Introduction and Basic Techniques of Fine Needle Aspiration CytologyCHAPTER 1

Needle aspiration cytology was first time described in 1847 by the French physician Kun. He described a new instrument for diagnosing palpable tumors. There are occasional case reports on needle aspiration cytology in nineteenth century. Leyden in 1883 applied needle aspiration technique to isolate pneumonic microorganisms¹ followed by Mentetrier in 1886 who applied needle aspiration cytology for the diagnosis of lung carcinomas.² In 1904, the British military surgeons Griegg and Grey diagnosed trypanosomiasis by aspirating swollen lymph nodes with the help of syringe and needle in sleeping sickness cases in Uganda, Africa.³ Very few pathologists were encouraged by this work and only few clinicians used this technique. The first practical report on fine needle aspiration cytology (FNAC) was submitted by Dudgeon and Patrick from UK in the year 1927.⁴ They proposed aspiration cytology as method for rapid microscopical diagnosis of tumors. Within a few years, Hayes E Martin, a surgeon, and Edward B Eliis, the chief histotechnologist at the memorial hospital in USA (presently Memorial Sloan-Kettering Cancer Center) published their paper on diagnosis of tumors with the help of needle aspiration cytology.⁵ Fred W Stewart was the chief surgical pathologist who interpreted the cytology smear and later on he compiled all the cases analyzed and recorded in his publication.⁶ The technique of FNAC was constantly and effectively followed in Memorial Hospital. Unfortunately, this technique generated little or better to say no interest among the other clinicians and pathologists in USA. Many clinicians, including James Ewing, the director of the Memorial Hospital for Cancer, USA, believed that aspiration cytology may spread the cancer through the needle tract. In contrast to USA, the technique of needle aspiration cytology was used since 1950 in many European countries, including Sweden, Holland, and France. Soderstrom⁷ and Franzen⁸ in Sweden and Lopes Cardozo⁹ in Holland studied large number of cases by needle aspiration. Joseph Zazicek in collaboration with Sixten Franzen and Torsten Lowhagen in Karolinska hospital, Sweden first enlisted the diagnostic cytological criteria of various lesions.¹⁰ The technique of FNAC flourished in the European countries and later on widely accepted in other continents, including Asia, Australia, and USA.

Box 1.1 highlights the advantages of FNAC over tissue biopsy.^11-14 The main advantage of FNAC is that, it can be done anywhere in the laboratory, in outpatient department, or in the hospital. This is a simple office procedure. In case of superficial swelling like lymph nodal enlargement, breast mass or thyroid swelling, the patient can be called in the FNAC clinic or outpatient department, and within jjust one hour, the report can be delivered.

No special precaution is needed for FNAC of a superficial mass. The success of this technique partly depends on the experience and skill of the operator and interpreter. Overall, FNAC is comparable or even better than the frozen section in breast lesion. It is a component of “triple test” in breast swelling. Triple test in breast FNAC provides similar specificity as that of histopathology section.¹⁵ Health care cost is very important in developing and underdeveloped country. FNAC technique can eliminate the patient stay in hospital and operation cost and thereby reduces the healthcare cost considerably. FNAC report provides a great psychological relief to the patient when he/she knows that the lesion is benign or infectious in origin. In case of malignancy, it can convince the patient to have an urgent treatment. Presently, with the help of ancillary technique on FNAC material, exact diagnosis is possible in most of the lesions. Therefore, FNAC can avoid tissue biopsy in many situations.

There is loss of complex tissue architecture in FNAC smear (Box 1.2). So, at time, it is difficult to categorize the lesion exactly particularly in soft tissue or skin lesion. In situ carcinomas are also difficult to diagnose by FNAC. Follicular pattern of lymphoma is important in diagnosis of follicular lymphoma. It is difficult to diagnose follicular lymphoma on FNAC smear alone. Similarly, the capsular invasion or lymphovascular emboli cannot be detected in FNAC. Interpretation of FNAC smear needs considerable training and practice.^16-21

The diagnostic accuracy of FNAC largely depends on the interpreter’s experience in this field. In fact, FNAC interpretation is also different from exfoliative cytology. Unlike exfoliative cytology, interpretation of FNAC needs more attention on the overall cellular pattern and background material (Table 1.1). Moreover, interpretation of FNAC sample is a bit different than tissue biopsy, and considerable skill is needed to interpret FNAC material.²²

An FNAC is usually free of significant complications (Box 1.3). There may be bleeding, hematoma, and pneumothorax (in lung FNAC). Rarely, anaphylactic reaction may occur due to accidental rupture of hydatid cyst during the procedure. However, these complications can be avoided if proper precautions are taken during FNAC. The direction of the needle should not be changed frequently, and to-and-fro movement of the needle should be gentle. Rarely, FNAC needle may cause rupture of an aneurysmal vessel or spleen. This can be avoided if ultrasonograph (USG) of the lesion is available before hand.

There is a serious concern in the group of physicians about tumor seeding through the needle tract during FNAC.^23-31 Tumor seeding have been reported by various workers in both intra-abdominal and superficial swelling.^{30, 31} In fact, the true incidence of needle tract seedling is very difficult to assess.

In a large study by Ito Y et al., ³² 10 cases (0.14%) of needle tract implantation were encountered out of 4, 912 patients who underwent FNAC. The time period from the FNAC to detection of tumor seeding was 2–131 months. The number of seeding after FNAC was almost negligible and the recurrences were managed by surgical treatment. In a review by Polvzo et al., ³³ the researcher concluded that needle tract seeding is a truth and not a myth. However, its incidence is rare, and the overall benefits of FNAC are far more than the potential risk of dissemination through the needle tract.

There are no absolute contraindications of FNAC. However, it should be avoided in severe bleeding diathesis (Box 1.4). It is better to avoid FNAC in known case of hydatid cyst, as severe anaphylaxis may occur in case of rupture of the cyst. The FNAC of ovarian cyst is not a contraindication. We routinely perform FNAC of ovarian tumor and have never faced any problem of dissemination of tumor cells.

The following equipment are needed for the FNAC procedure to do:

An FNAC clinic is used to perform FNAC of the superficial swelling. The patients are referred by the physicians and surgeons for FNAC. The location of the clinic is preferable in the hospital outdoor service (Box 1.5). However, it may be located in the pathology department or hospital indoor.

4The FNAC room should be well ventilated and well lighted. There should be an examination bed with access from both the sides, one writing table with chair, one working table, basic equipments for FNAC, and waste disposal baskets (Figure 1.2). It is also preferable to have rapid staining kit, and small binocular microscope. A dedicated cytotechnologist is needed to assist the cytopathologist. It is always better to have a small recovery room to manage any minor complication or to keep the patient for short observation.

A good clinical history is mandatory before performing FNAC procedure (Box 1.6). Chief complaints of the patient, site and size of swelling, and biochemical or radiological features provide additional help. Fixed or ulcerated swelling usually indicates malignancy. Lymph nodal swelling in Hodgkin’s lymphoma gives India rubber like feel.

Hard and fixed mass in the breast with retracted nipple usually indicates carcinoma of breast. Simple X-ray picture of the bone provides much important information. Similarly soft tissue tumor with bone infiltration is the feature of malignancy.

The tag of “biopsy” in the procedure often makes the patient afraid. Therefore, it is always preferable to have a good rapport with the patient, and this makes him/her relax. The operator should always ask the patient about his or her version of the swelling (Box 1.7). Many times, this may be a great help as the accuracy of the clinician’s version is also verified by this way. The whole procedure should be explained briefly to the patient. In addition, the purpose of FNAC, probable complications of the procedure and potential advantages of FNAC should be explained to the patient. In all cases, proper written consent should be taken. The informed consent should be written in simple language. The consent form should mention the procedure and its potential complications. Proper communication of the patient and written consent with explanation of the procedure usually avoids any medicolegal issue.

It is always preferable that cytopathologist himself/herself should perform FNAC (Box 1.8).^34-37 We routinely use 22–23 gauge needle, 20 mL syringe, and a pistol handle for FNAC technique.

The needle is connected to the plastic syringe, and the syringe is properly fitted with the pistol handle (Figure 1.3). The handle gives support and also helps apply suction during the procedure.

Occasional sample may show large amount of blood. The presence of large amount of blood makes the sample suboptimal and unfit for cytological examination. The bloody sample or clotted sample may be processed for cell block for histological study.

The material is collected in 10% buffered formalin. After fixation, the fluid is centrifuged and the cell pellet is processed for cell block.

Occasionally, fluid is aspirated from the cystic cavity. Clear fluid from a cystic lesion usually does not contain any significant cell population and many cytologists prefer not to process these samples. However, bloody fluid sample should always be processed. After evacuation of the cyst, a repeat examination of the swelling should always be done to evaluate for any solid lesion within the cyst. If a solid area is identified, then a repeat FNAC should be done from that area. If necessary USG-guided FNAC from the solid area could be done.

The direction of the long axis of the needle should make 45˚ angle with the slide and the initial placement of the material should be at least 1 inch away from the margin to keep space for labeling. The smear is made by gently pressing one clean slide over the other and moving the upper slide over the lower one to spread the material (Figure 1.8). Alternately, the smear can be made like a blood film by placing one edge of the upper slide on the material at 45˚ angle and then gently making the smear as the blood smear is prepared. The aspirated material is usually distributed in several slides to make multiple smears. Both air dried and alcohol fixed smears should be kept for staining. The residual material should be rinsed in citrate buffer solution and can be processed for ancillary techniques. A repeat FNAC could be done to get more material for the ancillary techniques.

The labeling of the smear is very important. In frosted slide the slide can be labeled by a lead pencil. Alternatively, the slide can be labeled or numbered by diamond pencil or any permanent marker.

The wrong labeling of the smear may cause significant problem and may lead to medicolegal case. It is the ultimate responsibility of the cytopathologist to get properly labeled slide. To avoid mix up, the slides of previous case should always be removed from the working space and any unmarked slide should be either removed or discarded.

Nonsuction fine needle sampling (FNS) is a helpful technique in certain situations like small swelling, thyroid swelling or breast lesions.^38-41 In this technique, the swelling or the area to be aspirated should be pressed in between the two fingers and a 23 gauge needle is gently introduced (Figure 1.9). The needle is moved to and fro in different directions. The material comes to the needle hub by capillary suction. The needle is gently withdrawn and the syringe filled with air is attached with the needle hub. The aspirated material is expelled gently on the slide by moving the piston of the syringe. The major advantage of FNS is to get abundant material in a bloodless background. FNS is particularly important in vascular organ like thyroid.

After FNAC, before releasing the patient, the puncture site should always be examined for any bleeding or hematoma. The patient should be strictly instructed to apply pressure on the puncture site. Adequate care should be taken for weak elderly and sensitive patient. Some little talk or additional time may raise the patient’s satisfaction level. The patients should be always asked for any discomfort. Usually no serious complications happen after superficial FNAC; however, the patient should never be kept unattended for any period of time.

FNAC is a simple procedure, but it needs good training to have good material. The experience can be gained by observing some experienced cytopathologist and then performing the procedure under someone’s guidance. Good number of FNAC procedure is needed to gain proper experience.

Wet fixation of the smear is needed for routine Papanicolaou’s stain or hematoxylin and eosin stain (Box 1.12). Ethanol (95%), methanol, or isopropyl alcohol may be used for fixation. The smears should be fully immersed in the fixative for at least 30 minutes. Adequate care should be taken to avoid transfer of the material from one slide to the other. It is preferable to use paper clips to prevent sticking of one slide with other. Ideally the smears of each patient should be kept in separate Koplin jar. This may prevent mix up. Commercially available spray fixatives can also be used for fixation. The major advantage of spray fixative is to avoid carrying bottles with liquid material. Two major functions of spray fixatives are to fix the cell and to have a protective covering of thin wax over the smear. The spray fixatives usually contain ethanol or isopropyl alcohol along with polyethylene glycol (carbowax). The spray fixative should be applied from an optimal distance of 10–12 inches away from the smear. Too close application of the spray fixative may damage the smear. Within a few seconds the smear is dried and carbowax provides a covering over the slide that prevents the shrinkage of the cell. Before staining, the smear should be dipped in ethyl alcohol for at least 15 minutes to remove the surface coating.

The air drying smear needs no additional step. It is preferable to dry the smear as quick as possible and the smear should be completely air dried before any stain. Air drying smears are generally used for Romanowsky staining.

May Grunwald Giemsa (MGG) or Romanowsky stain, or Diff-Quik stain is done on the air dried smear. In Romanowsky stain there is a mixture of methylene blue and Azure A, B or C. This is a metachromatic stain and thereby stains epithelial cells differently than stromal cells. This is also a good stain for mucin and extracellular matrix. May Grunwald Giemsa (MGG) stain gives excellent details of cytoplasmic characters. MGG is a convenient stain for FNAC smear. Table 1.2 compares the relative advantages and disadvantages of MGG stain over, Papanicolaou’s stain. The basic steps of MGG stains are as follows:

This is a commercial stain kit and is a variant of Romanowski stain. This stain is the modification of Wright Giemsa stain. Unlike MGG stain, Diff-Quick stain is much faster and gives equal details of the cells and stromal tissue. This is a metachromatic stain. The stain contains two solutions.

The solution 1 contains buffered eosin Y and the solution 2 contains methylene blue and Azure A.

On alcohol fixed smear, Papanicolaou’s (Pap) stain is used. Orange G component of Papanicolaou’s stain demonstrates keratin and thereby helps in the identification of squamous cells.

Here the intensity of the nuclear stain is done up to the desired level and the cytoplasm barely takes the dye.

Here the nuclei are deliberately over stained by hematoxylin dye and then excess stain is removed by diluted hydrochloric acid solution.

Hematoxylin : Harris hematoxylin for nuclear stain

Orange G : OG-6 is used for cytoplasmic counter stain. This dye specifically stains keratin component of the cytoplasm.

Eosin Azure (EA): It is a polychrome stain and consists of three dyes: Eosin Y, light green SF yellowish and Bismarck brown Y.

This is not a cytological stain and mainly used in histopathology section. It is usually used by surgical pathologists who intermittently practices cytopathology. H and E stained smear gives resemblance of the histology section. However, for routine use in the laboratory, H and E stained smear is not familiar to many cytopathologists and it is better to avoid this stain.

All the ancillary techniques used in tissue section can also be done in FNAC sample.²¹ Depending on the resource and the situation these tests should be done accordingly. 10However, most of the laboratories routinely do a cell block for immunocytochemistry and flow cytometry for immunophenotyping.

There are various causes of suboptimal material in FNAC. The most common cause of nonrepresentative material is faulty technique. Vigorous suction during FNAC in vascular organ like thyroid may yield blood. The best way to avoid this is to give intermittent suction during to-and-fro movement of the needle. In fact, the operator should always keep an eye that whether blood is in the needle hub or not. The suction should be immediately stopped when blood comes in the needle hub. There should be sufficient to-and-fro movement of the needle to dissociate the tissue. The negative suction should apply only when the tissue is sufficiently dissociated. At times, the mass may be too much sclerotic or hard, and therefore, even after adequate negative suction the material may not be obtained. In this case, thick bore needle can be used. The preparation of the smear is also very important. Too much pressure may cause crushing artifact, whereas, too much delay in the smear preparation may give rise to clotted material (Box 1.13).

Safety of the operator doing FNAC is essential and adequate precautions should always be taken (Box 1.14). The FNAC clinic should be properly ventilated with adequate air circulation. The room should be well lighted so that the direction of the needle should be properly traced. There should be proper container for the disposable of the waste material in the clinic.