Conventional Cytomorphology Research Articles

LC-1000 flow cytometry system complements intraoperative peritoneal cytology for pancreatic and biliary tract cancer.

The exfoliative cell analyzer, LC-1000, is medical device that utilizes the principles of flow cytometry, and might provide digital diagnostic information for cytology using a different approach from conventional cytomorphology. In this study, wae examined the usefulness of the LC-1000 as a diagnostic support system for intraoperative peritoneal lavage cytology and its prognostic impact for pancreatic (PC) and biliary tract cancer (BTC). Patients with PC and BTC who underwent surgical treatment were included. First, we identified useful indicators of LC-1000 and established cutoff values to discriminate positive cytology. Next, we verified the validity of these cutoff values. In the test set (n = 48), of the LC-1000 indicators examined, only MR-CPIx was significantly different between the negative and positive cytology groups, yielding a cutoff value of 0.86. In the validation set (n = 52), the sensitivity, specificity, positive and negative predictive value of the LC-1000 for cytology results was 1.0, 0.49, 0.11 and 1.0, respectively. In patients who had undergone radical resection, recurrence-free survival rate was significantly higher in the LC-1000 negative group than in the positive group in PC, but not in BTC. The LC-1000 was useful as digital support system for peritoneal cytology, and it might have potential as a prognostic factor for PC.

Issue Highlights — May 2021

Issue Highlights — May 2021

Enumeration of CD34+ blasts by immunohistochemistry in bone marrow biopsies from MDS patients may have significant impact on final WHO classification

The percentage of blasts cells in the bone marrow (BM) of MDS patients is one of the key parameters for MDS classification and for the differential diagnosis with acute myeloid leukemia (AML). Currently, the gold standard to determine the blast percentage is conventional cytomorphology. To assess the possible impact of blast cell enumeration in BM biopsies from MDS patients on the final WHO classification using CD34 immunohistochemistry (IHC) a total of 156 BM samples from MDS and MDS-AML patients were studied and compared to blast counts by cytomorphology (CM). Eighty-nine BM aspirates were also studied by flow cytometry (FCM). Percentages of CD34+ blasts by IHC were determined blindly by two hematopathologists. Automated CD34-cell count was performed in 25 cases. Good overall agreement was found for CM and FCM with respect to critical blast thresholds (5%, 10%, 20%) (p < 0.05). However, in 17% of patients, CD34+ blast counts by IHC were higher as compared to CM with possible impact on MDS subclassification. In 7 of 21 AML patients, diagnosis was established on BM histology, while the blast percentage by CM was below the AML threshold. The assessment of CD34+ cells by IHC showed high interobserver agreement (Spearman R 0.95, p < 0.01), while automated CD34 counts were not optimal due to interference with other cellular and stromal elements. BM histology including CD34 IHC improves the diagnostic accuracy in MDS and AML. The quantification of blast cells should be based on the integration of all three methods for reliable disease classification and risk assessment.

Prognostic value of initial bone marrow disease detection by multiparameter flow cytometry in children with neuroblastoma.

Multicolor flow cytometry (MFC) is widely available, fast and has an easy-to perform approach for finding neuroblastoma (NB) cells among normal bone marrow (BM) hematopoietic cells. Aim of the study was to investigate prognostic significance of initial MFC tumor cells' detection in BM of children with NB. 51 patients (24 boys and 27 girls) aged from 6days to 15years (median age 1year 3months) with NB were included in the study. BM samples at the time of diagnosis were obtained from 2 to 5 aspiration sites per patient. CD45(-)CD56(+)CD81(+)GD2(+)-cells were evaluated by MFC. NB cells were detected in BM by FC more frequently compared to conventional cytomorphology (49.0% and 29.4% patients, respectively, р = 0.043). Patients with NB cells detected in BM by MFC had significantly worse event-free survival and cumulative incidence of relapse/progression [0.24(0.08) and 0.60(0.10), respectively] compared to children with negative result of immunophenotyping [0.85(0.07) and 0.12(0.06), respectively, p < 0.001 in both cases]. BM involvement detection by MFC maintained its prognostic significance in various patients groups. In multivariate analysis, immunophenotyping proved to be an independent prognostic factor when analyzed jointly with other NB risk factors. In 42 patients BM involvement was also studied by RQ-PCR for PHOX2B and TH genes expression. Within groups of patients divided by RQ-PCR positivity, MFC-positivity retained prognostic significance. Thus flow cytometric BM involvement detection has very strong prognostic impact even stronger than RQ-PCR. It could be used in combination with other parameters for the treatment strategy choice in patients with NB.

Five Top Stories in Anatomic Pathology: Stories From the Faculty at UMass Medical School and UMass Memorial Medical Center, Worcester, Massachusetts

This month's issue of the Archives of Pathology & Laboratory Medicine introduces a new series of special sections featuring relevant and contemporary topics in Anatomic Pathology and Cytopathology, herein referred to as “Five Top Stories.”“Five Top Stories” intends to address developments and controversies in our specialty that I hope will be useful to the practicing pathologist. The selected stories are all written under the supervision of the faculty of the Department of Pathology at the University of Massachusetts Medical School, Worcester, Massachusetts. The expressed views and opinions are their own and not those of the Archives. Likewise, any mention of biologic products or instrumentation does not signify endorsement by the Archives. In their paper “Clinical Implications of Current Developments in Genitourinary Pathology,” Zhong Jiang, MD, and collaborators examine the role of fusion in the ETS gene family as it relates to adenocarcinoma of the prostate and the emergence of IMP-3 as a prognostic biomarker in cancers of the kidney and urinary bladder. Dr Jiang et al then go on to discuss the intriguing story of the so-called translocation renal cell carcinoma, the applications of the UroVysion FISH test in urine cytology, and the still timely subject of triple immunostaining for the diagnosis of prostatic cancer.For their part, Andrew Fischer, MD, et al reassess the diagnostic value of conventional cytomorphology in diagnosis and discuss the role of newer technologies, such as endoscopic ultrasound and endoscopic bronchial ultrasound, in quality sampling for cytologic examination. Dr Fischer and colleagues conclude their paper with comments on a new breed of cytopathologist, the “interventional cytopathologist,” and end up providing us with 2 added bonuses, sharing with us their views on thyroid molecular testing and targeting therapies based on cytology samples.All in all, I trust these collected stories will appeal to the pathology community in a constructive and meaningful way. More “Five Top Stories” in other disciplines (breast, endocrine, lung, and hematopathology) will follow in future issues of the Archives.

Flow Cytometric Leukemic Blasts Detection in Cerebrospinal Fuid of Children with Acute Leukemias

Abstract 1449Central nervous system (CNS) involvement is one of the important risk factors in childhood acute leukemia (AL). Tumor cells detection in cerebrospinal fluid (CSF) is one of the main signs of CNS lesion. Traditionally blasts presence in CSF is assessed by conventional cytomorphology (CM) of cytospin slides. However, sensitivity of this method is relatively low. Flow cytometry (FC) having a higher sensitivity could provide better diagnostic applicability for CSF blasts detection. Aim. To compare results of tumor cells detection in CSF of children with AL by flow FC and CM. Methods. 183 samples from 52 boys and 31 girls aged from 5 months to 15 years with different types of acute lymphoblastic leukemia (ALL) (77 patients), acute myeloid leukemia (AML) (5 patients) and acute biphenotypic leukemia (1 patient) were investigated. 17 positive samples obtained by traumatic lumbar puncture were excluded from analysis because tumor blasts were also detected in peripheral blood. Comparison between FC and CM data was performed in 166 samples. Among these samples 61 was taken at the time of initial diagnostics, 34 – during AL follow-up, 17 – at relapse and 54 – during relapse monitoring. Monoclonal antibodies panels were constructed according to immunophenotype of tumor cells in bone marrow. Results. In 24 out of 166 samples (14.5%) tumor cells were detected by CM. In all these cases blasts were also found by FC, while FC allowed finding blasts in other 35 samples. Thus the total number of FC-positive samples was 59 out of 166 (35.5%). This frequency was significantly higher than rate of CM-positive cases (g < 0.0001). Among initial diagnostics samples there were 20 FC-positive and only 10 CM-positive patients (32.8% vs. 16.1%, p=0.0585). At relapse 9 (52.9%) patients were FC-positive, while 6 (35.3%) were CM-positive (p=0.4897). In both B-lineage and T-lineage ALL, analyzed separately, FC detected blasts in CSF frequently than CM (p=0.0098 and p=0.0002 respectively). Absolute blast count in 1 ml in CSF samples, positive by both methods was significantly higher than in samples, positive only by FC (median = 418, range 8–158171 and median = 34, range 5–2762 respectively, g = 0.0002). Thus FC allows detecting tumor cells in CSF much more frequently than conventional CM, which could be explained mainly by higher FC sensitivity. Moreover FC is applicable also for qualitative and quantitative monitoring of CNS lesion. Nevertheless prognostic impact of FC CSF investigation is questionable. Among 13 patients in whom discordant results were obtained in initial diagnostics samples and at relapse, only for one patient risk stratification could have been changed. For all other patients there were other risk factors, that decreased significance of FC leukemic blast detection in CSF. Conclusion. Flow cytometry allows more frequent detection of tumor blasts in CSF of children with AL, while prognostic significance of these findings is still unclear and needs to be confirmed in large prospective trials. Disclosures:No relevant conflicts of interest to declare.

Thyrocyte morphomethric analysis significance in differential diagnosis of thyroid carcinoma

BACKGRAUND/AIM: Conventional cytomorphology of thyroid cell aspirates is limited in preoperative differential diagnosis of follicular adenomas (FA) and hyperplastic adenomatoid nodular goiters from well differentiated thyroid follicular carcinoma (FTC) and follicular variant of thyroid papillary carcinoma (PTC). This is the reason of inaccurate presurgical differential diagnosis and in the same cases of inadequate operative managament. The aim of the study was to evaluate the role of quantitative estimation of thyreocite nuclear features (cariomorphometry) in thyroid aspirated smears in preoperative differential diagnosis of benign from malignant thyroid lesions. A total of 48 patients with thyroid nodular disease underwent fine needle aspiration biopsy for cytomorphology, cariomorphometric analysis of the aspirates, and histopathologic explorations conducted fully postoperatively. On the basis of cytomorphology classification the patients were divided into three groups: benign (B), n = 8; malignant (M), n = 15, and suspicious for malignancy (S), n = 25. Using a microscope connected to a computerized video system, mean nuclear area, the nuclear area coefficient of variation (NACV) and anisocariosis ratio were measured and calculated. In all the 15 patients with cytologically malignant results the diagnosis of PTC was confirmed histopathologically. All cytologically benign lesions were confirmed histopathologically. Thyroid carcinoma was found in 15 out of 25 patients with suspicious lesions. The highest mean values of nuclear area were in the PCT (90.74 +/- 26.71 microm2), and were significantly different from all other groups (p < 0.001). The mean nuclear area in FTC was 69.20 +/- 27.31 microm2 and was significantly higher than in the benign adenomatous group (p < 0.01). There was no significant difference in mean nuclear area between FTC and FA, but there was a significant difference in NACV between these two groups (FTC: 39.46% vs FA: 23.42%,p < 0.001). In 27 out of 30 patients with thyroid carcinoma higher values of NACV than 18% were found. Preoperatively cariomorphometry is a useful method in differential diagnosis of thyroid carcinoma from benign lesions, as a complementary method to convencional cytodiagnostics. The NACV showed highest sensitivity as a parameter of malignant thyroid cell transformation.

Species-Specific Immunocytochemical Localization of Mycobacterium tuberculosis Complex in Fine Needle Aspirates of Tuberculous Lymphadenitis Using Antibody to 38 kDa Immunodominant Protein Antigen

To examine immunocytochemical localization of Mycobacterium tuberculosis (MTB) complex antigen in fine needle aspiration (FNA) smears of tuberculous lymphadenitis (TBLN) using species-specific monoclonal antibody MTSS to 38-kDa immnunodominant protein antigen as a diagnostic adjunct to conventional cytomorphology and its advantage over Ziehl-Neelsen (ZN) microscopy. Study Design FNA smears from 340 cases-174 TBLN; 34 negative controls from nontuberculous, positive controls of 13 known acid-fast bacilli (AFB)-positive sputum smears; 50 blind controls; and 69 other controls (smears from stock cultures of bacterial, atypical mycobacteria and fungal species) were subjected to ZN and immunocytochemical staining using MTSS by the streptavidin-biotin method.Immunocytochemical staining was positive in 59 of 61 (96.7%) archival and 110 of 113 (97.3%) fresh FNA smears; ZN positivity for AFB was observed in 27 of 61 (44.2%) archival and 48 of 113 (42.4%) fresh FNA smears of TBLN.The immunostaining using MTSS showed a definite advantage over conventional ZN staining for detection and specific diagnosis of TBLN in FNA smears with 0% false positive results. Immunostaining of cytosmears with species specific antibody to MTB would prove to be a good diagnostic adjunct to morphologic diagnosis.

Image Cytometry DNA‐Analysis of Fine Needle Aspiration Cytology to Aid Cytomorphology in the Distinction of Branchial Cleft Cyst from Cystic Metastasis of Squamous Cell Carcinoma: A Prospective Study

Frequently, the distinction between branchial cleft cyst and cystic metastases from squamous cell carcinoma is difficult by cytomorphology. In a prospective study, we investigated the need for, and the value of, image cytometry DNA-analysis as a complement to cytologic evaluation of cystic lesions in the neck. Image cytometry DNA-analysis was performed on the fine needle aspiration cytology smears from 50 patients, referred to our department, with a solitary cystic lesion in the lateral region of the neck. Smears from aspirates were Giemsa stained and cytologically evaluated. Ahrens image analysis was used for DNA analysis on smears stained with Schiff reagent, and lymphocytes were used as control cells. Epithelial cells with DNA values exceeding 5c were regarded as aneuploid, indicating malignancy. Nine lesions were diagnosed as squamous cell cancer metastases cytologically. DNA analysis showed aneuploidy in all of them except one. Three of these lesions had earlier been diagnosed as branchial cleft cyst at the referring hospital. Eight lesions were cytologically inconclusive and four of them were revealed as cystic metastasis at histopathologic analysis, and DNA analysis showed aneuploidy in all but one, which could not be analyzed. Two of these lesions were also diagnosed as branchial cleft cysts at the referring hospital. All benign lesions were diploid. Nine lesions were thyroid and salivary gland lesions. Image cytometry DNA-analysis was shown to help in the distinction between benign and malignant cystic lesions. Thus, when conventional cytomorphology does not suffice, DNA-analysis is clearly a valuable supplement.

Detection of Disseminated Tumor Cells in Neuroblastoma: 3 Log Improvement in Sensitivity by Automatic Immunofluorescence plus FISH (AIPF) Analysis Compared with Classical Bone Marrow Cytology

The sensitive detection of bone marrow involvement is crucial for tumor staging at diagnosis and for monitoring of the therapeutic response in the patient's follow-up. In neuroblastoma, only conventional cytomorphological techniques are presently accepted for the detection of bone marrow involvement, yet since the therapeutic consequences of the bone marrow findings may be far-reaching, the need for highly reliable detection methods has become evident. For this purpose, we developed an automatic immunofluorescence plus FISH (AIPF) device which allows the exact quantification of disseminated tumor cells and the genetic verification in critical cases. In this study, the power of the immunofluorescence technique is compared with conventional cytomorphology. 198 samples from 23 neuroblastoma patients (stages 4 and 4s) at diagnosis and during follow-up were investigated. At diagnosis, 45.6% of the samples (26 of 57) which were positive by AIPF investigation were negative by cytomorphology. During follow-up, 74.2% (49 of 66) of AIPF-positive samples showed no cytological signs of tumor cell involvement. False negative morphological results were found in up to 10% of tumor cell content. A tumor cell infiltrate below 0.1% was virtually not detectable by conventional cytomorphology. Using the sensitive immunofluorescence technique, the analysis of only two instead of four puncture sites did not lead to false negative results. Thus, the immunofluorescence technique offers an excellent tool for reliable detection and quantification of disseminated tumor cells at diagnosis and during the course of the disease.

Differential Immunocytochemical Staining Patterns of Uroplakin Observed on Neoplastic and Nonneoplastic Tissue Fragments Obtained from Upper Urinary Tract Brush Specimens

To discern any differences in the distribution of uroplakin expression on neoplastic and nonneoplastic upper urinary tract lesions. Thirty-seven representative 95% ethanol-fixed direct smears of brush specimens, which were subsequently diagnosed histologically as 10 reactive and 27 transitional cell carcinomas (TCCs), were stained with polyclonal uroplakin antibodies utilizing the avidin-biotin-peroxidase method. In order to ascertain any differences in diagnostic accuracy between conventional cytomorphology and uroplakin immunocytochemical staining, the results were compared to the original final cytologic diagnoses for all 37 cases. The linear staining pattern on the luminal surface of umbrella cells was the dominant pattern expressed on tissue fragments from all 10 reactive lesions. Tissue fragments from low grade TCC demonstrated a weaker and less continuous superficial membrane staining pattern along with a variably intense, diffuse, membranous staining pattern throughout the tumor cell groups. This staining pattern was seen in all 17 (sensitivity = 100%) histologically confirmed low grade TCCs, of which only 13 of the 17 (sensitivity = 76.5%) were diagnosed as TCC on the original final cytology report. Tissue fragments from 10 high grade TCCs lacked the superficial linear staining pattern seen in reactive cell groups. Instead, all 10 high grade TCCs displayed a strong diffuse membrane staining pattern in all the cells in the fragment and also demonstrated microluminal structures within the tumor cell groups. The distinctive patterns of uroplakin antigen expression observed in nonneoplastic and neoplastic upper urinary tract lesions in the present study can greatly enhance the accuracy of diagnostic interpretation of upper urinary tract lesions in conventional cytologic specimens.

E-cadherin expression is a sensitive and specific method for detection of carcinoma cells in fluid specimens.

The distinction between reactive mesothelial cells and carcinoma in pleural, peritoneal, and pericardial fluids is often difficult. We and others previously showed that E-cadherin, an epithelial-specific adhesion protein, can be useful for this distinction. In this study we tested the sensitivity and specificity of E-cadherin compared to, and in combination with, conventional cytology for assessment of carcinoma in fluids. Cytyc ThinPreptrade mark slides (Marlborough, MA) from 102 sequential fluids were evaluated for E-cadherin expression by routine immunologic techniques. No evidence of E-cadherin staining was seen in 71 cases, while 31 showed unequivocally positive staining. Sensitivity and specificity were independently determined for E-cadherin alone (72% and 97%, respectively), cytomorphology alone (62% and 100%, respectively), and both together (92% and 100%, respectively). We conclude that assessment of E-cadherin expression has sensitivity and specificity comparable to, or better than, conventional cytomorphology. If both cytomorphology and E-cadherin are used together, a definitive and correct diagnosis could have been made on nearly every case in this study.

Terminal Deoxynucleotidyl Transferase (TdT)-Positive Cells in Cerebrospinal Fluid and Development of Overt CNS Leukemia: A 5-Year Follow-up Study in 113 Children With a TdT-Positive Leukemia or Non-Hodgkin’s Lymphoma

AbstractWe investigated whether an indirect nuclear terminal deoxynucleotidyl transferase (TdT) immunofluorescence (IF) assay on single cells present in the cerebrospinal fluid (CSF) is more effective than conventional cytomorphology for early detection or exclusion of (minimal) meningeal leukemic infiltration in patients with a TdT+ malignancy. During a 5-year follow-up study, 1,661 consecutive CSF samples from 113 children with a TdT+ acute lymphoblastic leukemia (ALL) (n = 100), a TdT+ acute nonlymphoblas- tic leukemia (ANLL) (n = 8), or a TdT+ non-Hodgkin's lymphoma (NHL) (n = 5) were analyzed. In 1,511 (91.9%) of 1,643 evaluable CSF samples, the positive and negative findings of both cytomorphology and the TdT-IF assay were concordant. In 47 (2.9%) samples from 28 patients, the cytomorphology was suspect while the TdT-IF assay was negative; follow-up as long as 58 months revealed no CNS leukemia in any patient. In 85 (5.2%) samples, cytomorphology was negative (n = 70) or suspect (n = 15) but TdT+ cells were detected. RBC contamination seriously hampered evaluation in 31 of these 85 samples. From the remaining 54 TdT+ samples from 29 patients, 40 samples preceded overt CNS leukemia in 20 patients. Two consecutive findings of TdT + cells in the CSF were always followed by overt CNS leukemia. At initial diagnosis, 11 children had TdT+ cells in their RBC-free CSF. In one of these children, morphology was suspect; a repeated lumbar puncture was positive on both assays. Thus, initial CNS leukemia was diagnosed. In the other ten children, morphology was negative. In six of them, CNS leukemia was diagnosed 2 to 20 months later. In 32 other children examined at initial diagnosis, neither TdT* cells nor blasts were observed in the CSF. In none of these patients was a CNS leukemia diagnosed after a follow-up of 2.5 to 57 months (median 24 months). In 207 control CSF samples from 58 children with TdT“ oncologic, hematologic, or infectious diseases, no TdT+ cells could be detected. The TdT-IF assay is easy to perform and is a more reliable diagnostic tool for detection of CNS leukemia at an early stage than is cytomorphology. At initial diagnosis, the finding of TdT+ cells in a RBC-free CSF sample with a negative cytomorphology is highly predictive for development of overt CNS leukemia.

Terminal deoxynucleotidyl transferase (TdT)-positive cells in cerebrospinal fluid and development of overt CNS leukemia: a 5-year follow-up study in 113 children with a TdT-positive leukemia or non- Hodgkin's lymphoma

We investigated whether an indirect nuclear terminal deoxynucleotidyl transferase (TdT) immunofluorescence (IF) assay on single cells present in the cerebrospinal fluid (CSF) is more effective than conventional cytomorphology for early detection or exclusion of (minimal) meningeal leukemic infiltration in patients with a TdT+ malignancy. During a 5- year follow-up study, 1,661 consecutive CSF samples from 113 children with a TdT+ acute lymphoblastic leukemia (ALL) (n = 100), a TdT+ acute nonlymphoblastic leukemia (ANLL) (n = 8), or a TdT+ non-Hodgkin's lymphoma (NHL) (n = 5) were analyzed. In 1,511 (91.9%) of 1,643 evaluable CSF samples, the positive and negative findings of both cytomorphology and the TdT-IF assay were concordant. In 47 (2.9%) samples from 28 patients, the cytomorphology was suspect while the TdT- IF assay was negative; follow-up as long as 58 months revealed no CNS leukemia in any patient. In 85 (5.2%) samples, cytomorphology was negative (n = 70) or suspect (n = 15) but TdT+ cells were detected. RBC contamination seriously hampered evaluation in 31 of these 85 samples. From the remaining 54 TdT+ samples from 29 patients, 40 samples preceded overt CNS leukemia in 20 patients. Two consecutive findings of TdT+ cells in the CSF were always followed by overt CNS leukemia. At initial diagnosis, 11 children had TdT+ cells in their RBC-free CSF. In one of these children, morphology was suspect; a repeated lumbar puncture was positive on both assays. Thus, initial CNS leukemia was diagnosed. In the other ten children, morphology was negative. In six of them, CNS leukemia was diagnosed 2 to 20 months later. In 32 other children examined at initial diagnosis, neither TdT+ cells nor blasts were observed in the CSF. In none of these patients was a CNS leukemia diagnosed after a follow-up of 2.5 to 57 months (median 24 months). In 207 control CSF samples from 58 children with TdT- oncologic, hematologic, or infectious diseases, no TdT+ cells could be detected. The TdT-IF assay is easy to perform and is a more reliable diagnostic tool for detection of CNS leukemia at an early stage than is cytomorphology. At initial diagnosis, the finding of Tdt+ cells in a RBC-free CSF sample with a negative cytomorphology is highly predictive for development of overt CNS leukemia.