Detection of concurrent/recurrent non-Hodgkin's lymphoma in effusions by PCR

A subset of patients with non-Hodgkin's lymphoma (NHL), present with or subsequently develop lymphocytic effusions. Differential diagnosis between reactive lymphocytosis and recurrent low-grade NHL is difficult by cytology alone. We studied the use of polymerase chain reaction (PCR)-based techniques to detect concurrent/recurrent NHL. Both primary tumors and atypical lymphocytic effusions of 12 low-grade B-NHL patients and 4 T-NHL patients were studied. Six pleural effusions (reactive/carcinomatous), in patients with no history of NHL, were included. Samples were amplified by PCR, using Fr3, Fr2, LJH, and VLJH primers specific for the immunoglobulin heavy chain (IgH) gene and Vgamma-8, Vgamma9, Vgamma10, Vgamma11 and Jgamma1/Jgamma2 consensus primers specific for the T-cell receptor gamma (TCR-gamma) gene. IgH gene PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). TCR-gamma gene PCR products were analyzed using a novel nonradioactive single-strand conformational polymorphism (SSCP) procedure. IgH gene rearrangement analysis demonstrated monoclonality in 11/12 primary low-grade B-NHLs. Identical monoclonal bands were found in both primary tumor and effusion in 9 patients. TCR-gamma gene rearrangement analysis demonstrated monoclonality in 4 of 4 primary T-NHLs. Identical monoclonal banded patterns were found in both primary tumor and effusion in 3 patients. Our results strongly support the diagnosis of concurrent/recurrent NHL in 13 of 16 (81%) cases of atypical lymphocytic effusions. IgH/PAGE and TCR-gamma/SSCP analyses are useful tools in the diagnoses of lymphocytic effusions in patients with NHL.     

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.

AIMS: To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. METHODS: PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software. RESULTS: Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. CONCLUSIONS: These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.     

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.

AIMS: To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. METHODS: PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software. RESULTS: Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. CONCLUSIONS: These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.     

Pleural posttransplantation lymphoproliferative disorder following liver transplantation

A case of posttransplantation lymphoproliferative disorder (PTLD) involving the pleura is reported. The patient was a 57-year-old man who underwent liver transplantation 2 years prior to the development of PTLD. The PTLD was pleural-based and was first detected by radiologic studies as a pleural effusion. Transbronchial biopsy and cytologic examination of 2 pleural fluid specimens were nondiagnostic. Subsequent open-wedge biopsy revealed a monomorphic PTLD, composed of large immunoblasts with plasmacytoid differentiation. Immunohistochemical studies demonstrated B-cell lineage with expression of monotypic cytoplasmic immunoglobulin kappa light chain and CD79a, and absence of T-cell antigens. Immunohistochemical and in situ hybridization studies demonstrated Epstein-Barr virus protein and RNA, respectively. No evidence of human herpesvirus 8 DNA was detected by polymerase chain reaction. We report this case because pleural-based PTLD is rare. The diagnosis of this entity is made more difficult by the fact that PTLD is often underrepresented in pleural fluid cytology samples.     

Primary pleural effusion posttransplant lymphoproliferative disorder: Distinction from secondary involvement and effusion lymphoma

Pleural effusion presentation of posttransplant lymphoproliferative disorder (PTLD) is relatively uncommon. Most examples of effusion-based PTLD have been secondary to widespread solid organ involvement, and are associated with an aggressive clinical course. We report on a case of primary effusion PTLD in a 70-yr-old male liver transplant recipient with a history of hepatitis B infection. Cytomorphologically, the pleural fluid specimen showed a monomorphous population of intermediate to large-sized transformed lymphoid cells, with irregular multilobated nuclear contours and readily identifiable mitotic figures. Flow cytometric immunophenotypic studies revealed a CD5-negative, CD10-negative, lambda immunoglobulin light chain-positive, monoclonal B-lymphocyte (CD19-positive/CD20-positive) population. The immunocytochemical stain for CD30 antigen was negative. In situ hybridization study for Epstein-Barr virus (EBV) early RNA (EBER) and Southern blot analysis for EBV terminal repeat sequences were both positive. Southern blot analysis for human herpes virus-8 (HHV-8) was negative. No solid-organ PTLD was identified, and the cytologic results supported the diagnosis of primary effusion PTLD. Immunosuppression was decreased, and 8 mo following the diagnosis of pleural fluid PTLD, the patient was stable and his pleural effusion had markedly diminished. Recognition of primary effusion PTLD and its distinction from PTLD secondarily involving the body fluids and from other lymphomas is important, since the behavior and prognosis appear different.     

Application of a fluorescent PCR method for molecular diagnosis of posttransplant lymphoproliferative disorders on routine tissue sections.

Molecular detection of monoclonality can play an important role in the diagnosis of posttransplantation lymphoproliferative disorders (PTLD). To permit accurate molecular diagnosis of PTLD even on very small amounts of DNA extracted from routinely embedded histologic material, we adapted a commercially available PCR protocol (for FR-1, -2 and -3 regions), originally designed for use on fresh/frozen samples. We applied this approach on routine biopsy/surgical material of 10 PTLD (from nine patients). All three FR regions were always amplified, indicating that the extracted DNA was of medium quality. All five PTLD morphologically classified as lymphomas were monoclonal in at least one FR region. Thus, using the WHO histologic, immunohistochemical, and clinical criteria as the reference standard, the approach provided 100% sensitivity for detection of monoclonal malignancies, supporting the validity of the method. Of five specimens classified morphologically as polymorphic PTLD, three displayed a solitary IgH gene rearrangement peak, consistent with the presence of a monoclonal B-cell population (ie, monoclonal polymorphic PTLD). This rapid and straightforward procedure, which allows identification of a wide range of IgH rearrangements, could facilitate molecular analysis of PTLD in routine practice, while limiting consumption of valuable diagnostic material.     

Prospektiver Vergleich der diagnostischen Wertigkeit von Zytologie und Immunzytologie bei thorakoskopisch-bioptisch untersuchten Pleuraergüssen

Summary Two issues have been elaborated: (1) the value of immunocytochemistry in the diagnosis of pleural effusions, and (2) the reactivity of the investigated antibodies with different classes of cells in pleural effusions. Effusions of unknown origin from 38 patients were investigated using thoracoscopy, pleural biopsies, conventional cytology, and immunocytochemistry. The following antibodies were used: those monoclonal against various leukocyte antigens, macrophage antigens, epithelial membrane antigen (EMA), various cytoskeleton antigens, and melanoma antigens; those polyclonal against CEA and ferritin. All of the techniques used showed 18 patients (48%) as having a tumor-cell negative effusion. A pleural tumor with a malignant effusion showed in 13 patients (34%); in 12 of these immunocytochemistry also revealed tumor cells. Seven patients (18%) had a tumor of the pleura with a tumor-cell negative effusion; in 2 of these immunocytochemistry revealed a tumor-cell positive effusion. There was no difference with regard to the number of NK cells in patients with inflammation of the pleura and negative cytology and patients with tumor of the pleura and malignant effusion (3% vs 4.5%). Tumor cells were mainly stained by EMA, cytokeratin, and CEA. CEA was the only antibody to be tumor-cell specific, while EMA and cytokeratin were expressed by mesothelial cells also. The antibody against ferritin was a significant marker for mesothelial cells.

---

Abkürzungen BSA Bovines Serumalbumin - CEA Carcinoembryonales Antigen - DAB Diaminobenzidin - EMA Epitheliales Membranantigen - HE Haematoxylin-Eosin - LA Lymphangiosis carcinomatosa - LCA Leucoyte common antigen (= Pan-Leukozytenantigen) - NCA Nicht spezifische kreuzreagierende Aktivität - NK-Zellen Natürliche Killerzellen - PK Pleurakarzinose - undiff Ca undifferenziertes Karzinom     

Human herpesvirus-8 in lymphomatous and nonlymphomatous body cavity effusions developing in Kaposi's sarcoma and multicentric Castleman's disease

Human herpesvirus-8 (HHV-8) has been associated with Kaposi's sarcoma, multicentric Castleman's disease and primary effusion lymphoma. Kaposi's sarcoma and multicentric Castleman's disease patients may develop body cavity effusions that, unlike primary effusion lymphoma, are poorly characterized. To better define these effusions, pleural and peritoneal fluids derived from 12 human immunodeficiency virus-seropositive and one seronegative patients affected by Kaposi's sarcoma or multicentric Castleman's disease were analyzed by a combination of morphologic, immunophenotypic, and DNA analyses, including polymerase chain reaction amplification of HHV-8, Epstein-Barr virus, and immunoglobulin heavy-chain (IgH) gene sequences. In addition, HHV-8 serologic status was assessed by using an immunofluorescence assay. All patients were adult men with high antibody titers to HHV-8; 11 of the 13 patients were homosexual/bisexual. Effusions revealed monocyte/macrophage-rich infiltration (10 patients) or large-cell lymphoma with CD45(+)/non-T/non-B phenotype (three of 13 patients); polymerase chain reaction analysis showed the presence of HHV-8 sequences (nine of 13 patients), germline IgH (seven of 12 patients) or clonal IgH rearrangements (four of 12 patients), and rarely Epstein-Barr virus sequences (two of 12 patients). In the setting of HHV-8 infection, two effusion types may occur. One fulfills the criteria for HHV-8-positive PEL (lymphoma-morphology, HHV-8-DNA(+), IgH rearrangement). The other seems more reminiscent of an HHV-8-associated nonneoplastic process (monocyte-macrophage morphology, HHV-8-DNA(+/-), germline IgH). Interestingly, a single case of the latter effusion type harbored a B-cell monoclonal proliferation, which suggests the hypothesis that a prelymphomatous effusion may precede overt body cavity lymphoma.     

Recurrent Epstein-Barr virus-associated post-transplant lymphoproliferative disorder: report of a patient with histologically similar but clonally distinct metachronous abdominal and brain lesions.

A liver transplant patient developed a single central nervous system (CNS) intraparenchymal lesion 5 months after the diagnosis of an intraabdominal diffuse large B-cell post-transplant lymphoproliferative disorder (PTLD). Biopsy of the new CNS lesion showed a diffuse large B-cell PTLD morphologically and immunohistochemically indistinguishable from the abdominal lesion. In addition, both lesions were positive for Epstein-Barr virus (EBV) DNA by polymerase chain reaction (PCR) and for EBV-encoded RNA by in situ hybridization. Although these results were consistent with a metastatic origin for the CNS lesion, the finding of an intraparenchymal lesion without leptomeningeal or dural spread was suggestive of a new primary CNS lymphoma. Proof that the brain lesion was a second primary and not a metastasis was obtained by immunoglobulin gene rearrangement studies and assessment of EBV clonality. Multiple primary lymphoid neoplasms arise at higher frequency in the setting of immunosuppression, and molecular investigations of tumor clonality can provide clinically relevant staging and prognostic information.     

Bone marrow changes in anorexia nervosa are correlated with the amount of weight loss and not with other clinical findings

We describe a retrospective study of 4 cases of sporadic fatal infectious mononucleosis (IM), 1 case of fatal IM, and 1 case of sporadic severe IM. Patients were 26 months to 17 years old; 3 were male. Five died of complications of IM. All 5 of these patients had the Epstein-Barr virus (EBV) present in examined tissue specimens; EBV was monoclonal in 3 patients and biclonal in 1. EBV clonality studies were not performed in the remaining patient. All 5 patients also had monoclonal gene rearrangements. The sixth patient survived despite a life-threatening clinical course; EBV was oligoclonal, and gene rearrangements were not detected. EBV clonality and gene rearrangement studies may be usefulfor predicting which patients with clinically aggressive IM are at highest risk for fatal outcome. Patients in whom IM has a fatal outcome are more likely to have monoclonal or biclonal EBV and immunoglobulin heavy chain or T-cell receptor gene rearrangements. In contrast, patients with nonfatal IM may lack monoclonal EBV and monoclonal rearrangements of the aforementioned genes. The reasons EBV induces a monoclonal proliferation only in some patients remain to be elucidated.     

A case of leukemic pleural infiltration in atypical chronic myeloid leukemia

Pleural effusion in chronic myeloid leukemia (CML) is poorly understood and rarely reported in the literature. When the pleural effusion is caused by leukemic pleural infiltration, the differential white blood cell count of the effusion is identical to that of the peripheral blood, and the fluid cytology reveals leukemic blasts. We report here a case of bilateral pleural involvement of atypical CML in an 83-yr old male diagnosed with pancreatic cancer with abdominal wall metastasis and incidental peripheral leukocytosis. Based on bone marrow examination, chromosome analysis and polymerase chain reaction he was diagnosed with Philadelphia chromosome negative, BCR/ABL gene rearrangement negative CML. Following 3 months of treatment with gemcitabine for pancreatic cancer, he developed bilateral pleural effusions. All stages of granulocytes and a few blasts were present in both the pleural fluid and a peripheral blood smear. After treatment with hydroxyurea and pleurodesis, the pleural effusion resolved.     

肝移植术后淋巴组织增生性疾病的临床病理分析

目的探讨肝移植术后淋巴组织增生性疾病（PTLD）的临床病理特征。方法对3例肝移植术后PTLD行HE、免疫组织化学染色及TCR、IgH基因重排检测,同时复习其临床资料并随访。结果3例中,2例位于肝门部,1例位于肝动脉旁淋巴结。临床表现为发热和梗阻性黄疸。病理诊断例1为单形性T细胞PTLD的周围T细胞淋巴瘤,例2、例3为单形性B细胞PTLD的淋巴浆细胞样淋巴瘤和弥漫大B细胞淋巴瘤。基因重排检测例1 TCR阳性,例2、例3 IgH阳性。3例EBV免疫组化染色均阳性。行再次移植降低免疫抑制剂用量并配合抗病毒、抗CD20单抗和化疗等治疗,例1、例3分别于术后6个月和4个月死于淋巴瘤复发和肺感染,例2随访11个月无瘤生存。结论PTLD是肝移植术后严重的并发症之一,具有独特的形态和临床特征。病因可能与EB病毒感染和免疫抑制有关,需经病理组织学检查确诊。     

Common and HIV-related diffuse large B-cell lymphomas differ in their immunoglobulin gene mutation pattern

HIV-infected patients are at high risk of developing diffuse large B-cell lymphomas (DLBCL). It is currently unclear whether these lymphomas represent Epstein-Barr virus (EBV)-driven lymphoproliferations that develop in the setting of immunodeficiency, or whether these tumours are more closely related to the DLBCL seen in the general population. To clarify this issue, 12 HIV-related DLBCL from 11 patients were analysed for the presence of clonally rearranged and somatically mutated immunoglobulin heavy chain (IgH) genes and their association with EBV was determined. Eleven of the 12 tumour samples displayed monoclonal rearrangements of the IgH genes, with or without a moderate number of somatic mutations in the CDRII and in the FWIII regions (average four mutations). One patient presented two successive lesions; whereas the initial tumour showed an oligoclonal IgH rearrangement, the lymphoma at relapse proved to harbour a monoclonal B-cell population. Ten of 12 tumour samples expressed the EBV encoded small RNAs (EBERs), and six of these EBV-positive cases displayed, in addition, an expression of the EBV encoded nuclear antigen 2 (EBNA-2). The results obtained from HIV-related DLBCL are at variance to those described for DLBCL occurring in the general population, since the latter contain significantly more somatic IgH mutations in the CDRII and in the FWIII regions and are only rarely associated with EBV. It is concluded from these findings that HIV-related DLBCL represent a distinct group of B-cell lymphomas, a significant fraction of which most likely originates from EBV-driven lymphoproliferations, and that half of the cases derive from pre-germinal centre B-cells. Copyright © 1999 John Wiley & Sons, Ltd.     

Specificity of PCR-based clonality analysis of immunoglobulin heavy chain gene rearrangements for the detection of bone marrow involvement by low-grade B-cell lymphomas

A study was performed to investigate the utility of polymerase chain reaction (PCR)-based analysis of immunoglobulin heavy chain (IgH) gene rearrangements for the diagnosis of low-grade malignant B-cell lymphomas on formalin-fixed, EDTA-decalcified, and paraffin-embedded bone marrow trephine biopsies. On amplifying two DNA samples per biopsy, no reproducible monoclonal PCR result was found in 32 patients with reactive lymphoid hyperplasias. In contrast, 5/14 patients with known low-grade B-cell lymphomas, but histomorphologically and immunohistochemically lymphoma-free bone marrow, showed a reproducible monoclonal IgH gene rearrangement. In three of these cases, sequence analysis revealed completely different amplification products on comparing bone marrow and lymph node infiltrations, while in the other two cases the products were identical. In one of the latter biopsies, an unequivocal lymphoma infiltrate was found after step sectioning of the biopsy, while the other case remained lymphoma-free according to conventional criteria. A third group of three patients with known lymphomas and bone marrow findings that were suggestive but not diagnostic of bone marrow involvement showed monoclonal PCR results in all three cases, with identical sequences in bone marrow and extramedullary lymphoma infiltrates. These data suggest that a reproducible monoclonal IgH gene rearrangement is highly specific for the presence of malignant B-cells in bone marrow. In staging procedures for low-grade B-cell lymphomas, PCR yields no additional information in cases that are morphologically and immunohistochemically lymphoma-free after evaluation of representative sections. PCR may be useful in equivocal cases, provided that IgH gene rearrangements of extramedullary lymphoma and bone marrow are sequenced and compared.     

Epstein‐Barr virus related post‐transplant lymphoproliferative disorder prevention strategies in allogeneic hematopoietic stem cell transplantation

Epstein‐Barr virus associated post‐transplant lymphoproliferative disorders (EBV PTLD) are recognized as a significant cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). The number of patients at risk of developing EBV PTLD is increasing, partly as a result of highly immunosuppressive regimens, including the use of anti‐thymocyte globulin (ATG). Importantly, there is heterogeneity in PTLD management strategies between alloHSCT centers worldwide. This review summarizes the different EBV PTLD prevention strategies being utilized including the alloHSCT and T‐cell depletion regimes and the risk they confer; monitoring programs, including the timing and analytes used for EBV virus detection, as well as pre‐emptive thresholds and therapy with rituximab. In the absence of an institution‐specific policy, it is suggested that the optimal pre‐emptive strategy in HSCT recipients with T‐cell depleting treatments, acute graft vs host disease (GVHD) and a mismatched donor for PTLD prevention is (a) monitoring of EBV DNA post‐transplant weekly using plasma or WB as analyte and (b) pre‐emptively reducing immune suppression (if possible) at an EBV DNA threshold of >1000 copies/mL (plasma or WB), and treating with rituximab at a threshold of >1000 copies/mL (plasma) or >5000 copies/mL (WB). There is emerging evidence for prophylactic rituximab as a feasible and safe strategy for PTLD, particularly if pre‐emptive monitoring is problematic. Future management strategies such as prophylactic EBV specific CTLs have shown promising results and as this procedure becomes less expensive and more accessible, it may become the strategy of choice for EBV PTLD prevention.     

High-resolution analysis of gene rearrangements in lymphoid malignancies.

As T-cell receptor and immunoglobulin gene rearrangements provide specific clonal markers for lymphoid cell proliferations, analysis of these genes is useful for distinguishing between reactive and malignant disease. We have developed an automated, high-resolution analysis of PCR fragments to identify clonally rearranged TCR-gamma (TCR gamma) genes and IgH genes. Consensus primers are used to detect the majority of possible rearrangements in multiplex PCR assays, and the PCR products are fluorescently labelled for visualisation with ABI Genescan software. Polyclonal populations of lymphoid cells are represented by a spectrum of fragments, whereas a monoclonal population of cells is represented by one or two discrete bands, indicating rearrangement of one or both alleles. For TCR-gamma PCR, the rearranged DNA fragment from a monoclonal population of T-cells diluted to 0.1% in DNA from a polyclonal population of cells is still readily distinguishable from the polyclonal background. Similarly, for IgH PCR, the gene rearrangement from a monoclonal population of B-cells is still distinguishable to 0.5% in a polyclonal background. As this technique allows semi-quantitative resolution of fragments one base different in size, it is ideal for detecting monoclonal and oligoclonal populations of B- and T-cells. The accurate size determination of PCR fragments also minimises the risk of false positives resulting from contamination, as individual monoclonal rearrangements are frequently patient-specific on the basis of size alone.     

Cytologic detection of malignancy in pleural effusion: a review of 5,255 samples from 3,811 patients

A retrospective analysis of 5,255 pleural effusion specimens from 3,811 patients was undertaken to determine the accuracy of cytopathologic correlation with pleural biopsy, the detection rate of malignancy by cytology, and the frequency distribution of malignant effusions according to age group. The cytopathologic correlation was 96.5% accurate, with 0.1% false-positive results and 0.18% false-negative results by cytology. The sensitivity of cytologic detections was 6.7% higher than that of pleural biopsy. Frequency analysis showed that the incidence of carcinoma of the lung, the most common cause of malignant effusion, is not sex based. Adenocarcinoma of the lung was the most frequent type of malignancy found in pleural effusions. It represented 79% of lung carcinomas that metastasized to pleura, accounting for 40% of all malignant pleural effusions. In young adults, lymphoreticular malignancies were the most common cause of malignant effusions.     

The yellow nail syndrome. Light and electron microscopic aspects of the pleura

The yellow nail syndrome is a rare cause of recurrent pleural effusions. We studied a case of this entity, placing special emphasis on the microscopic and ultrastructural aspects of the pleural lymphatics. The patient had the classic symptoms of recurrent bilateral pleural effusions, yellow, dystrophic fingernails and toenails, and lower-limb edema. To control the pleural effusions, a left parietal pleurectomy was performed. Histologic study showed both pleura to be thickened with fibrosis and chronic inflammatory infiltration. The lymphatic capillaries in the visceral pleura were dilated. Electron microscopy confirmed the lymphatic nature of these capillaries. We believe that these ectatic lymphatic capillaries suggest a downstream obstruction to the lymph drainage.     

Effusion cytology in burkitt's lymphoma

The American form of Burkitt's lymphoma is a high-grade malignancy which usually involves the abdomen in children and young adults. There is only a limited literature which describes the cytologic features of Burkitt's lymphoma in serous effusions. We present three children with Burkitt's lymphoma initially diagnosed by effusion cytology. the first patient, an 11-yr-old boy, presented with bilateral pleural effusions, ascites, and abdominal masses and had diagnostic pleural fluid cytology without tissue confirmation (ultrastructural examination was performed on the effusion specimen). He died 7 months after the initial diagnosis. the second patient, a 9-yr-old boy, presented with ascites and abdominal masses and had diagnostic peritoneal fluid cytology with a subsequent confirmatory chest wall biopsy. the third patient, a 16-yr-old girl, presented with a 2-month history of irregular menses, a large pelvic mass, lymphadenopathy, and liver masses. Although an ovarian malignancy was clinically suspected, cytologic examination of her peritoneal fluid revealed Burkitt's lymphoma. Surgical exploration revealed involvement of her right ovary, cecum, and terminal ileum. the second and third patients are currently alive with no apparent disease following chemotherapy. in all three patients, effusion cytology revealed Burkitt's lymphoma, characterized by a uniform population of noncohesive lymphoid cells with noncleaved nuclei, prominent multiple nucleoli, and scanty-to-moderate basophilic cytoplasm. Cytoplasmic and/or nuclear vacuoles were also seen, more prominent in Diff-Quik—stained, air-dried smears. These cases demonstrate the importance of recognizing the cytologic features of Burkitt's lymphoma, as serous fluid may be the initial diagnostic specimen.     

Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.