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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2021  |  Volume : 64  |  Issue : 1  |  Page : 102-106
Modified improvised pre-embedding method for core needle biopsies: A clinicopathologic study


1 Department of Pathology, St. John's Medical College and Hospital, Bangalore, Karnataka, India
2 Department of Surgical Oncology, St. John's Medical College and Hospital, Bangalore, Karnataka, India

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Date of Submission01-Apr-2020
Date of Acceptance30-Apr-2020
Date of Web Publication8-Jan-2021
 

   Abstract 


Background: An optimal core needle biopsy (CNB) is expected to balance between tissue diagnosis, the accuracy of negative sampling, and concordance with reports from resected specimens to select the appropriate treatment. Though various techniques for CNBs are available, no guidelines exist for processing CNB, with practices varying from lab to lab for transport and processing. This prospective study aims to design a cost-effective, user-friendly pre-embedding method for CNBs to yield intact cores. Objective: To compare the outcomes of CNBs by a conventional method with those processed by the modified pre-embedded processing protocol over 2 years. Material and Methods: Presurgical CNBs from SOL in various organs were subjected to the conventional free-floating method in formalin (control) for histopathology diagnosis. CNBs from the corresponding, freshly resected SOLs (test) were taken, inked with coloring inks if multiple, placed between two 2 × 2 cm polyurethane foam meshes fitted inside cassettes, fixed in formalin, and transported to the laboratory. The two CNB groups were coded and scored independently for intactness, tissue processing, ease of embedding, and ease of cutting sections. Data obtained were statistically analyzed. Results: Test CNB cores were better processed, intact, linear, and aligned, compared to control CNBs. With four CNBs in one block, the number of blocks and sections were cut-down by one-fourth. Conclusion: CNBs processed using polyurethane foam and coloring inks were superior and economical against conventional free-floating CNBs. This technique can be practiced by surgeons at the bedside.

Keywords: Core needle biopsies, polyurethane mesh, pre-embedding histologic technique, tissue coloring inks

How to cite this article:
Sunny N, Thomas A, Manjunath S, Kini U. Modified improvised pre-embedding method for core needle biopsies: A clinicopathologic study. Indian J Pathol Microbiol 2021;64:102-6

How to cite this URL:
Sunny N, Thomas A, Manjunath S, Kini U. Modified improvised pre-embedding method for core needle biopsies: A clinicopathologic study. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jan 16];64:102-6. Available from: https://www.ijpmonline.org/text.asp?2021/64/1/102/306503





   Introduction Top


Core needle biopsies (CNBs), a minimally invasive screening and diagnostic tool play a vital role in tissue diagnosis, tumor typing, and staging to predict the patient outcome for treatment and management. These tissue cores are used to assess the grade and stage of malignant tumors based on the percentage of tumors involving the core, the absolute number of cores with a tumor and the overall percentage of tumors in all the cores put together.[1] This data also determines whether the patients are candidates for active surveillance.[2] The diagnostic yield of these cores is dependent on the intactness of the tissue cores which, in turn, is dependent on many variables,[3],[4] such as operator's skills, type of tissue sampled, the core length, core shape, core fragmentation, depth of sectioning of the core between slides, and its processing technique. Any pitfall results in the fragmentation of these CNBs which is one of the commonest problems and is the most challenging issue for the end-user, namely, the pathologist.

The practice of handling CNBs at the laboratory side varies from lab to lab. However, no established guidelines/protocols exist for handling and processing of CNBs and there is an imminent need for a technique to keep the tissue cores intact and in one plane throughout the processing and embedding of the tissue. Hence, this study was aimed at designing a cost-effective, user-friendly technique well-suited for a general pathology laboratory to keep CNBs well-supported and linear at all stations of tissue processing to get intact tissue cores.


   Material and Methods Top


This prospective observational study was conducted over 3 years and 1 month from January 2015 to January 2018 at the Translational research laboratory of the Department of pathology of a tertiary medical center after obtaining ethical clearance from the institutional ethical review board (IEC 23/2015). All CNBs (both freehand and guided) from in-patients admitted to the department of surgical oncology for space-occupying lesions in various organs and sent in 10% neutral buffered formalin by the conventional free-floating method and their corresponding resected specimens sent fresh to pathology laboratory were included in this study. Liver, kidney, brain, bone, and bone marrow biopsies, those biopsies sent during intraoperative consultation and referral cases from outside hospitals were excluded.

The main objective was to compare and contrast the conventional free-floating method of tissue fixation and transport (method A of the control group, Arm 1) with the proposed new technique (method B of the test group, Arm 2). A sample size of 100 (50 conventional and 50 test) was calculated and accepted to get 85% sensitivity, 10% precision, and a 95% confidence interval. The cases included were coded to circumvent bias. The methodology employed is described below and illustrated as a flow chart in [Figure 1].
Figure 1: Flow chart to describe the methodology for two arms for CNBs included in the study namely, Arm 1 for the conventional method and Arm 2 for the proposed method

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Method A of the control group (Arm 1) [Figure 1] and [Figure 2]
Figure 2: (a-f) Conventional free-floating method for CNBs: Pictures show core biopsies taken from a breast lump (a) fixed in 10% buffered formalin, floating free in a container, (b) after decantation, the cores are transferred from the container to a petri-dish, (c) for easy pick-up. The CNBs are picked, transferred on to filter paper, and stained with eosin (d-f). Note that the tissue cores after fixation are curved in various planes, are of varying lengths and fragment depending on the tissue consistency as there is no support

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The routinely practiced method is the conventional free-floating method. The CNBs taken by the surgeon at the bedside were immediately put in 10% neutral buffered formalin [Figure 1], floating free in a container for fixation. After overnight fixation, the formalin was decanted and the biopsies were picked to place them on to a filter paper. The tissues were colored with eosin/mercurochrome dye which was, in turn, conveniently placed in tissue cassettes as shown in [Figure 2].

Method B of the test group (Arm 2) [Figure 1] and [Figure 3]
Figure 3: Technique of the proposed technique: Using a 22 mm gauge Bard biopsy gun (a), three to four CNBs were taken from the SOL of the fresh specimen. The biopsy cores were placed between two 3 × 2 cm of 0.3 cm thick foam pieces cut from a larger sheet of locally available polyurethane foam (b). (c) illustrates the technique diagrammatically

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Using this modified improvised pre-embedding technique, the core biopsies were taken from corresponding resected specimens with space-occupying lesions (SOLs) from which CNBs had been taken earlier and processed by the method A (Arm 1) mentioned above.? The CNBs were thus obtained, by using a 22 mm gauge Bard biopsy gun [Figure 3]a. The biopsy cores, a minimum of three, and a maximum of four in number were placed directly on a 2 × 2 cm of 0.3 cm thick polyurethane foam piece cut from a large sheet obtained from the local market [Figure 3]b and [Figure 3]C and is covered by another piece of the foam to sandwich the core. When the CNBs were more than one, they were placed parallelly. If the cores were from specific sites, those were inked using various tissue coloring inks from Histolab/acrylic paint or Fevicryl acrylic hobby color groups (no. 6, 12, 22, 33) [Figure 4]a and [Figure 4]b.[5] Subsequently, the cassettes with tissue cores placed between the two foam pieces were sealed and dropped into containers containing 10% buffered neutral formalin for fixation overnight.
Figure 4: Inking of tissue cores: When the cores were site-specific, they were inked with different tissue coloring inks (a and b) and after overnight fixation, CNBs were embedded in parallel in paraffin wax (c). The prepared paraffin block (d) were serially sectioned at 4–5 μm and sections were subsequently stained with hematoxylin and eosin (e)

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Tissue cores subjected to both A and B methods were processed uniformly thereafter. Following dehydration and clearing processes, the core biopsies were embedded in paraffin wax. For those processed by method B, the foam pieces were separated and the tissue core was lifted with a forceps with ease and embedded placing the core parallel to the long length of the mold, parallel to each other and in one plane [Figure 4]c. The foam pieces were subsequently kept away for a day and later discarded.{Figure 4}

All paraffin blocks from both methods A [Figure 5]a and B [Figure 4]d and [Figure 5]d were serially sectioned at 4–5 μm without intervals using the conventional rotary microtome (Leica). After initial trimming of the block, complete sections thus obtained, were placed on a glass microscopic slide and were stained with hematoxylin and eosin (H and E) stain after heat fixation [Figure 4]e.
Figure 5: CNBs by conventional vs proposed pre-embedding method: Note that CNBs processed by the conventional free-floating method (Arm A) are fragmented, curved and placed haphazardly in various planes in paraffin block (a) and section, (b) with tissue loss appreciated at microscopy, (c, H and E × 40). In comparison, note the CNBs processed by modified improvised pre-embedding method (Arm 2) which are intact, linear, placed parallelly in a paraffin block, (d) and section, (e) and relatively less or no tissue loss at microscopy (f, H and E × 100)

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All the cores were independently evaluated at all levels including microscopy [Figure 5]b and [Figure 5]c and [Figure 5]e and [Figure 5]f for intactness of the cores during embedding, ease of embedding, degree of tissue processing, and ease of cutting sections and scored from 0 to 10. The results, thus, obtained were analyzed with descriptive statistics.


   Results Top


A total of 164 CNBs were included in this 2- year 1-month prospective study at the tertiary medical center. Sixty CNBs were excluded as their corresponding resected specimens were received fixed in formalin and hence not available for taking core biopsies to subject them for the modified improvised pre-embedding method. The CNBs were from various space-occupying lesions in breast, prostate, adrenal, thyroid, soft tissue, and so on.

The two methods of tissue embedding, one conventional and the other modified improvised pre-embedding methods with 52 samples each showed distinct differences in the shape and intactness of the core biopsies after formalin fixation and processing?. Tissue processing by the conventional free-floating method resulted in curved cores in various planes [Figure 5]a,[Figure 5]b,[Figure 5]c, thus, making it difficult for the technician to align the full length of the cores in one plane resulting in fragmentation at multiple points and hence tissue loss. In comparison, the cores of method B were generally linear, were well-processed, and easy to handle after impregnation and during paraffin embedding procedure [Figure 5d-f]. Hence, the latter could be placed straight in one plane and so were the other CNBs which were together. The cut sections were found to be a replica of the paraffin blocks and the microscopic slide showed less or no fragmentation and no loss of tissue. The first serial sections of four were complete and no serial sections were required thereafter in between the slides. Four CNBs could be placed in one block with ease thus, economizing on the number of paraffin blocks made. The tissue coloring system helped further in identifying each one of the cores to its specific sites of origin at gross and at microscopy.

The various scores, thus rated for the two methods against the four features, namely, intactness of the core during embedding, ease of embedding, ease of cutting sections, and degree of tissue processing are shown in [Table 1]. Method B scores were in the range of 7.3 ± 1.8 to 9.3 ± 0.5 vs 3.3 ± 2.1 to 6.8 ± 1.9. (Mean ± SD).
Table 1: Table to show the comparison in the scores obtained between the two embedding methods employed in this study

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The authors did find challenges while embedding the modified improvised method. The tissue cores would adhere to the foam and would find difficult to separate the cores from the foam packs. This problem was solved by wetting the foam before the placement of the tissue between the two foam packs and then allow them to fix in formalin. The authors also found the imprint of the grooves of the cassette on to the foam and subsequently on to the tissue core distorting tissue at microscopy. This problem was solved by placing the tissue on the non-grooved surface of the foam during the pre-embedding time. The polyurethane foam used remained inert and unaltered by the solvents and reagents used during tissue processing.


   Discussion Top


CNBs as a good screening and diagnostic tool serve as an interface between an invasive excisional biopsy and fine-needle aspiration cytology and they best contribute when they are intact. They are well-established as a minimally invasive tool in the management of patients with breast lumps and enlarged prostates.[6],[7] As more than 25% of CNBs contain only a tiny focus of carcinoma, adequate tissue processing is important to avoid the risk of failing to detect these carcinomas.[8],[9] Interestingly, there are no established guidelines for tissue processing and histologic sampling of core biopsies, and the practice varies considerably among different laboratories.

The results indicate that the pre-embedding procedure of tissue processing of CNBs has a great influence on the histologic yield. The biopsies were taken using the modified improvised technique found to have a far better scoring for the intactness of core, thus cutting down the number of blocks and number of serial sections required by one-fourth. This can easily be performed at the time of biopsy by the operator, be that be a physician, surgeon, or radiologist. It requires no special training or infrastructure. The polyurethane foam is easily available from any market. It was also found to be very economical as it cost approximately 10 INR per 1 × 1 m foam piece. The foam was also found to be inert to the solvents and reagent used during tissue processing. This technique may also best suited to embed minute biopsies to maintain their shape and orientation.

The use of various tissue coloring inks helped in reducing the number of paraffin blocks used and economize on the cost. If not, each core needs to be allotted an independent block to monitor its identity. As the tissue cores are in the right plane while embedding, the required number of complete sections comes down which represents the histologic overview of the biopsy. This will reduce the cost and manpower necessary both for slide preparation as well as reduces reporting time for the pathologist, thus, facilitating faster turnaround time (TAT) for these CNBs.

As the future perspective of this technique, the authors would like to forecast the potential improvement that can be foreseen in detecting malignant foci which could decrease the number of patients who would have to undergo repeat biopsies, thus making this modified improvised pre-embedding method valuable and cost-effective for both physicians and patients as well. This technique can also be practiced with ease by surgeons themselves at the bedside at the time of biopsy, thus simplifying the work thereafter.

This improvised pre-embedding method will be most beneficial in peripheral general pathology laboratories and healthcare centers in developing countries. A prospective study done on core biopsies with the operator at the patient bedside performing the orientation of the CNBs and their placement between the foam pieces immediately after biopsy on a routine basis is being undertaken. The results will further guide one to the next phase of this study, namely, the diagnostic assessment of the core biopsies by this new technique against the conventional free-floating method practiced routinely. At the end of this study, we note that the preformed cassettes with the mesh have just been made commercially available in the West but they are expensive and not a viable proposition for the Indian scenario.

Ethical approval

This study was conducted after the ethical approval at St John's Medical College, Bangalore IEC Study Ref. No. 23/2015. All procedures performed in the study involving human participants were as per the ethical standards of the concerned institutes and with the 1964 Helsinki declaration and its amendments. The study did not involve animals.

Declaration of patient consent

Informed consent was obtained from the participants enrolled in the study. Participants' information has not been disclosed in any format in the manuscript or abstract or the form of images or tables.

Financial support and sponsorship

Vision Group on Science and Technology, K-FIST II, GRD no. 337.

Conflicts of interest

There are no conflicts of interest.



 
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Carter HB, Kettermann A, Warlick C, Metter EJ, Landis P, Walsh PC, et al. Expectant management of prostate cancer with curative intent: An update of the Johns Hopkins experience. J Urol 2007;178:2359-64.  Back to cited text no. 2
    
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Fajardo DA, Epstein J. Fragmentation of prostatic needle biopsy cores containing adenocarcinoma: The role of specimen submission. BJU Int 2009;105:172-5.  Back to cited text no. 4
    
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Yadav L, Thomas S, Kini U. Improvised double embedding technique of minute biopsies: A mega boon to histopathology laboratory. Indian J Pathol Microbiol 2015;58:12-6.  Back to cited text no. 5
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Rogatsch H, Mairinger T, Horninger W, Gschwendtner A, Bartsch G, Mikuz G. Optimized pre-embedding method improves the histologic yield of prostatic core needle biopsies. Prostate 2000;42:124-9.  Back to cited text no. 6
    
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Rogatsch H, Moser P, Volgger H, Horninger W, Bartsch G, Mikuz G, et al. Diagnostic effect of an improvised pre embedding method of prostrate needle biopsy specimens. Hum Pathol 2000;31:1102-7.  Back to cited text no. 7
    
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Renshaw AA. Adequate tissue sampling of prostate core needle biopsies. Am J Clin Pathol 1997;107:26-9.  Back to cited text no. 8
    
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Garg P, Pathak P, Goyal R, Arora VK, Singh N. Current practice in handling and reporting needle biopsies: A hospital-based survey. Indian J Pathol Microbiol 2018;61:197-200.  Back to cited text no. 9
[PUBMED]  [Full text]  

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Correspondence Address:
Usha Kini
Translational Research Laboratory For Gut Motility Disorders, Department of Pathology, St. John's Medical College, Bangalore - 560 034, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_313_20

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