II Specimen Collection

Nongynecological Cytology Practice Guidlines

prepared by the American Society of Cytopathology, Cytopathology Practice Committee.

Adopted by the ASC Executive Board, March 2, 2004

II Specimen Collection and Submission

Nongynecological cytology specimens are collected from a variety of sites for the detection of malignant and benign processes. The site from which the sample is collected dictates the method of collection. The method of collection affects the morphology of the cellular samples. The importance of proper specimen collection and submission is essential. Clinical personnel should be trained in the appropriate submission of samples as well as in procedural techniques. The laboratory must provide instructions for proper collection of all nongynecological specimens. The instructions must be available to personnel at the location where the specimens are collected.6 The laboratory provides feedback on the adequacy of the sample via individual reports and may elect to provide summary information regarding patient sampling to its clients.

II.A. Collection

II.A.1 Fluids

Serous or “body cavity fluids” are usually collected with aseptic technique by needle puncture and aspiration of the body cavity fluid. Methods for obtaining the specimen vary depending upon the site. Fluids are best collected into a dry container and submitted in the fresh state to the laboratory.7 Adding 3-5 IU heparin/mL to a container prior to obtaining a bloody sample will usually inhibit clotting and not adversely affect morphology. If delay in transportation to the laboratory is unavoidable, most fluids may be kept refrigerated (4ºC) up to 72 hours. (The exception to this is cerebrospinal fluid, which begins to degrade shortly after collection if stored at room temperature or refrigerated.) A fluid sample intended for cytologic analysis should never be frozen. If a longer delay is expected, preservation at the time of collection with 50% ethanol equal to the volume of the specimen is suggested.8 Alternatively, proprietary transport medium supplied by the manufacturers of liquid based systems may be used. Any added fixative should be noted on the requisition. For small fluid accumulations the entire specimen is submitted for laboratory evaluation. For larger effusions, 50-200 mL of well-mixed fluid should be sent for cytologic examination; however, the entire specimen is also acceptable.8 All fluid specimens must be transported in accordance with OSHA regulations for biohazardous substances.9 Three separate specimens collected at different times are optimal for the diagnosis of malignancy.10

IIA.2 Washings

Washings can be collected from various body sites. Small aliquots of balanced saline solution are washed over a directly visualized area and removed immediately with suction. These washings are usually submitted unfixed to the laboratory. If a delay is expected, they may be partially fixed in 50% ethanol equal to the volume of the specimen, or in the proprietary transport medium supplied by the manufacturers of liquid based systems. Any added fixative should be noted on the requisition.

IIA.3 Brushings

Brushing specimens may be taken from any surface of the body. Direct smears, brush contents or a brush tip may all be submitted as brushing cytology.

IIA.3.a Direct smears

Prior to the procedure, slides should be labeled with the patient’s full name or other unique identifier, using pencil or solvent resistant marker. Some laboratories may require a second identifier such as date of birth, medical record number, social security number or collection date. The laboratory must have a written procedure that specifies the requirements for proper specimen identification.11

After the brushing is performed, the brush is rolled across the slide in an area approximately 2.5 centimeters in diameter (the size of a quarter) to produce a thin evenly layered smear.8 The slide should be fixed immediately. Immediate fixation of the cellular sample is necessary to prevent air-drying which obscures cellular detail and compromises specimen evaluation. Fixation can be by immersion (preferably) or spray fixation. Immersion fixation consists of placing the smear into 95% alcohol or its equivalent.12 If the specimen is immersed in alcohol, it may remain in the alcohol for transport to the laboratory. Alternatively, the specimen can be immersed in alcohol for 20-30 minutes, removed and allowed to air dry, then placed in a container/mailer to be transported to the laboratory. The immersion technique requires use of a separate fixative container for each body site sampled.

If a specimen is spray fixed, only quality controlled cytology fixatives should be used. Hair spray should NOT be used due to the fact that most modern products contain no alcohol. In the past it was the alcohol content of hair spray that allowed its use as a cellular preservative. The manufacturer’s instructions for fixation should be followed. Generally, spray fixatives should be held 6-10 inches (15-25 cm) from the glass slide when applied. 13

Alternatively, air-dried brushing smears may be submitted for staining with one of the Romanowsky stains or for rehydration prior to Papanicolaou staining.14,15,16

II.A.3.b Brush contents in liquid media

Material obtained from a brushing should be submitted in a physiologic transport solution, 50% ethanol equal to the volume of the specimen, or in the proprietary transport medium supplied by the manufacturers of liquid based systems. The brush may be submitted in the solution or discarded after vigorously removing the adherent cellular material into the medium. The container must be clearly labeled with the patient’s name or other unique identifier. It should be a leak proof container with enough fluid to cover the brush if submitted.8

IIA.4 Fine Needle Aspiration Biopsy (FNAB)

Fine needle aspiration biopsy in this document is distinct from core or cutting biopsies and refers to a procedure that procures cellular material for diagnosis using suction or non-suction techniques. Fine needle aspiration biopsy may be performed on any body site that can be reached with a fine needle. A fine or thin needle is defined as 22 or higher gauge. FNAB is a minimally invasive, cost-effective technique with high diagnostic accuracy (in the range of 90 to 99%).17

The accuracy of FNAB depends upon the site and type of lesion sampled, the operator’s experience, the quality of specimen preparation, and the pathologist’s diagnostic skills. Fine needle aspiration biopsy may be performed on palpable (superficial) lesions or with radiological, endoscopic or bronchoscopic guidance (deep lesions).

The aspirator should be aware of the pertinent history and clinical information, significant radiological studies and the clinical question that FNAB may answer. The procedure as well as the minor complications of bruising and bleeding should be explained to the patient. Site-specific complications for deep needle aspiration biopsy should be described to the patient if image guided FNAB is performed. Informed consent should be obtained from the patient, guardian or legal representative. Written consent should be obtained if the institution requires it where the procedure is performed.

The operator should be prepared to obtain material for ancillary tests, such as cell block preparations, molecular studies, flow cytometric studies or microbiologic studies if indicated. Prior to the procedure, slides should be indelibly labeled with the patient’s name or other unique identifier. Individual laboratories may require other information such as site of biopsy, date of birth, medical record number, social security number or collection date.

After a limited physical examination, palpable FNAB may be performed as clinically indicated. The operator should wear examination gloves as described in standard precautions.18 The skin should be cleansed with an alcohol swab prior to puncture for superficial FNAB. For percutaneous biopsy of deep lesions, sterile or aseptic technique is used.

Local anesthesia may be used for palpable lesions at the preference of the operator and the patient, but is usually used for percutaneous radiologically guided needle biopsies. Sedation is rarely needed for adult superficial FNAB, but may be used for pediatric patients, or deep-seated FNAB when the patient is uncomfortable or anxious. Rarely FNAB may be performed under general anesthesia, usually during surgery.

For solid lesions multiple passes with separate needles are performed. Staying within the lesion, the needle is moved in a cutting motion withdrawing cells into the needle hub. The force of the cutting motion needed to obtain an adequate sample must be adjusted for the body site and characteristics of the lesion. These biopsies may be performed with suction or by the “non-suction” technique.17 There are many styles of handles, syringes and needles that may be used, depending upon the operator’s preference. Once the cellular material is seen in the needle hub, suction is released and the needle is withdrawn.

The cellular material is expressed onto one or more slides and is smeared with a second slide or a cover slip. Often, a “paired” smear is used, by smearing the material between two slides, to make a mirror image pair. One of the slides is often fixed and the other air-dried. Alternatively, push smears, pull smears and one or two-step smearing techniques are used.17 Fixation techniques and fixatives vary, but should be stipulated by the laboratory that accepts the specimen for analysis. Smears are prepared from each needle pass. Two or more aspirates are often required for adequate sampling.

For subsequent analysis of the specimen, the needles may be rinsed into a physiologic solution, tissue culture medium, transportation medium or proprietary fixative for liquid based processing. Needles should not be re-sheathed using a two handed technique and instead should be discarded directly into an OSHA approved sharps disposal container.9

For cystic lesions, remove as much fluid as possible. The cyst fluid can be handled as a liquid specimen. If there is a residual mass, the procedure for solid lesions as described above should be followed. Ancillary testing can be performed as indicated. See section VIII.

Specimen adequacy may be assessed during FNAB by using a rapid stain such as a toluidine blue stained wet film, a Romanowsky stain or a modified Papanicolaou stain for microscopic analysis. Although there are no universal criteria for adequacy, a multiparameter assessment is usually performed which includes cellularity, site-specific architectural features, and cellular elements. Intraprocedural evaluation should be documented. At this time, additional material (or the needle rinses) may be designated for ancillary testing.

Local pressure is usually adequate to control post procedural bleeding for superficial sites. Patients who have undergone deep FNAB should be followed for complications as clinically appropriate. All patients who undergo FNAB should be observed during and following the procedure until they are stable.

A written report should follow the procedure. Depending on local practice this report may include a description of the procedure, any preliminary information obtained, complications and the disposition of the patient when released.

II.B. Test Requisition

A manual or electronic requisition must be generated for each specimen. The requisition should include the:

  • Full patient name
  • Unique identifier such as medical record number
  • Date of birth and age
  • Date and time of specimen collection
  • Source or site of the specimen
  • Type of specimen or examination requested
  • Collection method
  • The ordering clinician’s full name, and the name and address or other suitable identifier of the authorized provider ordering the test19
  • Methods of rapid communication such as telephone or FAX numbers of the ordering clinician
  • Clinical history, including pertinent physical findings, radiographic findings, history of environmental exposure, possible exposure to infectious agents, immunosuppression, chemotherapy, radiation therapy, surgical operations, history of cancer, previous histological or cytological abnormalities
  • ICD-9 code

Diagrams of the site sampled may be helpful, especially for fine needle aspiration biopsies.8

II.C. Specimen Submission

II.C.a Slides

Fixed smears should be submitted in containers that protect against breakage. Slide containers are available in a variety of shapes, volumes and material. Optimal design features include easily opened containers which stay closed during transport, shock resistant material, and enough room to prevent slides from adhering to one another or the container. The slides should be marked clearly with the patient’s name, as well as other identifiers if possible. If more than one site is sampled, the slides must be clearly marked as to their site. Enclosure in a transport bag indicating biohazardous contents is prudent if a courier system or manual delivery is planned. Fixed slides may be mailed according to transport guidelines.20 A paper or electronic requisition must accompany the specimen.21 Slides in fixative should be submitted in leak proof containers that protect against breakage and are clearly labeled with the patient’s name and specimen site(s). The slides in fixative may be placed in a secondary container or transport bag, to avoid leakage of fixative during transport. Paper requisitions that accompany slides in fixative should be placed in an outside pocket to avoid exposure to any leakage of fixative.

II.C.b Liquid transport

Each fluid specimen should be placed in a clearly labeled container that is leak proof. Enclosure in a transport bag indicating biohazardous contents is prudent if a courier system or manual delivery is planned. Paper requisitions that accompany slides in fixative should be placed in an outside pocket to avoid exposure to any leakage of fixative. Needles should never be transported with fluid specimens.9 Large glass collection containers should be avoided.

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6     College of American Pathologists. Commission on Laboratory Accreditation Inspection Checklist 2002 edition, Northfield, Illinois.

7     Naylor B.  Pleural, Peritoneal, and Pericardial Fluids. In Bibbo M(ed).  Comprehensive Cytopathology. Philadelphia: W.B. Saunders Company, 1997.

8     NCCLS.  Nongynecologic Cytologic Specimens:  Collection and Cytopreparatory Techniques; Approved Guideline. NCCLS document GP 23-A[ISBN 1-56238-380-9]. NCCLS, West Valley Road, Suite 14000, Wayne Pennsylvania 19087-1898 USA, 1999. 

9     Occupational Safety and Health Administration.  Bloodborne pathogens.  Standard 1910.1030.

10     Venrick MG, Sidawy MK.  Cytologic evaluation of serous effusions: Processing techniques and optimal number of smears for routine preparation.  Am J Clin Pathol 1993; 99: 182-86.

11     Clinical Laboratory Improvement Amendments of 1988; Final Rule. Federal Register. Jan. 24, 2003; 68: 493.1242(a).

12     Keebler CM.  “Cytopreparatory Techniques.”  In:  Bibbo, Comprehensive Cytopathology, Philadelphia: W.B. Saunders Company, 1991.

13     Somrak TM, Sorensen K, Abdul-Karim F.  Pap smear: Collection, handling and quality assurance.   Chicago: ASCP Press; 1990

14     DeMay RM,. “Stains.”  The Art and Science of Cytopathology.  Chicago:  ASCP Press, 1996

15     Yang GC, Alvarez II: Ultrafast Papanicolaou Stain: An Alternative Preparation for Fine Needle Aspiration Cytology. Acta Cytol 1995; 39: 55-60.

16    Yang GC: Ultrafast Papanicolaou Stain Is Not Limited to Rapid Assessment: Application   to Permanent Fine-Needle Aspiration Smears. Diagn Cytopathol 1995; 13: 160-162.

17     NCCLS.  Fine Needle Aspiration Biopsy (FNAB) Techniques; Approved Guideline—Second Edition. NCCLS document GP20-A2 [ISBN 1-56238-000-0].  NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002.

18     Guideline for Isolation Precautions in Hospitals. Infection Control and Hospital Epidemiology. CDC. 1996;Vol 17;1:53-80)

19     Clinical Laboratory Improvement Amendments of 1988; Final Rule.  Federal Register.  Jan. 24, 2003; Vol. 68:  493.1241(c)(1).

20     NCCLS. Procedures for the handling and transport of Diagnostic Specimens and Etiologic Agents -Third Edition: Approved standard (1994). NCCLS document H5-A3   NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 1994.

21     Clinical Laboratory Improvement Amendments of 1988; Final Rule.  Federal Register.  Jan. 24, 2003; Vol. 68: 493.1241(a)