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Fine needle aspiration: a changing paradigm
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Fine needle aspiration: a changing paradigm
While most immunohistochemical studies can be performed on the submitted and fixed FNA specimens, the recent developments in personalized (boutique) medicine and pharmacogenomics necessitate the availability in adequate quantities of well preserved and uncontaminated, fresh representative material. Most of the subcellular (molecular) highly sophisticated studies require special handling, preservation and processing of minute quantities of diagnostic materials available during the FNA procedures. The value of onsite, accurate interpretation of cellular samples in procurement and handling of such specimens cannot be over emphasized. This paper summarizes the present practices and recommendations for improvements both in the quality of specimens and delivery of FNA services.
Prabodh K Gupta
Abstract Fine needle aspiration (FNA) is used routinely for detection, diagnosis, therapeutics and follow-up of various tumours. This practice is recognized as an accurate, rapid and economical diagnostic procedure. In the rapidly changing healthcare paradigm, however, there are new challenges being imposed on the practice of fine needle aspiration cytopathology. The diagnosis of FNA specimens must not only be accurate it should be precise. This puts additional demands on the clinicians, the cytopathologists and the laboratory; slide preparations and reporting must meet not only the diagnostic but also the therapeutic challenges. This requires a multimodal approach and interaction among the various parties including the patient, the primary care, the radiology, molecular and cytopathology laboratory personnel.2 Recent developments in personalized medicine and pharmacogenomics necessitate the availability in adequate quantities of well preserved and uncontaminated, fresh representative material. The value of onsite, accurate interpretation of cellular samples in procurement and handling of such specimens cannot be over emphasized. This paper summarizes the present practices and recommendations for improvements both in the quality of specimens and delivery of FNA services.
FNA slides and specimen spread preparations Fine needle aspiration is performed using thin bore (22e27 gauge) needles whereas the core biopsies often use large bore (12e15) needles. The precise diagnosis of an FNA specimen mandates a number of prerequisite essential for proper specimen preparation and reporting. These include: Clean slide: glass surface should be clean and grease free. Common occurrence of the acellular blank areas in the smears results from holding the slides on the surface with a thumb and finger and leaving a grease mark that prevent cell adhesion (Figure 2a). This can be avoided by grasping the slides from the edges especially in the upper one-third, since the lower two-third is often utilized for smearing the samples and it should be clean, smooth and grease free.
Keywords FNAC methodology; FNAC techniques; organization of sampling
Local anaesthesia: injectable products (Lidocaine) result in flooding of fluid in the intercellular space in the tissues. This causes artificial separation of cells, shearing and morphological artifacts (Figure 2b). The use of injectable local anaesthesia for FNA procedures should be discouraged. If necessary the puncture should be made in the adjacent area. The topical anaesthesia or ice or cold pack in most situations can provide adequate numbness to the sensitive skin areas. The skin surface should be cleaned with alcohol pads and if necessary with iodine or NormsolÒ to avoid burning sensation during the FNA procedure.
In clinical medicine, cytodiagnoses of numerous disease processes have become the standard-of-care in the world. Fine needle aspiration (FNA) is used routinely for detection, diagnosis, therapeutics and follow-up of various tumours. This practice is recognized as an accurate, rapid and economical diagnostic procedure. In the rapidly changing healthcare paradigm, however, there are new challenges being imposed on the practice of fine needle aspiration cytopathology. The diagnosis of FNA specimens must not only be accurate it should be precise. The concept of precision in cytopathology is not new; it was emphasized by George Papanicolaou in the beginning of cytopathology practice1 (Figure 1). Precise and conclusive reporting of FNA specimens puts additional demands on the clinicians, the cytopathologists and the laboratory; slide preparations and reporting must meet not only the diagnostic but also the therapeutic challenges. This requires a multimodal approach and interaction among the various parties including the patient, the primary care, the radiology, molecular and cytopathology laboratory personnel.2
Specimen drying (curing, air wash, air drying) for Romanowsky stains: proper air drying of the aspirated specimen is critical for development of metachromasia observed by onsite staining using a Romanowsky or similar stained FNA smear preparations. Inadequate air fixation generally results in homogenous hematoxylinophilic or autochromatic bluish staining of the nucleus and the cytoplasm thus rendering the interpretation difficult (Figure 3a). The situation often becomes critical in an otherwise adequate representative samples that become suboptimal or unsatisfactory for interpretation resulting from inadequate air drying and poor fixation. Suboptimal air washing often occurs in smears which are extremely thick. Instant air drying of the FNA spreads is not recommended: very rapid slide drying such as by use of hair drier, results in uneven dehydration of the aspirate rendering the FNA specimens suboptimal for interpretation; there is discordant contraction of the cells
Prabodh K Gupta MBBS MD FIAC is Professor of Pathology and Laboratory Medicine, Director of Cytopathology and Cytometry and Director of the Cytopathology Fellowship Program at the University of Pennsylvania Medical Center, PA, USA. Conflicts of interest: none declared.
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Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 1 Precision in cytopathology reporting. Endometrial smear report by George Papanicolaou in 1952. Not only it gives a precise diagnosis but let the surgeon know “the diagnosis is well enough established to justify a major operation without curettage”. Adapted from e The Pap Smear; D. Erskine Carmichael, Charles C Thomas, Springfield, IL. 1973, p. 82.
and the surrounding intercellular material causing a pericellular clearing or a halo effect. Also, there is unusual degeneration and coagulation of cytoproteins producing hyperchromasia and loss of critical morphologic details (Figure 3b). Rapid air drying of a thyroid colloid material often results in disruption and beading of the colloid network. In such situations the cells of interest often accumulate as satellites at the nodes (Figure 3c). Alcohol fixation: it is a common practice to ethanol fix the smears for subsequent staining by polychromatic Papanicolaou stain. Although the use of 95% ethanol in Coplin jar and rapid immersion of the freshly prepared cell preparations is optimal, oftentimes commercially available, convenient spray fixatives are used for such purposes. Most of these products contain ethanol and or methanol and polyglycol. Often time the fixed slide will result in blobs of the cellular material giving a spongy, “Swiss cheese” appearance (Figure 3d), the diagnostic cells tend to accumulate along the edges of such clumps, they become overlapping, crowded and difficult to interpret. It is necessary that the manufacturer’s instructions be carefully followed while using the spray fixatives for FNA slides.
Figure 2 a Grease on slide surface, contamination from finger touch. Lubricants prevent specimen spread. b Effect of local anaesthesia injection. Disruption of cell and loss of metachromasia are evident (A), Lidocaine injection. Same specimen without local injection (B), Diff-Quik stain.
evaluation is available. Such a practice is painful and in our opinion unnecessary, uneconomical, unrewarding and wasteful (Figure 4a). As is obvious out of the 36 submitted slides only two smears (yellow labels) contain any cellular material. This whole specimen was considered unsatisfactory for diagnostic purposes. The excessive number of aspirations beyond three or five in most instances not only results in poor quality diagnosis. These preparations require additional supplies, screening, interpretation and diagnostic efforts. Two or three passes with onsite evaluation may be supplemented with additional material or a needle core biopsy if necessary for ancillary studies.
Number of punctures and slides: the number of needle pricks and slides prepared from each site during FNA procedures is extremely variable. It is not uncommon to perform 10e12 passes from one lesion.3 The situation is aggravated when no onsite
DIAGNOSTIC HISTOPATHOLOGY 17:7
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Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 3 a Effect of inadequate air drying. Poor staining (A); compare to the well dried cells (B), Diff-Quik stain. b Effect of rapid air drying. Notice the fried appearance of follicular cells. Insert shows higher magnification of the distorted cells, Diff-Quik stain. c Effect of rapid air drying of cell spreads. Notice the beaded appearance of colloid material. Insert shows epithelial cells aggregating at the tips. d Effect of improper spray fixation. Notice the clumping of the specimen giving a Swiss cheese appearance. Cells in such preparation collect at the margins, (green dot).
Cell blocks preparations: cell block slides should not be a substitute for good quality FNA spreads; they are complementary but not mutually exclusive. Cell blocks often produce variable cellularity and results (Figure 4b). They are valuable for ancillary studies. Some laboratories may not make smears preferring to collect the specimen in fixatives. As if obvious in this instance, even with nearly two dozen slides and over 50 cell block sections the diagnostic material is extremely scant and less than optimal for diagnosis. Superior cell block preparations using patented technique (CellientÔ) have been used with mixed results.4,5 It is recommended that only one cell block per site should be prepared from the FNA material. We generally stain with H/E the first and the fifth cut of the cell block to evaluate the diagnostic material adequacy and preservation. The adjacent or subsequent slides if necessary can be processed for additional immunohistochemical, ancillary and molecular studies.
Nitinol (Nickel Titanium Naval Ordnance Laboratory): this dark, granular carbon like material is commonly observed in the aspirated EUS FNA specimens.6 Nitinol is scraped by the stylus during the expulsion of the representative material (Figure 5b). It is important to realize that although considered foreign and often ignored, it may contain the diagnostic material wrapped around these aggregates and is necessary that it should be carefully examined. Cellulosic material: sometime cellular material associated with the disposable cytospin filters may disintegrate and contaminate the slide preparations. This material is generally acellular; it can cause cellular obscuring especially when cytospin preparations are utilized for immunohistochemical studies (Figure 5c).
Recommended FNA spread preparations Number of passes: generally, one to two or a maximum of three passes using fine (22/27) needle aspiration should be made for each site being sampled; thinner needle 25/27 is more appropriate for highly vascular organs such as the thyroid. Most other lesions can safely be sampled using 22 or 23 gauge needle by palpitation or under ultrasound or other imaging modalities and guidance.
Foreign material associated with fine needle aspiration Lubricating gel: one of the most commonly observed material in the ultrasound guided FNA’s is Aqua-Gel used with the transducer. This appears as granular platelet like material often obscuring the cells (Figure 5a). Recently, lubricating gel has been reformulated making it less toxic and viscous. While preferable for imaging studies; it tends to run and produce substantial foreign material in the background. In most situations, liquid soap, alcohol even saline can be substituted for this material during the US guided FNA procedures.
DIAGNOSTIC HISTOPATHOLOGY 17:7
Spread slide: it is necessary that the aspirated specimen be delicately spread on the slide and not smeared by rubbing or pressing the material with any force. The smearing often results in disassociation of low molecular weight cytoplasm resulting in numerous bare nuclei. This commonly results in suboptimal
281
Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 4 a Excessive number of slides. Only two preparations (yellow labels) have any epithelial cells. The specimen is unsatisfactory. Thyroid FNA, Pap stain. b Cell block slides. Notice the unnecessary number of slides with limited diagnostic value. Such a practice is expensive and not recommended.
preparations. Smearing may yield acceptable results when the specimen contains high molecular weight bearing cells such as keratinized squamous and transitional cells.
and the two slides are separated (Figure 5d). Such smears in many cases are quite adequate, satisfactory and of high technical quality, however, the preparations can be variable. These are generally not recommended for all sites.
Double touch (Butterfly) smear: in addition to specimen spread technique discussed above, contact (butterfly) smear is a common practice. A drop of the aspirated material is deposited on the pre-labelled, clean slide. A second slide is put on the drop
DIAGNOSTIC HISTOPATHOLOGY 17:7
Spatter smear: this can sometime be utilized especially when little or no visible specimen is obtained within the hub of the syringe. The needle is disconnected and the contents forcefully
282
Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 5 a Aqual gel. Notice the precipitated granular material obscuring epithelial cells. Liver US guided FNA Diff-Quik stain. b Nitinol material. Notice the dark acellular material. Insert shows details; epithelial cells are often associated with Nitinol. Pancreas EUS guided FNA, Pap stain. c Cellulosic material. Notice the fibres obscuring a cytospin preparation. CK7 immunoperoxidase stain. d Double touch (Butterfly) smear. Thyroid FNA, Diff-Quik and Pap stained slides. e Spatter smear. The specimen is not spread or smeared. Cells of interested are present in small groups (arrow). Cervical lymph node FNA Diff-Quik stain.
pushed onto the slide surface. In such preparations often time few groups of diagnostic cells may be present in the specimen droplets (Figure 5e). Obviously, such specimens are inadequate for any ancillary studies.
subsequent processing. Slide is slightly tilted in the direction of the label and the liquids are rapidly aspirated thus leaving most of the cellular components for smearing. This technique insures high quality FNA spread smears without loss of any diagnostic material or compromising the quality of preparation (Figure 6).
Specimen concentration: this is in our experience a worthwhile procedure.7 The expressed FNA specimen with fluid contents can be improved by aspirating the fluid or the blood by a hypodermic needle and a syringe or a disposable pipette. The aspirated material collected can safely be deposited in appropriate preservative for
DIAGNOSTIC HISTOPATHOLOGY 17:7
Thick cellular preparations: when onsite stained specimen is extremely thick, the material can be either scrapped or cut and scraped. The material can be deposited in a preservative for such when cell block or other preparation (Figure 7).
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MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 6 FNA specimen concentration technique (see text).
FNA specimens: sent-in, on-site and Point-of-care (POC): the bar graph (Figure 8) shows a comparison of the most common and critical variables observed in the three types of FNA specimens i.e. sent-in smears and slides, slides prepared onsite and the slides obtained at the POC. It is obvious that the freshly procured material has better preservation and is more valuable for ancillary especially molecular studies. FNA specimens have been considered superior to core biopsies for molecular studies in breast lesions.8 They are representative of the lesion, are more pure, and cost effective with reduced risk of complications. While studying thyroid FNA and core specimens the proportion of satisfactory specimens in thyroid case increases in core biopsies cases, but the diagnostic yield is lower by 25e30%.3 Most of the specimens submitted as slides to the laboratory or processed there have been collected in remote locations either by the clinician by palpation or under some imaging modality. These sent-in specimens include varying numbers of medium
quality smears and specimens collected in various fixatives and preservative. Diagnosis is severely compromised by lack of adequate additional information so very critical for interpretation of minute cell sample as and microbiopsies. It is preferably that the specimen be collected in the presence of a cytopathologist by the clinician or the cytopathologist him or herself. The procedure requires the presence of the cytopathologist staff along with necessary equipment and supplies for processing and examination of the samples. Onsite evaluation rendered by a qualified cytopathologist are billable activities in the United States and are cost effective.9 In some institutions there is a dedicated fine needle aspiration clinic where the patient may be referred for such procedures. The establishment and value of such a clinic is not universally accepted. In most places a fine needle aspiration cart equipped with necessary supplies and microscope is wheeled to the bedside. In our institution these carts have undergone a fair
Figure 7 Thick FNA cell spreads treatment. The excess material can be scrapped off a or the smear can be divided collecting a part of the material in appropriate preservative b. Material can be paraffin processed for ancillary studies.
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6
Sent-in On-site POC
7
8
9 Figure 8 Graphic comparison of the salient aspects of the sent-in, on-site and POC FNA specimens. 10
degree of refinement and now we are using the third generation cart (Penn-A-Cart).10
11
POC FNA service at Penn: presently, we have ten dedicated FNA stations in various clinics. Each location is equipped with stateof-the-art imaging, video microscopy, informatics, specimen collection and processing facilities. This permits an improve communication with the healthcare providers and the patient care. The cytopathologist is able to communicate freely with the clinical colleagues, examine the necessary investigational studies, perform the procedure and give onsite interpretation. It is generally cost effective, but depending on the individual situation, may require commitment of additional resources including personnel and space. There is an associated considerable initial investment in such endeavours. POC FNA service is ideal not only for providing a first rate patient care but also addressing the issues of rapidly changing healthcare paradigm requiring molecular and ancillary studies in order to provide personal medicine in each individual case.11 Penn-A-Cart offers a good alternative to the POC service. It can easily be wheeled to the bedside. Access to the internet and hi-definition imaging shall permit availability of high quality FNA service in remote locations where skilled cytopathologists may not be accessible. A
Practice points I C
C
C
The diagnosis of FNA specimens must be accurate, precise and conclusive Accurate, precise and conclusive reporting needs multimodal approach The personalized medicine and the development of pharmacogenomics necessitates also adequate quantities of well preserved, uncontaminated, fresh and representative material
II Important factors to be taken into consideration, which in general are regarded as “unimportant”: C The slides (clean and grease free) C The way of puncturing (how many punctures, who should perform it, etc.) C The way of work-up of the material (fixation, staining) C Cell block technique
REFERENCES 1 Carmichael DE. The Pap Smear life of George Papanicolaou. Springfield, IL: Charles C Thomas, 1973. 82. 2 Gupta PK, Baloch ZW. Intraoperative and on-site cytopathology consultations: utilization, limitations and value. Semin Diagn Pathol 2002; 19: 227e36. 3 Renshaw AA, Pinnar N. Comparison of thyroid fine-needle aspiration and core needle biopsy. Am J Clin Pathol 2007; 128: 370e4. 4 Anderson SR, Colasacco C, Mitchell J, Sherman JF, Warren S, Gibson PC. A comparative immunocytochemical study in body cavity fluids using the CellientÔ automated cell block system. Cancer Cytopathol 2008; 114: 353. 5 Wagner DG, Russell DK, Benson JM, Schneider AE, Hoda RS, Bonfiglio TA. CellientÔ automated cell block versus traditional cell block preparation: a comparison of morphologic features and
DIAGNOSTIC HISTOPATHOLOGY 17:7
immunohistochemical staining. Diagn Cytopathol October 2010; 14: 1097e339 (online). Vilmann P, S~aftoiu A. Endoscopic ultrasound-guided fine needle aspiration biopsy: equipment and technique. J Gastroenterol Hepatol 2006; 21: 1646e55. Gupta PK, Baloch ZW. IAC Atlas. In: Gupta PK, Baloch ZW, eds. Thyroid fine needle aspiration cytopathology, vol. 44. International Academy of Cytology Chicago, October 2002. Symmans WF, Ayers M, Clark EA, et al. Total RNA yield and microarray gene expression profiles from fine-needle aspiration biopsy and core needle biopsy samples of breast carcinoma. Cancer 2003; 97: 2960e71. Nasuti JF, Gupta PK, Baloch ZW. Diagnostic value and costeffectiveness of on-site evaluation of fine-needle aspiration specimens: review of 5,688 cases. Diagn Cytopathol July 2002; 27: 1e4. Gupta PK. University of Pennsylvania aspiration cart 9 (Penn-A-Cart) an innovative journey in fine needle aspiration service. Acta Cytol 2010; 54: 165e8. Gupta PK. Progression from on-site to point-of-care fine needle aspiration service: opportunities and challenges. CytoJournal 2010; 7: 1e8.
III Disturbing “background” materials to be avoided or at least always recognized: C Lubricating US gel C Nitinol C Cellulosic material IV Organization of FNA activity: C FNA clinic C Sent-in smears and slides C In site puncturing C POC (Points-of-care) technique C It is preferably that the specimen be collected in the presence of a cytopathologist by the clinician or the cytopathologist him or herself.
285
Ó 2011 Elsevier Ltd. All rights reserved.
Fine needle aspiration: a changing paradigm
While most immunohistochemical studies can be performed on the submitted and fixed FNA specimens, the recent developments in personalized (boutique) medicine and pharmacogenomics necessitate the availability in adequate quantities of well preserved and uncontaminated, fresh representative material. Most of the subcellular (molecular) highly sophisticated studies require special handling, preservation and processing of minute quantities of diagnostic materials available during the FNA procedures. The value of onsite, accurate interpretation of cellular samples in procurement and handling of such specimens cannot be over emphasized. This paper summarizes the present practices and recommendations for improvements both in the quality of specimens and delivery of FNA services.
Prabodh K Gupta
Abstract Fine needle aspiration (FNA) is used routinely for detection, diagnosis, therapeutics and follow-up of various tumours. This practice is recognized as an accurate, rapid and economical diagnostic procedure. In the rapidly changing healthcare paradigm, however, there are new challenges being imposed on the practice of fine needle aspiration cytopathology. The diagnosis of FNA specimens must not only be accurate it should be precise. This puts additional demands on the clinicians, the cytopathologists and the laboratory; slide preparations and reporting must meet not only the diagnostic but also the therapeutic challenges. This requires a multimodal approach and interaction among the various parties including the patient, the primary care, the radiology, molecular and cytopathology laboratory personnel.2 Recent developments in personalized medicine and pharmacogenomics necessitate the availability in adequate quantities of well preserved and uncontaminated, fresh representative material. The value of onsite, accurate interpretation of cellular samples in procurement and handling of such specimens cannot be over emphasized. This paper summarizes the present practices and recommendations for improvements both in the quality of specimens and delivery of FNA services.
FNA slides and specimen spread preparations Fine needle aspiration is performed using thin bore (22e27 gauge) needles whereas the core biopsies often use large bore (12e15) needles. The precise diagnosis of an FNA specimen mandates a number of prerequisite essential for proper specimen preparation and reporting. These include: Clean slide: glass surface should be clean and grease free. Common occurrence of the acellular blank areas in the smears results from holding the slides on the surface with a thumb and finger and leaving a grease mark that prevent cell adhesion (Figure 2a). This can be avoided by grasping the slides from the edges especially in the upper one-third, since the lower two-third is often utilized for smearing the samples and it should be clean, smooth and grease free.
Keywords FNAC methodology; FNAC techniques; organization of sampling
Local anaesthesia: injectable products (Lidocaine) result in flooding of fluid in the intercellular space in the tissues. This causes artificial separation of cells, shearing and morphological artifacts (Figure 2b). The use of injectable local anaesthesia for FNA procedures should be discouraged. If necessary the puncture should be made in the adjacent area. The topical anaesthesia or ice or cold pack in most situations can provide adequate numbness to the sensitive skin areas. The skin surface should be cleaned with alcohol pads and if necessary with iodine or NormsolÒ to avoid burning sensation during the FNA procedure.
In clinical medicine, cytodiagnoses of numerous disease processes have become the standard-of-care in the world. Fine needle aspiration (FNA) is used routinely for detection, diagnosis, therapeutics and follow-up of various tumours. This practice is recognized as an accurate, rapid and economical diagnostic procedure. In the rapidly changing healthcare paradigm, however, there are new challenges being imposed on the practice of fine needle aspiration cytopathology. The diagnosis of FNA specimens must not only be accurate it should be precise. The concept of precision in cytopathology is not new; it was emphasized by George Papanicolaou in the beginning of cytopathology practice1 (Figure 1). Precise and conclusive reporting of FNA specimens puts additional demands on the clinicians, the cytopathologists and the laboratory; slide preparations and reporting must meet not only the diagnostic but also the therapeutic challenges. This requires a multimodal approach and interaction among the various parties including the patient, the primary care, the radiology, molecular and cytopathology laboratory personnel.2
Specimen drying (curing, air wash, air drying) for Romanowsky stains: proper air drying of the aspirated specimen is critical for development of metachromasia observed by onsite staining using a Romanowsky or similar stained FNA smear preparations. Inadequate air fixation generally results in homogenous hematoxylinophilic or autochromatic bluish staining of the nucleus and the cytoplasm thus rendering the interpretation difficult (Figure 3a). The situation often becomes critical in an otherwise adequate representative samples that become suboptimal or unsatisfactory for interpretation resulting from inadequate air drying and poor fixation. Suboptimal air washing often occurs in smears which are extremely thick. Instant air drying of the FNA spreads is not recommended: very rapid slide drying such as by use of hair drier, results in uneven dehydration of the aspirate rendering the FNA specimens suboptimal for interpretation; there is discordant contraction of the cells
Prabodh K Gupta MBBS MD FIAC is Professor of Pathology and Laboratory Medicine, Director of Cytopathology and Cytometry and Director of the Cytopathology Fellowship Program at the University of Pennsylvania Medical Center, PA, USA. Conflicts of interest: none declared.
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Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 1 Precision in cytopathology reporting. Endometrial smear report by George Papanicolaou in 1952. Not only it gives a precise diagnosis but let the surgeon know “the diagnosis is well enough established to justify a major operation without curettage”. Adapted from e The Pap Smear; D. Erskine Carmichael, Charles C Thomas, Springfield, IL. 1973, p. 82.
and the surrounding intercellular material causing a pericellular clearing or a halo effect. Also, there is unusual degeneration and coagulation of cytoproteins producing hyperchromasia and loss of critical morphologic details (Figure 3b). Rapid air drying of a thyroid colloid material often results in disruption and beading of the colloid network. In such situations the cells of interest often accumulate as satellites at the nodes (Figure 3c). Alcohol fixation: it is a common practice to ethanol fix the smears for subsequent staining by polychromatic Papanicolaou stain. Although the use of 95% ethanol in Coplin jar and rapid immersion of the freshly prepared cell preparations is optimal, oftentimes commercially available, convenient spray fixatives are used for such purposes. Most of these products contain ethanol and or methanol and polyglycol. Often time the fixed slide will result in blobs of the cellular material giving a spongy, “Swiss cheese” appearance (Figure 3d), the diagnostic cells tend to accumulate along the edges of such clumps, they become overlapping, crowded and difficult to interpret. It is necessary that the manufacturer’s instructions be carefully followed while using the spray fixatives for FNA slides.
Figure 2 a Grease on slide surface, contamination from finger touch. Lubricants prevent specimen spread. b Effect of local anaesthesia injection. Disruption of cell and loss of metachromasia are evident (A), Lidocaine injection. Same specimen without local injection (B), Diff-Quik stain.
evaluation is available. Such a practice is painful and in our opinion unnecessary, uneconomical, unrewarding and wasteful (Figure 4a). As is obvious out of the 36 submitted slides only two smears (yellow labels) contain any cellular material. This whole specimen was considered unsatisfactory for diagnostic purposes. The excessive number of aspirations beyond three or five in most instances not only results in poor quality diagnosis. These preparations require additional supplies, screening, interpretation and diagnostic efforts. Two or three passes with onsite evaluation may be supplemented with additional material or a needle core biopsy if necessary for ancillary studies.
Number of punctures and slides: the number of needle pricks and slides prepared from each site during FNA procedures is extremely variable. It is not uncommon to perform 10e12 passes from one lesion.3 The situation is aggravated when no onsite
DIAGNOSTIC HISTOPATHOLOGY 17:7
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Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 3 a Effect of inadequate air drying. Poor staining (A); compare to the well dried cells (B), Diff-Quik stain. b Effect of rapid air drying. Notice the fried appearance of follicular cells. Insert shows higher magnification of the distorted cells, Diff-Quik stain. c Effect of rapid air drying of cell spreads. Notice the beaded appearance of colloid material. Insert shows epithelial cells aggregating at the tips. d Effect of improper spray fixation. Notice the clumping of the specimen giving a Swiss cheese appearance. Cells in such preparation collect at the margins, (green dot).
Cell blocks preparations: cell block slides should not be a substitute for good quality FNA spreads; they are complementary but not mutually exclusive. Cell blocks often produce variable cellularity and results (Figure 4b). They are valuable for ancillary studies. Some laboratories may not make smears preferring to collect the specimen in fixatives. As if obvious in this instance, even with nearly two dozen slides and over 50 cell block sections the diagnostic material is extremely scant and less than optimal for diagnosis. Superior cell block preparations using patented technique (CellientÔ) have been used with mixed results.4,5 It is recommended that only one cell block per site should be prepared from the FNA material. We generally stain with H/E the first and the fifth cut of the cell block to evaluate the diagnostic material adequacy and preservation. The adjacent or subsequent slides if necessary can be processed for additional immunohistochemical, ancillary and molecular studies.
Nitinol (Nickel Titanium Naval Ordnance Laboratory): this dark, granular carbon like material is commonly observed in the aspirated EUS FNA specimens.6 Nitinol is scraped by the stylus during the expulsion of the representative material (Figure 5b). It is important to realize that although considered foreign and often ignored, it may contain the diagnostic material wrapped around these aggregates and is necessary that it should be carefully examined. Cellulosic material: sometime cellular material associated with the disposable cytospin filters may disintegrate and contaminate the slide preparations. This material is generally acellular; it can cause cellular obscuring especially when cytospin preparations are utilized for immunohistochemical studies (Figure 5c).
Recommended FNA spread preparations Number of passes: generally, one to two or a maximum of three passes using fine (22/27) needle aspiration should be made for each site being sampled; thinner needle 25/27 is more appropriate for highly vascular organs such as the thyroid. Most other lesions can safely be sampled using 22 or 23 gauge needle by palpitation or under ultrasound or other imaging modalities and guidance.
Foreign material associated with fine needle aspiration Lubricating gel: one of the most commonly observed material in the ultrasound guided FNA’s is Aqua-Gel used with the transducer. This appears as granular platelet like material often obscuring the cells (Figure 5a). Recently, lubricating gel has been reformulated making it less toxic and viscous. While preferable for imaging studies; it tends to run and produce substantial foreign material in the background. In most situations, liquid soap, alcohol even saline can be substituted for this material during the US guided FNA procedures.
DIAGNOSTIC HISTOPATHOLOGY 17:7
Spread slide: it is necessary that the aspirated specimen be delicately spread on the slide and not smeared by rubbing or pressing the material with any force. The smearing often results in disassociation of low molecular weight cytoplasm resulting in numerous bare nuclei. This commonly results in suboptimal
281
Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 4 a Excessive number of slides. Only two preparations (yellow labels) have any epithelial cells. The specimen is unsatisfactory. Thyroid FNA, Pap stain. b Cell block slides. Notice the unnecessary number of slides with limited diagnostic value. Such a practice is expensive and not recommended.
preparations. Smearing may yield acceptable results when the specimen contains high molecular weight bearing cells such as keratinized squamous and transitional cells.
and the two slides are separated (Figure 5d). Such smears in many cases are quite adequate, satisfactory and of high technical quality, however, the preparations can be variable. These are generally not recommended for all sites.
Double touch (Butterfly) smear: in addition to specimen spread technique discussed above, contact (butterfly) smear is a common practice. A drop of the aspirated material is deposited on the pre-labelled, clean slide. A second slide is put on the drop
DIAGNOSTIC HISTOPATHOLOGY 17:7
Spatter smear: this can sometime be utilized especially when little or no visible specimen is obtained within the hub of the syringe. The needle is disconnected and the contents forcefully
282
Ó 2011 Elsevier Ltd. All rights reserved.
MINI-SYMPOSIUM: NON-GYNAECOLOGICAL CYTOLOGY
Figure 5 a Aqual gel. Notice the precipitated granular material obscuring epithelial cells. Liver US guided FNA Diff-Quik stain. b Nitinol material. Notice the dark acellular material. Insert shows details; epithelial cells are often associated with Nitinol. Pancreas EUS guided FNA, Pap stain. c Cellulosic material. Notice the fibres obscuring a cytospin preparation. CK7 immunoperoxidase stain. d Double touch (Butterfly) smear. Thyroid FNA, Diff-Quik and Pap stained slides. e Spatter smear. The specimen is not spread or smeared. Cells of interested are present in small groups (arrow). Cervical lymph node FNA Diff-Quik stain.
pushed onto the slide surface. In such preparations often time few groups of diagnostic cells may be present in the specimen droplets (Figure 5e). Obviously, such specimens are inadequate for any ancillary studies.
subsequent processing. Slide is slightly tilted in the direction of the label and the liquids are rapidly aspirated thus leaving most of the cellular components for smearing. This technique insures high quality FNA spread smears without loss of any diagnostic material or compromising the quality of preparation (Figure 6).
Specimen concentration: this is in our experience a worthwhile procedure.7 The expressed FNA specimen with fluid contents can be improved by aspirating the fluid or the blood by a hypodermic needle and a syringe or a disposable pipette. The aspirated material collected can safely be deposited in appropriate preservative for
DIAGNOSTIC HISTOPATHOLOGY 17:7
Thick cellular preparations: when onsite stained specimen is extremely thick, the material can be either scrapped or cut and scraped. The material can be deposited in a preservative for such when cell block or other preparation (Figure 7).
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Figure 6 FNA specimen concentration technique (see text).
FNA specimens: sent-in, on-site and Point-of-care (POC): the bar graph (Figure 8) shows a comparison of the most common and critical variables observed in the three types of FNA specimens i.e. sent-in smears and slides, slides prepared onsite and the slides obtained at the POC. It is obvious that the freshly procured material has better preservation and is more valuable for ancillary especially molecular studies. FNA specimens have been considered superior to core biopsies for molecular studies in breast lesions.8 They are representative of the lesion, are more pure, and cost effective with reduced risk of complications. While studying thyroid FNA and core specimens the proportion of satisfactory specimens in thyroid case increases in core biopsies cases, but the diagnostic yield is lower by 25e30%.3 Most of the specimens submitted as slides to the laboratory or processed there have been collected in remote locations either by the clinician by palpation or under some imaging modality. These sent-in specimens include varying numbers of medium
quality smears and specimens collected in various fixatives and preservative. Diagnosis is severely compromised by lack of adequate additional information so very critical for interpretation of minute cell sample as and microbiopsies. It is preferably that the specimen be collected in the presence of a cytopathologist by the clinician or the cytopathologist him or herself. The procedure requires the presence of the cytopathologist staff along with necessary equipment and supplies for processing and examination of the samples. Onsite evaluation rendered by a qualified cytopathologist are billable activities in the United States and are cost effective.9 In some institutions there is a dedicated fine needle aspiration clinic where the patient may be referred for such procedures. The establishment and value of such a clinic is not universally accepted. In most places a fine needle aspiration cart equipped with necessary supplies and microscope is wheeled to the bedside. In our institution these carts have undergone a fair
Figure 7 Thick FNA cell spreads treatment. The excess material can be scrapped off a or the smear can be divided collecting a part of the material in appropriate preservative b. Material can be paraffin processed for ancillary studies.
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6
Sent-in On-site POC
7
8
9 Figure 8 Graphic comparison of the salient aspects of the sent-in, on-site and POC FNA specimens. 10
degree of refinement and now we are using the third generation cart (Penn-A-Cart).10
11
POC FNA service at Penn: presently, we have ten dedicated FNA stations in various clinics. Each location is equipped with stateof-the-art imaging, video microscopy, informatics, specimen collection and processing facilities. This permits an improve communication with the healthcare providers and the patient care. The cytopathologist is able to communicate freely with the clinical colleagues, examine the necessary investigational studies, perform the procedure and give onsite interpretation. It is generally cost effective, but depending on the individual situation, may require commitment of additional resources including personnel and space. There is an associated considerable initial investment in such endeavours. POC FNA service is ideal not only for providing a first rate patient care but also addressing the issues of rapidly changing healthcare paradigm requiring molecular and ancillary studies in order to provide personal medicine in each individual case.11 Penn-A-Cart offers a good alternative to the POC service. It can easily be wheeled to the bedside. Access to the internet and hi-definition imaging shall permit availability of high quality FNA service in remote locations where skilled cytopathologists may not be accessible. A
Practice points I C
C
C
The diagnosis of FNA specimens must be accurate, precise and conclusive Accurate, precise and conclusive reporting needs multimodal approach The personalized medicine and the development of pharmacogenomics necessitates also adequate quantities of well preserved, uncontaminated, fresh and representative material
II Important factors to be taken into consideration, which in general are regarded as “unimportant”: C The slides (clean and grease free) C The way of puncturing (how many punctures, who should perform it, etc.) C The way of work-up of the material (fixation, staining) C Cell block technique
REFERENCES 1 Carmichael DE. The Pap Smear life of George Papanicolaou. Springfield, IL: Charles C Thomas, 1973. 82. 2 Gupta PK, Baloch ZW. Intraoperative and on-site cytopathology consultations: utilization, limitations and value. Semin Diagn Pathol 2002; 19: 227e36. 3 Renshaw AA, Pinnar N. Comparison of thyroid fine-needle aspiration and core needle biopsy. Am J Clin Pathol 2007; 128: 370e4. 4 Anderson SR, Colasacco C, Mitchell J, Sherman JF, Warren S, Gibson PC. A comparative immunocytochemical study in body cavity fluids using the CellientÔ automated cell block system. Cancer Cytopathol 2008; 114: 353. 5 Wagner DG, Russell DK, Benson JM, Schneider AE, Hoda RS, Bonfiglio TA. CellientÔ automated cell block versus traditional cell block preparation: a comparison of morphologic features and
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immunohistochemical staining. Diagn Cytopathol October 2010; 14: 1097e339 (online). Vilmann P, S~aftoiu A. Endoscopic ultrasound-guided fine needle aspiration biopsy: equipment and technique. J Gastroenterol Hepatol 2006; 21: 1646e55. Gupta PK, Baloch ZW. IAC Atlas. In: Gupta PK, Baloch ZW, eds. Thyroid fine needle aspiration cytopathology, vol. 44. International Academy of Cytology Chicago, October 2002. Symmans WF, Ayers M, Clark EA, et al. Total RNA yield and microarray gene expression profiles from fine-needle aspiration biopsy and core needle biopsy samples of breast carcinoma. Cancer 2003; 97: 2960e71. Nasuti JF, Gupta PK, Baloch ZW. Diagnostic value and costeffectiveness of on-site evaluation of fine-needle aspiration specimens: review of 5,688 cases. Diagn Cytopathol July 2002; 27: 1e4. Gupta PK. University of Pennsylvania aspiration cart 9 (Penn-A-Cart) an innovative journey in fine needle aspiration service. Acta Cytol 2010; 54: 165e8. Gupta PK. Progression from on-site to point-of-care fine needle aspiration service: opportunities and challenges. CytoJournal 2010; 7: 1e8.
III Disturbing “background” materials to be avoided or at least always recognized: C Lubricating US gel C Nitinol C Cellulosic material IV Organization of FNA activity: C FNA clinic C Sent-in smears and slides C In site puncturing C POC (Points-of-care) technique C It is preferably that the specimen be collected in the presence of a cytopathologist by the clinician or the cytopathologist him or herself.
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