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Cellient® Atlas IntroductionAndrew H. Fischer, M.D
In spite of years of research progress, the diagnosis of many diseases, especially cancer, still requires light microscopic evaluation of a sample of cells. To make a diagnosis, pathologists look for alterations in cell structure, and for changes in the composition and organization of tissues . It is obviously an advantage to be able to make a diagnosis, or to guide therapy, based on the smallest possible biopsy sample: the smaller the biopsy, the fewer are the risks and complications for the patient.
Cytology is the field that uses the smallest possible "microbiopsies" for diagnosis.The appeal of cytology is that it can provide a small but diagnostic sample. By minimizing risks and complications for detecting certain diseases, cytology can be used for the screening of disease.
When cellular level alterations alone are sufficient for a diagnosis, a minimal sample size is acceptable. For many diagnoses, however, it may be necessary to be able to recognize the larger-scale alterations in tissue architecture, or to study biochemical and molecular characteristics of the cells. Cell blocks fulfill this need.
Cell blocks are microbiopsies embedded in paraffin. A standard histologic section, measuring four or five microns in thickness, shows the organization and cellular composition of a microbiopsy fragment. Generally, diagnoses can be made with the most confidence when combinations of cellular and tissue level morphology are present.
Cell blocks are microbiopsies embedded in paraffin. They broaden the diagnostic value of cytology specimens and are complementary to cytology preparations.
Within the past couple decades, ancillary techniques have been developed to provide more diagnostic information than that which can be obtained solely from the morphology of cells and tissues. Immunohistochemistry (IHC) allows disease-specific antigens, or combinations of antigens, to be detected. Immunohistochemistry can be performed on cytology preparations of microbiopsy samples, however there are many advantages in using cell blocks. IHC on cytology can trap antibodies or reagents in large tissue fragments, giving the impression of a positive staining reaction. Paraffin sections allow each part of a microbiopsy to have equal access to IHC reagents. Another advantage of using cell blocks for IHC is the relative ease of scoring or quantifying positivity on a per cell basis.
Cell blocks are the ideal platform for IHC and molecular diagnostic ancillary studies.IHC on cell blocks also allows the staining reaction to be correlated with larger-scale tissue architecture. For example, the finding of hepatocytes growing more than a few cells away from the CD31 positive endothelial cells within a tissue fragment is virtually diagnostic of hepatocellular carcinoma (Figure 2). Serial sections that can be cut from cell blocks allow multiple IHC reactions to be studied in the same sample as in this Cellient cell block of a chordoma stained with pan keratin (Figure 3) and EMA (Figure 4). One tissue fragment 100 microns in diameter (i.e., only about half the diameter of a 25 gauge fine needle) could be used to study 20 IHC reactions in five micron serial sections, but it can only be studied in one immunohistochemical staining reaction in a cytology preparation. Cell blocks are a convenient and stable means for archiving biopsies at room temperature, with many advantages over freezing, storing glass slides, or storage of liquid fixatives. Defining individually optimal therapies is becoming an essential duty for pathologists with the advent of new molecular-based treatments. Paraffin embedded tissue has emerged as the standard platform to achieve this goal of "personalized medicine".
How small can a microbiopsy be? It obviously depends on the particular disease, but for many of the common questions that pathologists need to answer, a few hundred microns of tissue diameter are sufficient. Take the example of breast FNA. A 23 gauge "fine" needle has an internal diameter of about 320 microns. Two to three hundred microns is enough to recognize diagnostic changes of invasion in breast cancers, or to histologically diagnose the degree of proliferative changes [1,2].
If a diameter of a few hundred microns is sufficient to be able to diagnose a large number of breast lesions, why are core biopsies that are ten times this diameter used so widely? Contrary to popular belief, the answer is not related to the "amount" of tissue. Consider an average FNA pass with 2 drops (100 microliters) of material of which 50% of the sample is cells and 50% is blood. The volume of cells would be 50 microliters. An average 14 gauge core biopsy 2.1 mm in diameter and 1.4 cm long also has a volume of tissue of 50 microliters[3,4] . In a single 5 micron tissue section, the 14 gauge core biopsy would display up to 29 square mm of tissue. if all of the 320 micron fragments from a single FNA pass with a 23 gauge needle were placed side by side within the plane where a microtome can capture a histologic section, then there would be 156 square mm of tissue profiles, or more than 5 times that of the single core biopsy ! Such an FNA would still allow up to 60 five micron sections to be cut. A further advantage of an FNA is that sampling through a larger volume of target tissue is possible compared to core biopsies.
The major reason that core biopsies are bigger than they need to be is that current tissue processing methods lose tissue fragments and/or cannot place them consistently at the plane where a microtome will actually capture the section. A major limitation of other histologic techniques is the requirement to physically grasp the biopsy specimen at some point with forceps. If a large core biopsy is fragmented, it is physically difficult or impossible for the histologist to hold multiple fragments in place at the base of the wax mold while embedding. This often results in fragments that float to different planes (Figure 5). Physically holding onto tissue fragments also introduces the potential for cross-contamination, as shown in (Figure 6) in which gastric epithelium from one patient contaminated the paraffin block of a breast core biopsy.
For tissue fragments that are too small to be held by forceps, it has been necessary to concentrate and collect them together to bring them through the process of paraffin embedding (Figure 7). Two basic ways of collecting the fragments for "conventional" cell blocks have been used. The first binds the fragments together in a matrix of material such as fibrin or agar to allow a histologist to remove and place this bound collection as a whole in the wax mold. The limitation of this technique is that the tissue fragments are dispersed into a 3 dimensional space which is wider than the actual fragments (Figure 8). For example, if the FNA sample described above with 320 micron sized fragments is suspended in an agar button 3 mm in thickness, one tissue section would show about 10 fold less cells than if the cells were all within the same plane. Plasma, usually obtained from unused or outdated blood bank specimens, is commonly combined with thrombin to form a fibrin clot which acts as a carrier substance. In addition to the cellular and molecular cross contamination that could arise from introducing a different patient's biologic materials into a diagnostic sample, another problem with the plasma-thrombin cell block technique is the frequent occurrence of bacterial or fungal contamination of the plasma (Figure 9). The second method of grouping microsized biopsy particles together is to use a sectionable collodion "bag" to hold the particles . While there is no carrier substance diluting the cells, the limitation of this technique is that the cells are concentrated in a rounded three dimensional volume. If the same FNA had the cells concentrated into a sphere with a volume of 50 microliters, then a single tissue section would have a profile of cells measuring 4.5 mm in diameter, or about ten fold fewer cells than if all the fragments were placed in the same plane.
The Cellient® Automated Cell Block System has been developed to address these limitations of cell block preparation. This automated system for embedding cell samples and small tissue fragments in paraffin offers substantial benefits over existing manual methods for preparing cell blocks. A proprietary tissue cassette and filter assembly has been designed to capture cells or tissue fragments from residual ThinPrep® specimens, or other cell suspensions. The cassette, disposable filter and filter support permit the captured cells or tissue fragments to be correctly positioned in a plane for subsequent cutting by a microtome. Cells and microbiopsy fragments are deposited in a uniform layer until the filter is saturated. At this point, eosin stain can be added to improve visualization of the cells for the histologist. In the next steps, small aliquots of alcohol, xylene and paraffin are rapidly drawn through the sample. The filter region is then cooled. All of these steps are fully automated. Once the paraffin hardens, the filter pulls away leaving the cells embedded in one plane in the wax (Figure 10). The cassette is manually removed and placed into a disposable remolding base in the automated embedding station to add a layer of wax around the disk of embedded tissue. Once this step is complete, the block is ready for histologic sectioning. The layer of wax around the block provides the histologist with the ability to accurately identify the plane where the cells are located, minimizing loss of material during sectioning.
There are a number of advantages of the Cellient process compared to other cell block techniques. Cell loss during centrifugation and decanting is minimized. There is no additional loss of material due to incomplete transfer by the histologist from the cassette to the wax mold. Due to the efficiency of the flow-through extractions and elimination of carrier substances, complete processing uses a lower volume of reagents than standard tissue processors, without microwave radiation, and has a processing time that is significantly less than a standard tissue processor.
The advantages of the Cellient process are:
- Maximizes cellularity from scant samples.
- No loss of tissue during handling or centrifugation.
- Faster processing.
- Standard processing, with formalin-free option to maximize RNA preservation.
- Minimizes risk of cross contamination.
- Less technical help needed.
- Lower volume of reagents per block.
To achieve the goal of personalized medicine, paraffin-embedded tissue processing conditions need to be standardized. One of the key attributes of the Cellient process is the ability of the user to standardize processing conditions, using individual, identical, fresh reagents for each block. Formalin fixation can be standardized if desired. However, formalin fixation is not needed for producing crisp nuclear morphology in the sections, formalin does not facilitate histologic sectioning, and many labs have found that formalin fixation is not needed for immunhistochemistry . Processing quickly without formalin allows high quality RNA preservation to be achieved in paraffin . Major causes of cross-contamination are eliminated. Unlike batch processors, there is no chance for "floaters" to contaminate a specimen (Figure 6) . There is no need for handling of the sample by cytopreparatory technicians or histotechnologists, eliminating the chance of floaters to be introduced with forceps and reducing the time needed to process specimens.
In the following chapters, the broad utility of Cellient Cell Blocks is demonstrated. The long term hope is that by standardizing processing conditions and minimizing the size of biopsies that are diagnostic, Cellient will contribute to the development of novel screening tests for cancer detection, allow "personalized" optimal therapies for diseases, and provide a platform through which cancer researchers and cytologists can share some common ground to fight and help find a cure for cancer.
- Fischer AH, Zhao C, Li QK, Gustafson KS, Eltoum IE, Tambouret R., Benstein B, Savaloja LC, Kulesza P. The Cytologic Criteria of Malignancy. J Cellular Biochem 110:795-811, 2010
- Istvanic S, Fischer, A. H., Banner, B., Eaton, D., Larkin, A., Khan, A. Cell blocks of breast FNA's frequently allow diagnosis of invasion or histological classification of proliferative changes. Diagnostic Cytopathology 2006; In press.
- Burbank F, Parker SH, Fogarty TJ. Stereotactic breast biopsy: improved tissue harvesting with the Mammotome. American Surgeon 1996; 62:738-44.
- Jackman RJ, Burbank F, Parker SH, et al. Stereotactic breast biopsy of nonpalpable lesions: determinants of ductal carcinoma in situ underestimation rates. Radiology 2001; 218:497-502.
- Fahey C, Bedrosian, U. K. Collodion Bag: A cell block technique for enhanced cell collection. Laboratory Medicine 1993; 74:94-96.
- Gao H-G, Savas L, Woda B, Fischer A. Validation of Cellient-type processing for immunohistochemistry. Cancer Cytopathology 114(S5):404-5. Poster presentation at American Society of Cytopathology 56th annual meeting, November 2008.
- Fischer AH, Mou, Z., Woda, B. A. High-Quality preservation of RNA and morphology in paraffin. Modern Pathology 2006; 19:328A.