Use of allograft biopsies to assess thymopoiesis after thymus transplantation

Thymus allograft biopsies were performed in athymic infants with complete DiGeorge anomaly after thymus transplantation to assess whether the thymus allograft tissue was able to support thymopoiesis. Forty-four consecutive infants were treated with postnatal cultured thymus allografts. Thirty biopsies and six autopsies evaluating the allograft site were obtained in 33 infants, 23 of whom survive. The allograft was examined by immunohistochemistry for evidence of thymopoiesis. Grafted thymus tissue was found in 25 of 30 biopsies, 23 of which showed thymopoiesis. All 19 survivors with thymopoiesis on biopsy developed naive T cells and T cell function. Autopsies were done in six subjects, three of whom had biopsies. All autopsy samples contained thymus tissue including one for which the biopsy had not contained graft. Of the six autopsies, one had evidence of thymopoiesis. Epithelium without thymopoiesis was seen in two of 25 biopsies in which thymus tissue was detected and in five of six autopsies. Graft rejection was seen in one autopsy. Biopsies were important for showing the following: 1) the damaging effect of pulse steroids on thymopoiesis; 2) the need for adequate immunosuppression of atypical subjects; and 3) the presence of thymopoiesis in the presence of ongoing immunosuppression. In addition, the biopsy could rule out graft rejection in the atypical subjects who had oligoclonal T cells that could cause rejection. In summary, combining biopsy and autopsy data, allogeneic thymus tissues showed thymopoiesis in 24 of 29 (86%) evaluable transplants. The results of these biopsies led to improved care of these complex patients.     

Pulmonary cytology in the posttherapeutic monitoring of patients with bronchogenic carcinoma

A study of 113 patients with histologically confirmed bronchogenic carcinoma and survival for at least 18 months was performed. Posttherapeutic cytologic specimens were submitted for only 52.2% (59/113), and for only 8.5% (5/59) of the individuals was specimen procurement related to clinic visits. Yet, when utilized as a diagnostic procedure for the hospitalized patient, a positive cytodiagnosis was made on 55.6% (15/27) of the patients with recurrent carcinoma, in 61.1% (11/18) with persistent disease and in all 5 patients with a metachronous primary bronchogenic carcinoma. Cytologic diagnoses of cancer compared favorably with concurrent histologic interpretations of biopsies and radiologic observations of chest films.     

Utility of bronchoalveolar lavage in the diagnosis of drug-induced pulmonary toxicity

Two hundred consecutive bronchoalveolar lavages (BALs) performed at Mayo Clinic were retrospectively reviewed. The BAL specimens were evaluated for nuclear enlargement, hyperchromasia and increased cytoplasmic staining density as possible cytologic markers of cytotoxic drug effects. Of the 200 patients whose lavages were examined, 178 had sufficient clinical data for study. Thirty-six BAL specimens had cytologic changes consistent with drug-induced pulmonary toxic effects. Twenty-three of the 36 patients had concurrent or subsequent lung tissue available for evaluation; 10 of the 23 showed histologic evidence of cytotoxic effects. The results indicate that BAL is a safe and useful procedure, not only for the assessment of suspected pulmonary infection in immunocompromised patients, but also for the investigation of drug-induced pulmonary disease.     

Cytologic evaluation of bronchoalveolar lavage specimens in immunosuppressed patients with suspected opportunistic infections

The role of bronchoalveolar lavage cytology in the diagnostic evaluation of immunosuppressed patients with suspected opportunistic pulmonary infections was evaluated by comparing two groups of patients who underwent fiberoptic bronchoscopy. Bronchoalveolar lavage specimens were compared with other available diagnostic techniques, including bronchial washings, bronchial brushings, transbronchial lung biopsies and open lung biopsy. Prior to the initiation of a protocol for bronchoalveolar lavage, a specific etiology for the pulmonary infiltrate using the above combined modalities was identified in 23 of 47 cases, for an overall diagnostic rate of 49%. The combined bronchial washings and brushings (cytologic procedures) identified a specific etiology in 9 of 47 (19%) of the cases. There were ten cases in which a cytologically identifiable organism (Pneumocystis, virus or fungus) was not present in the bronchial washings and brushings and one missed case of malignancy, for a false-negative rate of 23%. With the addition of the lavage technique and better sampling of the distal airways, a specific etiology for the pulmonary infiltrate was identified in 32 of 48 (67%) of the cases. This is comparable to the values of 40% to 65% cited in the literature for diagnosis of infectious disease by open lung biopsy. The lavage cytologic procedure identified a specific etiology in 22 of 48 (46%) of the cases, and the false-negative rate was reduced to 6%. With the excellent sampling of the bronchoalveolar lavage and the improved cytology results, the need for transbronchial or open lung biopsy has been eliminated in immunosuppressed patients with suspected opportunistic pulmonary infections. This allows these patients to be studied on an outpatient basis.     

Diagnosing acute liver graft rejection: experimental application of an implantable telemetric impedance device in native and transplanted porcine livers

BACKGROUND AND AIMS: Diagnosis of acute rejection is a complex and persistent problem in liver transplantation. Focused on the use of proprietary impedance technology a porcine liver model was designed to provide immediate information for differentiation of normal and acute rejecting tissue by an implantable telemetric device. METHODS: Electrical impedance was analyzed by electrodes implanted in vitro and in vivo in the liver of pigs, where impedance is derived from measurements of voltage transients produced in response to programmed current pulses. Consequent electric recordings in porcine livers after transplantation and after mere laparotomy were evaluated in relation to biochemical parameters and histological results of liver biopsies. RESULTS: Acute rejection was correctly predicted in all cases and correctly excluded in the remaining 32 biopsy related impedance recordings (P<0.004). Impedance measurements not only correlated with the diagnosis from liver biopsy specimen (r=0.84, P<0.0001) but also exemplified the severity of histological acute rejection. CONCLUSION: Impedance analysis reveals evident physiologic relation of acute liver graft rejection and electrical organ properties. Electrodes implanted in transplanted porcine livers allow running less invasive monitoring and thus early detection of rejection. The technology may have broad value in providing an immediate diagnosis of acute rejection, reducing unnecessary patient anxiety and eliminating the significant expenses associated with multiple referrals, expensive sample handling and tissue analysis.     

Bronchoalveolar lavage results are independent of season, age, gender and collection site

Background

Clinical interpretation of bronchoalveolar lavage fluid results is dependent on the availability of reference values for healthy individuals. Only a few studies have published such reference values and the applicability of results is restricted by small sample sizes and the limited representativeness of the study population. We aim to investigate the influence of age, gender, collection site and season on bronchoalveolar lavage fluid results and to establish reference values for use in clinical practice.

Methodology/Principal Findings

Bronchoalveolar lavage fluid data from 295 healthy never-smoking volunteers, investigated during 1990–2009, were analyzed retrospectively. 47 volunteers had 2–5 repeat lavages during the course of several years. Fluid recovery, total number of cells, cell concentration, and differential cell counts on cytospin prepared slides were recorded. Reference values, as represented by the 5^th to the 95^th percentile, were 72–96% for macrophages, 2–26% for lymphocytes, 0–4% for neutrophils and 0–1% for eosinophils. Basophils and mast cells were rare. When repeat lavages were performed, there was a relatively large intra-individual variability, mainly for macrophages and lymphocytes. An age dependent decrease of lavage fluid return was present, but there was no age dependent correlation with any of the other BALF parameters. The BALF cell parameters were independent of gender, season and site (lingula vs. middle lobe).

Conclusions/Significance

Our data show that bronchoalveolar lavage fluid cell differential count is independent of age, gender, season and collection site (RML or lingua). It therefore seems acceptable to use the same reference values for all never-smoking individuals.     

Urinary Thromboxane B2 in Cardiac Transplant Patients as a Screening Method of Rejection

Noninvasive methods for regular monitoring of cardiac transplant patients for acute rejection are preferable to the only currently accepted method involving frequent endomyocardial biopsies. Thromboxane A₂ (TXA₂) is synthesized in large amounts by monocytes/macrophages during organ graft rejection. It enhances T-lymphocyte clonal expansion and cytotoxic function as well as upregulating the major histocompatibility class II expression on antigen presenting cells. Experimentally increased urinary excretion of TXA₂ metabolites is associated with cardiac transplant rejection. We therefore compared urinary immunoreactive thromboxane B₂ (i-TXB₂) levels to the rejection score of the endomyocardial biopsies. In addition we graded the degree of activated lymphocytes in peripheral blood. Urinary i-TXB₂ was significantly higher in patients exhibiting medium to severe rejection than in patients without rejection (1236 ± 372 vs. 526 ± 57 pg/mL). The urine i-TXB₂ (704 ± 48 pg/mL) of all patients who participated in this study, whose endomyocardial biopsy indicated rejection, was also significantly higher than in the non-rejecting group. Increased levels of urine i-TXB₂ were associated with increased biopsy scores. Circulating activated lymphocytes was also significantly increased in patients with moderate/severe rejection compared to patients with no rejection (66 ± 11 vs. 39 ± 4 per mm (3)) (p < 0.01). Further, this study shows that urine i-TXB₂ is associated with increased endomyocardial biopsy scores (acute rejection scores) and blood lymphocyte activation. Thus we conclude that urine i-TXB₂ may be of potential value as a diagnostic screening test for helping identify cardiac transplant patients undergoing acute rejection.     

Quantitative detection of HHV-6 and HHV-7 in transbronchial biopsies from lung transplant recipients

The occurrence and significance of HHV-6 and HHV-7 were investigated in pulmonary tissue from lung transplant recipients. Eighty-seven transbronchial biopsies from 30 patients were studied by quantitative real-time PCR; the association with histopathological features was investigated. HHV-6 and HHV-7-DNA were detected in 6.9% and 9.2% transbronchial biopsies, respectively. A significant association between HHV-6 detection on transbronchial biopsies and interstitial pneumonia was found, in contrast to the lack of association between viral detection on bronchoalveolar lavage and any histopathological feature. No association was evidenced in terms of acute and chronic rejection. The finding of HHV-6 and/or HHV-7-DNA positivity in all the cases with ischemia-reperfusion injury suggests a possible role in favouring ?-herpesviruses reactivation, as previously described for HCMV in renal transplantation.     

Bronchoalveolar lavage in the assessment of pulmonary Hodgkin's disease

Abnormal chest radiographs in patients with Hodgkin's disease are occasionally due to pulmonary Hodgkin's disease. The fluids recovered from bronchoalveolar lavages (BALs) from 50 patients prior to autologous bone marrow transplantation for advanced Hodgkin's disease were examined. Abnormal chest roentgenograms were present in 24 patients (48%); 4 (17%) of these had Reed-Sternberg cells or their mononucleated variants in the lavage fluid and an alveolar lymphocytosis averaging 31.4% (normal: 11.5%). The lymphocytes were small and monotonous. Of the 20 patients with abnormal chest roentgenograms but no Reed-Sternberg cells in the lavage fluid, the lymphocyte count was 10.88%, with only 3 patients exceeding 17%. Two patients with normal chest roentgenograms had Reed-Sternberg-like cells in their lavage fluids and averaged 23% lymphocytes in their lavage differential count. Eosinophils averaged 1% or less of the lavage differential and were not predictive of pulmonary Hodgkin's disease. This experience suggests that pulmonary Hodgkin's disease can be diagnosed by BAL. Reed-Sternberg cells and their mononucleated variants can be recognized by their characteristic cytomorphologic features, although care must be taken not to misinterpret reactive binucleated macrophages as neoplastic cells. In patients with Hodgkin's disease, Reed-Sternberg cells should be sought when an alveolar lymphocytosis is present.     

Fine needle aspiration diagnosis of lymphomatoid granulomatosis. A case report.

The diagnosis of lymphomatoid granulomatosis (LG) of the lung depends on obtaining adequate histologic material to demonstrate the characteristic angioinvasive, polymorphous, lymphoid infiltrate and normally requires an open lung biopsy. Fine needle aspiration biopsy (FNAB), if only smeared directly, does not allow an assessment of the lymphoid infiltrate in relation to blood vessels. However, we report a case diagnosed by FNAB in which the specimen was processed by an alternative method that allows cell blocks to be made from all visible particles. Percutaneous FNAB of a nodular pulmonary infiltrate was performed after bronchoscopy and transbronchial needle biopsy failed to yield a diagnosis. The FNAB specimen was placed in 50% alcohol and submitted for processing. The specimen was then filtered through a fine sieve, and all visible tissue was embedded in bacteriologic agar and processed as a standard surgical specimen. The filtrate was processed as standard fluid cytology. The atypical, angioinvasive, lymphoid infiltrate was clearly demonstrated on the cell blocks, and the diagnosis of LG was made. This diagnosis was confirmed by subsequent open lung and skin biopsies.     

Fine needle aspiration of breast lesions in women aged 30 and under

Fine needle aspiration (FNA) biopsies of 1,598 breast masses were performed between 1983 and 1989, and of them, 48 were from women aged 30 and under for whom a cytologic diagnosis was made by FNA and histologic follow-up was available. In 37 (77%) of the cases, both the cytologic and histologic diagnoses were benign. Fibroadenoma (20/37) and fibrocystic changes (14/37) were the most common benign lesions aspirated. Eight (17%) FNAs showed cytologic atypia. Four of these atypical lesions proved to be benign (two fibroadenomas, two fibrocystic changes). Epithelial proliferation in fibroadenomas and fibrocystic changes and cellular stroma in a fibroadenoma mimicking phylloides tumor were the causes of atypia in these biopsies. Four of the eight atypical lesions were shown to be carcinoma at biopsy (three infiltrating duct, one atypical medullary). Low cellularity, epithelial cohesiveness mimicking a fibroadenoma and background lactational changes in a pregnant patient were the causes of the atypical, rather than unequivocally malignant, diagnoses in these cases. In three patients (6%), a diagnosis of carcinoma was made by FNA and confirmed histologically (all were infiltrating duct carcinoma). Although most breast masses in women aged 30 and under are benign, cytologic atypia in a breast fine needle aspirate in this age group warrants a surgical biopsy. Clinical follow-up alone may be appropriate for young women with clinically nonsuspicious breast masses without cytologic atypia.     

Kidney graft rejection studies with labeled platelets and lymphocytes

The usefulness of In-111-labelled platelets and lymphocyte scintigraphy in acute kidney graft rejection is evaluated.One hundred fifty-five patients (36 treated with cyclosporine A) were studied with labelled platelets and 27 with labelled lymphocytes.Blood cells were labelled with 100–150 uCi of In-111-oxine and reinjected. Subsequently patients were scanned once daily from 2 hours post-reinjection up to a week. The graft / contralateral area activity ratio was calculated in all scans (Index I).Four groups of patients were established: Functioning grafts (FG); post-operative acute renal failure (p-ARF);acute rejection (AR) and nephrotoxicity (NTX), the last one only in patients under cyclosporine therapy.Results with labelled platelets showed similar index I mean values in FG, p-ARF and NTX patients I = 1.1 ± 0.1 and a significant increase (p <O.001), in acutely rejecting grafts I = 1.9 ± 0.4.Evolving controls showed a decrease of graft activity parallel to rejection resolution while the activity maintains or increases in patients with less or no response to treatment.Overall sensitivity was 97.2 %, specificity 90.2 % and accuracy 92.8 %.Results with labelled lymphocytes were similar to those with platelets. They showed a significant (p 0.001) difference of activity index between rejecting (I = 1.86 ± 0.3) and non rejecting grafts (I = 1.05 ± 0.1) Decrease of graft activity was only seen in patients with good response to treatment.It is concluded that In-111-labelled platelets scintigraphy is nowadys the method of choice for acute kidney graft rejection diagnosis, especially in patients under cyclosporine immunosuppression.     

Cell profiles in serial bronchoalveolar lavage after human heart-lung transplantation.

The aim of this study was to investigate serial changes in bronchoalveolar lavage (BAL) cell profiles after human heart-lung transplantation and to assess the clinical value of BAL cytology in the differential diagnosis of complications in the transplanted lung. BAL was performed serially on 23 occasions on four patients. Elevated counts of neutrophils (4-48%) were observed in all preparations, with peak values in the early postoperative phase, in bacterial infections and in cytomegalovirus pneumonitis. In the last condition, BAL cytology also showed relative lymphocytosis (less than or equal to 50%) with high proportions (less than or equal to 50%) of HLA DR-positive T lymphocytes. No characteristic light microscopic pattern was observed in acute pulmonary rejection. However, scanning electron microscopy revealed elevated counts (greater than 5%) of "villous" macrophages in BAL obtained during or shortly after episodes of rejection. BAL cytology may be helpful in differentiating viral and bacterial infections, while scanning electron microscopy seems to be more suitable to the diagnosis of acute pulmonary rejection.     

Hemangiopericytoma in a male breast. Report of a case with cytologic, histologic and immunochemical studies

A hemangiopericytoma in a male breast was studied by fine needle aspiration (FNA) biopsy. The FNA smears contained tissue clumps showing knob-like formations of atypical cells, spindle-shaped cells and fragments of capillaries lined by normal endothelial cells. Immunocytochemical study showed a positive reaction for vimentin, but a negative reaction for desmin and keratin. Staining for Factor VIII was positive only in the capillaries and endothelial cells. The cytodiagnosis was "mesenchymal tumor." Histopathologic study of the mastectomy specimen made the final diagnosis of hemangiopericytoma. While FNA cytology and immunocytochemistry cannot make a definitive diagnosis of this rare vascular tumor, they can be decisive in planning the surgical treatment, as in the present case.     

Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia

The time course of lung injury and recovery from a sublethal exposure to 100% O2 was investigated in adult rabbits. Animals were exposed to 100% O2 for 64 h and then returned to room air for varying periods of time up to 200 h. By the end of the exposure period, the alveolar permeability to solute increased significantly, and biochemical analyses of bronchoalveolar lavages showed a 30% decline in phospholipid content and a threefold increase in protein levels. However, other parameters such as wet-to-dry lung weight ratios, blood gas values, and pressure-volume mechanics were not significantly different from control levels after 64 h of hyperoxia. Twenty-four hours postexposure, alveolar phospholipid levels had declined even further (51% of control), and mean protein levels in lavage increased to eight times control values. These lavages exhibited severely impaired dynamic surface activity at 37 degrees C and 100% humidity in an oscillating bubble apparatus. In addition, total lung capacity, lung compliance, and arterial O2 partial pressure declined greatly at this time. Between 12 and 48 h postexposure, animal mortality was 35%; the remaining animals survived, and physiological parameters returned to normal by 200 h postexposure. Bronchoalveolar lavages from the recovered animals contained protein levels equal to those of controls and phospholipid levels approximately twice those in control lavages. Lavage surface activity also returned to normal by the 200 h postexposure time point.     

Colonic cytology. A retrospective study with histopathologic correlation

Three hundred sixty cytologic specimens obtained by colonoscopic brushing from 336 patients were compared with biopsy specimens simultaneously obtained for histologic examination. Of the cytologic specimens, 160 (44%) were positive for malignant cells, 37 (10%) contained suspicious cells, 54 (15%) had atypical glandular cells, 107 were cytologically negative, and 2 were considered unsatisfactory. Eight-four percent of the patients with cytologically positive smears and 54% of those with suspicious smears had malignant neoplasms in the simultaneously obtained tissue biopsies. Of the patients with follow-up, all with cytologically positive findings and nine with suspicious findings on the initial cytologic examination and simultaneous negative tissue biopsies, were subsequently found to have carcinoma of the colon. Cytology proved to have a sensitivity of 0.73 and a specificity of 1.00 while tissue biopsy showed a sensitivity of 0.81 and a specificity of 1.00. By combining the two methods, the sensitivity increased to 0.92. It is concluded that cytologic examination of colonic brushings is a highly accurate and reliable technique for the detection of malignant neoplasms of the colon and can preempt the use of biopsy forceps.     

Cytohistologic correlation of urothelial lesions secondary to photodynamic therapy

Qualitative cytologic evaluations of urinary bladder washings were performed on a selected population following photodynamic therapy for recurrent transitional cell carcinoma of the bladder. The seven patients were monitored trimonthly by cystoscopy, multiple biopsies and cytopreparations. Cancers reappeared in two of the five patients who initially responded to therapy. In the remaining two patients, the recurrent neoplasms were therapeutically refractory. Cytology detected recurrent cancer prior to biopsy confirmation and/or cytoscopic identification. Exfoliative cytology was correlated with the histopathology of the concurrent biopsies; a possible source for a false-positive cytodiagnosis was the cellular atypia of reepithelialized bladder mucosa. Dysplasia was not identified cytologically or histologically.     

Bronchoalveolar lavage in liver transplant patients

Because immunosuppression is required to control rejection, liver allograft recipients are susceptible to a variety of opportunistic pathogens. A total of 191 bronchoalveolar lavage (BAL) specimens from 89 patients (53 adults and 36 children) who underwent orthotopic liver transplantation was reviewed. One case each of cytomegalovirus (CMV), staphylococcal and Enterobacter pneumonia was diagnosed with the aid of pretransplant BAL. The pretransplant BAL in 62 patients showed rare yeasts in 24.2%; these probably represent oropharyngeal contaminants since the patients involved had no symptoms of Candida pneumonia. Among 54 patients who developed respiratory symptoms and underwent posttransplant BAL, 23 (42.6%) were infected with opportunistic pathogens, including Pneumocystis carinii (22.2%), CMV (22.2%) and herpes simplex virus (HSV) (7.4%). Frequently, infection with multiple organisms was present. Adults constituted 100% of the HSV-infected group, 69.2% of the CMV-infected group and 16.6% of the group infected with P carinii. The diagnosis of these infections was aided by a combination of cytology, microbial culture and in situ hybridization techniques. Although BAL permitted the diagnosis and treatment of opportunistic infections, high mortality (62.5%) occurred with CMV and HSV pneumonia. Further studies into methods that permit earlier diagnoses of these infections are necessary.     

Generalized immune response to Pneumocystis carinii infection in the lung.

We studied inflammatory cells retrieved by bronchoalveolar lavage (BAL) from immunocompromised patients with or without Pneumocystis carinii pneumonia (PCP). Twenty-four patients with PCP, and 20 patients without PCP underwent lavages of both an uninvolved lobe and the lobe involved in pulmonary infection. Patients without P. carinii, had a significant increase (p less than 0.02) in the percentages of neutrophils (22 +/- 7.1%, mean +/- SEM) and lymphocytes (16 +/- 3.8%) in the involved lobe compared to those in the uninvolved area (neutrophils: 9 +/- 4.8%; lymphocytes: 10 +/- 2.4%). Patients with PCP, had no differences between the % neutrophils or % lymphocytes in the involved vs. uninvolved lobes. Patients with PCP had more (p less than 0.01) P. carinii in the upper lobe (23 +/- 4.6 P. carinii clusters/500 cells) than the middle lobe (11 +/- 3.6). In PCP, despite regional infections, there was a diffuse inflammatory response.     

Fine needle aspiration cytologic findings in four cases of pilomatrixoma (calcifying epithelioma of Malherbe).

Four cases of pilomatrixoma studied by fine needle aspiration (FNA) biopsy prior to resection and histologic diagnosis were reviewed to identify the cytologic features useful in making the FNA cytodiagnosis. Cytopathology had initially correctly diagnosed two cases while misdiagnosing one as a carcinoma and one as a suspected carcinoma. The aspirates from all cases contained either clustered or isolated basaloid cells, with variably sized nuclei and prominent nucleoli. "Ghost cells" were also present in most smears stained with the Giemsa stain and could thus be very helpful for making the FNA diagnosis of pilomatrixoma. The occurrence of either foreign-body giant cells or calcium deposits in the aspirates could also contribute to suggesting the correct diagnosis of pilomatrixoma in some cases.