Total 90 archival slides were chosen for the prospective study which was conducted in Department of Pathology for the duration of eight months. Distyrene, Plasticizer and Xylene (DPX) were the mounting media in all slides. They were grouped according to duration into three major group as 1 to <5-year-old, 5 to <10-year and >/=10-year-old. According to coverslip size, they were divided into three subgroups; one pair of slide was taken with coverslip size 22x22 mm, 22x30 mm and 22x40 mm. In total six slides were included in each of these subgroups and total 18 slides under each major group. Five methods used for removal of coverslip were; Freezing, Xylene at room temperature, Xylene at 56°C, Petrol and Diesel. The chemicals were taken into glass beaker which were labeled and had proper lid for closure. The slides were labelled with diamond pencil and a unique number was allotted to each slide. First alphabet designated method used, middle number represented duration of slide and last number represented size of coverslip. Number 0, 1, and 2 were designated for slide duration 1 to <5-year-old, 5 to <10-year and >/=10-year-old, respectively. Similarly, number 0, 1, and 2 were given for coverslip size 22×22 mm, 22×30 mm and 22×40 mm. For example, F00 slide number means Freezing method was used, slide is 1 to <5-year-old and coverslip size is 22×22 mm.

In freezing method, the slides were kept for 10 minutes in freezing chamber (Temperature 0° to -4°C) of domestic refrigerator with coverslip facing downwards. Then after every five minutes disposable microtomy blade was passed in between the slide and coverslip to check whether coverslip was popping up easily or not. Time was noted from slide kept in freezer compartment and coverslip popped out easily. To avoid cut related injury cut resistant gloves were worn [Table/Fig-1]. For xylene at 56°C, water bath was used. The slides were kept in a glass jar and immersed inside the water bath maintained at a temperature of 56°C. Time was noted immediately after inserting jar containing slides in water bath till coverslip fell off from slides. For petrol and diesel, plastic airtight jar with cap was used. The time was noted immediately after immersing slide in chemical until coverslip detached out themselves. After the coverslips got separated, the slides were taken for restaining. Broken slides, slides with broken coverslip and slides with tissue loss during processing were excluded from study.

[Table/Fig-1]:

Removal of coverslip in freezing method with disposable microtomy blade.

In conventional xylene method (Room temperature), the mean time for removal of coverslip was 52 hour 10 minutes. Mean duration in which coverslip came-off was 13 minutes 7 seconds by freeze method, 14 hours 7 minutes with xylene at 56°C and in 20 hours 2 minutes with petrol method. Slide with duration 1 to <5-year old, 5 to <10-year and >/=10-year-old, coverslip came off in 9 minutes 2 seconds, 15 minute 8 seconds and 15 minute 8 seconds with freezer method while with xylene at room temperature it took 41 hours 20 minutes, 56 hours and 59 hours respectively and 9 hours, 12 hours 5 minutes and 39 hours respectively with petrol method. The duration of slides and time taken to remove coverslip is shown in [Table/Fig-2]. While the mean time taken for coverslip coming off the slide is shown in [Table/Fig-3]. With the diesel method, coverslip did not come out even after 120 hours. Hence, they were excluded from the study group.

Traditionally, xylene is widely and most commonly used solvent for coverslip removal [1]. The turn around time with xylene is more, sometimes it might take 72 to 94 hours for coverslip removal [7,8]. Most common need for coverslip removal is for extraction of DNA for molecular analysis for research purpose. In the era of molecular testing where there is demand for target therapy, even a smaller specimen can be useful. Determining mutations in Epidermal growth factor receptor, BRAF and KRAS genes which is useful for target therapy [1,3]. Sometimes the archival cytological slides may be the only source for such studies like in lung adenocarcinomas, thyroid lesions and other soft tissue tumors. Other research fields where coverslip removal is needed is for neuronal cultures [5,9,10].

There is paucity of literature regarding untraditional methods for coverslip removal. In this study, methods which are easily available and feasible were used. This is the first kind of extensive study which has described the usage of different methods for removal of coverslip. Few studies have also described use of liquid nitrogen, ultrasonic vibrations and scratching coverslip along with application of ice block for coverslip removal.The problem with these methods are either they are not easily available at the setup, like liquid nitrogen or may cause damage to the tissue section like in ultrasonic or with scratching method [3-6].

Mechanism of action of Diesel, Petrol and Xylene at room temperature is same i.e., they all are solvents and dissolve the DPX. Heating aids in penetration as well as fastens melting of DPX. Impurities and high density of diesel might be a reason for poor penetration as well as ineptitude in dissolving DPX. On the contrary, Petrol which is also a product of crude oil, has low density and undergoes fractional distillation, which might be a reason for robust solvent properties compared to diesel.

In the present study, it was found out that the most efficient and fastest method for coverslip removal was freezing method. The mean time in which the coverslip popped out was 13 minutes 7 seconds. For 5 to <10 years and more than or equal 10-year-older slide it took 15 minutes 8 seconds each, while for less than five-year-old slide it took 10 minutes. Similar findings were seen in the study conducted by Treece AL et al., and Da Cunha Santos G et al., where they used freezer method for coverslip removal [1,2].

In other methods, for 1 to <5-year-old slide, coverslip came out within 9 hours with petrol method. Mean time was 20 hours 2 minutes, as it took more time for >10-year-old slides and with Xylene at 56°C, the mean time in which coverslip came out was 14 hours 7 minutes. In both of these methods, turn around time was significantly less compared to routine xylene method. No significant changes in terms of quality of staining was found on microscopic examination of slide.

Further studies should be carried out to determine the loss of any cells with different methods of coverslip removal by molecular analysis and immunohistochemistry which was constraint of this study. However there was no loss of part/whole tissue in any of the methods in this study.

Alternative methods especially freezing, xylene at high temperature and petrol method can be used for removal of coverslip. These methods are reliable within available resources and remarkably reduce the turn around time for restaining of faded old stained slides.