Smear Specimens Research Articles

Prevalence of tuberculosis in small pulmonary nodules obtained by video-assisted thoracoscopic surgery

The prevalence of tuberculosis in small solitary lesions of the lung obtained by video-assisted thoracoscopic surgery (VATS) is still unclear. Of 103 lung lesions resected by VATS in 98 patients (47 men, 51 women), 19 were identified macroscopically as inflammatory changes, 78 were neoplastic, and 6 were undefined. Presumptive diagnosis based on microscopic analysis of fresh specimen smears treated with Papanicolaou stain was performed in 19 lesions. Of these, 11 lesions had epithelioid cells, granulomas with caseous necrosis and Langerhans-type giant cells. The 6 undefined lesions were non-inflammatory benign changes. Isolation and identification of tuberculosis were based on microscopic findings of fresh material smears and sections of fixed specimens stained with Ziehl-Neelsen's dye, cultivation using egg-based Ogawa medium, and in situ hybridization between polymerase chain reaction (PCR) products of each of the 11 lesions and specific DNA sequences for Mycobacterium tuberculosis, M. avium, and M. intracellulare. Of these 11 lesions, M. tuberculosis was confirmed in one (0.96%) by PCR and M. avium was confirmed in four by culture and PCR. Of the 78 malignant lesions, final pathologies were primary lung cancer (n=59, 70.2%) and pulmonary metastatic cancer (n=19, 22.6%). The most frequent primary malignant cancer was adenocarcinoma, which was found in 19 men and 28 women in the present study. Eight lesions in 8 men were squamous cell carcinomas. The results of the present study suggested that even though the prevalence of lung tuberculosis is low, attention should be paid to the presence of M. tuberculosis in specimens obtained by VATS.

Intérêt de la télépathologie dans le diagnostic rapide des spécimens neurochirurgicaux

To determine whether still digital images of neurosurgical specimens sent by e-mail are an effective and safe way of obtaining an emergency pathological diagnosis, representative images from 48 neurosurgical smear specimens were digitized, compressed and sent by electronic mail to our consultant neuropathologist with relevant clinical and radiological information. The time to select, digitize, compress and send the images for each case was recorded. Comparison was made between diagnosis made from digital images (DI) and historical smear (HS) using the original permanent section (PS) diagnosis as the gold standard. A diagnosis of normal vs. abnormal, neoplastic vs. non-neoplastic, primary vs. secondary and tumor grade was made in each case. The mean time for selection and digitalization of each case was 9.51 minutes. The number of correct pathological diagnosis was 41/48 using DI and 48/48 using HS, giving an accuracy rate of 85.4% and 100% respectively. DI was 100% sensitive in recognizing neoplastic tissue but only 98% specific. Most inaccuracies were in grading and caused by problems with field selection and familiarity with the technique. We conclude that static digital images are reliable and safe method of interpreting tissue during neurosurgical procedures. This corresponds with the findings of previous studies.

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.

Mantle-cell lymphoma of colon

and the pelvis demonstrated no specific findings. At colonoscopy, the ileocecal valve showed an ulcerated growth extending to adjacent cecal wall. This growth showed multiple, irregular, marginated shallow ulcerations (A and B). The histopathology of the biopsied tissue from the ileocecal growth revealed a proliferation of large, atypical lymphoid cells with ovoid vesicular nuclei and prominent nucleoli (C; H&E, orig. mag. !400). Immunohistochemistrywas positive for CD20 andnegative for CD3. Examination of a peripheral blood smear and bone marrow biopsy specimen revealed no specific findings. A diagnosis was made of malignant lymphoma, diffuse, large, B-cell type. The patient underwent resection of the tumor and adjuvant chemotherapy. Hae Jung Song, MD Bong Min Ko, MD Young Koog Cheon, MD Chang Beom Ryu, MD Joon Seong Lee, MD Moon Sung Lee, MD Chan Sup Shim, MD Institute for Digestive Research Digestive Disease Center Soon Chun Hyang University College of Medicine Seoul, Korea

Recovery Rate and Characteristics of Nontuberculous Mycobacterial Isolates in a University Hospital in Korea

Background : The incidence of nontuberculous mycobacterium (NTM) infections in Korea is increasing. This retrospective study was performed to examine the recovery rate of NTM from respiratory specimens as well as the isolated NTM colony characteristics, and to assess the clinical significance of a NTM isolation. Methods : The results of the respiratory specimens requested for an acid-fast bacilli (AFB) examination during 2002 at Asan Medical Center, along with the patients clinical characteristics were analyzed. Results : A total 26,820 respiratory specimens were requested for the acid-fast bacilli (AFB) smear and culture during the study period. The proportion of M. tuberculosis and NTM isolation was 5.7% and 2.2%, respectively. Among the AFB smear and culture positive specimens, 12.2% were found to be NTM. The scotochromogen showing a low colony count

Laboratory detection and identification ofAspergillusspecies by microscopic observation and culture: the traditional approach

Molecular and immunologic tests promise better, faster laboratory diagnosis of aspergillosis, but microscopy and culture remain commonly used and essential tools. Procedural changes, as well as adequate training of laboratory professionals, can enhance the value of these traditional tools. Using Blankophor or Calcofluor for microscopic examinations; improving recognition of morphologic characteristics of opportunistic fungi in stained smears of specimens; maximizing the growth rate and production of conidia by Aspergillus spp. in culture; and recognizing atypical variants of common aspergilli can improve the laboratory's contribution to rapid diagnosis. Surveys indicate that the number of laboratory professionals is declining as the demand for healthcare is rising. Effective recruitment, retention, and training of personnel must be concurrent with advances in technology.

The interpretation of nucleic acid amplification tests for tuberculosis: do rapid tests change treatment decisions?

Objectives. To describe changes in treatment decisions after receipt of nucleic acid amplification (NAA) test for the diagnosis of M. tuberculosis. Methods. Retrospective notes review of treatment decisions in patients receiving a NAA test for suspected pulmonary or non-pulmonary tuberculosis at the Royal Free Hospital in London between March 2001 and February 2002. Notes were sought on a 50% random sample of patients with both smear and NAA negative specimens and all patients with other specimen results. Results. Two hundred and fifty patients were tested with NAA; clinical details were obtained on 138; 61 were ever treated. Seventeen (17/18) smear-negative patients were started on treatment after a positive NAA; none of six smear-negative patients treated prior to a negative NAA result had treatment stopped. Seventeen (17/21) smear-positive patients were treated prior to NAA result and all were NAA positive; treatment was delayed in four smear-positive patients until receipt of an NAA and one NAA-negative patient was not treated. Conclusions. In routine practice a positive test in an untreated smear-negative patient leads to decision to treat in almost all, but the proportion testing positive is low (8% or 17/219). In patients already on treatment negative tests did not lead to decisions to stop.

Fine-needle aspiration cytology of mucinous tumors of the pancreas.

Tumors of the pancreas associated with extracellular mucin production include mucin-producing ductal adenocarcinoma, mucinous cystic neoplasm (MCN), and intraductal papillary mucinous tumor (IPMT). Fine-needle aspiration (FNA) is used as an adjunct to radiologic analysis for the preoperative categorization of these tumors. The current study was designed to identify distinctive cytomorphologic features that would be useful for the categorization of mucinous tumors of the pancreas. The authors evaluated Papanicolaou smears and Diff-Quik-stained smears of specimens obtained by computed tomography-guided and endoscopic ultrasound-guided FNA of the pancreas in 51 cases of mucinous tumors. In the 19 cases in which the patients underwent surgical excision after FNA, the cytologic features were compared with the histopathologic findings. The remaining cases were categorized as one of the three above-mentioned types of mucinous tumors on the basis of cytomorphologic features identified as indicative of subtype among the cases in which a cytologic-histologic correlation was performed. Among the 19 cases with cytologic-histologic correlation, 2 cases of serous cystadenoma and 2 cases of gastrointestinal duplication cyst were misdiagnosed on cytologic analysis as low-grade MCN. Among the remaining 15 cases, cytologic features by histologic diagnosis were as follows: ductal adenocarcinoma (n = 4): moderate to high cellularity, mild to moderate background mucin, three-dimensional clusters, high nuclear cytoplasmic ratios, and mild to moderate nuclear membrane irregularities; low-grade MCN (n = 5): mild to moderate cellularity, abundant background mucin, small clusters, and flat sheets of relatively bland glandular cells; mucinous cystadenocarcinoma (n = 1): similar to ductal adenocarcinoma but more abundant background mucin; and IPMT (n = 5): moderate to high cellularity, abundant background mucin, and prominent papillary arrangement of tall columnar cells with mild to moderate nuclear atypia. The remaining 32 cases were categorized based on cytology alone as adenocarcinoma with mucin production (n = 24) and low-grade MCN (n = 8). IPMT and low-grade MCN possess distinctive cytologic features that can be used to diagnose them correctly and distinguish them from one another and from other cystic tumors. Duplication cysts closely mimic low-grade MCN, which can lead to false-positive diagnoses. Because of substantial overlap in cytologic features, mucin-producing ductal adenocarcinoma was unable to be distinguished from mucinous cystadenocarcinoma cytologically.

Distribution of human papillomavirus types in cervicovaginal washings from women evaluated in a sexually transmitted diseases clinic.

Women evaluated for sexually transmitted diseases (STDs) may be at increased risk for human papillomavirus (HPV) infection, a condition associated with cervical dysplasia. The distribution of HPV types in such a population is unknown. The goal was to determine the prevalence of HPV infection, the distribution of HPV types, and the type distribution in relation to cervical dysplasia in women in an STD clinic. Cervicovaginal lavage and Papanicolaou smear specimens were obtained from 295 women. Lavage specimens were analyzed for HPV by polymerase chain reaction/reverse blot strip assay. Cervical cytologic findings were abnormal for 19.7% of women. HPV DNA was detected in 49.2% of women (high-risk HPV in 42.4%). HPV positivity correlated with the degree of cytologic abnormality. In women with dysplasia, HPV types 16, 66, 83, 56, 52, and 59 were commonly detected. Specimens containing abundant HPV DNA occurred most often in women with dysplasia. HPV infection was common in women attending an STD clinic. Numerous individual HPV types were associated with cervical dysplasia, including "low-risk" types.

Gram Stains – Beyond the Basics

As Barenfanger and Drake illustrated in their recent article,1 direct microscopy of Gram-stained smears of clinical specimens is an extremely important part of diagnostic microbiology. In addition to what can be considered the basics, an awareness of other possibilities may be of diagnostic value. This present article illustrates some of these possibilities. Some of the findings are self-evident if the observer knows the morphological features or structures, eg, Giardia lamblia and Trichomonas vaginalis. Others are less obvious, eg, toxoplasma trophozoites, “negatively-stained” organisms, and possibly Pneumocystis carinii. For these, suspicions must be confirmed with other stains and/or techniques. Whatever the source of the specimen, heat fixation of smears is not recommended. Fixation with methanol or the ethanol/acetone mixture used as the decolorizing reagent gives less distortion and better results with the Gram stain.2,3

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.

Application of the membrane filter technique to bromodeoxyuridine immunochemistry for exfoliative cytology

The membrane filter technique for smear specimens of tumors in bromodeoxyuridine (BrdU) immunochemistry is described. The staining results of Raji cells processed using the filter technique was compared with that obtained by the conventional cytospin method. Although the BrdU mean labeling index (LI) for in cytospin specimens was almost the same as the LI in membrane filter specimens, filter specimens showed excellent staining and less cell destruction compared with those processed by cytospin. Small amounts of tumor specimens such as squamous cell carcinoma and polymorphous low-grade adenocarcinoma also were processed using the membrane filter appliance. For squamous cell carcinoma, the LI for the filter specimens was 5.36 ± 0.38 and that of the paraffin sections was 5.56 ± 0.38. The membrane filter technique provided relatively undamaged specimens for exfoliative cytology and will be useful for immunohistochemical evaluation of tumor cells and for routine, noninvasive cytological screening.

Multiple Oblique Illumination and High-Definition Microscopy for Breast Fine-Needle Aspirates

The ability of multiple oblique illumination (MOI) and high-definition microscopy (Edge R-400 3-D microscope) to improve resolution of cellular detail in the evaluation of cytopathological specimens of Pap smears and thyroid fine-needle aspirates (FNAs) has been demonstrated. However, previous experiments showed that the advantages of MOI and high-definition stereo microscopy were less certain for the breast FNAs. We hypothesized that these findings were due to the lack of sample thickness for the breast FNA specimens. To test this hypothesis, we analyzed breast FNA specimens that were significantly thicker (10.5 microm). The number of lights (1, 2, 3, 4) and the angle of light (+1.5, 0, -3) were varied independently, creating 12 groups. Three images at each combination of settings were digitally captured and analyzed to obtain a histogram. The coefficient of resolution (Cr) was calculated to mathematically evaluate the grayscale histograms for intensities (0-255), where Cr = [¿IM - IN¿ x (N)] (IM, median pixel intensity; IN, measured pixel intensity; and N, number of pixels at given intensity). Mean Cr values demonstrated that the angle of light obliquity was not a factor in altering the resolution and contrast (p = .9). However, there was a significant increase in resolution, as measured by mean Cr values, as the number of lights was successively reduced from four lights to one light. Thus, the thicker specimen did show that increases in resolution were a significant function of the number of lights utilized.

Significance of heterogeneous nuclear ribonucleoprotein B1 as a new early detection marker for oral squamous cell carcinoma.

The development of an early tumor detection marker for oral cancer is an obvious need due to the high recurrence rate and poor survival rate. Based on our previous report that overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) B1 protein was found in 100% of squamous cell carcinomas of human lung, we applied the same immunohistochemical method, using anti‐hnRNP B1 antibody, to human oral squamous cell carcinoma (OSCC). Seven human tissue sections of OSCC showed strong staining with anti‐hnRNP B1 antibody, and hnRNP B1 protein of 37 kDa was identified in protein fractions isolated from six of the cancerous tissue sections, while it was not found in adjacent noncancerous tissue. Moreover, three non‐homogeneous (nodular) leukoplakia sections showed significant anti‐hnRNP B1 staining. The results suggest that this antibody detects precancerous lesions as well as advanced lesions (stages I to IV) of OSCC. We also present positive results of cytodiagnosis for two smear specimens. All of the above results indicate that hnRNP B1 is a new and useful marker for early detection of OSCC.

Genital human papillomavirus infection among women recruited for routine cervical cancer screening or for colposcopy determined by Hybrid Capture II and polymerase chain reaction.

The purpose of this study was to evaluate the clinical use of the Hybrid Capture (HC)-II system for the detection of human papillomavirus (HPV) DNA to identify women at risk of progression to high grade squamous intraepithelial lesions (HGSIL) and carcinomas by differentiating low risk (LR) HPV types (6, 11, 42, 43, 44) and high/intermediate risk (HR) HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). Five hundred and ninety-six women were enrolled in the study. Among them, 466 attended the hospital for routine cytologic screening and 130 were referred for colposcopy because of an abnormal Pap smear. The presence of HPV DNA was tested in cervical samples collected with the Digene Cervical Sampler in Digene Specimen Transport Medium (Digene Corporation, Silver Spring, MD, U.S.A.) using the HC-II assay. Results were compared with those obtained by polymerase chain reaction (PCR) using the MY09-MY11 primers followed by several hybridizations with specific probes. The overall HPV positivity was 32.9% by HC-II and 37.8% by PCR. Among cytologically normal smears, 19.5% were positive by HC-II (14.3% HR) and 25.1% by PCR. Of the atypical squamous cells of undetermined significance samples, 52.9% were positive by HC-II (41.1% HR) and 55.9% by PCR. Of the low grade SIL, 64.5% were positive by HC-II (59.4% HR) and 68.7% by PCR. The HPV positivity rate was found identical by both techniques in high grade smears (81.6%) and squamous cervical carcinomas (100%). By using PCR as the reference method, the sensitivity of HC-II was higher among women with abnormal cytology than with normal cytology (87.3% vs. 70%). Specificity was 80.8% and 97.5%, respectively. In summary, these results indicate that the HC-II method and MY-PCR identified nearly equivalent prevalences of HPV in cervical smear specimens.

Loss of chromosome 10 in glioblastoma: Relation to proliferation and angiogenesis

Loss of chromosome 10 was assessed in 17 specimens of glioblastoma (GBM) by fluorescence in situ hybridization (FISH) technique using the centromere probe for chromosome 10. Cytospinned smear specimens were prepared from paraffin-embedded specimens. The percentage of nuclei containing a single fluorescent signal ranged from 19.2 to 88. 0% (mean, 49.3%). Thirteen tumors (76.5%) were designated as monosomy 10 because the proportion of single-signal nuclei exceeded the cut-off value (31.5%: mean of five control materials +3 standard deviations). The results confirmed the importance of the loss of chromosome 10 for the development of GBM, although no significant correlation was demonstrated between the loss of chromosome 10 and survival. In addition, proliferation potential and angiogenesis of GBM were immunohistochemically analyzed using antibodies against Ki-67 antigen (MIB-1), factor VIII-related antigen (FVIII R/Ag) and vascular endothelial growth factor (VEGF), respectively. The labeling indices of MIB-1 (1.5-57.8%) and the number of blood vessels immunoreactive for FVIII R/Ag (18-279/10 high-power fields) were not significantly related to the loss of chromosome 10. Vascular endothelial growth factor immunoreactivity in areas microvessels were counted was seen in 12 cases. However, neither the loss of chromosome 10 nor number of vessels was not correlated with VEGF expression. Other genetic abnormalities as well as loss of chromosome 10 may be involved in the cell proliferation and angiogenesis of GBM.

Clinical utility of Amplified Mycobacterium tuberculosis Direct Test in the Diagnosis of Pulmonary Tuberculosis

Background: Acid-fast stain and cultures for diagnosis of pulmonary tuberculosis are primary and essential method, but have their limitation : low sensitivity and time consuming. The objective of this study is comparison of amplified Mycobacterium tuberculosis direct test(MTD) by the conventional AFB smears and cultures in the detection of Mycobacterium tuberculosis in respiratory specimens. Methods: During the period between November, 1997 and May, 1998 a total of 267 respiratory specimens (sputum 173, bronchial washing 94) from 187 patients suspected pulmonary tuberculosis were subjected to AFB smears, cultures and MID test. MID is based on nucleic acid amplification. We compared the MID with 3% Ogawa culture method. In positive AFB smear and negative MID specimen, positive culture identification between nontuberculous mycobacterium and M.tuberculosis was assesed by using Accuprobe M.tuberculosis complex probe. In negative AFB smear and negative AFB culture, MTD results are assessed by clinical follow-up. Results : 1) Compared with culture in sputum and bronchial fluid specimens, sensitivity and specificity of MTD in positive AFB smear is 79.7% and 20.0%, sensitivity and specificity of MTD in negative AFB smear specimens is 75.0% and 79.7%. 2) Discrepant analysis is assessed by clinical follow-up and other specimen results beyond study. Culture negative but MTD positive specimens were proved to be true positive and gave MTD sensitivity 79.2%, specificity of 84.4%, positive predictive value 80.5% and negative predictive value 83.2%. 3) 14 out of 31 specimens in negative AFB smear, negative AFB culture and positive MTD showed pulmonary tuberculosis diagnosed on clinical follow-up and sensitivity is 45.2%. 4) 2 out of 13 specimens in positive AFB smear, positive AFB culture and negative MID diagnosed as non tuberculous mycobacterium by Accuprobe culture. Conclusion: This study suggested that MID in respiratory specimens is simple and rapid diagnostic method, but considered adjuvant method rather than replace the conventional AFB smear and culture.

Is Schistosomicidal Chemotherapy Sub-curative? Implications for Drug Resistance

Parasitology is still much in use for the diagnosis of schistosomiasis. Kato–Katz smears of stool specimens[ 1 Katz N. Chaves A. Pellegrino J. Rev. Inst. Med. Trop. Sao Paulo. 1972; 14: 397-400 PubMed Google Scholar ]are widely used to detect eggs of the two hepato-intestinal schistosome species, Schistosoma mansoni and S. japonicum, while filtration of eggs from urine is used for S. haematobium diagnosis[ 2 Braun-Muntzinger R.A. Parasitol. Today. 1986; 2: 82-83 Abstract Full Text PDF PubMed Scopus (7) Google Scholar ]. However, parasitological methods (particularly those for detecting eggs in faeces) are frequently acknowledged to be insensitive. Some of the consequences from this conclusion and some possible remedies have been outlined elsewhere[ 3 de Vlas S.J. Gryseels B. Parasitol. Today. 1992; 8: 274-277 Abstract Full Text PDF PubMed Scopus (225) Google Scholar , 4 de Vlas S.J. et al. Parasitol. Today. 1993; 9: 305-307 Abstract Full Text PDF PubMed Scopus (51) Google Scholar , 5 Gryseels B. de Vlas S.J. Parasitol. Today. 1996; 12: 115-119 Abstract Full Text PDF PubMed Scopus (68) Google Scholar ]; one factor that has not received much attention is the effect that insensitive parasitological diagnosis may be having on schistosomicidal chemotherapeutic practice.

Polymerase chain reaction in the diagnosis of bacterial endophthalmitis

BACKGROUNDMicrobiological investigations of vitreous fluid (VF) and aqueous humour (AH) specimens have often failed to detect the infecting agent in infectious endophthalmitis, resulting in a clinical dilemma regarding therapy. In...

Retrospective analysis of clonality and detection of residual disease in myeloid leukemia by FISH on long-term stored bone marrow smears.

Fluorescence in situ hybridization (FISH) has allowed the detection of numerical chromosomal aberrations in interphase nuclei on fresh or frozen smears of leukemia. To analyze clonality and residual disease in myeloid leukemia retrospectively, we applied FISH to bone marrow smears stored at ambient temperature for up to 9 years. When hybridization efficiency was investigated on stored control smears from patients without hematological malignancy, more than 96% of nuclei showed the expected number of signals using DNA probes specific for chromosome 7, X or Y. In combination with cell morphology, we observed much higher hybridization efficiency in blasts and granulomonocytic cells compared with lymphoid and erythroid cells. On the basis of good hybridization efficiency for old smear specimens, we applied FISH to stored bone marrow smears of myeloid leukemias, in which either loss of chromosome 7 or loss of sex chromosomes had been verified previously by conventional cytogenetics (one patient with chronic myelomonocytic leukemia (CMML) and four with acute myeloid leukemia (AML; three M2 and one M7)). As a result, the loss of chromosome was detected in blasts from all patients and was observed in mature granulocytes, except in M7. In the CMML patient and one AML (M2) patient with t(8;21), lymphoid and erythroid cells also showed the loss of chromosomes, suggesting that it should occur at stem-cell level. A high amount of residual disease was detected in the morphological remission samples in one AML (M2) patient after induction therapy. The patient eventually succumbed to relapse. Thus, the present FISH technique is useful to analyze the clinical significance of clonality and the residual disease in myeloid leukemia, retrospectively.