This article identifies key fundamentals of tissue acquisition, sample preparation, and staining. It defines the understanding of different aspects of sample preparations, such as types of smear-preparation techniques, touch preparations, types of fixative, and newer technologies such as liquid-based preparations.
Key points
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A successful cytology practice mandates teamwork and close interaction between a skillful tissue procurer and an experienced cytopathologist.
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The goal of cytologic preparation is to obtain the representative cells in an appropriate and optimum state for microscopic examination; without too much overlapping or too much spreading of the cells over a large area.
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Many different staining protocols have been used in cytology practices.
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Cell blocks should always be used in conjunction with analysis of smears and other cytologic preparations.
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Telepathology has rapidly evolved from a simple model of capture, store, and forward concept of static images to a very complex and dynamic process, which holds promise to significantly change the way anatomic pathology is being practiced today.
Introduction
Fine-needle aspiration (FNA) and small-tissue sampling and analysis are complex and highly coordinated processes. The main objective of tissue sampling of a focal lesion by endoscopic ultrasonography (EUS)-guided fine-needle aspiration biopsy (FNAB) and/or core-needle biopsy (CNB) is to provide a rapid, accurate, and cost-efficient diagnosis for informed decision making and appropriate patient management. Samples obtained using these modalities, however, should be handled with care because whichever modality is used (FNA or CNB), the samples procured are limited in volume. The main components required to optimize cell yield are interlinked with each other, and include (1) sample acquisition, (2) sample handling and preparation, and (3) analysis. A preliminary understanding of how samples are prepared for cytology and histology, as noted in Table 1 , would help in understanding the differences between the types of assessments offered by 2 relatively different tissue-processing methodologies. Aspirates are routinely handled as cytologic specimens; however, cell blocks and micro biopsies can be simultaneously prepared from the FNAB material for histologic examination. From core biopsies, one can obtain touch imprints for cytologic assessment as well.
| Cytology Technique | Histology Technique | |
|---|---|---|
| Fixation | Air drying Ethanol | 10% Neutral buffered formalin |
| Transport | Special medium for liquid-based sample processing (eg, Preservecyt, SurePath, ThinPrep, Hanks balanced salt solution) | Saline, formalin |
| Processing | Direct smear/centrifugation related preparations/liquid-based preparations | Gross examination Tissue processing Paraffin embedding Microtome sectioning |
| Staining | Papanicolaou/Romanowsky stains | Hematoxylin-eosin (H&E) |
| Product | Smears and liquid-based preparations: whole cells are analyzed Cell-block preparations: H&E-stained sections are analyzed | Sections (part of cells) |
A successful cytology practice mandates teamwork and close interaction between a skillful tissue procurer and an experienced cytopathologist.
Introduction
Fine-needle aspiration (FNA) and small-tissue sampling and analysis are complex and highly coordinated processes. The main objective of tissue sampling of a focal lesion by endoscopic ultrasonography (EUS)-guided fine-needle aspiration biopsy (FNAB) and/or core-needle biopsy (CNB) is to provide a rapid, accurate, and cost-efficient diagnosis for informed decision making and appropriate patient management. Samples obtained using these modalities, however, should be handled with care because whichever modality is used (FNA or CNB), the samples procured are limited in volume. The main components required to optimize cell yield are interlinked with each other, and include (1) sample acquisition, (2) sample handling and preparation, and (3) analysis. A preliminary understanding of how samples are prepared for cytology and histology, as noted in Table 1 , would help in understanding the differences between the types of assessments offered by 2 relatively different tissue-processing methodologies. Aspirates are routinely handled as cytologic specimens; however, cell blocks and micro biopsies can be simultaneously prepared from the FNAB material for histologic examination. From core biopsies, one can obtain touch imprints for cytologic assessment as well.
| Cytology Technique | Histology Technique | |
|---|---|---|
| Fixation | Air drying Ethanol | 10% Neutral buffered formalin |
| Transport | Special medium for liquid-based sample processing (eg, Preservecyt, SurePath, ThinPrep, Hanks balanced salt solution) | Saline, formalin |
| Processing | Direct smear/centrifugation related preparations/liquid-based preparations | Gross examination Tissue processing Paraffin embedding Microtome sectioning |
| Staining | Papanicolaou/Romanowsky stains | Hematoxylin-eosin (H&E) |
| Product | Smears and liquid-based preparations: whole cells are analyzed Cell-block preparations: H&E-stained sections are analyzed | Sections (part of cells) |
A successful cytology practice mandates teamwork and close interaction between a skillful tissue procurer and an experienced cytopathologist.
Sample acquisition
Before other sections highlight types of needs and core biopsies, it is important to understand some differences in these terminologies.
Fine-Needle Aspiration
Aspiration of tissue sample with a needle size smaller than 21 gauge is considered as a fine-needle aspiration. For the purposes of EUS, for a long time 22-gauge, 25-gauge, and now 27-gauge needles have been used to obtain samples from various organ sites. Using such thin needles have proved to be less invasive, and has the advantage of obtaining adequate samples from target lesions to provide an accurate diagnosis. By comparison, larger-bore needles (19-gauge) have often been used to obtain samples. Such needles offer less flexibility and are associated with greater tissue trauma. This type of sampling offers acquisition of cytology as well as micro biopsy samples.
Core-Needle Biopsy
Core biopsy needles allow acquisition of tissue core biopsies. Innovation has allowed acquisition of core biopsy samples with a needle size as small as 25-gauge, 22-gauge, and 19-gauge. A sample acquired using this modality offers core biopsies and the advantage of reviewing tissue architecture. Such a sample often helps to reduce the number of passes. It also helps general surgical pathologists who are more familiar with such samples to render their opinions. This modality, however, is more morbid and may lead to an increased number of adverse events. It is recognized that cutting needles provide long, adequate samples without fragmentation. Large-bore aspiration-type needles may occasionally lead to tissue fragmentation. The moving and cutting mechanics of the various large-bore needles underlie the procurement of the tissue samples, which often include tumor and host tissue as well.
In view of the differences, the 2 modalities (FNA and CNB) complement each other when performed at the same sitting.
Key Features: Sample Acquisition
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The operator is the most important team member, whose task is to provide representative and sufficient samples.
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The fundamentals for successful aspiration are negative pressure, precision, and intralesional placement of the needle at all times when negative pressure is applied.
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The fundamentals for successful core biopsy are needle type and moving mechanics, precision, and positioning of the needle in the vicinity and into the mass.
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Route of acquisition may be the source of contaminants picked up en route that may influence interpretation.
Handling a cytology sample
The goal of cytologic preparation is to harvest the representative cells in an appropriate and optimum state for microscopic examination, without too much overlapping or too much spreading of the cells over a large area.
Direct smears can be sourced from aspirates and touch imprints. Direct smears can be made either by gently spreading the material using another spreader slide, or via a butterfly technique whereby samples are spread on both slides. Generally touch imprints are made from core biopsies to assess adequacy. Cellularity in touch preparations will depend on the type of lesion and the force applied to the cores when touched on the slides.
Handling of Cytology Samples
The factors involved in proper handling of cytology samples are (1) appropriate fixation, (2) appropriate number of slides, and (3) appropriate handling of excess blood.
Appropriate fixation
Wet fixation
Smears still wet should be directly immersed using 95% ethanol fixative, which can be achieved by immersing slides in alcohol, placing alcohol on slides laid on a plane surface, or spraying the smears using spray fixatives. Such preparations are usually stained using Papanicolaou stain. Air drying of cells when one uses this protocol will lead to lack of staining characteristics for cells.
Dry fixation
The air-dried preparation requires the cytologic smears to be immediately dried. Here, air drying serves as a fixation. In general, these slides are stained with Romanowsky stains. In instances where the smears are still wet, staining will be uneven and will lose crisp cellular details.
Appropriate number of slides
The number of slides prepared should correspond to the volume of the aspirates. There are 4 smearing methods: leveling, pushing (butterfly, facing), sliding, and crushing.
Leveling is performed when the volume of the aspirate is merely sufficient for only one slide.
Pushing is good for lymph node aspirates and granular cytologic samples. A drop of sample is expressed onto the center of the slide, and another slide is placed on it such that the material on the slide spreads by capillary action from the center to the periphery.
Sliding is more appropriate for aspirates with some fluid or for large-volume samples. If the sample is voluminous and 1 pair of slides would result in thick smears, 2 pairs or more are recommended, by dividing the samples using another opposing slide and repeating the sliding method for the divided samples.
The crushing method has to be avoided at all costs.
Appropriate handling of excess blood
Blood clots usually trap and hide the incriminating cells. One suggestion is to remove the blood clot before making the smears. Cell blocks should be prepared if there is substantial admixed tissue.
It cannot be overemphasized that sample preparation holds the key to the success of FNAB interpretation. Making a direct smear on site is one of the most basic steps that should be learned by any practitioner of cytopathology. Practice makes perfect.
Potential Pitfalls
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When aspirating from vascular organs, often there will be a significant amount of blood within the needle. In such instances, once a drop of material is placed on the slide, the sample may be concentrated with a syringe and needle to avoid making too many slides with cells entrapped in clotted blood, making them uninterpretable.
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One should also avoid the use of force while making smears, as this may lead to shearing of cells. Such smears will demonstrate many naked hyperchromatic nuclei with prominent nucleoli.
Liquid-Based Technologies
Liquid-based methodologies for preparing samples use either membrane filter or cytocentrifugation. The former uses membranes with pores small enough to filter out fluid and debris. The cells are captured on the membrane that is transparent under light microscopy. The latter basically uses centrifugation to concentrate cells in a limited area. These techniques are suitable for cytologic samples, collected in liquid. The most significant benefit over conventional cytology is the even fixation and preparation.
Cytospin and Millipore filter are 2 basic methods still performed in laboratories for liquid-based samples. These methods are useful for needle rinses and cyst contents. Cytospin, the trade name for cytocentrifugation, offers concentrated samples placed in a defined area on a slide, thereby improving cell yield as well as providing evenly distributed cells. Millipore membrane filters are made of biologically inert mixtures of cellulose acetate and cellulose nitrate, and serve as sieves. The advantage of these membranes is that they help to enrich cells on the membrane while allowing passage of very small particles to be eliminated from cytologic evaluation.
In recent years newer modalities of liquid-based commercial technologies, such as ThinPrep and SurePath, have been replacing older technologies. Their usage in nongynecologic cytology is gradually expanding. These different features might affect the cellular details and, hence, influence the interpretation. Table 2 highlights salient differences between these 2 technologies.
Related posts:
New Methods to Obtain Better Tissue Samples with EUS-guided FNA
Preface: EUS-Guided Tissue Acquisition
The Changing Paradigm in EUS-Guided Tissue Acquisition
Techniques for EUS-guided FNA Cytology
Techniques for Endoscopic Ultrasound-Guided Fine-Needle Biopsy
Tips to Overcome Technical Challenges in EUS-guided Tissue Acquisition
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