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Fine Needle Aspiration Cytology

INTRODUCTION
David B. Kaminsky, MD, FIAC

The world of cytopathology was defined by the advent of cervical cancer screening conceived and promoted by Dr. George Papanicolaou. It was revolutionized by the introduction of fine needle aspiration biopsy and liquid based cytology. The marriage of the two provides a powerful diagnostic tool that facilitates molecular and ancillary studies in oncologic and infectious diseases. Both represent state-of-the-art and standard of practice. Together they are the reinforcement of the foundation for modern cytopathology and are indispensable.

Fine needle aspiration biopsy [FNAB] had its origins in Stockholm, at the Karolinska and Radiumhemmet, in the hands of the pioneers Franzen, Zajicek and Esposti whose seminal work, corroborated by biopsy data, validated that criteria for cell diagnosis were faithful to tissue patterns and that the needle probe was a practical diagnostic tool that could provide minimally invasive tissue-equivalent information.


A skeptical American medical community repudiated Martin's attempt in the 1930s to introduce FNAB which competed with the new technology, frozen sections and exfoliative cytology. The provocative technique found its way into certain university-based medical centers where pathologists learned to interpret cell patterns and clinicians transcended their ambivalence and skepticism.

Under the influence of committed pathologists like Koss and Frable, aspiration biopsy began to flourish and the American Society of Cytopathology introduced courses at its annual scientific meeting. Dr. Frable and I collaborated in hands-on workshops to teach the aspiration technique, criteria, and correlation with histologic patterns, assuring our colleagues of the accuracy, efficacy and security of the procedure. Other pathologists were enthusiastic about FNAB and ultimately it became an accepted element in residency programs, course curricula, and eventually general practice. Collaboration with interventional radiologists assured its ultimate success when computed tomography, ultrasonography and fluoroscopy assured visually controlled access to deep visceral lesions with acceptable risk and optimal outcomes. An informed society with a challenging tumor burden and escalating financial responsibility for its management was receptive to a precise, cost-effective, minimally invasive procedure adaptable to an outpatient environment and conducive to their participation in the choice and expedient implementation of therapy. The technique was a perfect fit with quality enhancement endeavors because it offered a measurable data-based, outcome-oriented effort. The introduction of liquid based cytology provided an added dimension; the needle can be rinsed in the collection fluid to provide a reservoir of cells for sample integrity and preservation and for augmented cytologic analysis and ancillary techniques.

Fine needle aspiration biopsy has earned its recognition as an irrevocably essential protagonist in patient care and a mediator of relationships among cytopathology professionals, clinicians, interventional radiologists, patients and third party payors. It is the bridge from cytoarchitecture to tissue anatomy, immunocytochemistry, flow cytometry, culture, molecular diagnostics, targeted therapy and clinical correlation. It makes financial sense, offers low risk and achievable results. It has character, personality, a life of its own.

Indications
The presence of a palpable mass at the surface of the body, or an unexplained lesion detected by radiographic imaging are indications to perform a fine needle aspiration biopsy under direct visualization or image-guidance. More specifically, the technique is utilized to:
  • Provide a reliable, expedient, cost-effective diagnostic modality
  • Differentiate benign from malignant disease with minimal invasion
  • Rescind clinical judgement of non-resectability
  • Confirm metastatic disease
  • Obtain tissue-equivalent information in medically restricted patients
  • Triage by cellular composition: assignment to radiation, chemotherapy, immunotherapy, surgery or surveillance
  • Acquire material for culture, electron microscopy, flow cytometry, immunocytochemistry, DNA image analysis and DNA probes
Contraindications:
There are few contraindications to performing fine needle aspiration biopsies in clinical practice, and among these are hemorrhagic diathesis, anticoagulant therapy, arteriovenous malformations and hydatid cyst disease. If the aspiration target is thorax, additional contraindications include pulmonary hypertension, uncontrolled cough, advanced emphysema, and inability of the patient to cooperate with breath-holding instructions.

Complications
Aspiration biopsy of palpable masses under direct visualization may be complicated by minor bleeding [controlled by pressure] and rarely by infection. If appropriate antiseptic technique is utilized, infection is exceedingly rare. Complications associated with image-directed aspiration biopsies of deep lesions may include hemorrhage, infection, and translocation of tumor cells [rare event], and thoracic probes could result in pneumothorax, air embolism and subcutaneous emphysema.

With transthoracic percutaneous pulmonary aspirates as a point of reference, hemorrhage is expected when the needle traverses the pleura and enters the lung parenchyma and is generally visible as an enhancement of the target in post-puncture chest films.
It is occasionally associated with self limited hemoptysis as a clinical expression of the intraparenchymal seepage. Pneumothorax is an expected accompaniment of transthoracic aspiration biopsy because the needle violates the pleura in transit to its parenchymal target resulting in some degree of lung collapse; it is symptomatic pneumothorax which is considered a complication, generally requiring insertion of a chest tube to achieve lung reinflation.
Factors which influence the occurrence of symptomatic pneumothorax include the patient's age, size, location and depth of the lesion, number of punctures, presence of emphysema, intensity of scrutiny with post-procedural films and the experience of the aspirator.

Air embolism is avoided when the radiologist utilizes a three-way stopcock or is conscientious in preventing air from entering the needle during the short interval between removal of the stylet and connection of the syringe. Subcuatneous emphysema may occur when air percolates through the soft tissue causing a crepitant suffusion that is self-limited.

In exhaustive studies of large numbers of patients undergoing aspiration biopsy at the Karolinska Institute, translocation of malignant cells along the needle tract was a rare, if only anecdotal event, representing an occurrence of perhaps one in many thousand cases, usually associated with a highly malignant tumor predicted to disseminate spontaneously.

Most fatalities are due to use of cutting rather than fine needles and are truly rare events with 22 gauge needles. Lung fatalities are due to endobronchial hemorrhage from cutting needles.

The Technique for Superficial FNAB's Under Direct Visualization:
Fine needle aspiration biopsy is a safe and efficient method of obtaining cells for diagnostic cytologic evaluation of palpable superficial masses from breast, thyroid, salivary glands, lymph nodes, cysts and metastatic tumors, utilizing a 20 cc syringe, 22 gauge needle and optional syringe holder ["gun"].

Patient preparation is minimal, consisting of an explanation of the procedure; declaration of its complications with recitation of side-effects, limitations and expected outcomes; annotation of history including allergies, anticoagulant therapy or bleeding tendencies; acquisition of signed informed consent; respect for all HIPAA and ethical considerations, including the presence of an assistant if the patient is female; physical examination of the target area and achievement of antisepsis. Local anesthesia is optional.

Following palpation and antiseptic treatment, the needle is inserted into the target, the plunger is evacuated to create a negative pressure while the needle is oscillated several times in different directions, and re-equilibration is achieved before the needle is removed from the patient. The procedure is terminated when the aspirator assesses adequate penetration or material appears in the hub of the syringe. The syringe is then removed from the needle and filled with air to serve as an expressive force when reconnected to the needle. The syringe may be held manually or optionally by a syringe holder.

Cells obtained from the aspiration are expressed onto labeled glass slides by placing the needle, bevel side down, in contact with the glass, and expressing a droplet. Utilizing a second slide, the droplet is dispersed by surface tension into a circular monolayer. The slides are separated in a quick, perpendicular fashion, avoiding smearing, and immediately immersed in 95% ethanol. Air dry artifact must be avoided unless slides are intentionally air dried for Romanowsky stains. The needle may then be rinsed in saline or in the ThinPrep® vial. A cell block may be prepared from solid material.


If a lymph node is aspirated, additional "passes" may be performed to provide slides for Diff Quick staining and samples for RPMI transport medium for flow cytometry. A preliminary diagnosis is communicated expediently to the referring physician or care provider and documented.

The Technique for Image-Guided FNABs of Deep Lesions.
Non-palpable, deep lesions may be accessed by guiding the 22 gauge needle through a trajectory to its target under the guidance of ultrasound, fluoroscopy or computed tomography. It is generally the interventional radiologist who performs the puncture in collaboration with the cytopathology professional who is responsible for assessing specimen adequacy, providing an immediate diagnosis when possible, and deciding on the requisite ancillary techniques to assure optimal specimen processing and evaluation. The presence of a cytopathologist during the procedure assures specimen adequacy, statistically significantly increases diagnostic accuracy, reduces the number of "passes" and secondary pneumothorax in chest aspirates, and offers immediate triage decisions. The radiologist orients the patient to the procedure, plans the trajectory, administers the local anesthetic, positions the needle, acquires the sample and provides therapy for complications. The radiologist benefits because he/she is assured of an optimal sample and appropriate processing; the cytopathology professional benefits by better understanding of the clinical problem and anatomical relationships of the lesion subjected to biopsy. The surgeon can plan the operative approach and better strategize scheduling of operating room time and office management. The patient benefits from a collaborative team approach.


Materials and reagents on an FNAB tray should include an antiseptic solution, 1% Xylocaine with injection syringes/needles, 20 cc syringes, 22 gauge flexible spinal needles with stylets, optional syringe holder, sterile gauze pads, clear glass slides, 95% ethanol, physiologic saline, sterile empty capped tubes, ThinPrep® vials, bottle with 10% formalin.


Following explanation of the procedure as it is described above for aspirates obtained under direct visualization from palpable lesions, including acquisition of informed consent, the radiologist establishes the position of the lesion and plans his trajectory utilizing the selected imaging modality. For thyroid aspirates, ultrasound is the preferred method to guide the needle in real time and establish the position of its tip in the target. A still photo-document can be obtained to verify that the needle tip is in position.


For lung and abdominal lesions, CT-scans are often employed for precise and exquisite location guidance of the needle and documentation of the cellular source from the intended target.


Fluoroscopy is the least often utilized modality for lung lesions because advances in CT images and the rapidity of image generation have facilitated the efficiency of aspiration. A conventional fluoroscopically-directed FNAB with needle in target is noted below:


After the establishing [baseline] images are completed, the patient is cleansed with an antiseptic and draped to create a sterile field. Local anesthetic is introduced, and then the 22 gauge flexible spinal needle, stylet in place, is inserted through the skin, to a pre-estimated depth, into the lesion [thoracic and abdominal lesions], or the 22 gauge needle, attached to 20 cc syringe, is positioned within the target under ultrasonographic control. The needle tip is verified. If a stylet is used, it is removed. The syringe is attached to the needle, and the plunger evacuated while the needle is oscillated several times in various planes. Following equilibration of pressures, the needle is removed from the patient. It is then disconnected from the syringe; air is introduced into the syringe which is then reconnected to the needle. Material is expressed from the needle onto glass slides, dispersed, then immediately immersed in 95% ethanol. The needle is then rinsed in saline, the ThinPrep® vial, or in RPMI. Generally for thoraco-abdominal lesions and lymph nodes, the patient is retained while slides are rapidly stained and interpreted for adequacy, triage and diagnosis. Thyroids are generally subjected to three "passes" per target with the material stained and evaluated following conclusion of the ultrasound procedure.


Types of Samples Acquired by Fine Needle Aspiration Biopsy
Any and every body site can be accessed for sampling with a 22 gauge needle, including uncommon considerations like brain, ocular orbit, testicle and penis. The approach is modified according to site but the technique remains consistent. Virtually FNAB of any tumor will provide material that contains cells which reflect histoarchitectural patterns classifiable as carcinoma, sarcoma, lymphoma, melanoma, and undifferentiated malignancy.


If pattern recognition does not immediately result in a specific diagnosis and categorization, ancillary techniques such as immunocytochemistry may provide molecular information that assists with classification. For infectious processes, it is possible to isolate specific etiologic agents of infection demonstrable with the Papanicolaou stain, Giemsa stains for bacteria, and AFB stains for acid fast bacilli and PAS and GMS for fungal organisms.


The sample presentation may vary subtly depending on whether the cells are placed directly on slides or processed through liquid based ThinPrep® technology. The direct smears will contain background debris and blood; nuclei of malignant cells will generally appear more hyperchromatic and dense. With ThinPrep cellular aggregation is preserved, background blood is lysed, and nuclei of malignant cells may appear less dense and hyperchromatic. One exception may be with the ground glass vesiculation characteristic of papillary thyroid carcinoma in which the optical changes of the nuclei are similar in both conventional and liquid based presentations.

Direct Smear

ThinPrep®



The concept of sample adequacy is being addressed by the American Society of Cytopathology and the Papanicolaou Society through FNA Guidelines for specific organ sites. FNA Guidelines for breast are under development by the ASC and the Papanicolaou Society has provided organ-specific guidelines for thyroid and lung. There is a certain subjectivity about sample adequacy without definitive standardization that varies with the experience of the interpreters. Hologic is accruing experience on what constitutes adequate samples collected and processed by liquid based technology. Hologic references examples of unsatisfactory, for instance, when there is blood only, fibrin without epithelium from cyst contents, inflammation only, thyroid colloid without cells. A rapid interpretation at the time the aspiration biopsy is performed will provide an immediate assessment of adequacy and expedite clinical management if a definitive diagnosis can be rendered.

Financial Considerations
When a cost comparison of FNAB with conventional biopsy is made, immediate spectacular savings are obvious, particularly when the FNAB is performed in the outpatient setting. Effective cost-containment in the delivery of health care is dependent on productivity and in this context needle aspiration offers the advantage. The procurement of cells by a relatively inexpensive technique that bypasses hospitalization and triages patients to non-surgical therapeutic protocols, including targeted therapy, offers a financial edge as well as more immediate access to care in an overwhelmed health care delivery system. From a hospital's perspective, operating room resources can be more effectively managed, freeing the system for non-biopsy oriented surgeries that are otherwise postponed, transferred, or, in some cases, never done. In community hospitals where bed space is limited, keeping biopsy patients out of pre-surgical beds frees space for patients who clinically require hospitalization. FNAB releases surgeons, anesthesiologists, nurses, histotechnologists and pathologists to pursue post-biopsy therapeutic and analytic responsibilities. Reducing the workload of technologists can result in holding down staffing levels without a sacrifice in quality of patient care.

Medicare and third party payors will reimburse both professional and technical components for peforming the aspirate, providing an immediate assessment for sample adequacy and diagnosis, interpreting the aspirate, examining accessory preparations such as ThinPrep® processing and cell blocks and for specific numbers of markers by immunocytochemistry and flow cytometry. Although multiple "passes" from a single site, such as unilateral thyroid nodule are reimbursed as one fee, FNAB of separate sites, such as bilateral thyroid nodules, are reimbursed according to the number of separate sites of access. The following are CPT Codes for the facilitation of billing:

CPT Code Description
10021 FNA without image guidance
88172 FNA immediate evaluation for adequacy
88173 FNA interpretation and report
88305 Cell block
88173 ThinPrep, Cytospin
88180 Flow cytometry
87070 Routine culture
88305 Core biopsy
87102 Culture for acid fast bacilli
87116 Culture for fungi
88342 Immunocytochemistry
88312 Special stains

Concluding Remarks:
We are privileged to practice cytopathology is a modern era of medical achievement that relates cellular morphology to microanatomy, molecular diagnostics and quality clinical management. Cytology continues to be a driving force in cancer screening and diagnosis, classification and improved outcomes. Fine needle aspiration biopsy is a unique tool that provides safe, minimally-invasive, immediate, accurate, cost-conservative, tissue-equivalent diagnostic information with low risk for complications and with universal patient satisfaction. It is complementary to radiographic imaging in exploring the human body and to research in molecular diagnostics, genetics and targeted therapy. Its application to liquid based cytology holds promise of even greater diversity and accuracy.

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