence of EBV had positive EBV tissue PCR results. Conclusion: Increased EBV load was found in more than 50% of patients, pointing to a previously underrecognized importance of EBV detection in tissues from transplant recipients. The presence of EBV in tissue correlated with the presence of viremia, whereas tissue PCR had 100% specificity. EBV load should be included in biopsy evaluation. Am J Kidney Dis 41:212-219. © 2003 by the National Kidney Foundation, Inc.

Chronic sinusitis, otitis media with effusion, and upper respiratory tract infections are commonly found in patients with Down syndrome. These diseases are generally felt to be secondary to depressed immune function and altered craniofacial dimensions. Recently, a cilia ultrastructure abnormality was found in a child with Down syndrome. This study is the first to be carried out to determine if cilia ultrastructure abnormalities are prevalent in the population with Down syndrome. Four of 10 patients had documented cilia abnormalities, but these were present in the background of normal cilia, suggesting that they were the result rather than the cause of chronic sinusitis. Similarly, nasal epithelium metaplasia was detected in 50% of the patients. Chronic sinusitis, otitis media with effusion, and recurrent upper respiratory tract infections in children with Down syndrome cannot generally be attributed to primary cilia ultrastructure abnormalities.

Management of meningo-encephalitis often involves the need for antibiotic and antiviral treatment. We report a retrospective analysis over a 6-month period of 17 patients (age range 1–14 years) who were treated with combination therapy of ceftriaxone and acyclovir. Mean acyclovir and ceftriaxone doses were 1,222±304 and 2,315±509 mg/m2 per day, respectively. Three patients developed acute renal failure with a peak creatinine of up to 865% above baseline, occurring 2–3 days after starting combination therapy. Patients revealed a tubular proteinuria pattern. Renal biopsy of 1 patient showed a tubulotoxic picture but no evidence of crystals. In 12 of 17 patients (70%) there was a significant increase in serum creatinine. This was significantly greater than literature reports of 16% with acyclovir monotherapy. The degree of renal impairment in our patients correlated significantly with the acyclovir dose, while no correlation was found with the ceftriaxone dose. We conclude that the addition of a second nephrotoxic drug aggravated the extent of renal injury in our patients. The mechanism is tubulotoxicity. Caution should be exercised when using this potentially nephrotoxic cocktail, with clear criteria established for the initiation of combination therapy and close monitoring of serum creatinine.

Objective: Adequate biopsy specimens that clearly demonstrate cilia, and therefore enable the determination of the presence or absence of primary cilia dyskinesia, may be difficult to obtain. This study is an attempt to identify the optimal sampling technique to best examine respiratory tract cilia.Design: A prospective comparison of the four sampling techniques was carried out: nasal brushing, nasal biopsy, bronchial brushing, and tracheal biopsy.Setting: Tertiary care pediatric hospital: Children's Hospital of Eastern Ontario.Methods: Ten consecutive patients booked for bronchoscopy and tracheal biopsy underwent all four procedures. Specimens were examined under light microscopy for an assessment of quality.Results: The nasal brushing and tracheal biopsy specimens provide superior quality (p = .22); however, nasal brushing is more cost efficient. Nasal biopsy samples frequently are metaplastic and therefore are inferior to nasal brushing samples (p = .02).Conclusion: With equal efficiency demonstrated, the reduction in potential morbidity and health care costs suggests nasal brushings to be the optimal initial investigation for primary ciliary dyskinesia.

A revised silver impregnation method has been developed to show nerve fibers, axons, neurons, and pathological changes such as neurofibrillary tangles and plaques of Alzheimer's disease. This method used a sensitization step in 0.1% silver nitrate with a small quantity of citric acid included. This step was performed in a 65 degrees C waterbath to enhance the demonstration of the above entities. This was followed by a physical developer consisting of hydroquinone, gum mastic and silver nitrate. The entire procedure takes under 30 min to perform and gives similar results to the Bielschowsky method for less than one-hundredth of the cost.

Ewing sarcoma and other peripheral primitive neuroectodermal tumors (pPNETs) display limited neural differentiation and are thought to have a neural crest origin. Greater than 95% of these tumors share common t(11;22) (q24;q12) or t(21;22) (q22;q12) chromosomal translocations leading to EWS/FLII or EWS/ERG gene fusions, respectively. The resulting chimeric oncoproteins seem to function as aberrant transcription factors. However, whether these molecules contribute to the limited neural differentiation observed in pPNETs or actually inhibit differentiation remains unclear. We report a Ewing sarcoma case from the forearm of a 10-year-old girl which expressed EWS/FLI1 fusion transcripts. The tumor was treated with surgery, chemotherapy, and local radiation, but residual tumor was detected within a year as a well-differentiated peripheral neural tumor lacking detectable EWS/FLI1 expression. Further studies suggested that the primary and residual tumors were clonally related. This association between apparent therapy-induced differentiation in Ewing sarcoma and absence of detectable fusion transcripts in the residual tumor provides presumptive evidence that EWS/FLI1 expression may inhibit differentiation in tumour cells.

The periosteum in children and especially infants has significant osteogenic potential, To determine the efficacy of periosteal flaps to assist in improving acetabular coverage in children with acetabular dysplasia, a series of experiments were designed using young rabbits. Three groups of five rabbits each had periosteal flaps fashioned and brought down from the anterolateral aspect of the innominate bone superior to the acetabulum and sutured to the capsule of the hip, The study was designed to examine the effects of the periosteal cambium layer in the formation of new bone to augment the acetabulum and to determine the effects of a periosteal flap plus cancellous bone graft. A control group of five rabbits underwent a sham operation of an open arthrotomy of the hip. Radiographic and histologic examination at 12 weeks revealed augmentation of the acetabulum with periosteal flaps that resulted in an average improvement of the acetabular index of 3.5 degrees and 6.6 degrees, without and with bone graft, respectively. New bone formation from the rim of the acetabulum averaged 3.9 mm with periosteal flaps alone and 4.6 mm with bone graft added. Periosteal augmentation of the acetabulum in conjunction with established procedures for augmenting acetabular coverage would appear to be a useful procedure for improving coverage of the femoral head in children with acetabular dysplasia.

A modified Gomori technique for the assessment of frozen skeletal muscle and all paraffin embedded sections is presented. The modified Gomori solution consists of 0.015% fast green FCF, 0.12% chromotrope 2R, 0.65% phosphotungstic acid and 1.0% acetic acid in 100 ml distilled water. Frozen skeletal muscle staining of nuclei is done with celestine blue followed by hematoxylin; paraffin sections are stained with a modified Verhoeff hematoxylin. This method allows demonstration of nemaline rods, cytoplasmic inclusion bodies and other abnormalities in frozen skeletal muscle and takes about 12 minutes to perform. In paraffin sections the staining results are similar to the Masson trichrome method with less manipulation, cleaner background and staining time is about 17 minutes. (The J Histotechnol 19:329–333, 1996)

Parosteal fasciitis is a pseudosarcomatous benign lesion of fibrous tissue that has often been confused with malignancy. The purpose of this study was to document the clinical and pathological features of four children with this condition and compare the results with the world literature. The ages of the patients whose cases were reviewed ranged from 9 to 15 years. There were three girls and one boy. All the lesions occurred in the lower extremities, and histologically were characterized by a fibroblastic proliferation with inflammatory cells, diffuse capillary network, and varying quantities of metaplastic bone. All patients were treated by local excision of the lesion. One child had a recurrence of the lesion requiring another surgical removal. A set of clinicopathological criteria has been formulated for the diagnosis of parosteal fasciitis in children. Parosteal fasciitis is uncommon in children but manifests the same clinicopathological features as the adult lesion.

Three connective tissue methods are presented: modifications of Masson trichrome, Movat pentachrome and a fibrin method. A modified Verhoeff hematoxylin preheated and applied in the 60-degrees-C paraffin oven was used for all methods. The Movat pentachrome modification additionally included staining with alcian blue before application of Verhoeff hematoxylin, and the fibrin method was stained with lissamine fast yellow before application of the working red stain. All sections were stained with a working dilution of Biebrich scarlet and acid fuchsin, rinsed, and differentiated with phosphotungstic acid in a 60-degrees-C paraffin oven. Demonstration of collagen in the modifications of Masson and fibrin was done with either light green or aniline blue; saffron was used in the Movat pentachrome.All 3 techniques were improved in quality and precision with the aid of heat. Although fibrin was demonstrated in all techniques, minute quantities were better seen in the fibrin stain because the red cells were stained in a different color. These modified stains demonstrated several entities in a single slide preparation in about 20 min.

Two improved silver methods for fungi and Pneumocystis carinii that use a modified oxidizing agent are presented. The sections were first oxidized with a solution consisting of chromic, sulfuric, and glacial acetic acids for 1 min in the 60-degrees-C paraffin oven, thoroughly washed in running tapwater, and treated with sodium metabisulfite before they were impregnated in modified silver methenamine or ammoniacal silver nitrate solution. The sections were treated with sodium thiosulfate before and after gold chloride. They were counter-stained with light green.This modified oxidation prevented staining of elastic and collagen fibers. The entire procedure was performed in approximately 40 min or less, depending on the temperature used in the silver impregnation. All fungi tested and P. carinii were stained in the same amount of time in both silver nitrate and gold chloride.

A modified periodic acid-Schiff method is presented. The sections were oxidized with 1.0% periodic acid, then stained with a modified Schiff solution. The modified solution contained pararosaniline hydrochloride in a concentration of about one-tenth of that normally used for basic fuchsin. The concentration of sodium meta-bisulphite was doubled, and sodium hydrosulfite was included to decolorize effectively the pararosaniline hydrochloride. Concentrated hydrochloric acid was added. The solution was treated with charcoal and filtered. This reagent was stored in special dropping bottles to prevent loss of the sulfurous acid content and diluted just before use with an equal volume of water to maximize its stability. A modified baking tin was used in the staining of up to 20 slides at a time in a paraffin oven at 60-degrees-C. The stained sections were developed in warm water and counter-stained in modified Mayer hematoxylin. Microwave and room temperature methods were effective, as well.This modified Schiff can be combined with other techniques. No sulfite rinses are required, and the background staining is light with PAS staining entities clearly demarcated.

A simplified version of the modified Verhoeff's elastic-van Gieson stain is presented. This method uses a timed differentiation of the elastic tissue stain coupled with van Gieson counterstain. The van Gieson counterstain is balanced so that the picric acid completes the differentiation without loss of staining of the finer elastic fibrils. The entire procedure takes about 20 min to perform.

Brown, Martha Ph.D.; Rossier, Edmond M.D.; Carpenter, Blair M.D.; Anand, C. M. M.D. Author Information

A simple, reliable, consistent and economical nonbuffered acid acid method for melanin is presented. After a short presensitization in silver, the sections are treated in gum mastic and then immersed in a physical developer consisting of hydroquinone, gum mastic, and a very low concentration of silver nitrate. The sections are placed in thiosulfate and counterstained with nuclear fast red. The entire procedure takes under 40 minutes to perform.

A modified alizarin red S and a silver nitrate method for the demonstration of calcium are described. In the alizarin red S method, isopropyl alcohol is used to dehydrate the sections, thus minimizing the solubility of the calcium lake. The sensitive silver method, based on sensitization and physical development, demonstrates minute traces of calcium deposits mainly in the form of phosphates and is comparable to the alizarin red S. (J Histotechnol 12:225, 1989).Keywords: Calcium depositsdye-metal complexmetal substitution techniquesilver impregnation