2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: medical indications.

The clinical indications for diagnostic flow cytometry studies are an evolving consensus, as the knowledge of antigenic definition of hematolymphoid malignancies and the prognostic significance of antigen expression evolves. Additionally the standard of care is not routinely communicated to practicing clinicians and diagnostic services, especially as may relate to new technologies. Accordingly there is often uncertainty on the part of clinicians, payers of medical services, diagnostic physicians and scientists as to the appropriate use of diagnostic flow cytometry. In an attempt to communicate contemporary diagnostic utility of immunophenotypic flow cytometry in the diagnosis and follow-up of patients with hematolymphoid malignancies, the Clinical Cytometry Society organized a two day meeting of international experts in this area to reach a consensus as to this diagnostic tool. This report summarizes the appropriate use of diagnostic flow cytometry as determined by unanimous approval of these experienced practitioners.

Radiotherapy in the management of orbital lymphoma.

PURPOSE: This retrospective study reviews the treatment technique, disease outcome, and complications of radiotherapy used in the management of lymphoma involving the orbits. PATIENTS & METHODS: Thirty-eight patients were treated between May 1969 and January 1995, with a median follow-up of 8.3 years. All patients had biopsy-proven orbital lymphoma. Twenty patients who had limited disease were treated with curative intent, and 18 patients who had known systemic disease were treated with palliative intent. Of the 20 patients treated with curative intent, 14 had low-grade and 6 had intermediate- or high-grade disease. None received chemotherapy. Most patients received treatment with 250 kVP or 60Co radiation, using either an en face anterior field or wedged anterior and lateral fields. Median treatment dose was 25 Gy. Lens shielding was performed if possible. For patients treated for cure, cause-specific survival and freedom from distant relapse were calculated using the Kaplan-Meier method. RESULTS: Control of disease in the orbit was achieved in all but 1 patient, who developed an out-of-field recurrence after irradiation of a lacrimal tumor and was salvaged with further radiotherapy. In the patients treated curatively, the 5-year rate of actuarial freedom from distant relapse was 61% for those with low-grade and 33% for those with intermediate/high-grade disease (p = 0.08). Cause-specific survival at 5 years was 89% for patients with low-grade and 33% for those with intermediate/high-grade disease (p = 0.005). Two patients with low-grade disease had contralateral orbital failures; both were salvaged with further irradiation. Acute toxicity was minimal. Cataracts developed in 7 of 21 patients treated without lens shielding and 0 of 17 patients treated with lens shielding. No patient developed significant late lacrimal toxicity. CONCLUSION: Radiotherapy is a safe and effective local treatment in the management of orbital lymphoma.

Fine-needle aspiration of a presacral myelolipoma: a case presentation with flow cytometry and immunohistochemical studies.

Myelolipomas are benign soft-tissue tumors predominantly found in the adrenal gland. Extra-adrenal myelolipomas are rare, with about 30 previous cases reported. Approximately half of the reported cases were located in the presacral region. These were usually found at autopsy or during the workup of vague abdominal symptoms. Histologically, these lesions show bone marrow elements, with adipose tissue and scattered lymphoid aggregates. Radiologically-guided fine-needle aspiration (FNA) is helpful in establishing the diagnosis, thus obviating resection in some patients. We report on a case of an incidental presacral myelolipoma that underwent examination by computer tomography, magnetic resonance imaging, FNA, and immunohistochemical staining. This lesion was also analyzed by flow cytometry. To our knowledge, the use of the latter technique in the characterization of such tumors has not been previously reported.

Systemic infection of mice by wild-type but not Spv- Salmonella typhimurium is enhanced by neutralization of gamma interferon and tumor necrosis factor alpha.

The spv genes of the virulence plasmid of Salmonella typhimurium and other nontyphoidal serovars of S. enterica are involved in systemic infection by increasing the replication rate of the bacteria in host tissues beyond the intestines. We considered the possibility that the Spv virulence function is to evade suppression by the host response to infection. To examine this possibility, gamma interferon (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) were neutralized in BALB/c mice by intraperitoneal administration of monoclonal antibodies. Neutralization of IFN-gamma and/or TNF-alpha resulted in increased splenic infection with wild-type salmonellae after oral inoculation; however, Spv- salmonellae were defective at increasing splenic infection in cytokine-depleted mice. The use of a temperature-sensitive marker plasmid, pHSG422, indicated that neutralization of IFN-gamma caused less killing of wild-type S. typhimurium, while neutralization of TNF-alpha resulted in an increased in vivo replication rate for wild-type salmonellae. These results demonstrate that the Spv virulence function is not to evade suppression of bacterial infection normally mediated by IFN-gamma or TNF-alpha.

Fluorescence in situ hybridization assessment of the telomeric regions of jumping translocations in a case of aggressive B-cell non-Hodgkin lymphoma.

We report a jumping translocation involving a donor chromosome 1 long arm in a case of aggressive B-cell non-Hodgkin lymphoma (NHL). Conventional cytogenetic banding studies demonstrated a breakpoint distal to the heterochromatic region of the donor 1q chromosome. Characterization by fluorescence in situ hybridization (FISH) of the jumping translocation demonstrated an apparent telomeric sequence loss of the recipient chromosomes. Additional cytogenetic aberrations, including the t(18;22) translocation associated with non-Hodgkin lymphoma, were also observed in this case. Cytogenetically similar cases of jumping translocations reported in the literature have implicated a preferential involvement of the donor chromosomes' heterochromatic regions and the telomeric regions of the recipient chromosomes. Jumping translocations are still considered rare and their appearance is associated with a poor prognosis. The presence of these specific findings for this case are discussed and compared with those previously reported in other hematologic disorders.

Rapid polymerase chain reaction-based detection of the causative agent of cat scratch disease (Bartonella henselae) in formalin-fixed, paraffin-embedded samples.

Bartonella (formerly Rochalimaea) henselae (Bh) plays a central role in cat scratch disease. A polymerase chain reaction (PCR)-based assay that can detect Bh DNA in formalin-fixed, paraffin-embedded (FF-PE) samples would have utility in the evaluation of processed lymph nodes suggestive of this disorder. Fresh or FF-PE cultures of Bh and related species were analyzed. Thirteen lymph nodes (12 FF-PE and one fresh cell suspension) with necrotizing suppurative granulomatous inflammation and seven FF-PE negative control lymph nodes were also evaluated. PCR was performed using a novel, hemi-nested protocol. Amplified products were analyzed by gel electrophoresis. The fresh and FF-PE Bh cultures showed a specific PCR product with an analytical sensitivity of 0.5 pg bacterial DNA. Seven (54%) of 13 clinical lymph node samples with morphological features suggestive of cat scratch disease also had detectable Bh DNA, whereas none of the seven negative control lymph nodes yielded positive results. We have designed a rapid and sensitive PCR test that can reliably detect Bh DNA in fresh and FF-PE samples. Our findings indicate that this assay has clinical utility in the diagnosis of cat scratch disease.

Mantle cell lymphoma. Rapid polymerase chain reaction-based genotyping of a morphologically heterogeneous entity.

BACKGROUND: Mantle cell lymphoma is heterogeneous at the morphologic level. Since this B-cell lymphoma may be confused with other entities, ancillary molecular testing may be necessary for definitive diagnosis. A polymerase chain reaction-based method, which is less complicated and more rapid than that generally available for the detection of immunoglobulin heavy chain (IgH) and bcl-1 gene rearrangements, would be helpful in this process. METHODS: Thirty-one mantle cell lymphoma samples (frozen or ethanol-preserved) from 29 patients were studied with two separate polymerase chain reaction assays using an air thermocycler and a low-volume, capillary-tube format for rapid DNA amplification. The reverse primer, JH, was common to both assays. The forward primers were directed to the IgH framework III variable region (VH-FRIII) and the bcl-1 gene major translocation cluster. Agarose gels were used to evaluate amplicon. Additional product verification was also performed. RESULTS: Immunoglobulin heavy chain and major translocation cluster bcl-1 gene rearrangements were detected in all 29 (100%) and in 12 (41%) of 29 mantle cell lymphoma samples, respectively. Each VH-FRIII/JH assay required 26 minutes to complete, whereas the major translocation cluster bcl-1/JH reaction required only 21 minutes. The seemingly low yield of bcl-1 gene rearrangements is not unexpected since this assay only detects major translocation cluster breakpoints. CONCLUSIONS: Presented is an extremely rapid, nonisotopic polymerase chain reaction-based method that detects IgH and major translocation cluster bcl-1 gene rearrangements in mantle cell lymphoma. Each polymerase chain reaction amplification was complete in 26 minutes or less, required only a 10-microL reaction volume, and exhibited adequate and specific product yield. This approach permits superior turnaround time and is thus advantageous in the clinical setting.

Optimal primer selection for clonality assessment by polymerase chain reaction analysis. III. Intermediate and high-grade B-cell neoplasms.

Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JH. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) (P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F= 85% [P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment-Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms.

The cell surface antigen and DNA content distribution of lymph nodes with reactive hyperplasia.

Few studies have comprehensively examined the expression and distribution of cell surface antigens and DNA content in non-neoplastic, reactive lymph nodes. We have performed a flow cytometric immunophenotypic and DNA content analysis of 64 lymph nodes from 62 patients (23 male; 39 female; ages 21 months to 80 years) with typical reactive lymphoid hyperplasia as assessed by histology. CD45, a pan-leukocyte marker, was detected on virtually all cells (99 +/- 4%). All pan-T-cell-associated antigens studied were expressed within a narrow range of average values (54 - 64%), and no loss of individual T-cell antigens was observed in any case. Intercase variation of fluorescence intensity for each antigen was minimal. The mean CD4:CD8 ratio was 3.9 +/- 2.6, with only one case (HIV+) showing a marked CD4:CD8 inversion (0.4). The number of cells coexpressing CD4 and CD8 was very low (3 +/- 3%), consistent with the mature phenotypic nature of the majority of T lymphocytes. B-cells, as defined by CD19 and CD20 antibodies, accounted for 36 +/- 16% and 43 +/- 18% of cells analyzed, respectively, and, like the pan-T-cell antigens, displayed minimal intercase variability in antigen expression. Cells coexpressing CD20 and CD5 were noted, although their frequency could not be calculated accurately. IgD- and IgM-bearing cells were both generally well-defined populations: 24 +/- 10% and 31 +/- 14%, respectively. On the other hand, IgG- and IgA-bearing cells displayed broad fluorescence intensity, precluding an exact calculation of their frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

A knowledge-based system for the interpretation of flow cytometry data in leukemias and lymphomas.

Flow cytometry immunophenotyping and analysis of DNA ploidy and proliferative activity have become extremely helpful techniques for diagnosing and subclassifying hematopoietic cell populations in a modern, full-service hematopathology laboratory. The number of physicians with special training in the interpretation of these studies is limited. A knowledge-based computer system has been designed to aid in the interpretation of immunophenotyping and DNA flow cytometry results in hematopoietic disease. The system, known as "Professor Fidelio," is a heuristic classification system that reasons on the basis of defined diagnostic patterns. In this study, Fidelio was tested as a stand-alone system on 366 specimens from two large tertiary medical centers. Fidelio's interpretation was considered to be appropriate in all cases. In 300 of 366 (82%) specimens, the system's interpretation agreed with the diagnosis of record. Many of the disagreements could be traced to errors in the recording of the original diagnosis and minor differences in diagnostic criteria between Fidelio's knowledge base and the criteria in use at the medical centers. When used in a stand-alone mode, Fidelio's interpretation was less specific than the diagnosis of record in certain lymphoproliferative disorders that require morphologic information for subclassification. Professor Fidelio is one module in a workstation for the diagnostic hematology laboratory. This workstation is designed for interpretive reporting, education, and database functions for clinical research. Clinical and morphologic information are shared between Fidelio and the other modules for peripheral blood analysis, bone marrow morphology, and lymph node interpretation by means of a relational database. The system will be useful in hospitals that lack individuals specially trained in flow cytometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of perfusion of oral rehydration solutions containing glucose polymers from corn on disaccharidases and mucosal morphology in rat small intestines.

This study aims to determine the effect of glucose and glucose polymers (GP) from corn in oral rehydration solutions (ORS) on disaccharidases and morphometric measurements in small intestinal mucosa of rats. ORS containing standard composition of salts as in WHO ORS and 2, 5, or 10 per cent glucose or GP [initial glucose polymers, long chain (> 9 molecules) and short chain (2-9 molecules) glucose polymers] from corn were infused into the duodenum of 72 Sprague-Dawley rats (250-350 g). Six rats were sham operated as controls. The levels of lactase, sucrase, maltase, palatinase, and glucoamylase enzymes were higher in rats infused with ORS-containing glucose or GP than control rats. Villus height, villus width, and crypt height in corresponding segments of duodenum, jejunum, and ileum were not significantly different between rats perfused with ORS containing glucose polymers from corn and those with ORS containing glucose. ORS containing GP from corn have no adverse effects on small intestinal enzymes and morphometric measurements.

Sarcomatoid collecting duct carcinoma: a clinicopathologic and immunohistochemical study of five cases.

Sarcomatoid renal cell carcinoma is a well-known entity, but sarcomatoid collecting duct carcinoma has not been reported. We recently encountered five cases. The patients were men whose ages ranged from 59 to 82 years (mean age, 68 years). All presented with gross hematuria and three had abdominal fullness. Tumor size ranged from 6 to 9 cm in greatest dimension. The Fuhrman's nuclear grade of the carcinomatous components was 3 in three cases and 4 in two. The sarcomatoid areas were composed of pleomorphic spindle cells forming a malignant fibrous histiocytomatous pattern in four cases and a fibrosarcomatous pattern in one. The immunohistochemical findings in the carcinomatous and sarcomatoid components were identical. Wide-spectrum anti-cytokeratin cocktail, epithelial membrane antigen, and vimentin antibodies demonstrated immunoreactivity, while Leu-M1 did not react in all five cases. Three of the five tumors were positive for Ulex europaeus agglutinin I lectin. One sarcomatoid carcinoma reacted with monoclonal antibody to high molecular weight keratins, and all five tumors reacted with a monoclonal antibody to low molecular weight keratins. Two patients died at 5 months and 13 months after diagnosis, two are alive with metastatic disease at 1 and 14 months, and one is alive with no evidence of disease at 36 months.

Absorption of glucose polymers from rice in oral rehydration solutions by rat small intestine.

BACKGROUND: This study aims to determine the effect of replacing glucose in oral rehydration solution (ORS) with starch hydrolysates from rice on absorption in the small intestine and levels of glucose in portal venous blood and on disaccharidase levels and morphometric measurements in intestines of rats. METHODS: ORS containing standard composition of salts and 2% glucose (WHO ORS) or 2%, 5%, or 10% starch hydrolysates were infused into duodena of 60 Sprague-Dawley rats (250-350 g). Portal venous blood glucose levels were determined at 0, 30, 60, 90, and 120 minutes. RESULTS: Significantly larger areas under the curve of glucose absorption (AUCs) were produced by ORS containing 10% unfractionated starch hydrolysates (123.2 +/- 3.8), 2%, 5%, and 10% starch hydrolysates with long-chain ( > 9 molecules) glucose polymers (109.5 +/- 10.6, 109.3 +/- 7.4, and 115.3 +/- 7.1, respectively), and 5% and 10% starch hydrolysates with short-chain (2-9 molecules) glucose polymers (124.4 +/- 6.1 and 128.1 +/- 6.8). ORS with 2% and 5% unfractionated starch hydrolysates and 2% short-chain glucose polymers produced AUCs comparable with those of WHO ORS (96.48 +/- 5.7). Disaccharidase levels and morphometric measurements were not significantly different. CONCLUSIONS: Starch hydrolysates from rice containing glucose polymers can be used in ORS in higher concentrations than glucose to provide higher caloric density without increased osmolality.