Air-dried Smears Research Articles

Thin core needle biopsy crush preparations in conjunction with fine-needle aspiration for the evaluation of thyroid nodules

Fine-needle aspiration (FNA) is widely accepted as the initial test to evaluate thyroid nodules; however, inadequate and suboptimal specimens have been 1 of its limitations. Unsatisfactory rates of 4.1% to 43% have been reported, but suboptimal specimens with adequate epithelial cells and other limiting factors, such as clotting, often are not addressed. The authors' institution has a low unsatisfactory rate, especially for thyroid biopsies performed under ultrasound in the Interventional Radiology (IR) Department. In addition to on-site evaluation for all cases, they concomitantly use thin, 22/20-gauge core needle biopsy (CB) crush preparations (CP) for unsatisfactory/suboptimal FNAs. The CB usually is done after 2 FNA passes and, in most cases, is exhausted by making an air-dried CP, which is evaluated on site for adequacy; any residual tissue is processed for tissue sections. Experience is required to interpret CP on air-dried smears. In this report, the authors describe a complementary approach to thyroid biopsy that has worked well. All thyroid FNA and CB/CP that were performed in the IR Department during the year 2005 were reviewed. Follow-up histology and records of all procedural complications were retrieved. Seven hundred thirteen thyroid biopsies qualified, 225 biopsies (31%) had FNA with CB/CP (85% had only CP for evaluation), and 488 biopsies (69%) had only FNA. The final unsatisfactory rate in IR for FNA was 8.7%; this was reduced to 3.4% with the use of CB/CP. The addition of CB also helped to obtain a more definitive diagnosis in suboptimal FNA specimens. Cytologic-histologic correlation was comparable for FNA only cases and FNA/CB cases. There were no significant procedural complications in CB cases. FNA in conjunction with a thin CB/CP performed during the same procedure is a safe technique that can reduce the rate of unsatisfactory and suboptimal thyroid biopsy.

CD163: A Specific Immunohistochemical Marker for Acute Myeloid Leukemia With Monocytic Differentiation

Acute myeloid leukemias (AMLs) with monocytic differentiation with or without recurring cytogenetic abnormalities have prognostic significance. Therefore, it is important to recognize such cases. Monocytic differentiation is identified by morphology and confirmed by cytochemical stains or flow cytometry. However, the materials for such analyses are not always available (air-dried aspirate smears, touch preparations, fresh cells). Thus, the utility of CD163, a hemoglobin scavenger molecule present on monocytes/macrophages, in identifying a monocytic component in AML in fixed, paraffin-embedded bone marrows was assessed. Using tissue microarrays, 31 cases of AML with monocytic differentiation and 35 cases without monocytic differentiation were evaluated in a blinded fashion for CD163 positivity and compared with CD68. Diagnoses were previously well established using cytochemistry and flow cytometry techniques. CD163 immunoreactivity was seen in 49% of AML cases with and 6% (2/35) without monocytic differentiation. CD68 was positive in 81% of AML cases with and 17% without monocytic differentiation. CD163 showed superior specificity for a monocytic component in AML compared with CD68 (94% vs. 83%). However, the suboptimal sensitivity (49%) limits its utility as a definitive marker of monocytic differentiation if negative in paraffin sections. These findings demonstrate that CD163 is helpful for the evaluation of a monocytic component in AML. When positive, correlation and evaluation for an associated recurring cytogenetic abnormality is warranted.

Method-Related Estimates of Sperm Vitality

Comparison of methods that estimate viability of human spermatozoa by monitoring head membrane permeability revealed that wet preparations (whether using positive or negative phase-contrast microscopy) generated significantly higher percentages of nonviable cells than did air-dried eosin-nigrosin smears. Only with the latter method did the sum of motile (presumed live) and stained (presumed dead) preparations never exceed 100%, making this the method of choice for sperm viability estimates.

Cell Blocks from Scraping of Cytology Smear

To compare cytomorphology preservation and immunohistochemistry results between conventional cell blocks (CCB) and cytoscrape cell blocks (SCB).Fine needle aspiration (FNAC) was done in 17 consecutive cases. Air-dried smears for May-Grünwald-Giemsa stain and wet-fixed smear for hematoxylin-eosin (H-E) stain were prepared. Simultaneously another pass was made in each case for preparation of material for CCB. One of the H-E-stained smears was spared for SCB. SCB was compared with CCB for cell morphology. Immunostaining was performed both cell blocks, as well as on FNA smears in 8 cases. Results were evaluated for intensity of staining and percentage of cells showing positivity.CCB and SCB sections showed adequate cellularity in all cases. Morphologic preservation was good in SCB sections. There was good architectural and nuclear preservation in all cases of SCB. Immunostaining results showed better and clear intensity of staining with little background in all cell block cases.SCB is a valuable technique in cell blocks from stained FNA smears. The cytomorphologic details are equally good in SCB and CCB. Additional panels of immunostaining can be done on SCB for better diagnosis and classification, particularly in cases in which repeat FNA is not possible.

Air-Dried Smears for Optimal Diagnostic Immunocytochemistry

To exploit formalin-postfixed, air-dried smears for diagnostic immunocytochemistry (ICC).A series of 144 cases of diagnostic fine needle cytology samples in which air-dried, supplementary smears were available was used to exploit postfixation in the process of antigenic stabilization and rescue for immunocytochemical staining.Postfixation with formalin and with a formalin/ethanol solution gave comparable results as far as recovery and immunocytochemical detection of most monoclonal and polyclonal antibodies. The visualization of the antibody reactions was often superior to that obtained with wet-fixed slides, probably due to the interaction of slow cell dehydration with their consequent optimal flattening observed with formalin postfixation after short rehydration in physiologic saline.Although wet fixation of cytopathologic slides in 95% ethanol represents a common standard for ICC, the usage of formalin-postfixed air-dried smears proved reliable and efficient for antigenic rescue and may enter routine usage in cytopathology laboratories. Moreover, in some instances, the visual evaluation of results was easier in the larger, well-flattened cells obtained in air-dried cells.

Automated immunostaining of cell smears: an alternative to flow cytometry

Aims:To assess the applicability of an automated tissue immunostainer machine for the phenotypic analysis of peripheral blood and bone marrow aspirate smears.Methods:Air-dried smears of peripheral blood and bone marrow from...

Wertigkeit der konventionellen oralen Bürstenbiopsie

This study evaluated the performance of oral brush biopsies using standard morphological analysis and haematoxylin and eosin (HE) staining for detecting oral squamous cell carcinomas and their respective precursor lesions Brush biopsies were obtained in 169 consecutive patients who underwent routine biopsies and histological examination for clinically suspicious oral lesions. Air-dried smears were processed by acetone fixation and HE staining. Cytological assessment used well-established criteria of atypia to classify the specimen as either "tumor negative" (no signs of atypia, no malignant cells) or "tumor positive" (malignant cells, any sign of atypia or doubtful cells). Despite a sufficient number of cells, a definite cytological diagnosis could not be established in six cases. According to the criteria specified above, these specimens were classified as "tumor positive." The cytological analysis identified 49 out of 62 oral malignancies (sensitivity 79%). Seven out of 107 benign lesions were classified as false positive (specificity 93%). The positive and negative predictive values were each 88%. Oral brush biopsies will identify only about 80% of oral malignancies when the smears are processed by routine HE stains and are analysed via standard morphological criteria. Thus, this technique should not be used for diagnostic proof or to exclude malignant cells in a lesion suspicious for cancer. However, oral brush biopsy provides a versatile back-up strategy to uncover the true nature of the disease if a lesion is clinically considered benign by mistake.

Validation of the use of Pap‐stained vaginal smears for diagnosis of bacterial vaginosis

Papanicolaou-stained cervicovaginal smears (Pap smears) are used to screen for cervical cancer. Since there is a lack of consensus in published reports respecting the efficacy of Pap-stained smears in BV diagnostics, there is a need to validate their use for diagnosis of BV. Slides from the international BV00 workshop were Pap stained and independently analyzed by four investigators under a phase-contrast microscope. All workshop slides--whether Pap-stained, Gram-stained or rehydrated air-dried smears--were scored according to the same Nugent classification. The diagnostic accuracy of Pap smears for diagnosis of BV had a sensitivity of 0.85 and a specificity of 0.92, with a positive and negative predictive value of 0.84 and 0.93, respectively. The interobserver weighted kappa index was 0.86 for Pap-stained smears compared to 0.81 for Gram-stained smears, and 0.70 for rehydrated air-dried smears using the mean Nugent score as the criterion standard. Provided that the samples are taken from equivalent locations (the vaginal fornix) and analyzed according to the same scoring criteria, there is no discernable difference in the diagnostic accuracy of the three smear-staining methods. The Pap-stained vaginal smears can be used as a wholly adequate alternative to Gram-stained smears for BV diagnosis.

Cytomorphology and B72.3 labeling of benign and malignant ductal epithelium in pancreatic lesions compared to gastrointestinal epithelium

Endoscopic ultrasound-guided pancreatic fine-needle aspiration biopsy very frequently produces gastrointestinal epithelial contamination (GIC). We studied the cytomorphology and B72.3 immunoreactivity of lesional epithelium of benign and malignant ductal lesions of the pancreas and compared the findings to our previously established template of GIC. Air-dried smears, fixed smears, and ThinPrep (TP) specimens were obtained using a cytobrush, directly from benign and malignant ductal lesions of 18 Whipple specimens, to ensure purity of the epithelium studied. Smear background, cell architecture, and cellular features were analyzed. Immunocytochemical staining with B72.3 was performed in 14 cases. Epithelium of ductal carcinoma was distinguished from benign ductal epithelium in chronic pancreatitis and GIC primarily by crowded architecture and atypical cellular features, including high nuclear-to-cytoplasmic ratio, irregular nuclei, nucleoli, and vacuolated cytoplasm. Benign ductal and GIC epithelium were only distinguished by architecture (goblet cells and brush borders), but not consistently, especially gastric epithelium that lacked these features. B72.3 shows promise in the differentiation between GIC and benign and malignant ductal epithelium, with no staining supporting benign ductal cells, fine punctate perinuclear staining correlating with GIC, and strong cytoplasmic staining supporting malignancy.

A technically simple method for staining of acid‐fast bacilli in cytology smears: An evaluation

To study the effects of modifications in the Ziehl-Neelsen staining procedure on predictive accuracy for acid fast bacilli in comparison to the conventional technique. Simplicity of procedure and reagent economy were the factors taken into consideration. Comparative evaluation between thick and thin air-dried smears stained conventionally and thick ethanol-fixed smears stained by the modified technique was done. Positive predictive accuracy of all the three smears, that is, thick air-dried, thin air-dried and thick ethanol-fixed, was 100%. Negative predictive accuracy for thick air-dried, thin air-dried and thick ethanol-fixed smears was 36.36%, 32.33% and 34.78%, respectively. Overall predictive accuracy was 66.67% for thick air-dried, 61.90% for thin air-dried and 64.29% for thick ethanol-fixed. These differences were found to be statistically insignificant. The modified method offers an accuracy comparable to the conventional technique, is simpler and with improved reagent economy. It is of special importance to diagnostic facilities in rural set-ups.

Leishman-Giemsa Cocktail

To develop an economical, quick, readily available and simple alternative to May-Grünwald-Giemsa (MGG) stain and so to explore the combination of Leishman and Giemsa stain (LG cocktail).One wet-fixed and 2 air-dried smears were prepared from 720 cases during the period January 2003-November 2004. The LG cocktail and MGG stain were used on the air-dried smears and Papanicolaou stain in wet-fixed smears. Diagnoses of the cases were made using the LG cocktail-stained smears, and then its diagnostic efficacy was cross-checked with the MGG- and Papanicolaou-stained smears by the same cytopathologist. A comparative study of the LG cocktail and MGG-stained smears was done.The results achieved with the LG cocktail-stained smears were comparable to or even better than those with the MGG-stained smears, with excellent diagnostic efficacy.As compared to MGG stain, the 1-step LG cocktail is cheaper, easier to standardize and quicker.

Fast drying of Fine Needle Aspiration slides using a hand held fan : Impact on turn around time and staining quality

To analyze the impact of using a hand held fan to speed the air-drying process during immediate adequacy evaluation of Fine Needle Aspirations. The effect on turn around time and staining quality is evaluated.Two mirror image air-dried smears for each pass were prepared. One was subjected to a small hand-held fan with a fan diameter of 7 cm held an average distance of 3 to 5 cm from the slide. The other smear was left to dry without a fan. A total of 93 consecutive pairs were evaluated over a 2-month duration.The average time needed for air-drying using the fan was 73 seconds (range 10–300 seconds, standard error 6.986), while it was 200 seconds (range 15–645 seconds, standard error 17.799) for those without fan. This difference was statistically significant (p < 0.001). Smears were then evaluated for single cells, cell clusters and background material and no appreciable difference in stain quality was noted between the 2 groups.The use of a small hand-held fan for air-drying shortened the drying time for FNA adequacy by an average of 127 seconds (63% time reduction) for each pass. The quality of staining was comparable. Using a fan is highly recommended.

FNA diagnosis of recurrent sebaceous carcinoma

We describe fine-needle aspiration (FNA) diagnosis in a case of sebaceous carcinoma presenting as recurrent swelling eyelid. The patient, a 35-yr-old woman, presented with a gradually increasing painless mass in the left upper eyelid. She had a history of excisions of swelling at the same site over a period of 5 years. On FNA, the smears were cellular and demonstrated sheets, three dimensional clusters, and singly scattered polygonal tumor cells having centrally located hyperchromatic and pleomorphic nuclei and cytoplasmic microvacuolations. Oil red-O stain performed on air-dried smears showed positivity, confirming the presence of intracellular and extracellular lipid globules. A final impression of sebaceous carcinoma of the left upper eyelid was rendered, based on which the eyelid mass was radically excised. Subsequent histopathology confirmed the diagnosis. Despite wide excision, such tumors not uncommonly recur and metastasize. An early and accurate diagnosis of such lesions can be established on minimally invasive needle aspiration.

Tuberculoma of the heart

1. Case report A 38-year-old woman was admitted in the emergency services department in a gasping state. She had developed progressive dyspnea, orthopnea, bluish discoloration of finger tips, mild fever, dry cough with streaky hemoptysis, and jaundice in the past 4 days. She had a previous admission 1 month back for low-grade intermittent fever and Grade II dyspnea. A two-dimensional echocardiography then revealed minimal pericardial effusion and global hypokinesia suggestive of dilated cardiomyopathy. The patient died within 4 h of admission. At autopsy, the subject’s heart appeared massively enlarged and weighed 470 g. There was extreme thickening of both layers of the pericardium, enclosing confluent foci of creamy caseous material. These were prominently seen over various grooves and the left ventricular apex. However, at the anterior and lateral surfaces of the right atrium, the caseation had a thickness of 3 cm. It had infiltrated the atrial wall to produce three smooth-surfaced, broad-based polypoidal masses, tuberculomas (Fig. 1) that had almost obliterated the atrial cavity. The epicardial coronary arteries were surrounded by caseation but were patent. Few acid-fast bacilli could be demonstrated in the air-dried smears. The left ventricle was small. Microscopically, the areas of caseation were surrounded by the typical granulomatous reaction. In addition, there were tuberculous bronchopneumonia and lymphadenitis with miliary granulomas in the liver, spleen, and bone marrow and hemorrhagic hepatic necroses. No bacillus was demonstrated in the tissue sections. No culture was done.

Neural tumours of the neck presenting as thyroid nodules: a report of three cases

Neural tumours of the neck may at times secondarily involve the thyroid and manifest clinically as thyroid nodules. On cytological evaluation these nodules may be confused with other spindle lesions of the thyroid. We report two cases of schwannoma and one case of a malignant peripheral nerve sheath tumour (MPNST) of the neck, which presented as thyroid nodules and evaluate the role of cytology in identifying these tumours. The thyroid nodules in all the three cases were sampled by the non-aspiration technique using a 23-gauge needle. Both alcohol-fixed and air-dried smears were prepared and stained by the Papanicolaou and May-Grünwald-Giemsa stains. Cytology smears and histology sections from the resected specimens were reviewed, and the findings noted. Both the cases of schwannoma were correctly identified on cytology while the case of MPNST could only be typed as a spindle cell tumour. However, on cytology it was not possible to state whether the tumours were thyroidal or extrathyroidal in origin. Schwannomas of the neck are easily identifiable on cytology compared with MPNST. However, cytology alone is not helpful in identifying the origin of these tumours. As primary neural tumours of the thyroid are rare, the possibility of a soft tissue neural tumour extending into the thyroid should always be ruled out while evaluating these cases.

Reduced expression of Claudin‐7 in fine needle aspirates from breast carcinomas correlate with grading and metastatic disease

To study the immunocytochemical expression of the tight junction protein Claudin-7 in smears from breast carcinomas and correlate with grading, nodal status, locoregional and distant metastases and the cellular cohesion. The material consisted of 52 air-dried smears from fine needle aspirates of breast carcinomas, both primary and metastatic and smears from seven benign lesions. A primary antibody to Claudin-7 was used for immunocytochemical staining. The degree of staining was recorded as negative, reduced or full, with full expression meaning equivalent to the staining pattern found in the fibroadenomas used as benign control. Staining intensity and the percentage of stained cells were evaluated. The control smears revealed a strong membrane and cytoplasmic positivity in all luminal epithelial cells. Cellular cohesion was graded as: (1) mainly cohesive groups, (2) groups and single cells and (3) mainly single cells. All primary and recurrent/metastatic breast lesions expressed Claudin-7. Full expression was demonstrated in 46% of the cases. Reduced expression was found in 54%. In cases with reduced expression, the percentage of stained cells were usually high, and no smear showed <50% stained tumour cells. The staining pattern was heterogeneous and always mixed membrane/cytoplasmic. Claudin-7 expression showed a significant correlation (P < 0.05) with grading, locoregional and distant metastases, nodal involvement and cellular cohesion in invasive carcinomas, but not with tumour size or subtype. Reduced expression of Claudin-7 correlated with higher tumour grade, metastatic disease, including loco-regional recurrences and with cellular discohesion.

Genome size and endonuclear DNA replication in spiders

Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development.

Cytomorphology of gastric and duodenal epithelium and reactivity to B72.3: A baseline for comparison to pancreatic lesions aspirated by EUS-FNAB

Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) has become a widely used method of pre-operatively evaluating pancreatic masses. This technique introduces gastrointestinal contamination into the specimen, which poses a diagnostic pitfall. The cytomorphologic features of these contaminants have not been fully characterized. The current study was designed to systematically document the cytomorphology of gastric and duodenal epithelium on fixed and air-dried smears, as well as ThinPrep (TP) preparations, and to assess the reactivity of the epithelial cells to the tumor marker B72.3 so as to establish a baseline for future comparative studies with EUS-FNAB of the pancreas. Air-dried and fixed smears and TP specimens were obtained using a cytobrush from gastric (GM) and duodenal (DM) mucosa from 14 Whipple specimens. Cytologic features of cell architecture, cellular features and smear background were analysed. Immunocytochemical staining with B72.3 was performed in 10/14 cases. Mucin was present in all preparations except one case from the duodenum. It was consistently present as isolated thick to thin clumps but never as diffuse 'colloid-type' mucin; it was most prominent on air-dried smears, and most abundant from the GM. Epithelial cells were admixed with mucin, but degenerated cells and inflammation were not present within the mucin. Mucin on TP appeared as fragmented wisps of pale blue staining material. Background inflammation and debris were not significant findings. The epithelial cells were arranged in large and small monolayered sheets. Smaller groups were more common from GM on smears and more abundant on TP than smears. Luminal edges (DM>GM) were a prominent feature, with a brush border noted in DM. The nuclei of GM and DM were round, evenly spaced and without atypia, and dense, non-vacuolated cytoplasm was the rule, with the exception of goblet cells and occasional gastric foveolar cells noted in one case. In both GM and DM, B72.3 stained goblet cells with a strong, coarsely granular pattern and stained epithelial cells focally in a finely granular, punctate, perinuclear distribution; mucin also stained strongly in all cases. These baseline characteristics provide a template for assessing mucin and epithelial GM and DM contamination on pancreatic EUS-FNAB specimens.

Cytomorphology and morphometry of small round-cell tumors in the region of the kidney

Small round-cell tumors (SRCTs), with malignant cell components measuring 10 m or less in diameter with scanty cytoplasm in alcohol-fixed smears, pose a diagnostic challenge at fine-needle aspiration cytology (FNAC), especially when they are situated in and around the kidney and need facilities such as electron microscopy, immunohistochemistry, tissue culture, and cytogenetics for their subtyping. A precise cytodiagnosis of SRCTs is important because a definite diagnosis is mandatory in preoperative diagnostic workup for presurgical chemotherapy in these cases. With this view in mind, an attempt has been made to diagnose SRCTs in the region of the kidney based on cytomorphology and morphometry alone so as to facilitate its diagnosis in a simple cytology laboratory of a developing country where facilities for auxiliary techniques are not easily available. Of 2,028 abdominal aspirates in a 12-yr period, 36 SRCTs were diagnosed in the region of the kidney by correlating with histology, radiology, and clinical features. The smears were studied for cellularity, morphology, pattern of cell arrangement, and smear background and morphometrically analyzed using an ocular micrometer. An aspirate with preponderant malignant round cells that were larger or double the size of red blood cells in air-dried smears or measured less than 10 micro in diameter in alcohol-fixed smears was considered as a small blue-cell tumor. Twenty-one were diagnosed as Wilms' tumor (WT), 10 were diagnosed as neuroblastoma (NB), 3 were ganglioneuroblastoma (GNB), 1 was a cellular congenital mesoblastic nephroma (CMN), and 1 was an adrenocortical carcinoma (ACC). Cell clusters with neuropil and cytoplasmic processes were diagnostic of NB, ganglion cells of GNB, and blastema with tubular differentiation in WT. Aspirates from CMN and ACC were considered as simulators/mimickers of SRCT because they had superficial resemblance to SRCT and their differentiating cytomorphological features observed at histology were too subtle to be noted at cytology. The latter were appreciated only on retrospective analysis after histological confirmation.Thus, morphometry in correlation with cytology, clinical history, physical findings, and radiological data is helpful in guided FNA for a definite diagnosis of SRCT in the region of the kidney. One needs to keep in mind the mimickers of small round-cell lesions at this anatomic site.

EGFR gene copy number heterogeneity in fine-needle aspiration cytology from breast carcinomas determined by chromogenic in situ hybridization

Most studies have shown epidermal growth factor receptor (EGFR) overexpression to be associated with poor prognostic factors in breast carcinomas. The relationship to EGFR gene copy number is unclear. The aim of our study was to investigate the heterogeneity of the EGFR gene copy number in breast carcinomas. The material consisted of air-dried smears from 29 breast carcinomas and 3 breast cancer cell lines (MCF-7, SKBR3, and T47D). Chromogenic in situ hybridization (CISH) was done using chromogenic detection. The mean signal numbers for EGFR gene and chromosome 7 as well as the EGFR gene/chromosome 7 centromere probe (CEP7) ratio were recorded. Immunohistochemical (IHC) staining was done on the corresponding paraffin sections. The copy number of the EGFR gene in each tumor/cell line ranged from 1.2 to 5.6. The EGFR gene/CEP7 ratio showed a biological continuum ranging from 0.59 to 1.94 with a mean of 1.04. EGFR gene copy loss was found in 16.6% of cases whereas copy gain was demonstrated in 19.4%. There was no relationship between IHC protein expression of EGFR and EGFR gene copy number or EGFR gene/CEP7 ratio.In conclusion, most breast carcinomas had a balanced EGFR gene/CEP7 copy number with a mean ratio of 1.04. Almost equal subpopulations revealed limited copy gain and copy loss. EGFR high dosage amplification, like in HER-2, was not demonstrated. Demonstration of EGFR gene copy loss might have a potential as a surrogate marker for EGFR gene mutation and/or deletion.