Diagnosis of B-cell lymphoma. Utility of the polymerase chain reaction for detecting clonality from archival cytologic smears

OBJECTIVE: To apply the polymerase chain reaction (PCR) to detect clonality for potentially helping to establish a definitive diagnosis of lymphoma in cytologic material. STUDY DESIGN: In this retrospective study, Papanicolaou-stained cytologic smears and formalin-fixed, paraffin-embedded tissues from 17 cases of B-cell lymphoma were examined to investigate their clonality by a PCR technique using three different approaches (FR3, FR3A and FR2) for amplification of immunoglobulin heavy chain genes. Cytologic smears from 10 cases of nonneoplastic lymphoid tissues and T-cell lymphomas served as negative controls. RESULTS: Monoclonality was detected in 9 of 17 cases (53%) of B-cell lymphoma in cytologic smears as compared with 8 of 16 cases (50%) in tissue sections. Semi-nested PCRs (FR3A/FR2) were superior to the single PCR (FR3) in the detection rate (41% vs. 18%). Five of seven cases (71%) of marginal zone B-cell lymphomas showed monoclonality, whereas only 4 of 10 cases (40%) of diffuse large B-cell lymphomas did so. Monoclonality was demonstrated in none of the negative controls. CONCLUSIONS: Clonality detection in B-cell lymphomas by PCR using cytologic smears is specific and equal in sensitivity to that using formalin-fixed, paraffin-embedded tissues. The detection rate is especially excellent in marginal zone B-cell lymphoma, in which the cytologic diagnosis is particularly challenging. Combined seminested PCRs for FR3A and FR2 are advocated for a reliable assessment of clonality.     

Role of Immunohistochemistry in Staging Diffuse Large B-cell Lymphoma (DLBCL)

The use of immunohistochemistry (IHC) in staging bone marrow in non-Hodgkin''s lymphoma (NHL) is largely limited to ambiguous cases, particularly those with lymphoid aggregates. Its role in routine clinical practice remains unestablished. This study aimed to determine whether the routine use of IHC in diffuse large B-cell lymphoma (DLBCL) would improve the detection of lymphomatous involvement in the bone marrow. It also sought to determine the impact of IHC on predicting survival compared with routine histological diagnosis using hematoxylin and eosin (H&E), Giemsa, and reticulin staining. The bone marrow trephines of 156 histologically proven DLBCL cases were assessed on routine histology, and IHC using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a), and κ and λ light chains. IHC detected lymphomatous involvement on an additional 11% cases compared with histology alone. Although both routine histology and IHC were good predictors of survival, IHC was better at predicting survival on stepwise multivariate Cox regression analysis. IHC performed routinely on bone marrow trephines has the ability to improve detection of occult lymphoma in experienced hands. Furthermore, it is a better predictor of survival compared with routine histological examination alone. (J Histochem Cytochem 56:893–900, 2008)     

PCR clonality detection in Hodgkin lymphoma

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein–Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.     

Primary mediastinal B-cell lymphoma: detection of BCL2 gene rearrangements by PCR analysis and FISH

Primary mediastinal large B-cell lymphoma (PMBCL) has a characteristic clinical presentation, morphology, and immunophenotype, representing a clinically favorable subgroup of diffuse large B-cell lymphoma (DLBCL). By gene expression profiling (GEP), PMBCL shares features with classical Hodgkin lymphoma (cHL). Of further interest, BCL6 gene mutations and BCL6 and/or MUM1 expression in a number of PMBCLs have supported an activated B-cell (ABC) origin. Several studies, including GEP, have failed to detect BCL2 gene rearrangements (GRs) in PMBCL. An index case of t(14; 18)+ PMBCL prompted our study of the incidence of BCL2 GRs in PMBCL by polymerase chain reaction (PCR)/fluorescence in situ hybridization (FISH) analyses and its possible clinical impact. Twenty-five retrospectively identified, well-defined PMBCLs (five with cytogenetics) from three institutions were analyzed for a BCL2 GR by PCR/FISH analyses. The formalin-fixed, paraffin-embedded tissue blocks of 24 available cases were also analyzed by BCL2 immunohistochemistry (IHC). Of the five with cytogenetics, two had a t(14; 18) (q32; q21). Of the 25 analyzed by PCR, 2 had no amplifiable DNA (aDNA), including 1 t(14; 18)+ case. Of those with aDNA, two showed a BCL2 GR; by FISH analysis, three demonstrated a BCL2 GR. BCL2 protein expression by IHC analysis was variably detected in 21 out of 24 (strongly, uniformly expressed: 6, including all with a t(14; 18) or a BCL2 gene rearrangement; moderately weakly expressed in a subset of the malignant cells: 15). Available clinical follow-up of this BCL2+ subset showed a similar course to the other PMBCL cases. Our results imply that a subset of PMBCL [(4 out of 24 analyzed) in our series] may be of GC origin. A larger study is necessary to determine any clinical significance.     

Cytologic evaluation of prognostic markers in breast carcinoma

To type breast carcinomaon on fine needle aspiration cytology (FNAC) material and correlate the results with histologic typing, to grade breast carcinoma on FNAC material and correlate the findings with Bloom-Richardson histologic grading, and to determine the estrogen receptor (ER) status in cases of breast carcinoma by immunocytochemical (ICC) staining of FNA cytologic material and correlate the findings with ER status, as determined by immunohistochemical (IHC) staining of tissue sections. STUDY DESIGN: Seventy-seven cases of breast carcinoma diagnosed on FNAC formed the basis of this study. Typing was done in all cases on the basis of cytologic features and grading in 62. (Fifteen cases were special types of breast carcinoma). In all cases, ER status was determined by immunostaining of cytologic smears. Results of tumor typing, grading and ER status on cytologic material were compared with the results of histologic typing, grading and immunostaining of histologic material obtained from mastectomy or wide excision specimens. RESULTS: Tumor typing was accurate in 73 of 77 cases (94.8%). Fifteen of 18 cases that were cytologically grade 3 were confirmed on histology, while 3 proved to be grade 2. Of 40 cytologic grade 2 cases, 26 were confirmed on histology, while 14 cases were grade 3. Three of 4 cytologically grade 1 cases were confirmed on histology while 1 was grade 2. The overall accuracy for cytologic grading was 71% (44 of 62 cases). Thirty-seven of 40 ER-positive cases (92.5%) were labeled ER positive on ICC. One case was ER negative on cytology, while in 2 cases the cellularity of the cytologic smear was insufficient to assess ER expression. Thirty-seven cases were negativefor ER on IHC. Nine of these showed ER positivity on ICC, 26 were negative, and 2 had cellularity that was inadequate for assessment of ER. Sensitivity and specificity rates for ER detection on ICC were 97.4% and 74.3%, respectively. CONCLUSION: Tumor typing, grading and evaluation of ER status on FNA C material in breast carcinomas are simple, quick and moderately reliable techniques that compare and correlate favorably with histologic typing, grading and ER status on IHC.     

Serous fluid cytology of multicentric Castleman’s disease and other lymphoproliferative disorders associated with Kaposi sarcoma‐associated herpes virus: a review with case reports

C. Lobo, S. Amin, A. Ramsay, T. Diss and G. Kocjan Serous fluid cytology of multicentric Castleman’s disease and other lymphoproliferative disorders associated with Kaposi sarcoma‐associated herpes virus: a review with case reports Objective: The aim of this study is to describe and review the cytological features of Kaposi sarcoma‐associated herpes virus (KSHV) related entities, such as multicentric Castleman’s disease (MCD), plasmablastic‐lymphoma (PBL) and primary effusion lymphoma (PEL), which all may present as body cavity effusions. Serous fluid cytology of MCD and PBL has not, to our knowledge, thus far been described. Although different in nature, MCD, PBL and PEL are characterized by similar morphological features. Materials and methods: Body cavity effusions from four different patients with previously known or unknown KSHV‐related lymphoproliferations have been examined by routine cytology, immunocytochemistry (IC) and polymerase chain reaction (PCR). Results: MCD, PBL and PEL are all characterized by increased cellularity, comprising mainly lymphoid and plasmacytoid cells with variable proportions of immunoblasts. Immunocytochemistry and PCR results show the MCD to be CD138 and KSHV positive, CD30 negative, IgM, IgH and lambda restricted but IgH polyclonal. PBL was CD138 positive, kappa restricted, weakly positive with VS38 and over 80% positive with MIB 1. PEL was CD45, EMA, CD138, KSHV, p53 and CD3 positive, CD20, EBV, CD30, CD2, CD4, ALK1, epithelial and mesothelial markers negative, and PCR monoclonal B‐cell expanded (Ig‐kappa bands). Conclusion: Cytological examination of effusions in KSHV‐related lymphoproliferative disorders may show similar morphological features but clonality studies and immunocytochemistry are very helpful in distinguishing between these rare benign and malignant lymphoproliferative diseases.     

Induction of anti B-cell malignance CTL response by subfamily-shared peptides derived from variable domain of immunoglobulin heavy chain

The variable domain of immunoglobulin heavy chain (Ig HV) is well-characterized tumor associated antigen expressed in B-cell malignancies, which may function as a T-cell target. However, T-cell epitopes derived from shared framework regions (FRs) of each IgHV subfamily capable of inducing cytotoxic T lymphocytes (CTLs) against the B-cell malignancy, have not been identified. Using the specific PCR primers of seven IgHV gene subfamilies, we amplified the IgHV gene rearrangement for 108 cases of B-cell acute lymphoblastic leukemia (B-ALL) patients. The IgHV gene rearrangement fragments of B-ALL patients were directly sequenced then classified into seven different subfamilies. The T-cell epitopes encoded by the IgHV gene in the B-ALL patients were predicted by SYFPEITHI and BIMAS programs and compared with those from 56 representative germline IgHV sequences in the genebank. For the HLA-A*0201 locus, we found 1 or 2 top score shared epitopes from each subfamily and got 12 epitopes altogether. Results showed that ten of them were in the FRs. Using an antigen-specific T-cell expansion system, we generated the peptide-special CTLs in vitro, which were capable of killing B lymphoma cell lines that belonged to the same IgHV subfamily in a peptide-specific and HLA-restricted manner. Furthermore, we proved that the cytotoxicity of CTLs was IgHV subfamily-specific. These data indicate possible immunotherapy approaches for B-cell malignances patients based on IgHV gene subfamilies.     

CD22-Binding Peptides Derived from Anti-CD22 Ligand Blocking Antibodies Retain the Targeting and Cell Killing Properties of the Parent Antibodies and May Serve as a Drug Delivery Vehicle

CD22 is a B-cell specific membrane glycoprotein that mediates homotypic and heterotypic cell adhesion; it also regulates B-cell receptor (BCR)-mediated signals. Monoclonal antibodies (mAb) directed at the ligand binding domain of CD22 initiate CD22-mediated signal transduction and apoptosis in B-cell lymphomas (NHL). Amino acid analysis of the complimentary determining regions (CDRs) of six different anti-CD22 ligand blocking mAb revealed a high level of sequence conservation. The heavy chain CDRs 1, 2, and 3 are 85, 40, and 38% conserved, respectively; light chain CDRs 1, 2, and 3, are 95, 90 and 90% conserved, respectively. Based on these conserved sequences, five peptides were designed and synthesized. Only the sequence derived from heavy chain CDR2 (Peptide 5) demonstrated significant B-cell binding. Peptide 5 bound to both malignant and primary B-cells with very little T-cell binding. The affinity had a Km of 5 × 10⁻⁶ M. Peptide 5 mediated killing of several NHL cell lines to a degree similar to that of the parent mAb (HB22.7). Peptide 5’s loop structure was shown to be crucial for B-cell binding and ligand blocking. Mutational analysis revealed that most Peptide 5 amino acids were critical for B cell binding. Using a CD22 transfected COS cell line, we demonstrated CD22-specific binding and CD22 ligand blocking to a degree similar to HB22.7. Finally Peptide 5 was used as a vehicle to deliver a pro-apoptotic peptide into NHL cells. Peptide 5 was fused to a BH3 death domain-containing peptide which demonstrated more effective NHL cell killing than the parent peptide.     

Two-color immunostaining of liver fine needle aspiration biopsies with CD34 and carcinoembryonic antigen. Potential utilization in the diagnosis of primary hepatocellular carcinoma vs. metastatic tumor

OBJECTIVE: To examine immunohistochemical staining of cell block material with antibodies against vascular marker CD34 and polyclonal carcinoembryonic antigen (pCEA) for their clinical utility as part of a 2-color staining protocol in fine needle aspiration (FNA) biopsy of liver masses to distinguish metastases from primary hepatocellular carcinoma (HCC). STUDY DESIGN: The authors obtained cell block material from 96 liver FNAs and performed simultaneous (i.e., "dual-color") immunohistochemical staining utilizing antibodies against vascular marker CD34 and pCEA. Cases were blinded and evaluated by the authors for staining pattern and intensity. A consensus was obtained, the results were unblinded, and the diagnoses were correlated. RESULTS: After staining, 89 cases had sufficient tissue for evaluation. Of the 19 HCC cases, 16 (84%) showed peripheral staining with CD34, and 13 (68%) showed a canalicular or mixed canalicular-cytoplasmic staining pattern for pCEA. Thirteen cases (68%) showed staining for both antigens. All HCC exhibited immunostaining for at least 1 antibody in an appropriate staining pattern. Of the 67 cases of metastatic malignancy, 5 (7%) showed a predominantly transgressing pattern of CD34 staining, 43 (64%) showed a predominantly cytoplasmic or mixed cytoplasmic-canalicular pattern of pCEA staining, and 2 cases (3%) showed staining for both antigens in a transgressing CD34 pattern and cytoplasmic pCEA pattern. None of the 3 normal liver tissue blocks showed staining with either antigen. CONCLUSION: Two-color immunohistochemical staining of liver cell block material obtained by FNA with antibodies to CD34 and pCEA can be helpful in differentiating metastatic tumors vs. primary HCC.     

Analysis of clonality in cytologic material using the polymerase chain reaction (PCR)

Analysis of clonality in cytologic material using the polymerase chain reaction (PCR) Immunoglobulin heavy chain (IgH) gene rearrangement analysis was performed on 27 fine needle aspiration (FNA) specimens (13 reactive hyperplasia, 11 B cell non-Hodgkin's lymphoma (B-NHL), one Hodgkin's disease and two suspicious of non-Hodgkin's lymphoma). Satisfactory amplification was achieved in 23/27 cases. A polyclonal pattern was seen in 14 cases (11 reactive hyperplasia, one B-NHL, one suspicious of lymphoma, one Hodgkin's disease). A monoclonal band was seen in nine cases (eight B-NHL, one reactive hyperplasia). Amplification was unsuccessful in four cases. Clonal analysis by PCR-based IgH gene rearrangement analysis can be successfully applied to FNA material and can be useful in diagnosis, but the results must be interpreted in conjunction with morphology and other ancillary information. Analyse de la clonalité en utilisant la PCR sur matériel cytologique L'analyse des réarrangements géniques des chaînes lourdes des immunoglobulines (IgH) a été réalisée sur 27 produits de ponction à l'aiguille fine (13 cas d'hyperplasie réactionnelle, 11 cas de lymphome non hodgkinien de type B (LNH-B), un cas de maladie de Hodgkin et deux cas suspects de lymphome malin non hodgkinien. Une amplification satisfaisante a été obtenue dans 23 cas sur 27. Un aspect polychonal a été observé dans 14 cas (11 hyperplasies réactionnelles, 1 lymphome malin non hodgkinien de type B, 1 cas suspect de lymphome, 1 cas de maladie de Hodgkin). Une bande monoclonale a été observée dans 9 cas (8 LNH-B, 1 hyperplasie réactionnelle). L'amplification n'a pas réussi dans quatre cas. L'analyse de la clonalité par analyse des réarrangements géniques par PCR peut être appliquée avec succès au matériel cytologique qui est obtenu par ponction à l'aiguille fine et elle peut être utile au plan diagnostique, mais les résultats doivent étre interprétés en intégrant les informations morphologiques et les autres données complémentaires. Polymerase Chain Reaction an zytologischem Material Die Analyse schwerer Immunglobulinketten (IgH) wurde an 27 FNP (13 reaktive Hyperplasien, 11 B-NHL, 1 Hodgkin und und zwie verdächtige Fälle) analysiert. Eine ausreichende Amplifikation wurde in 23/27 Fällen erzielt. Ein polyklonales Muster wurde in 14 Fällen beobachtet (11 reaktive Hyperplasien, 1 B-NHL, 1 Hodgkin sowie 1 verdächtiger Fall). Ein monoklonales Band wurde 9 mal gefunden (8 B-NHL, 1 reaktive Hyperplasie). Die Amplifikation war ungenügend in 4 Fällen. Die PCR-Analyse kann für die Diagnostik von Nutzen sein muss jedoch zusammen mit der Morphologie und anderen Informationen interpretiert werden.     

Analysis of KIT mutation and protein expression in fine needle aspirates of gastrointestinal stromal/smooth muscle tumors

OBJECTIVE: To determine if sequencing the KIT gene could facilitate more definitive FNA diagnosis. STUDY DESIGN: Sixteen cases of gastrointestinal stromal/smooth muscle tumor (GIST) in which fine needle aspiration (FNA) was performed (mean age, 67; M/F = 12/4) were studied. DNA was extracted from cytologic preparations from all patients (15 cell blocks, 1 alcohol-fixed smear) and seven subsequent resection specimens. DNA was amplified by polymerase chain reaction, using primers designed to amplify a segment of the KIT gene exon 11 and sequenced on an ABI Prism 377 DNA sequence analyzer (Applied Biosystems, Indianapolis, Indiana, U.S.A.). Immunocytochemical staining for CD 117 (the KIT gene product) was performed on sections from 12 cell blocks and 7 surgical resections. RESULTS: In-frame deletion of exon 11 was detected in eight cases (7 monoalleic, 1 bialleic); a point mutation was found in one case. Mutation was found only in histologically malignant (6 of 10 cases) and borderline GISTs (3 of 4 cases). No mutation was identified in benign tumors. In three cases, scant cellularity or blood precluded sequencing. CD 117 was expressed in 12 of 15 cases. CONCLUSION: Immunocytochemical staining for CD 117 is useful in confirming a cytologic diagnosis of GIST but does not facilitate diagnosis of malignancy. FNA biopsy specimens are suitable for KIT gene sequencing; detection of a KIT mutation favors a malignant diagnosis, though absence of mutation does not preclude malignancy.     

抗 CD20融合蛋白在 CHO 细胞中的表达及其纯化与鉴定

目的：构建CD20胞外区与Igβ胞外区和人IgG1 Fc融合基因的表达载体,并在CHO细胞中表达。方法与结果：根据已知的IgM的CH2区域和Igp胞外区序列,分别设计PCR引物并进行PCR扩增,然后用重叠PCR法扩增得到900bp的Igp-CH2序列,插入本实验室构建的pIRIS-EGFP-Fc载体,转化大肠杆菌,得到pIRIS-Igβ-CH2-Fc重组质粒,将其转染CHO-K1细胞,在G418抗性培养基中培养,荧光显微镜观察结合ELISA法筛选高表达细胞系,细胞系扩大培养后,通过亲和层析rProteinA柱纯化得到纯度融合蛋白,SDS-PAGE显示目的蛋白去糖基化后,相对分子质量约为42×10^3。结论：得到了Igβ-CH2-Fc重链抗体样分子,并鉴定为糖蛋白;须进一步对所得蛋白的生物学活性进行检测,验证其是否能够通过胞外区蛋白定位于B细胞淋巴瘤细胞表面对B淋巴瘤细胞进行杀伤。     

PCR-based clonality assessment in patients with lymphocytic leukaemias: a single-institution experience

PCR-based clonality testing can be performed in all lymphoproliferations by analysing gene rearrangements of antigen receptors, rearrangements that are unique for each kind of lymphocyte. Reactive lymphoproliferations have polyclonally rearranged Ig/TCR genes, whereas malignant proliferations (leukaemias and lymphomas) show clonal rearrangements. The aim of this study was to assess the clinical benefits of clonality testing with previously evaluated consensus primers in leukaemia patients. The study included peripheral blood and bone marrow samples of 67 leukaemia patients (32 B-CLL, 24 B-ALL and 11 T-ALL). Clonality testing was based on PCR amplification of rearranged IgH and TCR genes. During diagnosis, monoclonal pattern was found in all analysed B-CLL and T-ALL samples. Testing in B-ALL patients showed positive results in all bone marrow and one peripheral blood samples. Results of clonality testing in B-CLL patients during follow-up were concordant between peripheral blood and bone marrow. Obtained results corresponded to clinical course in all but one patient. In B-ALL group, results of molecular testing in peripheral blood and bone marrow confirmed remission estimated according to clinical criteria in all except one patient. Before any clinical sign of relapse, monoclonal pattern was found in six/seven patients by bone marrow and in three/seven patients by peripheral blood analysis, respectively. Results of molecular monitoring in T-ALL patients did not confirme clinical evaluation in two patients. Obtained results indicate high accuracy of re-evaluated primers for clonality assessment in ALL and CLL patients at the time of diagnosis. Results of clonality testing in B-ALL patients indicate that bone marrow analysis has higher sensitivity compared to analysis of peripheral blood.     

Biallelic IgH rearrangements in patients with indolent lymphoproliferative disorders: molecular and practical implications

We report a group of patients (pts) with indolent lymphoproliferative disorder who had both alleles for the immunoglobulin heavy chain genes rearranged (biIgH). This group of 17 pts consisted of 9 small lymphocytic lymphomas (SLL) and 8 chronic lymphocytic leukemia (CLL). The polymerase chain reaction (PCR) amplification of clonal immunoglobulin heavy (IgH) rearrangement using the complementarity determining region III (CDRIII) constantly retrieved two distinct bands in all PCR informative samples of those pts. To rule out biclonality, we evaluated samples by fluorescein activated cell sorting (FACS) analysis and sequenced the PCR products. We were able to obtain both IgH sequences from 12 patients. FACS suggested biclonality in one case, which also correlated with sequencing results as both IgH rearrangements were in-frame. Recently, we reported a patient who sustained transformation into an aggressive disease after biIgH was detected in the setting of monoclonal disease (Cerny et al., 2003b, Haematologica 88(05):ECR15 B.). We decided to compare clinical characteristics and prognosis of 17 pts with biIgH and 37 pts with monoIgH rearrangements. Although we found some minor differences in disease characteristics between both groups, these did not translate into a significantly different overall survival. Our findings suggest that true biclonal cases of CLL are rare.     

Galectin-3 is not useful in thyroid FNA

INTRODUCTION: Previous studies have suggested that galectin-3 immunohistochemistry may be useful in the fine needle aspiration (FNA) diagnosis of thyroid carcinoma as it has been reported to selectively stain carcinomas and not adenomas or goitres. METHODS: Fifty-one patients were included in a prospective study of galectin-3 in thyroid FNA; 88.2% were female and 11.8% male, mean age 53 years, range 25-87 years. Cell blocks were prepared and stained for galectin-3 if any cells were present in needle washings from the respective FNAs. RESULTS: Twelve of 51 (23.5%) of cell blocks contained epithelial cells. One benign and one inadequate FNA were negative for galectin-3 staining. One of five non-diagnostic FNA cases, a papillary carcinoma on final histology showed positive staining. Four follicular neoplasm/suspicious of carcinoma cases showed negative staining. One malignant FNA case, a papillary carcinoma showed positive staining with galectin-3 but three further carcinomas, two papillary and one follicular were galectin-3 negative. CONCLUSION: Galectin-3 immunohistochemistry does not appear to be a useful adjunct to diagnosis in thyroid FNA as it does not reliably distinguish malignant and benign lesions. Many thyroid aspirates are of low cellularity and are not suitable for cell block immunohistochemistry.     

An approach to diagnosis of T-cell lymphoproliferative disorders by flow cytometry

T-cell lymphoproliferative disorders are among the most challenging diagnoses in hematopathology. Unlike the more common B-cell disorders, in which clonality is often readily discernible by surface immunoglobulin light chain restriction, there is no specific immunophenotypic signature that is diagnostic of a clonal T-cell population. Immunophenotypic criteria that are helpful in the diagnosis of T-cell neoplasms include T-cell subset antigen restriction, anomalous T-cell subset antigen expression, deletion or diminution of one of the pan T-cell antigens, a precursor T-cell phenotype, and expression of additional markers (e.g., CD30, CD20, major myeloid antigens, and TCRgammadelta). Analysis of the inherent forward and orthogonal light scatter properties of the cell can also provide important diagnostic clues. None of these features is 100% specific, however, for aberrant expression of pan-T antigens may be seen in viral infections, B-cell malignancies, or in reactive changes following administration of certain medications. An increased CD4:CD8 ratio is often observed in Hodgkin's lymphoma. Based on the analysis of 87 neoplastic and 80 control cases, we conclude that flow cytometric features that are most suspicious for malignancy include the loss or markedly dim expression of CD45; complete loss of one or more pan-T antigens; diminished expression of more than two pan-T antigens in conjunction with altered light scatter properties; and CD4/CD8 dual-positive or dual-negative expression (except thymic lesions).     

Optimized multiplex IgH/ras PCR: a tool for quantitative monitoring of B-lymphoproliferative disorders

The use of quantitative PCR is recommended to monitor the level of residual hematological malignancies. The proposed multiplex IgH/ras PCR uses a co-amplification of the clonal CDR3 rearrangement of the immunoglobulin heavy chain gene (IgH) as a disease marker and a segment of the Hras 1 gene containing codon 61 (ras) as a control gene. Serial dilutions of stored diagnostic DNAs are examined together in the same PCR at a sub-plateau phase and, after analysis by densitometry, the amount of CDR3 product is related to the ras product. An increase of this ratio at comparable amounts of DNA is viewed as an increase of malignant cells. This endpoint PCR quantifying approach appears to be applicable in monitoring B-lymphoproliferative disorders as was shown to be true in B-cell non-Hodgkin's lymphoma and may provide information on disease activity and treatment outcome.     

Minimal peripheral blood cells carrying clonal markers of B cell disorders: evidence for monoclonality of circulating lymphocytes in patients with multiple myeloma

Peripheral blood lymphocytes (PBL) of 20 patients with multiple myeloma (MM) were assayed for clonality by Southern blot and cell surface marker analysis. Eight samples showed monoclonal origin of circulating lymphocytes by demonstrating rearrangements of the heavy chain immunoglobulin gene (IgH). In selected experiments, comparison of IgH rearrangements of bone marrow plasma cells and peripheral blood-derived mononuclear cells, highly enriched for B lymphocytes, proved to be identical. However, monoclonal circulating cells could not be detected in samples with rearranged IgH genes by surface marker phenotyping using one-color immunofluorescence analysis and a panel of monoclonal and polyclonal antibodies to various B lineage-associated antigens. These results indicate that in a substantial proportion of MM, monoclonal growth involves circulating B lymphocytes and underscores the clinical usefulness of Southern analysis of IgH gene rearrangements for monitoring this disease.