CYTOPATHOLOGY, Issue 2006
M. Salto-Tellez
Molecular diagnosis is the application of molecular biology techniques and knowledge of the molecular mechanisms of disease to diagnosis, prognostication and treatment of diseases. Molecular Diagnosis is, arguably, the fastest growing area of diagnostic medicine. The US market for molecular testing generated $1.3 billion in 2000, which was predicted to increase to about $4.2 billion by 2007.1 We proposed the term Diagnostic Molecular Cytopathology to define the application of molecular diagnosis to cytopathology2. Diagnostic Molecular Cytopathology is essential for the following reasons: (i) Molecular testing is sometimes indispensable to establish an unequivocal diagnosis on cell preparations; (ii) Molecular testing provides extra information on the prognosis or therapy of diseases diagnosed by conventional cytology; (iii) Molecular testing provides genetic information on the inherited nature of diseases that can be directly investigated in cytology samples, by either exfoliation or by fine needle aspiration; (iv) Sometimes the cytopathology sample is the most convenient (or the only available) source of material for molecular testing; (v). Direct molecular interrogation of cells allows for a diagnostic correlation that would otherwise not be possible. Parallel to this direct diagnostic implication, cytopathology is increasing important in the validation of biomarkers for specific diseases, and in therefore of significant importance in the overall translational research strategies. We illustrate its application in some of the main areas of oncology molecular testing, such as molecular fingerprinting of neoplasms,3 lymphoreticular diseases,2 sarcomas4 and lung cancer,5 as well as translational research using diagnostic cytopathology techniques. The next years will see the consolidation of Diagnostic Molecular Cytopathology, a process that will lead to a change of many paradigms. In general, diagnostic pathology departments will have to reorganize molecular testing to pursue a cost-efficient operation. Sample preparation will have to take into account optimal preservation of nuclear acids. The training of technical staff and the level of laboratory quality control and quality assurance would have to follow strict clinical (not research) laboratory parameters. And, most importantly, those pathologists undertaking molecular diagnosis as a discipline would have to develop their professional expertise within the same framework of fellowships and professional credentials that is offered in other sub-specialties. The price to pay if this effort is not undertaken is too important for the future of diagnostic pathology in general. The increasing characterization of molecular biomarkers with diagnostic, prognostic or therapeutic value is making the analysis of tissue and cell samples prior to treatment a more complex exercise. If cytopathologists and histopathologists allow others to take charge of molecular diagnosis, our overall contribution to the diagnostic process will be diminished. We may not become less important, but we may become less relevant. However, those within the discipline of diagnostic pathology who can combine the clinical background of diseases with the morphological, immunocytochemical and molecular diagnostic interpretation will represent bona fide diagnostic specialists. Such ,molecular cytopathologists' would place themselves at the centre of clinical decision-making. Reference:, 1. Liz Fletcher. Roche leads molecular diagnostics charge. Nature Biotechnol 20, 6,7; 2002 2. Salto-Tellez M and Koay ESC. Molecular Diagnostic Cytopathology - Definitions, Scope and Clinical Utility. Cytopathology 2004; 15:252,255 3. Salto-Tellez M, Zhang D, Chiu LL, Wang SC, Nilsson B, and Koay ESC. Immunocytochemistry Versus Molecular Fingerprinting of Metastases. Cytopathology, 2003 Aug; 14(4):186,90. 4. Chiu LL, Koay SCE, Chan NL and Salto-Tellez M. Molecular Cytopathology: Sequencing of the EWS-WT1 Gene Fusion Transcript in the Peritoneal Effusion of a Patient with Desmoplastic Small Round Cell Tumour. Diagnostic Cytopathology, 2003 Dec; 29(6): 341,3. 5. TM Chin, D Anuar, R Soo, M Salto-Tellez, WQ Li, B Ahmad, SC Lee, BC Goh, K Kawakami, A Segal, B Iacopetta, R Soong. Sensitive and Cost-Effective deptection of epidermal growth factor Receptor Mutations in Small Biopsies by denaturing High Performance Liquid Chromatography. (In press). [source]

NON-GYNAECOLOGICAL CYTOLOGY: THE CLINICIAN'S VIEW

CYTOPATHOLOGY, Issue 2006
I. Penman
There is increased recognition of the importance of accurate staging of malignancies of the GI tract and lung, greater use of neoadjuvant therapies and more protocol-driven management. This is particularly important where regional lymph node involvement significantly impacts on curability. Multidetector CT and PET scanning have resulted in greater detection of potential abnormalities which, if positive for malignancy, would change management. There is also a greater recognition that many enlarged nodes may be inflammatory and that size criteria alone are unreliable in determining involvement. In other situations, especially pancreatic masses, not all represent carcinoma as focal chronic pancreatitis, autoimmune pancreatitis etc can catch out the unwary. A preoperative tissue diagnosis is essential and even if unresectable, oncologists are increasingly reluctant to initiate chemotherapy or enroll patients into trials without this. The approach to obtaining tissue is often hampered by the small size or relative inaccessibility of lesions by percutaneous approaches. As such novel techniques such as endoscopic ultrasound (EUS) guided FNA have been developed. A 120cm needle is passed through the instrument and, under real-time visualisation, through the gastrointestinal wall to sample adjacent lymph nodes or masses. Multiple studies have demonstrated the safety and performance of this technique. In oesophageal cancer, confirmation of node positivity by has a major negative influence on curative resection rates and will often lead to a decision to use neoadjuvant chemotherapy or a non-operative approach. Sampling of lymph nodes at the true coeliac axis upstages the patient to M1a status (stage IV) disease and makes the patient incurable. In NSCLC, subcarinal lymph nodes are frequently present but may be inflammatory. If positive these represent N2 (stage IIIA) disease and in most centres again makes the patient inoperable. Access to these lymph nodes would otherwise require mediastinosocopy whereas this can be done simply, safely and quickly by EUS. Overall the sensitivity for EUS , FNA of mediastinal or upper abdominal lymph nodes is 83,90% with an accuracy of 80,90%. In pancreatic cancer performance is less good but pooled analysis of published studies indicates a sensitivity of 85% and accuracy of 88%. In a recent spin-off from EUS, endobronchial ultrasound (EBUS) instruments have been developed and the ability to sample anterior mediastinal nodes has been demonstrated. It is likely that this EBUS , FNA technique will become increasingly utilised and may replace mediastinoscopy. The development of techniques such as EUS and EBUS to allow FNA sampling of lesions has increased the role of non-gynaecological cytology significantly in recent years. Cytology therefore remains important for a broad range of specialties and there is ongoing need for careful and close co-operation between cytologists and clinicians in these specialties. References:, 1. Williams DB, Sahai AV, Aabakken L, Penman ID, van Velse A, Webb J et al. Endoscopic ultrasound guided fine needle aspiration biopsy: a large single centre experience. Gut. 1999; 44: 720,6. 2. Silvestri GA, Hoffman BJ, Bhutani MS et al. Endoscopic ultrasound with fine-needle aspiration in the diagnosis and staging of lung cancer. Ann Thorac Surg 1996; 61: 1441,6. 3. Rintoul RC, Skwarski KM, Murchison JT, Wallace WA, Walker WS, Penman ID. Endobronchial and endoscopic ultrasound real-time fine-needle aspiration staging of the mediastinum ). Eur Resp J 2005; 25: 1,6. [source]

O-12 BLAND DYSKARYOSIS: A NEW PITFALL IN THINPREP® LIQUID BASED CYTOLOGY

CYTOPATHOLOGY, Issue 2006
M. A. Lynch
Liquid based cytology (LBC) has improved cell visualization and preservation in cervical cytology. There has been a reduction in inadequate rate and some data to suggest an increase in sensitivity for dyskaryosis. Training for LBC has focused on differences in distribution of abnormal cells, but in most cases the morphological appearance of the dyskaryotic cells themselves is similar to that seen in conventional cytology. We are describing a new presentation of dyskaryosis which may be a cause of false negative cytology. We have referred to this as ,Bland dyskaryosis' because cells appear deceptively bland on low power examination, and can be misinterpreted as metaplastic or endocervical cells. Bland dyskaryosis cells are seen in groups. The architecture of the group is very disorganized, and adjacent cells show variation in size. Cells have a high nuclear/cytoplasmic ratio and smooth nuclear membranes. Chromatin is finely granular and evenly distributed. This is an unusual presentation of high-grade dyskaryosis and we feel that there is a learning curve in laboratories converting to liquid based cytology. The spectrum of appearances of squamous dyskaryosis needs to be delineated to allow further increases in sensitivity for dyskaryosis. [source]

O-13 ENDOMETRIAL CARCINOMA DETECTED WITH SUREPATH LIQUID BASED CERVICAL CYTOLOGY: COMPARISON WITH CONVENTIONAL CERVICAL CYTOLOGY

CYTOPATHOLOGY, Issue 2006
C. J. Patel
Introduction:, Conventional Pap Smear (CPS) has had little impact on the detection of endometrial carcinoma (MC). Although Liquid Based Cytology (LBC) is replacing CPS in the UK, experience with identification of endometrial cancers with this is limited. A few studies of ThinPrep LBC show promise with reported increased detection rate, but to date, there has been no reported study of detection with SurePath LBC. Aim:, The purpose of this 2-year retrospective study was to compare the accuracy of the SurePath LBC with that of conventional smear in detecting endometrial cancers. Methods:, Our study group consisted of all SurePath cases of endometrial atypia/carcinoma diagnosed between 1st Jan 2004 and 31st Dec 2005, following 100% conversion of our laboratory to the SurePath system in 2001. Conventional smears reported over a 6-year period (1993,1998), comprised the control group. Histological follow up was obtained. Results:, Endometrial lesions were reported in 95 (0.07%) of 130352 SurePath LBC smears. These included 70 (0.053%) reports of endometrial atypia, 05 (0.003%) suspicious and 20 (0.015%) diagnostic of endometrial carcinoma. A total of 58 (0.014%) cases of 409495 CPS were diagnosed as endometrial carcinoma. Adequate histological follow up was available in 47 (49.5%) SurePath LBC and 52 (89.6%) conventional cases. In these, the positive predictive value (PPV) for endometrial carcinoma of SurePath LBC was 73.3% compared to 55.4% of CPS. The PPV for endometrial carcinoma of the atypical and suspicious LBC categories was 14.3% and 40% respectively. No categorisation as atypical or suspicious in the conventional study was available for comparison. The sensitivity of the SurePath LBC, calculated from retrograde analysis of histologically diagnosed endometrial cancers during the same period was 40%. Conclusion:, The SurePath LBC is at least an as accurate and sensitive method for detecting endometrial cancer as CPS. [source]

P-11 THE IMPACT OF INTRODUCING LIQUID BASED CYTOLOGY INTO A ROUTINE SCREENING LABORATORY

CYTOPATHOLOGY, Issue 2006
L. Gregory
With the exception of information from the liquid based cytology (LBC) pilot site there has been limited data to date on the impact of the introduction of SurePathÔ LBC in the NHSCSP. We will present data to show the impact on a medium sized laboratory (42 000 requests per annum) over the first phases of rollout. Data from before, during and post conversion, a period of 28 months, shows the following trends: (1) A significant fall in the inadequate rate (2) A slight decrease in the borderline / mild dyskaryosis rate (3) A small increase in the high-grade squamous dyskaryosis reporting rate (4) An increase in both the number and specificity of glandular lesions reported (5) A largely unchanged positive predictive value for high-grade abnormalities (6) A significant increase in laboratory productivity, unrelated to inadequate rate but through increased speed of screening. Although our experience is not directly comparable to the experience of the LBC pilot, our observations may well reflect that we were fortunate to be trained by staff from one of the LBC sites and thereby benefited directly from their experience. [source]

IS IT REALITY OR AN ILLUSION THAT LIQUID-BASED CYTOLOGY IS BETTER THAN CONVENTIONAL CERVICAL SMEARS?

CYTOPATHOLOGY, Issue 2 2002
Robin Moseley
No abstract is available for this article. [source]

IS IT REALITY OR AN ILLUSION THAT LIQUID-BASED CYTOLOGY IS BETTER THAN CONVENTIONAL CERVICAL SMEARS?

CYTOPATHOLOGY, Issue 1 2002
J. Linder
No abstract is available for this article. [source]

THREE CASES OF NODULAR FASCIITIS: PRIMARY DIAGNOSES BY FINE NEEDLE ASPIRATION CYTOLOGY

CYTOPATHOLOGY, Issue 5 2001
O. Aydin
No abstract is available for this article. [source]

PROBLEMS WITH AUDIT IN CERVICAL CYTOLOGY

CYTOPATHOLOGY, Issue 3 2000
C. Womack
[source]

THE IMPACT OF SYNOPTIC CYTOLOGY REPORTING ON FINE-NEEDLE ASPIRATION CYTOLOGY OF THYROID NODULES

ANZ JOURNAL OF SURGERY, Issue 11 2007
Cyril J. L. Tsan
Background: Fine-needle aspiration cytology (FNAC) is integral to the diagnosis and management of patients with thyroid nodules. We introduced synoptic cytology reporting for thyroid nodules in 2004. The aim of this study was to examine the effect of synoptic cytology reporting in our institution. Methods: A comparative study of two 2-year periods (1 August 2002 to 1 August 2004 and 2 August 2004 to 2 August 2006) before and after the introduction of synoptic reporting was conducted from a prospectively collected database of patients presented with thyroid nodules. The only change during these periods was the format of FNAC reporting. We used the same radiological practice and cytopathology service throughout the study period. All patients are still being followed up. Results: There were a total of 660 patients. Of these, 376 were operated and 284 non-operated. The female to male ratio was 7:1. Comparing the two periods, the overall FNAC sensitivities were 60% versus 79.1%; specificities, 83.7% versus 79.4%; accuracy, 76% versus 79.3%; false-positive result, 16.3% versus 20.6% and false-negative result, 40% versus 20.9%. The non-diagnostic rates were 7.4% versus 3.15%. FNAC prompted surgery in 66.7% versus 100% in carcinoma and 56.4% versus 73.6% in adenoma. A benign FNAC prompted surgery in 15% versus 19.8% of cases. There was no thyroid cancer detected in the current follow up. Conclusions: Synoptic cytology reporting has resulted in an overall improvement in all measures of the tests. It is a simple and effective tool to use. Synoptic cytology reporting is therefore recommended for all endocrine surgical units. [source]

SHOULD MOLECULAR TECHNOLOGY REPLACE URINE CYTOLOGY?

BJU INTERNATIONAL, Issue 10 2008
Vijay K. Sangar
No abstract is available for this article. [source]

Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow

CYTOMETRY, Issue 6 2006
Elena A. Jones
Abstract Background: To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony-forming unit-fibroblast (CFU-F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry-based methods for their evaluation. Methods: Pre-enrichment with D7-FIB-conjugated microbeads, cell sorting for CD45lowD7-FIB+LNGFR+ cells, and CFU-F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three-color flow cytometry following pre-enrichment or by direct four-color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases. Results: In normal BM, a significant correlation was found between the frequencies of CFU-Fs and CD45lowD7-FIB+LNGFR+ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, ,15% of these cells were clonogenic. The same cells were enriched using LNGFR-based positive selection, CD45/Glycophorin A-based depletion, or plastic adherence. CD45lowD7-FIB+LNGFR+ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45lowD7-FIB+LNGFR+ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age-matched controls (n = 26 and n = 31, respectively). Conclusions: This study demonstrates the usefulness of flow cytometry-based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease. © 2006 International Society for Analytical Cytology [source]

A European interlaboratory testing of three well-known procedures for immunocytochemical detection of epithelial cells in bone marrow.

CYTOMETRY, Issue 6 2006
Results from analysis of normal bone marrow
Abstract Background: This investigation intended to study the unspecific background to be expected in normal bone marrow (BM), comparing three well recognized protocols for immunocytochemical detection of disseminated carcinoma cells. The interlaboratory variation in screening and evaluation of stained cells was analyzed and different screening methods were compared. Methods: BM mononuclear cells (BM MNC) from 48 normal BMs were immunostained in parallel by three participating laboratories. The protocols, based on three different anti-cytokeratin antibodies, have all been in common use for detection of disseminated carcinoma cells: the A45-B/B3 protocol (Hamburg), the CK2 protocol (Augsburg) and the AE1AE3 protocol (Oslo). For all protocols, the immunostained cells were visualized by the same alkaline-phosphatase (AP) detection system (APAAP) followed by detection of the cells by manual screening and by two different automated screening systems (ACIS from Chromavision and MDS1 from Applied Imaging). Detected AP-visualized cells were morphologically classified into unambiguous hematopoietic (Uhc) and questionable cells (Qc, potentially interpreted as tumor cells). Results: Seven of 48 BMs (15%) harbored ,1 AP-visualized cell(s) among 1 × 106 BM MNC, both for the A45-B/B3- and for the AE1AE3 protocol, while for CK2 a higher proportion of BMs (21 BMs; 44%) harbored AP-visualized cells (P < 0.01, McNemar's test). The number of Qc was, for all protocols, 1 log lower than the total number of AP-visualized cells. On average, the frequency of Qc was 0.04, 0.08, and 0.02 per 106 BM MNC with A45-B/B3, CK2 and AE1AE3, respectively, and the number of Qc-positive BMs 1, 4, and 1. The MDS1 screening sensitivity was similar to manual screening, while ACIS detected fewer cells (P < 0.001, McNemar's test). Conclusions: All protocols resulted in AP-visualization of occasional hematopoietic cells. However, morphological classification brings the specificity to a satisfactory high level. Approximately 10% of AP-visualized cells were categorized "questionable". The CK2 protocol turned out less specific than the A45-B/B3 and AE1AE3 protocols. © 2006 International Society for Analytical Cytology. [source]

Routine immunophenotyping in acute leukemia: Role in lineage assignment and reassignment

CYTOMETRY, Issue 5 2006
Misbah Qadir
Abstract Diagnostic evaluation of acute leukemia at Roswell Park Cancer Institute has routinely included immunophenotyping by multiparameter flow cytometry. In a retrospective analysis of 646 cases, morphology and cytochemistry established lineage in 612, but not in 34 (5%), of which 26, 5, and 3 were myeloid, undifferentiated, and lymphoid, respectively, based on immunophenotyping. In addition, immunophenotyping changed the lineage assigned based on morphology and cytochemistry in 11 cases (2%); 8 changed from lymphoid to myeloid, and 3 from myeloid to lymphoid. The data support routine inclusion of at least limited immunophenotyping in the diagnostic evaluation of acute leukemia. © 2006 International Society for Analytical Cytology [source]

Flow cytometry for ZAP-70: New colors for chronic lymphocytic leukemia

CYTOMETRY, Issue 4 2006
Adrian Wiestner
Abstract ZAP-70 has become one of the most studied prognostic markers in Chronic Lymphocytic Leukemia (CLL). ZAP-70 is remarkable in many ways: ZAP-70 has been identified as the best discriminating gene between prognostically distinct CLL subtypes using large scale gene expression profiling; ZAP-70 has been shown to enhance signal transduction in CLL B-cells and therefore could contribute to disease progression; and ZAP-70 is one of the rare examples of an intracellular target considered for clinical flow cytometry. This issue attests to the enormous effort and the steady progress made in overcoming technical challenges of testing for ZAP-70 expression and sets the foundation for a successful translation of this important marker into clinical practice. Despite the best effort, one will likely have to accept that not all cases can be clearly assigned to one or the other group, given that ZAP-70 expression between CLL patients falls along a continuum from absent to high. Nevertheless, ZAP-70 expression could become a key parameter to guide patients towards risk adapted treatment strategies in prospective clinical trials. © 2006 International Society for Analytical Cytology [source]

Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLL

CYTOMETRY, Issue 4 2006
Mark Shenkin
Abstract Background: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity. Methods: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS. Results: Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling. Conclusions: Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. © 2006 International Society for Analytical Cytology [source]

An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia

CYTOMETRY, Issue 4 2006
T. Vincent Shankey
Abstract Background: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. Methods: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4,17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. Results: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. Conclusions: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory. © 2006 International Society for Analytical Cytology [source]

Comparison of bone marrow and peripheral blood ZAP-70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia

CYTOMETRY, Issue 4 2006
Rachel Sheridan
Abstract Background: The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. Methods: We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19+ B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t -test. Results: A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. Conclusions: These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. © 2006 International Society for Analytical Cytology [source]

Remission induction chemotherapy induces in vivo caspase-dependent apoptosis in bone marrow acute myeloid leukemia blast cells and spares lymphocytes

CYTOMETRY, Issue 3 2006
J.-P. Vial
Abstract Background The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response. Methods We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment. Results We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk-VAD-FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk-VAD-FITC, in the d15 residual bone marrow blast cells, correlated with lower disease-free survival probability. Conclusion More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction. © 2006 International Society for Analytical Cytology [source]

Unsupervised immunophenotypic profiling of chronic lymphocytic leukemia

CYTOMETRY, Issue 3 2006
Luzette K. Habib
Abstract Background Proteomics and functional genomics have revolutionized approaches to disease classification. Like proteomics, flow cytometry (FCM) assesses concurrent expression of many proteins, with the advantage of using intact cells that may be differentially selected during analysis. However, FCM has generally been used for incremental marker validation or construction of predictive models based on known patterns, rather than as a tool for unsupervised class discovery. We undertook a retrospective analysis of clinical FCM data to assess the feasibility of a cell-based proteomic approach to FCM by unsupervised cluster analysis. Methods Multicolor FCM data on peripheral blood (PB) and bone marrow (BM) lymphocytes from 140 consecutive patients with B-cell chronic lymphoproliferative disorders (LPDs), including 81 chronic lymphocytic leukemia (CLLs), were studied. Expression was normalized for CD19 totals, and recorded for 10 additional B-cell markers. Data were subjected to hierarchical cluster analysis using complete linkage by Pearson's correlation. Analysis of CLL in PB samples (n = 63) discovered three major clusters. One cluster (14 patients) was skewed toward "atypical" CLL and was characterized by high CD20, CD22, FMC7, and light chain, and low CD23. The remaining two clusters consisted almost entirely (48/49) of cases recorded as typical BCLL. The smaller "typical" BCLL cluster differed from the larger cluster by high CD38 (P = 0.001), low CD20 (P = 0.001), and low CD23 (P = 0.016). These two typical BCLL clusters showed a trend toward a difference in survival (P = 0.1090). Statistically significant cluster stability was demonstrated by expanding the dataset to include BM samples, and by using a method of random sampling with replacement. Conclusions This study supports the concept that unsupervised immunophenotypic profiling of FCM data can yield reproducible subtypes of lymphoma/chronic leukemia. Expanded studies are warranted in the use of FCM as an unsupervised class discovery tool, akin to other proteomic methods, rather than as a validation tool. © 2006 International Society for Analytical Cytology [source]

JC-1, a sensitive probe for a simultaneous detection of P-glycoprotein activity and apoptosis in leukemic cells

CYTOMETRY, Issue 3 2006
Driss Chaoui
Abstract Background JC-1 probe has been successfully used for the analysis of either apoptosis or P-glycoprotein (P-gp) activity. Therefore, we wanted to see if JC-1 could also simultaneously assess both, P-gp activity and apoptosis, in acute myeloid leukemia (AML) cells. Methods P-gp activity was measured using JC-1 and compared to the results of the Rhodamine 123 (Rh 123) assay in P-gp negative and P-gp positive cell lines, and 12 AML samples. For apoptosis, spontaneous apoptosis, as well as, apoptosis induced by Cytosine Arabinosine and Homoharringtonine were analyzed. Both mitochondrial red fluorescence and cytoplasmic green fluorescence of JC-1 with and without a P-gp inhibitor (Cyclosporine A : CsA) were used for the identification of apoptotic cells, and this was compared to Annexin V/PI staining. Results (1) We found a good correlation between JC-1 and Rh 123 in viable cells. Even in a small population of viable cells, P-gp positive cells emitting low red fluorescence, gained on red fluorescence after P-gp inhibition with CsA permitting an evaluation of P-gp activity. (2) We found a good correlation between the Annexin V/PI staining and JC-1 (P < 0.0001) in the assessment of apoptotic cells. Most importantly, the apoptotic cells could be distinguished by the loss of red fluorescence and the increase of green fluorescence without any change after P-gp inhibition with CsA. Conclusions JC-1 can simultaneously evaluate two important parameters involved in drug resistance in AML cells, P-gp activity and apoptosis. © 2006 International Society for Analytical Cytology [source]

Flow cytometric measurement of circulating endothelial cells: The effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations

CYTOMETRY, Issue 2 2006
Rebecca Gusic Shaffer
Abstract Background: Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition, numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD), we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs), EPCs, and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset. Methods: Blood was collected from young healthy subjects (N = 9, mean age 33 ± 8 years), older healthy subjects (N = 13, mean age 66 ± 8 years), and older subjects with PAD (N = 15, mean age 69 ± 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later. Results: Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings. Conclusions: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status. © 2005 International Society for Analytical Cytology [source]

Increased immature hematopoietic progenitor cells CD34+/CD38dim in myelodysplasia

CYTOMETRY, Issue 2 2006
Mariela B. Monreal
Abstract Background Myelodysplastic syndromes (MDS) are clonal disorders affecting hematopoietic progenitor cells (HPC). Despite the relevance of clonal CD34+ cells in developing MDS, only few studies analyze the phenotype of this cell population. The aim of this study was to evaluate phenotypic changes on HPC in MDS that could reflect abnormalities in the differentiation process of stem cells. Methods We analyzed the expression of CD38 and HLA-DR on CD34+ cells by flow cytometry in 36 patients with MDS, as well as in healthy donors (n = 12) and patients with other hematological disorders: non-Hodgkin lymphomas and multiple myeloma, both in complete remission (CR) (n = 32); acute lymphoblastic leukemia in CR (n = 17); de novo acute myeloblastic leukemia (AML) at diagnosis (n = 22) and in CR (n = 37); and AML secondary to MDS at diagnosis (n = 19). Cases with available karyotype were grouped according to the International Prognostic Scoring System (IPSS). Results Compared to normal BM, the fraction of immature HPC, characterized as CD34+bright, intermediate FSC/SSC, and CD38dim, was significantly increased in high risk MDS and secondary AML, but not in low risk MDS, (P , 0.001, P = 0.03, and P = 0.7). De novo AML showed decreased immature HPC. High numbers of immature HPC correlated with higher IPSS risk groups (P = 0.05) and showed significant impact on disease progression (P = 0.03). Conclusion Our study confirms that evaluation of CD38 expression pattern on HPC is an easy and reproducible test that allows evaluating the immature subset of progenitor cells. Increased immature HPC in high risk MDS and secondary AML may reflect blocked differentiation of CD34+ cells in these diseases. © 2006 International Society for Analytical Cytology [source]

The BSCC Code of Practice , exfoliative cytopathology (excluding gynaecological cytopathology)

CYTOPATHOLOGY, Issue 4 2009
A. Chandra
Exfoliative cytopathology (often referred to as non-gynaecological cytology) is an important part of the workload of all diagnostic pathology departments. It clearly has a role in the diagnosis of neoplastic disease but its role in establishing non-neoplastic diagnoses should also be recognised. Ancillary tests may be required to establish a definitive diagnosis. Clinical and scientific teamwork is essential to establish an effective cytology service and staffing levels should be sufficient to support preparation, prescreening, on-site adequacy assessment and reporting of samples as appropriate. Routine clinical audit and histology/cytology correlation should be in place as quality control of a cytology service. Cytology staff should be involved in multidisciplinary meetings and appropriate professional networks. Laboratories should have an effective quality management system conforming to the requirements of a recognised accreditation scheme such as Clinical Pathology Accreditation (UK) Ltd. Consultant pathologists should sign out the majority of exfoliative cytology cases. Where specimens are reported by experienced biomedical scientists (BMS), referred to as cytotechnologists outside the UK, this must only be when adequate training has been given and be defined in agreed written local protocols. An educational basis for formalising the role of the BMS in exfoliative cytopathology is provided by the Diploma of Expert Practice in Non-gynaecological Cytology offered by the Institute of Biomedical Science (IBMS). The reliability of cytological diagnoses is dependent on the quality of the specimen provided and the quality of the preparations produced. The laboratory should provide feedback and written guidance on specimen procurement. Specimen processing should be by appropriately trained, competent staff with appropriate quality control. Microscopic examination of preparations by BMS should be encouraged wherever possible. Specific guidance is provided on the clinical role, specimen procurement, preparation and suitable staining techniques for urine, sputum, semen, serous cavity effusion, cerebrospinal fluid, synovial fluid, cyst aspirates, endoscopic specimens, and skin and mucosal scrapes. [source]

Cytological diagnosis of basal cell carcinoma and actinic keratosis, using Papanicolaou and May,Grünwald,Giemsa stained cutaneous tissue smear

CYTOPATHOLOGY, Issue 5 2008
E. Christensen
Objective:, Cytology may become the diagnostic method of choice with the advent of new non-invasive treatments for non-melanoma skin cancer, as the sampling technique for cytology entails little tissue disfiguration. The aim of this study was to compare and evaluate the diagnostic performance of scrape cytology using two different cytological staining techniques, and to evaluate additional touch imprint cytology, with that of histopathology of basal cell carcinoma (BCC) and actinic keratosis (AK). Methods:, We investigated 50 BCC and 28 AK histologically verified lesions, from 41 and 25 patients, respectively. Two separate skin scrape samples and one touch imprint sample were taken from each lesion. The smears were stained with Papanicolaou (Pap) or May,Grünwald,Giemsa (MGG) stains. All cytological specimens were examined in random order by pathologists without knowledge of the histology. Cytodiagnostic results were compared with the histopathological report. Results:, Scrape cytodiagnosis agreed with histopathology in 48 (Pap) and 47 (MGG) of the 50 BCC cases, and in 26 of 28 (Pap) and 21 of 26 (MGG) AK cases, yielding sensitivities of 96%, 94%, 93% and 81%, respectively. No significant difference in sensitivity between the two staining methods was found but a trend towards higher Pap sensitivity for AK was noted (P = 0.10). Touch imprint cytology confirmed histopathology in 38 of the 77 cases of BCC and AK. Conclusion:, Cytological diagnosis with either Pap or MGG stain for BCC and AK is reliable, and differentiates well between BCC and AK. Imprint cytology proved to be non-diagnostic in half of the examined cases. [source]

Diagnostic accuracy of cytology and immunocytology in carcinomatous effusions

CYTOPATHOLOGY, Issue 4 2008
G. Metzgeroth
Background:, Immunocytology substantially improves the diagnostic accuracy of conventional cytology in the diagnosis of carcinomatous effusions. Due to the unequivocal characterization of the various cell populations, a sensitivity of 92% and specificity of 100% was achieved by immunocytology, examining samples of 1234 serous effusions. Objective:, Cytology plays a central role in the aetiological clarification of serous effusions. The sensitivity of this method for the diagnosis of carcinomatous effusions varies between 40% and 80%. The aim of the present study was to investigate whether immunocytology substantially improves the diagnostic quality of the cytological examination in the diagnosis of carcinomatous effusions. Method:, Consecutive serous effusions were examined by conventional cytology and by immunocytology. The immunocytological examination was performed on smears, using a standard panel of three antibodies against pancytokeratin, human epithelial antigen 125 and calretinin. Results:, Altogether, 1234 effusion samples were examined. A total of 603 effusions were caused by carcinomas, five by malignant mesotheliomas, 11 by malignant lymphomas and 615 by non-malignant disorders. In conventional cytology, carcinomatous effusions were correctly diagnosed in 314 samples, corresponding to a sensitivity of 52%. In 31 specimens (5%) tumour cells without further specification were described and in 161 samples (27%) the presence of tumour cells was suspected (84% overall sensitivity). A total of 97 carcinomatous effusions (16%) were diagnosed false-negatively and 50 (8%) of the 615 non-malignant effusions false-positively (92% specificity). In immunocytology, 561 carcinomatous samples were correctly diagnosed, representing a sensitivity of 93%. In six cases (1%) the presence of tumour cells was suspected. A total of 36 carcinomatous effusions (6%) were diagnosed false-negatively (94% over-all sensitivity). Out of the 615 non-malignant specimens, there were no false-positive diagnoses (100% specificity). Conclusion:, Immunocytology is a simple, cost-effective, routinely practicable method which substantially improves the diagnostic accuracy of conventional cytology in the diagnosis of carcinomatous effusions. Therefore, we recommend the use of immunocytology in all those cases where cytology on its own is not completely unequivocal. [source]

O-11 Proposal for extending the role Of ABMSPS in reporting cervical loops

CYTOPATHOLOGY, Issue 2007
K. Ellis
Introduction:, The advanced biomedical scientist practitioner (ABMSP) in Cervical Cytology was established in the NHS cervical screening programme (NHSCCSP) in 2001 and there are approximately 60 ABMSPs in post. The aim of this study was to explore the potential for further expansion of their role in the NHSCSP by reporting the histology of loop excision biopsies of the cervical transformation zone (LLETZ). Methods:, The initial study included LLETZ specimens from 55 sequential patients, which, according to standard local practice had the diagnosis of CIN confirmed by cervical punch biopsy prior to the procedure. All the cases were independently examined by an ABMSP and a consultant histopathologist and reports complying with the Royal College of pathologists (RCPath) minimum data sets were assembled. The cases were reviewed at the discussion microscope and ABMSP reports were compared to the final reports issued by the histopathologist. Results:, In the preliminary findings, total agreement between ABMSP and consultant histopathologist was reached on just under 90% of cases. Of those cases that did not reach total agreement, none varied by more than one grade. There was agreement on other parameters from the RCPath minimum data sets. Discussion:, Based on our preliminary findings, it appears there may be scope for extending the role of ABMSPs to report LLETZ samples under the supervision of a histopathologist. We plan to increase the number of cases both in our department and through collaboration with other UK centres and to present evidence to the RCPath, with a view to adoption of this role by ABMSPs and development of an appropriate training scheme. [source]

Cytology of epithelioid sarcoma

CYTOPATHOLOGY, Issue 5 2006
I. Yildiz
No abstract is available for this article. [source]