Two main types of NCBs are in use: cutting core type and vacuum-assisted type. It is notable that the diameter (bore) of the needle is inversely proportional to the number of needle gauge (e.g., 7-gauge is larger than 14-gauge needle). In current practice, fine needle aspiration (FNA) cytology procedures of breast typically utilize 25-gauge needles.

The cutting (non-vacuum-assisted, spring-loaded gun) NCB is typically used for sampling breast masses using 14-gauge needles. Needles of wider bore are used less often. The cutting NCB system is a simple, but noisy, guillotine-type device. Drawbacks of the system include the need for multiple insertions if a larger volume of tissue is to be obtained and procurement of relatively small artifact-prone specimens. The procedure is relatively inexpensive and typically takes approximately 15 minutes.

Vacuum-assisted NCB is the method of choice to sample suspicious microcalcifications without an accompanying palpable mass, and for investigating lesions considered suspicious on breast ultrasound or MRI. This technique utilizes larger (7-12 gauge) needles than those used in cutting-type biopsy instruments. An inner rotating cutting cannula is advanced into the target where it cuts a core of tissue. Vacuum delivers the sampled tissue through the needle into the collection chamber. Multiple biopsies are taken by rotating the needle without the need for multiple insertions. Vacuum-assisted biopsies yield specimens with minimum artifact. The procedure typically takes 30 to 60 minutes to perform—depending upon which guidance (stereotactic, ultrasound, or MRI) system is utilized, and is comparatively more expensive (2,3).

Stereotactic guidance is typically used for NCB performed to investigate suspicious calcifications detected on mammograms. Ultrasound-guided core biopsies are usually performed for solid masses or complex cystic lesions. Ultrasound guidance is particularly helpful for patients with mammary implants. MRI-guided biopsies are useful for lesions that are not detectable on clinical examination or on mammographic and ultrasonic evaluation. MR biopsies have a high sensitivity but poor specificity, and require sophisticated and specialized equipment and the use of contrast media (4,5).

The number of cores removed for optimal sampling should depend on the nature of the targeted lesion (i.e., calcifications, mass, etc.), particular radiographic technique employed for guidance (ultrasound, stereotactic, MRI), and size of the needle used (Figs. 26.1 and 26.2). An interdisciplinary group recommended at least 20 cores with 11-gauge needles for vacuum-assisted stereotactic breast biopsy (6), and at least 24 cores with 11-gauge needles for MRI-guided NCB (7). For ultrasound-guided vacuum-assisted NCB, another consensus paper recommended the removal of at least 10 cores with an 11-gauge needle and at least 6 cores with 8-gauge needles (8). Preibsch et al. (9) have devised a matrix that facilitates the implementation of German recommendations vis a vis required number of vacuum-assisted NCBs to be taken for different needle sizes. In summary, the authors calculated that the required minimum number of cores obtained to conform to the latest German guidelines is 20, 14, 9, and 5 for 11-, 9-, 8-, and 7-gauge needle sizes, respectively. The current German guidelines recommend a sample number of at least 12 cores with 10-gauge needle for stereotactic vacuum-assisted biopsies (9). Of note, 14-gauge needles (the least invasive needle that can be used for core biopsy purposes) are usually used in handheld ultrasound-guided vacuum-assisted NCB.

Immediately after procurement, the NCB specimen should be placed in 10% neutral buffered formalin (10). Prompt formalin fixation preserves cytologic and architectural detail,
and ensures optimal immunohistochemical (IHC) staining. Bouin fixative is known to degrade DNA and reduces immunoreactivity for estrogen receptor (ER) and progesterone receptor (PR). Alcohol fixative can interfere with hormone receptor and HER2 testing.

Ischemic time is the period of time between the time of acquisition (i.e., loss of blood supply) to the time when the biopsied sample is placed into fixative. The ischemic time could be measured in seconds for some NCBs; however, it does not typically exceed 15 minutes in cases even when specimen radiography is performed. Prolonged ischemic time (>60 minutes) should be documented, because an extended ischemic period can affect the results of tests that utilize protein, mRNA, and DNA. Delayed tissue fixation impairs HER2 protein expression (11).

Fixation time is defined as the time from the sample being placed into fixative to commencement of tissue processing. Cross-linking occurs during the fixation period, and this process inhibits deterioration. The fixation time should be at least 6 hours and not more than 72 hours before tissue processing starts. Under-fixation (<6 hours) and over-fixation (>72 hours) can lead to suboptimal histology, false-negative results on immunohistochemistry, and problems in performing other ancillary tests. Short fixation time results in poor preservation of antigens for IHC. Prolonged fixation time results in alterations of proteins in the tissue. Extended periods of fixation may also result in the radiographic disappearance of calcifications (12).

The American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) practice guidelines recommend a minimum of 6 hours of formalin fixation for breast tissue specimens including NCB specimens (13), although some reports have suggested that shorter fixation time for NCB have no negative impact on the reliability of IHC—at least for ER and Ki67 testing, if not for all others (14,15).

Decalcification of NCB specimens may be necessary for some highly calcified specimens; however, every attempt must be made to separately process any noncalcified portions of the specimen, and minimize time in decalcifying solution. Immunostains performed on decalcified tissue ought to be interpreted with caution.

The requisition form submitted with the NCB specimen should include the following information: patient name, age and gender, laterality of the specimen, indication for the procedure, clinical diagnosis, and sampled site(s). The name of the submitting physician and the date of the procedure must also be provided. The specimen container must be labeled with patient and specimen identification information that must match identifying information on the accompanying requisition form.

Specific information that should ideally accompany NCB specimens targeted for mass or imaging abnormality is listed in Table 26.1. The sampled site is generally indicated by a clock-face designation and distance from the nipple (e.g., right breast, 2 o’clock, N4) indicating that the specimen was taken from the upper-inner quadrant of right breast at the 2 o’clock position from a site 4 cm from the center of the nipple. Multiple palpable as well as impalpable lesions may be simultaneously sampled via NCB, safely and efficiently, and this practice favorably influences patient management (16,17).

The pathologic findings in any previously performed breast biopsy procedure must be conveyed in the requisition form. Relevant history of prior treatment (e.g., surgery, radiation, hormone modulation therapy, or chemotherapy) that could affect the histology of the breast should be provided (Fig. 26.3). Information regarding any known systemic disease that may also affect the breast (e.g., neoplasm at another site, diabetes mellitus, sarcoidosis, vasculitis, etc.) should be noted. Family history of breast or ovarian carcinoma, or of BRCA1 or BRCA2 mutations, should be included. Ideally, the instrument (cutting or vacuum-assisted) type utilized to procure NCB specimens
should be stated. The image modality used for guidance to the target (e.g., stereotactic, ultrasound, or MRI) ought to be included.

As per ASCO-CAP guidelines, the ischemic time, that is, time between specimen procurement and its placement in fixative, must be recorded in the requisition form (18).

A gross description should be recorded for each specimen with documentation of the number of samples, the range (and aggregate extent) of their lengths, as well as any other notable feature (e.g., color). The entire specimen, including any accompanying blood clot, must be processed for histologic evaluation. The bottom surface of the lid of the specimen container should be routinely examined for tissue that may be stuck to it. If the material in a sample is too abundant to be placed in one tissue cassette (i.e., >10.0 cm in aggregate length), the cores should be separated into groups of approximately equal number and size (Fig. 26.2B). Formalin fixation causes minimal shrinking of NCB samples (the shrinkage effect has been estimated to be 7% for 16-gauge tru-cut biopsy samples from the liver) (19). No more than four intact NCB should be placed in one cassette. The number of cassettes corresponding to each sample should be recorded, and each cassette should be labeled with a unique identifier.

Dipping of NCB specimens in dyes that are routinely available in a surgical pathology laboratory, such as methylene blue or eosin, increases the visibility of the embedded tissue in the paraffin block (Fig. 26.4). Inking of breast NCB specimens at the time of gross examination has been proposed as a relatively simple, inexpensive, and effective way to reduce the possibility of specimen mix-up during the processing of the tissue in the pathology laboratory (Fig. 26.5). All NCB specimens from a patient are inked with a single color. The next set of NCB specimens from another patient is inked with a different color, and so on. The color of the ink used for a case should be noted in the gross description. Three discrepancies were discovered in a study of 1,000 core biopsies that were inked sequentially with six different colors. In one instance, the error was related to switching of a tissue block. In another case, the error was related to incorrect labeling, and in a third, the error was typographic (20). Of course, no laboratory procedure can guard against the misidentification of specimens in the radiology office where NCB samples are usually obtained.

Some pathology and radiology departments weigh the NCB specimens as an objective measure of the volume sampled. In this regard, it must be kept in mind that tissue weight is proportional to tissue volume only if tissue density (i.e., weight divided by volume) is constant. Mammary tissue density, of course, is variable and depends upon the ratio of adipose, glandular, and fibrous tissue in any sampling. In spite of the foregoing, Park and Kim (2) have reported that while the 14-gauge needle collects 40 mg of tissue in each sample, the 11-gauge needle obtains 100 mg, and 8-gauge needle acquires at least 250 mg.

In general, NCB material taken for diagnostic purposes should not be taken for research studies until slides are prepared from that material. Harvesting of tissue for research should use formalin-fixed, paraffin-embedded NCB tissue rather than “fresh” tissue.

Routine methods of paraffin embedding, sectioning, and staining with hematoxylin and eosin (H&E) can be used for NCB specimens from the breast. A “fast-track” method for rapid processing of NCB specimens has been described (21). However, compliance with regulatory processing standards and achievement of optimal histologic and IHC staining should be ensured before the adoption of this technique (22).

The NCB samples must be embedded in a manner that positions them at approximately the same plane in the paraffin block.
Histologic sections should be 4 to 5 µm thick. The evaluation of multiple levels (at least three “interval” levels, 50 µm apart) for NCB is standard practice in most pathology laboratories. Sectioning at lesser intervals is appropriate for samples obtained with smaller needles. Evaluation of three-step sections reportedly maximizes the chances of visualizing microcalcifications in NCB samples (23). Examination of a minimum of five levels has been recommended to ensure maximum sensitivity for detecting “atypical foci” (24) and of six levels to ensure “accurate” diagnosis (25).

The value of obtaining multiple levels for NCB performed to investigate mammographically detected calcifications has been well established (Fig. 26.6); however, the routine examination of levels for NCB taken for lesions other than calcifications are of limited value. Lee et al. (26) demonstrated that the diagnosis after examining three levels was different from that in the initial level in 4 of 272 (1.5%) NCBs taken for reasons other than calcification, and in 13 of 103 (13%) NCBs taken to investigate calcifications.

It is important not to exhaust the NCB tissue in the preparation of initial histologic sections to preserve material for IHC studies that may be necessary to establish or refine a diagnosis. If laboratory resources allow, intervening sections cut between the various stained levels can be mounted unstained on labeled slides and saved for possible IHC or other ancillary studies. Such a protocol saves tissue, time, and effort that may be subsequently spent in the retrieval and processing of tissue blocks. If recuts are made at a second sitting for immunostains, one new recut slide should always be submitted for H&E staining (27).

Findings on various imaging modalities in a particular case are often communicated in the requisition. NCBs are being increasingly performed under some form of image guidance; thus, pathologists
ought to be acquainted with the fundamentals of breast imaging and reporting. Imaging techniques commonly employed to study the breast include mammography (including digital mammography), ultrasound, MRI, and positron emission tomography (PET). The ACR BI-RADS (American College of Radiology’s Breast Imaging-Reporting and Data System) is used in reporting findings on mammography and has also been applied to the reporting of findings on other imaging modalities (Table 26.2).

NCB can be performed under stereotactic image (i.e., mammographic) guidance. Stereotactic NCB is generally used for calcifications, masses, and architectural distortion. Mammography using low-dose ionizing radiation can detect masses, architectural distortion, or calcifications. For a mammographically detected mass (or lesion causing architectural distortion), the radiology report usually states its density, shape, and borders. On mammography, a mass suspicious for malignancy may be dense and irregular with spiculated edges. For mammographically detected abnormal calcifications, the radiology report usually describes their morphology and distribution. Calcifications suspicious for malignancy may be linear (“casting-type”), branching, and/or pleomorphic. Digital breast tomosynthesis is an evolving, enhanced three-dimensional mammographic technique that increases lesional visibility by detecting subtle changes in the texture of parenchyma.

The specimen radiograph corresponding to the NCB specimen, particularly in cases wherein the target lesion is calcification, should accompany the specimen. A brief description of the abnormality seen in the specimen radiograph should be a part of the gross description.

Ultrasound imaging utilizes high-frequency sound waves to detect lesions through varying echo patterns. It is useful for determining the size and shape of masses and identifying cysts. The echogenicity of a lesion vis a vis that of subcutaneous adipose tissue and the orientation of the lesion in relation to the skin of the breast are usually reported in ultrasound reports. A lesion may be “isoechoic” (having the same echogenicity as adipose tissue, e.g., a lipoma), “anechoic” (e.g., a cyst), “hyperechoic” (normal fibrofatty breast tissue), or “hypoechoic” (most clinically significant lesions). On ultrasound, a lesion suspicious for carcinoma may be hypoechoic with a “taller than wide” orientation. Ultrasound is often employed to further study lesions identified on mammography and MRI. An ultrasound-guided biopsy procedure is relatively simple and quick to perform.

MRI screening is based on the premise that neoplasms incite neovascularity, which results in locally increased blood flow and permeability. MRI-guided biopsies are performed for lesions that cannot be identified by other methods. Injection
of contrast (intravenous gadolinium) leads to enhanced and accelerated deposition of contrast in the region of the tumor (“wash-in”) and accelerated loss of contrast (“washout”). MRI can evaluate lesional morphology (shape and border) and the kinetics of contrast enhancement (initial and delayed). On MRI, a lesion suspicious for carcinoma may be irregular in outline with rim enhancement and can exhibit characteristic kinetics. MRI of the breast has diagnostic and screening applications (e.g., evaluation of occult tumor, extent of tumor, multifocality, multicentricity, response to neoadjuvant chemotherapy, recurrence, and in the screening of high-risk women). In a study of 445 MRI-guided biopsies, all performed on high-risk patients, 79% were benign (28). The technique requires sophisticated equipment, including open coil MRI and MRI compatible needles.

PET screening of the breast assesses the level of glycolysis in tissues after injecting a patient with a radiotracer with an unstable nucleus. PET scans of the breast have been used in a limited fashion with mixed results for screening in high-risk patients, for evaluating recurrences, and for evaluating response to chemotherapy or hormonal therapy.

The concordance of the clinical impression, imaging results, and pathologic findings is often referred to as the “triple-test.” It is important to ensure that the clinical and radiographic findings are consistent with the pathologic findings on NCB. Re-biopsy with NCB or an excisional biopsy is usually recommended for discordant cases (i.e., cases that fail the “triple-test”).

The histopathologic diagnosis ought to be based entirely on the microscopic appearance of the sampled tissue in the NCB specimen. The results of a pathologic interpretation that is not consistent with the clinical impression should be discussed with the submitting radiologist or responsible clinician to ensure that the sample is representative of the lesion. A written note of this discussion should be kept with the pathology records of the case. The repeated procurement of minuscule or otherwise inadequate samples (e.g., blood only) should be discussed with the appropriate clinician.

NCB specimens derived from a target with calcifications, as demonstrated by mammography, should undergo specimen radiography immediately after the procedure, and the presence of calcification in the sampling should be confirmed. This process makes it possible to identify and segregate the NCB samples containing calcifications from those without visible calcifications before submission to the pathology laboratory. The cores with and without calcifications from each biopsy site can then be placed in fixative in separately labeled containers. Alternatively, the two sets of cores can be placed into separate tissue cassettes, differentiated by color and/or label, and submitted in a single container. The method chosen to separate specimens before submission to the pathology laboratory should be standardized within a given institution. The practice of separating the specimens with and without calcifications is useful for correlation with the specimen radiograph. The diagnostic yield has been reported to be higher in the segregated cores containing calcifications, although equally careful attention must be paid to samples with and without calcifications. A commercially available “tray” has been devised to facilitate radiology-pathology correlation mainly by allowing the usually fragile tissue samples to maintain their orientation and integrity (Fig. 26.7). Calcifications can be visualized in X-ray images of paraffin blocks, and they remain detectable in this condition for an indefinite period.

Calcifications that are less than 100 µm (0.1 mm) in maximum dimension are unlikely to be radiographically visible (29).
Consequently, histologically detected calcifications of minuscule proportions cannot be assumed to represent the calcifications seen in a clinical mammogram. Whenever a biopsy procedure is performed for calcifications, the pathology report should specify whether calcific deposits are microscopically evident and the type of breast tissue in which they are located (Fig. 26.8).

One possible explanation for the occasional lack of histologic visualization of calcification in NCB material obtained for mammographically detected microcalcification is their loss during histologic sectioning. This may occur either due to discarding of shavings containing calcifications in the microtome or “fracturing” of the calcifications when they are hit by the microtome blade, resulting in ejection (“chipping”) of shattered calcific debris, in the course of preparation of levels (Fig. 26.9). Radiography of histologic shavings has provided evidence for both eventualities (30,31). “Chipping” occurs more often with larger deposits of calcification (such as those in sclerotic fibroadenomas) rather than with microcalcifications. Other explanations for “missing” calcification are inadequate sampling, mislabeling of samples, and failure to recognize calcium deposits in histologic sections. This is
more likely to occur with calcium oxalate than with calcium phosphate calcifications.

If calcifications are described in the radiograph of the NCB specimens and none are initially evident histologically, the slides should be examined for calcium oxalate (“weddelite”) crystals. These crystals do not stain with the H&E stain but are birefringent with polarized light (32). Calcium oxalate crystals are usually located in cysts lined by apocrine epithelium and may rarely elicit a foreign body-type giant cell reaction in the cyst or in periductal stroma. Less-common types of calcifications are shown in Figure 26.10.

Correlation with imaging findings is crucial to the reporting of NCB specimens, as exemplified even by the seemingly innocuous finding of histologically unremarkable adipose tissue—an instance that may represent either fatty breast parenchyma, a lipoma, or a missed target. It must also be kept in mind that several noncalcium elements in breast tissue can radiologically simulate microcalcifications. In this context, suture material from prior surgical procedure is commonly encountered. Tattoo pigment used for cutaneous adornments can simulate calcifications, especially when the pigment is carried into intramammary lymphatic channels. Hemosiderin (from
hemorrhage at an earlier date) has been known to simulate calcifications. Injection of material into breast tissue, such as gold (injected into breast tissue for therapeutic use) and various substances (used to “lace” or “cut” recreational drugs) can also mimic calcifications radiographically.

Occasionally, calcifications are not identified in the routine slides prepared from NCB that had targeted calcifications. In such cases, the source of the specimen should be verified. This step should be followed by review of the specimen radiograph (which should ideally accompany the specimen). In most cases, radiography of the tissue block(s) can identify calcifications that have not yet been sectioned. Additional deeper levels (at least three “shallow” recuts) should be obtained from those tissue blocks that show calcifications on radiography. In exceptionally rare cases, calcifications within cysts (“milk of calcium”) can be lost. This can happen by mechanical drainage of the contents
when the cyst is sectioned either at the time of biopsy or at the microtome. Occasionally, the “missing” calcifications are found in the stroma (amid fibroelastic tissue) or within arterial vessels (in the pattern of Monckeberg sclerosis). Calcifications can rarely appear as minuscule “vesicles” within the stroma of some sclerotic fibroadenomas.

Well-prepared and optimally stained H&E-stained sections are crucial to rendering the correct interpretation. A definitive diagnosis should not be made on slides that are not “full face” or present extremely fragmented samples (Fig. 26.11). Additional “deeper” levels should be obtained in these cases, which are sometimes helpful.