Introduction

Precise Macroscopic and microscopic analysis of resected specimens by pathologist is necessary for proper diagnosis of a pathology [1]. In tumour cases, involvement of surgical margins of resected specimens has significant therapeutic and prognostic implications. Malignant lesions may extend beyond the macroscopic limits of surgical resection, which if not diagnosed accurately can result in treatment failure and poor prognosis [1]. In the case of surgically excised specimens, it is often desirable to identify the orientation of the specimen permanently for both further gross examination and microscopic identification of resected surgical margins [2]. Several methods have been used for proper identification and orientation of resected margins in surgical pathology such as inking of specimens, sectioning method, stitches and suture marker method [3].

Inking of surgical margins is the most reliable and safe method. Methods other than inking like usage of stitches can damage microtome knife and sutures can cause artifacts with loss of tissues [4]. Several materials have been used as surgical ink for inking of specimens. India Ink is the traditional marking ink in the field of surgical pathology because of its resistance to tissue processing schedule and good visibility under microscope [3]. No material is 100% reliable to be used as surgical inks. Inspite of many advantages of India Ink, it has few disadvantages like more drying time, monochrome, toxicity and cost. Other than India ink, acrylic pigments, alcian blue, coloured gelatin, starch were also tried as surgical inks [5].

Therefore, the aim of this study is to systematically review the studies which compares various materials used for inking of surgically resected specimen.

Materials and methods

Articles included in this study were extracted from PubMed, Cochrane, Google search, manual search and back references of the articles. Studies which assessed and compared the efficacy of different surgical inks were included.

Search criteria — Search: ((((((inking[MeSH Terms]) OR (tissue marking dyes[MeSH Terms])) OR (surgical margin[MeSH Terms])) OR (tumour margin[MeSH Terms])) AND (surgical pathology[MeSH Terms])) OR (grossing[MeSH Terms])) OR (gross specimens[MeSH Terms])

Inclusion criteria for the literature review were original studies on Inking in histopathology, comparative studies and human studies. Animal studies, studies in foreign language were excluded. After screening three articles were included in this study. The entire search methodology is depicted in figure.

The process of selection of articles from PubMed, Cochrane, Google search, manual search and cross reference of articles.

Results

Three studies which compare different types of surgical inks and their properties were a part of this systematic review. Three articles with a total sample size of 1325 were considered in this review.

In the study conducted by Sachin C Sarode et al, India Ink was analysed based on the efficacy of Ease of application, Drying Time, Visibility on paraffin block, Visibility on microscopy, Contamination of other tissue processing fluids and Interference with cellular and nuclear detail. It was observed that India ink was good with respect to the ease of application. Drying time of India Ink was observed to be around 2.3 minutes. Visibility on paraffin blocks for India Ink specimens were moderate and only 56.66% of the samples marked with India Ink showed good visibility. All specimens of India Ink showed good visibility on microscope. 5% of the samples marked with India ink showed slight contamination of other tissue processing fluids. No Interference were observed with cellular and nuclear detail of the specimen while using India ink. In yet another similar study conducted by Divya Pursnanai et al, India ink was compared based on the Ease of application, Drying Time, Visibility on paraffin block, Visibility on naked eye examination of slides, Visibility on microscopy, Contamination of tissue processing fluids and Penetration in to tissues and Interference with cellular and nuclear detail. It was also observed in this study that India ink was good with respect to the ease of application. The drying time of India Ink was observed to be around 2.44 minutes. Visibility on paraffin blocks were found to be good for India ink. India ink showed good visibility on naked eye and microscopic examination for all the samples. India ink showed no contamination of tissue processing fluids and penetration in to tissues and Interference with cellular and nuclear detail. In another study conducted by Kemal Kosemehmetoglu et al, five different colours of India Ink (Black, Blue, Yellow, Green and Red) were compared based on Spread Surface Area, Penetration depth, Surface Area Stained, Mean Density and Intensity. The tissue penetration level was low for all the four colours of India ink except for the yellow colour. The different types of India ink showed mixed results for the visibility under microscope. Black and blue colours of India ink showed good visibility under microscope.

The usage of acrylic colours as an alternative to India Ink were analysed in the study Sachin C Sarode et al. Acrylic colours were tested for its Ease of application, Drying Time, Visibility on paraffin block, Visibility on microscopy, Contamination of other tissue processing fluids and Interference with cellular and nuclear detail. Ease of application was good for acrylic colours. Acrylic colours were observed to have a comparatively lower drying time of 1.3 minutes when compared with India Ink. Acrylic colours have good visibility on paraffin blocks. 86.66% of the samples inked with acrylic colours showed good visibility on paraffin blocks. Acrylic colours were found to be good on the visibility on microscope for all the samples. Acrylic colours showed no contamination of other tissue processing fluids and no interference with cellular and nuclear detail for all the samples. In the study conducted by Divya Pursnanai et al, Acrylic colours were compared based on the Ease of application, Drying Time, Visibility on paraffin block, Visibility on naked eye examination of slides, Visibility on microscopy, Contamination of tissue processing fluids and Penetration in to tissues and Interference with cellular and nuclear detail. Acrylic colours were observed to be good with respect to the ease of application. Drying time of acrylic colours were found to be 1.28 minutes. All the samples of acrylic colours were good visibility on paraffin blocks. 97.7% of all the samples of acrylic showed good visibility on naked eye. Out of all the acrylic colour samples, 98% showed good visibility on microscope. 2.5% of Acrylic colours samples showed contamination of tissue processing fluids and 9.2% of samples showed penetration in to tissues and Interference with cellular and nuclear detail.

Tissue marking dyes were compared for its usage as a replacement for India Ink based on the Spread Surface Area, Penetration depth, Surface Area Stained, Mean Density, Intensity and cost in the study conducted by Kemal Kosemehmetoglu et al. TMD looked more smudgy than India Ink. The tissue penetration level was high when marked with TMD. Blue, red and yellow colours of TMD showed good visibility under microscope. On economical aspect, TMD were comparatively more expensive than India Ink.

Discussion

Three articles with a total sample size of 1325 were considered in this review. 1320 samples each for India Ink and acrylic colours and 5 samples each for India ink and tissue marking dyes were compared. All the three types of Ink were review based on various functional parameters which are discussed below.

Ease of application

1320 samples were reviewed in studies (Table: Serial no 2&3) to find the effectiveness of India ink and acrylic colours. All the samples reviewed in these studies clearly indicate that both the types of Ink are equally good while considering the ease of application.

Articles included in the systematic review

Serial No	1st Author Name	Year	Ink Used and sample size	Parameter Studied	Limitation
1	Kemal Kosemehmetoglu	2010	India Ink (Rotring) [n=1×5=5] 5 colours —Blue, Green, Black, Red, Yellow Tissue Marking Dye- Thermo handon [n=1×5=5] 5 colours —Blue, Green, Black, Red, Yellow	1. Spread Surface Area 2. Penetration depth 3. Surface Area Stained 4. Mean Density 5. Surface Area Stained x Mean Density 6. Cost	No statistical data
2	Sachin C Sarode	2015	India Ink [n=15×4=60] Acrylic Colours [n=15×4=60] Blue, Green, Red, Brown	1. Ease of application 2. Drying Time 3. Visibility on paraffin block 4. Visibility on microscopy 5. Contamination of other tissue processing fluids 6. Interference with cellular and nuclear detail	No statistical data
3	Divya Pursnanai	2016	India Ink [n=1260] 2) Fevicryl Acrylic hobby colours [n=1260] 13 colours used	1. Ease of application 2. Drying Time 3. Visibility on paraffin block 4. Visibility on naked eye examination of slides 5. Visibility on microscopy 6. Contamination of other tissue processing fluids 7. Penetration in to tissues and Interference with cellular and nuclear detail 8. Cost	No statistical data

Drying Time

Acrylic colours are having approximately 40% less drying time as compared to India Ink. Acrylic colours have drying time in the range of 1.28—1.3 minutes for all the 1320 samples reviewed in two studies. India Ink have drying time in the range of 2.3—2.44 minutes (Table: Serial no 2&3). Acrylic colours thus have a clear upper hand in terms of drying time when compared with India Ink.

Visibility on paraffin block

Out of all the 1320 samples analysed in the studies, 1312 samples showed good visibility on paraffin block for acrylic colours. For India Ink,1294 samples out of total samples showed good visibility on paraffin block (Table: Serial no 2&3). Though acrylic colours were having slight edge over the number of samples with good visibility, both the inks are good with respect to the visibility on paraffin block.

Visibility on naked eye examination of slides

Visibility on naked eye for examination of slides is good for all the 1260 samples tested with India ink. 1231 samples out of 1260 gave good visibility on naked eye for acrylic colours (Table: Serial no 2&3). Though India ink was having slight edge over the number of samples with good visibility on naked eye, both the inks are good with respect to the visibility on naked eye.

Visibility on microscopy

Visibility on microscope is good for all the 1320 samples tested with India ink. 1295 samples out of 1320 gave good visibility on microscope for acrylic colours (Table1: Serial no 2&3). When India Ink is compared with tissue marking dyes (Table: Serial no 1), black, blue colours of India Ink and blue, red and yellow of tissue marking dyes were better under microscope when compared to the other colours of both these inks.

All the three inks were good with respect to the visibility on microscope though India Ink have a slight edge over the visibility on microscope with respect to the study results.

Contamination of tissue processing fluids

3 samples out of 1320 resulted in contamination of tissue processing fluids when India Ink were used (Table: Serial no 2&3). 33 samples out of 1320 resulted in contamination of tissue processing fluids when acrylic colours were used. India ink is having better advantage over acrylic colours with respect to contamination of tissue processing fluids. However, the performance of acrylic colours is not very poor to be not considered as an alternate.

Interference with cellular and nuclear detail

All the 1320 samples marked with India ink showed no interference with cellular and nuclear detail of tissues. 116 samples out of 1320 showed interference with cellular and nuclear detail of tissues when marked with acrylic colours (Table: Serial no 2&3). India ink clearly have the edge over acrylic colours with respect to this property.

Penetration into tissues

None of the samples marked with India Ink showed penetration to tissues. 116 samples out of 1260 showed penetration to tissues when marked with acrylic colours (Table: Serial no 2&3). Tissue penetration was Less for India Ink for all colours except yellow (Table: Serial no 1). India Ink is having a clear advantage over acrylic colours and TMD with respect to penetration in to tissues.

Cost

Thermo Shandon tissue marking dyes were costlier than India ink. And acrylic colours were more economical than India ink.

By analysing all the above factors, it is evident that acrylic colours could prove to be future alternate for India ink. However, interference with cellular and nuclear detail and tissue penetration of acrylic colours are still a matter of concern. More studies are being carried out with different types of acrylic colours which could prove as a key for usability of acrylic colours as an alternate. India ink still retains its place as the best tissue marking dye due to its ruggedness.

The main limitations of this systematic review were less availability of original researches in the field of inking in surgical pathology, less amount of sample size

Conclusion

In conclusion, acrylic colours have the potential to be widely used as a tissue marking dyes except for the few disadvantages. Tissue marking dyes are not economical when compared to acrylic colours. More studies are being done with various variants of acrylic colours. However, more studies need to be done with acrylic colours to improve the properties and to ascertain its usage in Immuno- histochemical staining. India Ink will continue to dominate as the best surgical ink till newer studies are available for acrylic colours or other dyes.

The authors declare no conflicts of interest.