Buccal Cells As DNA Source For Forensic Essay, Aiding Identification.

Appreciate the importance of experimental design and the scientific presentation of experimental work. 

Collection procedures for buccal cells

Buccal cells (squamous epithelial cells from the oral cavity) are used extensively as a source of DNA for the forensic identification of individuals using the DNA profiling technique known as ‘DNA17’. The interest of getting clearer concept of genetics and various diseases and various body processes have increased, requiring the isolation of DNA. Over the years blood samples have been an excellent source of obtaining DNA, however extraction of DNA from the buccal cells has brought new perspective in DNA analysis, (Benschop et al., 2010 pp. 118).

There are currently two types of procedures which are currently available for the buccal cell collection, dry and wet collection methods are currently available. Wet method entails swishing liquids in mouth and spitting them into a collecting cup. This process has been shown to yield higher amounts of longer fragments of DNA which requires more cumbersome and higher costs. Dry procedures use buccal swabs and cytobrushes. These methods are often simpler and cheap and less sensitive to effect of long term storage methods at room temperature. According to recent studies , the utilization of cytobruches have appeared to be more appropriate in facilitating collection of human DNA genome exhibiting good quality  and high security, (Thomasma & Foran, 2013, pp 466).

However, the microscopic comparison of these buccal cells remains an under-evaluated area of study.  A study by Donald et al. in 2013, compared the buccal mucosal cells from 400 females to determine changes related to the hormonal status of the donors and this indicated that it may be possible to distinguish cells from different age ranges and menstrual cycle stages of females.  This was achieved through measurement of the average Cell Diameter (CD) and Nucleus Diameter (ND) and the ratio of these two measured values (ND:CD).

Various factors have been advanced on DNA degradation as it contains in buccal cells. DNA degradation occurs when the samples are exposed to varied environmental insults and chemical, (Dowlman et al, 2010 pp 168). These include factors such as light, humidity which is attributed to more effect on quality rather the quantity, elevated temperature and moisture content  which degrades DNA and make it difficult for profiling of fungal contamination and interval post mortem. The survival of these DNA however is dependent on various factors and varied environmental conditions, (Verdon, Mitchell & Van, 2013 pp 168).

Obtaining DNA genome from buccal epithelial cells has overcome many problems with venipuncture which require extraction of blood through phlebotomist to draw blood. Self collection of buccal cells can be undertaken. This collection is less invasive technique which often requires less invasive technique to obtain the DNA samples, (Nandita et al, 2014 pp 59).

Various staining process have been undertaken with regard to buccal cells. Use of hematoxylin and eosin has been used over a century and it is still essential in identifying various tissue types and different morphological changes in cells. The stain works well with many varied fixatives, (Verdon, Mitchell & Van, 2014 pp 1080). Hematoxylin has a deep purple color which offers staining on the nucleus through complex reaction. On the other end Eosin is pink in color and offer staining to proteins non specifically. In typical tissues, nuclei are stained to be blue while the cytoplasm and the extracellular matrix have varied degrees of staining. Hence staining are very essential in disclosing the abundant structure of structural information which have specific functionalities.

Focus on exfoliative cytology has been undertaken with key focus on microscopy examination being undertaken, (Kumar et al, 2011 pp 293). Mucous membrane has been focused with this approach. Haematoxylin and Eosin (H&E) stains used in the buccal swabs have been effective in using different color on the cellular differentiation.  With advancements used in quantitative exfoliative cytology, oral cytology process has been enhanced and used as an emerging diagnostic tool in diagnosing oral lesions, (Marshall et al, 2014 pp 192).

Assistance of computer morph metric analysis have been shown to improve on the overall parameters such as the nuclei diameter, cell diameter and nuclear area, cytoplasm area and nuclear to cytoplasm ratio, (Goregen, Ankul & Gundogdu, 2011 pp 10). The yield of the buccal cells have shown to vary in terms of quantity from those obtained from blood cells and vary in term s of how the cells have been acquired, stored and transported. Thus this experimental study seeks to investigate the effect of Haematoxylin and Eosin (H&E) stains on heat treatment conditions through microscopic observation. 

The present study has been conducted on four participants in the academic institution. The subjects were of age group 25-30 years and it entails 2 males and 2 females. This study utilized experimental design research to investigate the effects of temperature on storage of buccal cells under Haematoxylin and Eosin (H&E) stains and with buccal swabs.

Ethical clearance was sought from the department of Biological sciences at the university. Patient data sheet was obtained to explain to the participant on the required information of the study, there after a count was sought from the participants.

The participants were asked to rinse their mouth to remove debris in the oral cavity. Buccal cells were obtained and transferred using buccal swap on a dry glass slide. There after staining was done. Rapid PAP kit of biolab was used for staining. The smears were fixed in tap water followed by rapid PAP in 45 s. The stained smears were observed under 50X microscope, with measurements of cell. Different temperature ranges were subjected to the buccal cells; photo micrographic images were under taken. The captured images captured for further analysis for calibration of image. Assessment of morph metric parameters was done. Nuclear diameter and cell diameter was undertaken using digitalized interactive measurement tool used for tracing the perpendicular lines.

Effect of temperature and staining on buccal cells

Statistical methods applied on this experimental study included mann Whitney U test comparison for group analysis for nuclear diameter and cytplasmic diameter. 

The results of the experiment revealed the following results when the buccal cells were subjected to varying temperatures

Cells	Positive Control	4 degrees	Room temp	40 degrees
1	CD :25 ND:3	CD:28  ND:4	CD:52  ND:8	CD:10   ND:2
2	CD:30  ND:4	CD:16  ND:4	CD:32  ND:5	CD:12  ND:2
3	CD:21  ND:3	CD:16  ND:3	CD:15  ND:3	CD:15  ND:3
4	CD:35  ND:4	CD:36 ND:4	CD:35  ND:4	CD:11  ND:2

Table 1: showing the cytoplasm and nuclear diameter under different temperature conditions 

Cell	Positive Control	4 degrees	Room Temp	40 degrees
1	CD: 40 ND:5	CD:12  ND:2	CD:18  ND:2	CD:10  ND:2
2	CD:34  ND:4	CD:8 ND:2	CD:12  ND:3	CD:7    ND:2
3	CD:30  ND:4	CD: 15 ND:2	CD:14  ND:4	CD:12  ND:1
4	CD:38  ND:3	CD:16  ND:1	CD:19  ND:2	CD:10  ND:2

Table 2: showing the cytoplasm and nuclear diameter under different under alcohol treatment

The result above shows that when the temperature is increased, the positive control had an average of cell diameters of 27.75mm while the nuclear had diameter was 3.5 mm. On room temperature, the average temperature was 33.5mm while the nuclear diameter was 5mm with standard deviation of +-10.5. When the temperature was lowered there was decreased cell and nuclear diameter at 24 mm.

On alcohol media, the positive control showed cell diameter of 35.5 mm, while under lowered temperature in alcohol media, there was 12.75 mm, when the temperature was increased there was 12 mm observed. In room temperature the cell diameter was higher with 33.5mm and nuclear diameter being 15.75. When the temperature was lowered, there was observed low cells and nuclear diameter having 12.75 mm and 1.75 mm respectively. 

Our investigations as shown above show that there were variables yields with regard to DNA obtained, there were substantially lower quantities for cases than in controls. Cell diameter and nuclear diameter were shown to have high lengths compared to subjecting the buccal cells to vary temperatures of 4, 40 and room temperature.

Temperature incubation and variation on buccal cells showed that when treated alcohol, there were more observed observation, with the buccal cells being incubated at various temperature conditions of 4, room temperature and 40 degrees Celsius . Numerous reports have shown that different sampling techniques with different stage conditions could have an effect on the quality and quantity of isolated DNA in buccal cells. Transport of samples and field work factors have an effect on the quality of samples of DNA analysis. The testing of the prolongation of storage time in buccal swabs at the three tier temperature ranges shows that are that DNA quantity can have an effect, (Thomasma & Foran, 2013 pp 468).

Permeability of DNA was further observed with the treatment of alcohol on the buccal cells. This was exhibited by the large proportion of DNA in the bacterial growth. Compared to positive control treatment on alcohol, there was significant difference on the different temporary treatment son the samples.

Result and statistical analysis

Forensic DNA analysis has often utilized buccal swabs for extraction of DNA cells. Testing done have shown that significant quantities of the DNA are often retained in the swab and later get lost. Testing assessment has indicated that variation of temperature plays key role. The incubation time protocol and temperature with added stains are key in enhancing permeability of the cell and cytoplasm nucleus. Incubations process in this study under various temperature states and subjecting it to alcohol staining. Samples incubated under alcohol showed increase permeability and observation of DN in the buccal cells, (Zech, Malik & Thali, 2012 pp 1038).

Other studies linking buccal cells incubation with temperature have shown varied results. Increasing the temperature at the incubation with buccal cells that there were lower quantity of buccal cells at 65°C. When incubation was done at shaking of 900 rpm at 65°C, the average quantification time had lowered than the 55°C. The buccal cell quantification and difference conditions were shown to indicate significant difference in this study. There was an average increase in cell diameter and cytoplasm diameter for the positives controls compared to variation of the different temperature states.

DNA available in buccal cells can be rapidly degraded by post mortem which initial enzymes of the cells dies thus losing its structure and overall integrity which affect later the environmental conditions. Temperatures play crucial role and degradation of DNA and its overall survival. This is relying on the integrity conditions of the biological conditions of the location, (Suvarna et al, 2012). When temperature increases often damage occurs this is rapid over time. 

Microbes often don’t have any way of regulating internal temperature so they evolve temperature and environmental adaptations. Changes in temperature have an effect on the enzymes and their activity. Temperatures below the optimal often leads to faster metabolism which yields decrease enzyme activity and lowered metabolism rate, while higher temperature have been shown to have denaturing effect on the proteins such as the enzymes. Microbes have growth curve which have an optimal temperature which the growth peaks and minimum and maximum temperature which exhibit growth, (Sumanthi et al, 2012).

Humans exhibit mesophile microbes , which have growth optima of 37 degrees Celsius and ranges for 20-45 degrees Celsius. Nearly all human micro flora fall in this category with other human pathogens. This mesophiles have same occupation of environment which humans have.  On the other end , thermophiles have same spectrum as they prefer higher temperature. Thermophils have temperature ranges ranging from 45degrees to 80 degrees with growth optimum of 60 degrees Celsius, (Warnakulasuriya et al, 2014 pp 268).

Conclusion

Further other factors have been assessed with regard to humidity and the survival of the buccal cells DNA. Exposure of biological material to the external environment with high quantity of water and elevated temperature often leads to growth of micro organisms such as bacteria and fungi.

Thus from the results it is evident that, temperature has effect on permeability for buccal cells. At room temperature the buccal cells exhibited increased permeability as it was observable with the cell and cytoplasm diameters compared to lowered temperature and higher temperature environments. Thus storage temperature of buccal cells plays critical factor in its cell mediation and permeability. When the cells are subjected to alcohol conditions, the positive control showed increased permeability of the cells compared to normal room temperature, at lower temperature and at an increased environmental condition. 

References

Benschop, C.C., Wiebosch, D.C., Kloosterman, A.D. and Sijen, T., 2010. Post-coital vaginal sampling with nylon flocked swabs improves DNA typing. Forensic science international: genetics, 4(2), pp.115-121.

Donald, P.M., George, R., Sriram, G., Kavitha, B. and Sivapathasundharam, B., 2013. Hormonal changes in exfoliated normal buccal mucosal cells. Journal of Cytology/Indian Academy of Cytologists, 30(4), p.252.

Dowlman, E.A., Martin, N.C., Foy, M.J., Lochner, T. and Neocleous, T., 2010. The prevalence of mixed DNA profiles on fingernail swabs. Science and Justice, 50(2), pp.64-71.

GÖREGEN, M., AKGÜL, H.M. and GÜNDO?DU, C., 2011. The cytomorphological analysis of buccal mucosa cells in smokers. Turkish Journal of Medical Sciences, 41(2), pp.205-210.

Kumar, S., Vezhavendhan, N. and Priya, S., 2011. Role of oral exfoliative cytology in oral leukoplakia and squamous cell carcinoma. International Journal of Clinical Dental Science, 2(1).

Marshall, P.L., Stoljarova, M., Larue, B.L., King, J.L. and Budowle, B., 2014. Evaluation of a novel material, Diomics X-Swab™, for collection of DNA. Forensic Science International: Genetics, 12, pp.192-198.

Nandita, K.P., Boaz, K., Srikant, N., Lewis, A.J. and Manaktala, N., 2014. Oral epithelium in diabetics: A cytomorphometric correlation. Dental Hypotheses, 5(2), p.59.

Sumanthi, J., Reddy, G.S., Anuradha, C.H., Sekhar, P.C., Prasad, L.K. and Reddy, B.R., 2012. A study on cytomorphometric analysis of exfoliative buccal cells in iron deficiency anemic patients. Contemporary clinical dentistry, 3(Suppl 2), p.S156.

Suvarna, M., Anuradha, C., Kumar, K.K., Sekhar, P.C., Chandra, K.L.P. and Reddy, B.R., 2012. Cytomorphometric analysis of exfoliative buccal cells in type II diabetic patients. Journal of Dr. NTR University of Health Sciences, 1(1), p.33.

Thomasma, S.M. and Foran, D.R., 2013. The influence of swabbing solutions on DNA recovery from touch samples. Journal of Forensic sciences, 58(2), pp.465-469.

Verdon, T.J., Mitchell, R.J. and Oorschot, R.A., 2014. Swabs as DNA collection devices for sampling different biological materials from different substrates. Journal of forensic sciences, 59(4), pp.1080-1089.

Verdon, T.J., Mitchell, R.J. and van Oorschot, R.A., 2013. The influence of substrate on DNA transfer and extraction efficiency. Forensic Science International: Genetics, 7(1), pp.167-175.

Warnakulasuriya S, Tilakaretne WM. Potentially malignant disorders. In: Silverman S Jr, Thongprasom K, Warnakulasuriya S, editors. Oral Medicine and Pathology: A Guide to Diagnosis and Management. New Delhi: Jaypee Brothers Medical Publishers; 2014. pp. 268–70.

Zech, W.D., Malik, N. and Thali, M., 2012. Applicability of DNA analysis on adhesive tape in forensic casework. Journal of forensic sciences, 57(4), pp.1036-1041.