CHAPTER 2 The techniques of FNA cytology
Basic techniques
Svante R. Orell
Although severe complications are rare1,2 the possible benefits of a cytological diagnosis should be weighed against risks and patient discomfort. Risk factors such as age, coagulation disorders, respiratory failure, etc. should be taken into account. FNB of superficial lesions can safely be carried out as an office procedure. Biopsies of most deep sites (transpleural, transperitoneal, etc.) are better performed in hospital so that patients can be sedated if necessary and kept under observation for a few hours after the procedure. The pathologist should be consulted before the procedure to give advice on feasibility, the likely informative value of the test, the need for and choice of ancillary techniques, etc.
Preparation for biopsy
Equipment
Needles
If the purpose of the biopsy is to obtain a core of tissue for paraffin embedding and sectioning, a cutting core needle is used. A range of small-gauge cutting core needles is commercially available. Core needle biopsy (CNB) fragments allow the study of tissue architecture and provide more tissue for ancillary tests. However, CNB is more traumatic and more costly, has a slightly greater risk of complications, and must be processed in a laboratory, precluding an immediate, on-site assessment of adequacy or a preliminary diagnosis.3,4
Syringes and syringe holder
Standard disposable plastic syringes mounted in a syringe holder/pistol grip are suitable for conventional aspiration biopsy. The Cameco Syringe Pistol (Cameco AB, Taby, Sweden) is made to fit 10-cc plastic syringes. The holder leaves one hand free to fix and to feel the target, which allows better precision in placing the needle (Fig. 2.1).
Patient preparation
The procedure should be clearly explained to the patient to assure his/her consent and cooperation.5 A formal written consent may be required, at least for deep biopsies. The procedure is usually carried out with the patient supine on an examination couch with easy access from either side. A couch with stirrups is preferable for transrectal and transvaginal biopsy, and an examination chair with adjustable headrest for biopsy of lesions in the head and neck.
The biopsy procedure
Insertion of the needle
The use of radiological imaging techniques to guide deep biopsies is described in Chapter 3. Ultrasonographic (US) guidance may be of value even in palpable lesions. It defines the lesion exactly, gives the optimal depth for biopsy, guides the needle to a solid portion of a complex lesion, and shows the relationship to other anatomical structures such as major vessels, pleura, etc.
FNB with aspiration (Figs. 2.1 and 2.2)
The aspiration technique is illustrated diagrammatically in Figure 2.2. The negative pressure does not tear cells from the tissue but merely holds the tissue against the sharp cutting edge of the needle, which scrapes or cuts softer tissue components along the track as the needle advances through the tissue.6 Highly cellular tissue components are softer and more friable than the supporting stroma and are selectively sampled. Fibrous stromal components are poorly represented, whereas myxoid stroma is more easily sampled. To increase the yield, the needle should be moved back and forth within the lesion with the negative pressure maintained, more vigorously in fibrous tissues with low cell content. Several passes may be necessary to sample a sufficient number of cells. In highly cellular and vascular tissues such as spleen, lymph nodes, liver and thyroid, a few rapid passes usually suffice. Additional passes mainly increase the amount of blood aspirated, causing dilution of the cellular component. Admixture with blood tends to be less if the needle is moved along the same track rather than in multiple directions. One should never wait to see material enter the hub of the needle, except when evacuating a cyst or an abscess. The ideal aspirate has a creamy consistency due to high cell content in a small amount of fluid and remains inside the needle.
The negative pressure must be released before the needle is withdrawn. Even so, part of the aspirate is often drawn up into the hub of the needle (see below). A maintained negative pressure may draw the aspirate into the syringe, which must then be rinsed with fluid to recover the specimen. It can also cause contamination by material aspirated along the track during withdrawal of the needle. Aspiration of US gel in guided FNB of breast lesions (Chapter 7) is a good example.
Fine needle sampling without aspiration (Figs 2.3 and 2.4)
As mentioned, the negative pressure plays a relatively minor role compared to the scraping or cutting effect of the advancing oblique needle tip. Fine needle biopsy without aspiration was introduced by Zajdela in 1987.7 This technique is based on the observation that the capillary pressure in a fine needle is sufficient to keep the detached cells inside its lumen. A 27–23-gauge standard needle is held directly with the fingers, inserted into the target tissue, moved back and forth in several directions for a few seconds depending on the cellularity and the vascularity of the tissue, and is then withdrawn. Using this technique, the operator gets an excellent feel of the consistency of the tissues. This is a valuable piece of diagnostic information and improves precision when sampling small lesions. Admixture with blood is generally less than with aspiration. The technique is particularly well suited for biopsy of the thyroid and other vascular tissues. The cell yield may be smaller than with aspiration but not significantly so.8,9 We use sampling without aspiration routinely in superficial biopsies except in cystic lesions and in fibrotic paucicellular tumors in the breast and soft tissues. Aspiration with 22-gauge needles is used in most deep biopsies in order to obtain a maximum volume of cells with a minimum number of passes, in view of the frequent demand for ancillary tests. However, non-aspiration biopsy sometimes produces better samples in highly vascular lesions, for example in renal tumors.
![image](https://i0.wp.com/basicmedicalkey.com/wp-content/uploads/2017/06/B9780702031519000025_f002-003-9780702031519.jpg?w=960)
Fig. 2.3 FNB without aspiration (thyroid)
Needle held with finger tips, other hand feels and fixes the target.
Failure to obtain a representative sample
Possible causes of failure to obtain a representative sample are illustrated diagrammatically in Figure 2.5. If the needle narrowly misses the target (Fig. 2.5B), only the adjacent tissues are sampled. An inflammatory reaction around the tumor may lead to an erroneous diagnosis. If the sample is derived from a central focus of necrosis, hemorrhage or cystic change (Fig. 2.5C), no diagnostic elements may be included. A dominant benign lesion like a cyst or lipoma can hide a small adjacent malignancy (Fig. 2.5D), for example in the breast or thyroid. Repeat biopsy of any remaining palpable abnormality after evacuation of a cyst is important. Finally, adequate samples may be difficult to obtain from desmoplastic tumor tissue in which cells are firmly held in a dense collagenous framework (Fig. 2.5E).
Processing the sample
The sample contained in the needle is expelled on to a clean and dry microscopy slide using air in a syringe. Care must be taken to avoid splashing. Not infrequently, the best part of the sample is found in the hub of the needle and is not easily expelled. In this case, the sample can be aspirated from the hub using another needle (Fig. 2.6).
![image](https://i0.wp.com/basicmedicalkey.com/wp-content/uploads/2017/06/B9780702031519000025_f002-006-9780702031519.jpg?w=960)
Fig. 2.6 Collecting a sample trapped in the hub of the biopsy needle
The sample is aspirated with another needle mounted on a syringe.
Direct smearing (Figs 2.7–2.12)
Smear quality is highly dependent on the smear being thin and evenly spread, ideally as a monolayer of cells. Perfect smearing is not easily learned. This is one of the reasons why FNB generally has a higher success rate when the biopsy procedure is attended by laboratory staff. Nuclear detail is poorly shown and confusing artifacts are common if smears are thick, uneven and dry slowly (Figs 2.9–2.11). Sometimes, even well prepared smears, particularly of lymphoid cells, may show a peculiar raisin-like distortion of the nuclei probably caused by moisture on the slide (Fig. 2.12). Exposure of air-dried smears to formalin vapor, which can occur during transport of material to the laboratory, can affect nuclear staining and cause loss of morphologic detail.10
The optimal sample, obtainable from cell-rich tissues, has a creamy consistency due to high cellularity with little or no blood or fluid (’dry’ sample). A ‘dry’ sample is best smeared with the flat of a second slide exerting a light pressure as it is moved along the specimen slide (Fig. 2.7, top). The pressure must be carefully adjusted to achieve a thin, even spread without causing disruption of tissue fragments with loss of micro-architecture, or smudging artifacts as in Figure 2.11A. Optimal smearing is a fine balance between too thick and too thin. Smears of ‘dry’ aspirates dry quickly, resulting in a milky, finely granular film on the slide.
A ‘wet’ aspirate consists mainly of blood or fluid containing smaller numbers of cells. The cells can be concentrated and separated from the fluid using a two-step smearing technique as illustrated in Figure 2.7. The smearing slide is held against the specimen slide at a blunt angle near one end of the slide, allowing the fluid to accumulate in the angle. The smearing slide is then rapidly moved along the specimen slide, half way or all the way depending on the amount of fluid. Most of the fluid is left behind while the cells tend to follow the smearing slide. The concentrated cells are then smeared with the flat of the slide as for a ‘dry’ aspirate, either on the same specimen slide, or swiped to another slide.
Examples of the macroscopical appearances of smears are shown in Figure 2.8. Figure 2.8A is an optimal smear of carcinoma showing numerous cell aggregates seen as granules spread evenly over the slide. Figure 2.8B is a two-step smear of carcinoma showing a film of blood at the top and concentrated cells at the middle, seen as a granular material. Figure 2.8C and 2.8D are examples of unsatisfactory smears from external sources.
Indirect smearing
Thin watery samples are processed by centrifugation in a cytocentrifuge. Millipore or Nucleopore filtration is an alternative but has been less satisfactory in our hands. Some laboratories prefer to rinse needles and syringes routinely with saline or with a fixative, which is then centrifuged or filtered onto slides.11,12 More recently, the ThinPrep technique developed for gynecological cytology has been increasingly applied also to FNB specimens.13–16 These techniques offer alternative solutions to the frequent problem of suboptimal samples received from distant sources, when the laboratory has no control over the biopsy procedure. However, the ThinPrep technique has its specific problems, and established diagnostic criteria may have to be redefined for FNB samples.17,18 In our opinion, direct smears expertly prepared by an experienced cytopathologist remain the optimal basis for FNAC diagnosis available today, and our first priority is to perfect this technique. ThinPrep preparations are a valuable supplement, particularly for immunocytochemical staining (see below).
Monolayered smears with optimal cell preservation are particularly important in the diagnosis of malignant lymphoma. For lymph node aspirates, we recommend that a cell suspension be prepared in addition to direct smears. Hank’s balanced salt solution with the addition of 10–20% fetal calf serum is ideal for this purpose. The suspension is spun on the cytocentrifuge at low r.p.m. Dilution may be necessary to achieve optimal dispersion of cells on the slide and to avoid clumping (Fig. 2.13). A number of slides can usually be made from one aspirate to allow immunocytochemical studies. Further details on techniques suitable for lymph node samples are given in Chapter 5.
Tissue fragments and cell blocks
Sometimes, a thin core or fragments of tissue may be obtained with a standard 22-gauge needle (Fig. 2.14). Tissue fragments are fixed in 5–10% buffered isotonic formalin and processed as for routine histology.
Some laboratories recommend the routine preparation of cell blocks for paraffin embedding of FNB samples. Cell blocks may give a better idea of tissue architecture and allow multiple sections for panels of immune markers with controls.19–21 However, they are relatively time consuming and costly compared to routine smears.22 We use cell blocks selectively, mainly if a need for immunocytochemistry is anticipated. Cell blocks are helpful if samples are heavily admixed with blood. Surprisingly good tissue fragments are often found in a cell block even when smears show only blood.
More recently, we have developed a simplified technique for cell blocks that we call ‘cell buttons’, shown diagrammatically in Figure 2.15. It is applicable to cell-rich tissues such as lymph nodes and cellular neoplasms. A drop of thick, creamy material obtainable from such tissues using a 27–25-gauge needle without aspiration is gently expelled onto a glass slide as usual, but is not spread or smeared. After a few seconds to allow the drop to adhere to the slide, the slide is carefully immersed in 90% ethanol. The sample remains stuck to the slide as a drop (‘button’). Alcohol-fixation, unlike formalin, holds the sample together. After fixation, the ‘button’ is gently detached with a scalpel blade and processed like a small biopsy. The amount of tissue obtained in this way can be substantial, cell preservation and fixation is excellent, and the material is well suited to immunocytochemical studies (Fig. 2.16). An advantage over a conventional cell block is that the cell material is concentrated, whereas multiple sections may be necessary to find scanty tissue fragments in a cell block.
Fixation and staining
Two fundamentally different methods of fixation and staining are used in FNAC: air-drying followed by a Romanowsky-type stain such as MGG, Jenner-Giemsa, Wright’s stain or Diff-Quik (Harleco, Philadelphia); and alcohol-fixation followed by Papanicolaou (Pap) or hematoxylin and eosin (H&E) staining. Both methods have their advantages and deficiencies. The effect produced on cells by air-drying and wet-fixation is easily understood if one compares the three-dimensional shape of a fried egg with that of a boiled egg (Fig. 2.17). Air-drying causes the cell, both cytoplasm and nucleus, to flatten on the slide just like an egg flattens in the frying pan. It therefore appears larger than a cell fixed in ethanol, which maintains its three-dimensional rounded shape. Nuclear enlargement and variation in nuclear size are exaggerated in air-dried smears. This enhances the difference between normal and abnormal cells (see Fig. 2.9A).
Optimal fixation of air-dried smears depends on rapid drying. This can be enhanced by using a hair dryer with moderate heat. Slow drying of thick bloody smears tends to produce artifacts, in particular shrinkage of cells and nuclei, which may render diagnosis impossible (see Fig. 2.10). The main problem with wet-fixed smears is that highly cellular smears dry so quickly that drying artifacts can be difficult to avoid (Fig. 2.18).
Pathologists trained in gynecological cytology usually prefer alcohol-fixation and Pap staining also for FNB smears while those trained in hematology choose air-dried MGG-stained smears. If sufficient material is available, the two methods should be used in parallel since some features of cells, cell products and stroma are better demonstrated by one than by the other. The differences between air-dried MGG smears and alcohol-fixed Pap smears and the features highlighted by each method are listed in Tables 2.1 and 2.2. The two methods obviously provide complementary diagnostic information. If only air-dried smears are available, some can later be rehydrated and stained with Pap or H&E.23 However, additional material processed as a cell block may be required to allow special stains, multiple immune markers or other ancillary tests.
Table 2.1 Comparison of airdried and wet-fixed smears – general properties
Airdried MGG | Wet-fixed Pap | |
---|---|---|
Dependence on smearing technique | Strong | Moderate |
The ‘dry’ smear | Good fixation | Drying artefacts common |
The ‘wet’ smear | Arifacts common | Good fixation |
Tissue fragments | Cells poorly seen due to heavily stained ground substance | Individual cells usually clearly seen |
Cell and nuclear size | Exaggerated, differences enhanced | Comparable to tissue sections |
Cytoplasmic detail | Well demonstrated | Poorly demonstrated |
Nuclear detail | Pattern different from the familiar Pap stain | Excellently demonstrated |
Nucleoli | Not always discernible | Well demonstrated |
Stromal components | Well shown and often differentially stained | Poorly demonstrated |
Partially necrotic tissue | Poor definition of cell details | Good definition of single intact cells |
Table 2.2 Comparison of airdried and wet-fixed smears – tissues-specific properties
Tissue | Feature emphasized by MGG | Feature emphasized by Pap |
---|---|---|
Epithelial tissues | ![]() Stay updated, free articles. Join our Telegram channel![]() Full access? Get Clinical Tree![]() ![]() ![]() |