The use of isolated peripheral lymphocytes and human whole blood in the comet assay

doi:10.21203/rs.3.nprot-5207/v2

Method Article

The use of isolated peripheral lymphocytes and human whole blood in the comet assay

https://doi.org/10.21203/rs.3.nprot-5207/v2

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The Comet assay, a highly sensitive method for detecting DNA damage, is used to measure DNA breaks and alkali-labile lesions in eukaryotic cells. This study outlines the application of whole blood in the alkaline gel electrophoresis method. A total of 1,200 blood samples from various individuals were analysed, comprising 300 samples from healthy individuals, 300 from suspected or pre-cancerous individuals, and 600 from cancer patients. Each sample was divided into two identical volumes in separate falcon tubes. The blood samples were prepared and preserved by adding an equal amount of RPMI medium and 10% DMSO. Analysis of the data using Student’s t-Test revealed a p-value of 0.59 for Olive tail moment (OTM) and 0.16 for % tail DNA, indicating no statistically significant differences between the two methods, whether with or without treatment. In conclusion, employing whole blood instead of isolated lymphocytes not only saves time but also maintains a high level of sensitivity, requiring less than 20 µL of blood from each individual.

Cell biology

Drug discovery

peripheral lymphocytes

whole blood

Comet assay

The single-cell gel electrophoresis (SCGE), also known as the comet assay, is a straightforward technique used to assess DNA damage and repair in single-cell suspensions derived from various cell types, including protozoa, yeast, plants, invertebrates, and mammals (Olive and Banath, 2006). The single cell gel electrophoresis assay, commonly known as the Comet assay, is a highly sensitive, rapid, and visual method used for both quantitative and qualitative evaluation of DNA damage in individual cells (Tice et al., 2000, Najafzadeh et al., 2012, Kurzawa-Zegota et al., 2012). In this technique, cells are immobilized in agarose on a microscope slide and subsequently lysed. The nucleoids, which protect DNA supercoils, are tethered to the nuclear matrix. To facilitate electrophoresis, the cells need to be treated with detergent and high-salt solutions (Collins, 2004). The assay is capable of measuring single- and double-strand DNA breaks as well as alkali-labile sites, based on the pH of the electrophoresis buffer (apurinic/ apyrimidinic sites), DNA cross-links, base/ base-pair damage and apoptotic nuclei in the cells (Nandhakumar et al., 2011, Clementi et al., 2021). Different pH levels, whether high or low, can lead to the formation of structures resembling comets, while DNA loops with a break lose their supercoiling and become free to extend towards the anode. The final step involves the assessment of the intensity of the comet tail in comparison to the head, utilizing parameters such as Olive Tail Moment and % Tail DNA. These parameters reflect the extent of DNA breaks that can be visualized through fluorescence microscopy (Collins, 2004).

The alkaline Comet assay and γH2AX were employed to evaluate induced DNA damage following a genotoxic insult using white blood cells. These assays serve as highly sensitive and reliable biomarkers of DNA damage, capable of detecting single and double-strand breaks, respectively (Heylmann and Kaina, 2016, Roos et al., 2016, Laubenthal et al., 2012). Double strand breaks can also be detected at lower pH levels in the neutral comet assay (Roy et al., 2021, Olive et al., 1998). In in vivo studies, whole-blood techniques have been utilised to administer rats with the carcinogenic compound Ferric nitrilotriacetate (Fe/NTA) and to evaluate DNA strand breaks in whole blood by comparing them with isolated lymphocytes (Chuang and Hu, 2004). In the current protocol, whole blood and isolated lymphocytes were exposed to UVA+B treatment to induce DNA damage. The results were then compared with the lymphocyte samples obtained from the same individuals before and after the treatment.

Overview of the protocol

Following treatment, it is essential to assess the viability of lymphocytes to rule out any potential cytotoxic effects. A viability of ≥ 75% in the trypan blue dye exclusion test is required for the tested compound to mitigate the risk of false positive results in the comet assay stemming from toxicity (Henderson et al., 1998). Untreated lymphocytes may show DNA damage ranging from 0 to 10% in the tail (Collins, 2004). Blood samples were collected into heparin-coated tubes and then diluted with 0.9% saline at a 1:1 ratio. The diluted blood was gently layered on top of Lymphoprep (Axis, Norway) at a 2:1 ratio without mixing the two layers. Subsequently, the tubes were centrifuged at 800 g for 20 minutes at room temperature using a Mistral 3000 centrifuge. After centrifugation, the thin, cloudy lymphocyte layer above the transparent Lymphoprep layer was carefully transferred into universal tubes containing 0.9% saline using a Pasteur pipette. These tubes were then centrifuged at 770 g for 15 minutes at room temperature. Following centrifugation, the supernatant was removed, and the lymphocytes were resuspended in RPMI medium for use in experiments. Additionally, some of the lymphocytes were preserved in liquid nitrogen for long-term experimentation. The cellular pellet was suspended in 1 ml of fetal bovine serum (FBS) containing 10% dimethyl sulfoxide (DMSO) in a 1.5-ml cryovial and stored in liquid nitrogen vapor overnight before being completely immersed in the storage Dewar for final storage. The comet assay, as outlined by Tice et al. 2000, was performed using ethanol-flamed BDH SuperfrostTM glass slides coated with 1% normal melting point agarose. The cell suspension was combined in a 1:1 ratio with 1% low-melting point agarose (< 40°C) and applied to the dry agarose-coated slides. Subsequently, a top layer of 0.5% low-melting point agarose was applied. Two replicate slides were produced for each concentration point. The slides were horizontally immersed in a container with cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10) and incubated at 4ºC overnight. Following lysis, the slides were placed horizontally in the tray of an electrophoresis tank filled with cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH <13) and incubated in the dark at 4ºC for 30 minutes to allow DNA unwinding and the expression of alkali-labile damage. Electrophoresis was conducted at the same temperature for 30 minutes at 25 volts and an adjusted current of 300 mA (by raising or lowering the buffer level). After electrophoresis, the slides were removed from the tank and soaked three times in neutralizing buffer (400 mM Tris, pH 7.5) for five minutes each (Collins et al., 2019, Tice et al., 2000). The cells were treated with a solution containing 20 µg/ml of ethidium bromide and subsequently analyzed using a fluorescence microscope that was outfitted with a monochrome CCD camera. The fundamental Comet assay on lymphocytes has been detailed since its origin by Olive and Banath in 2006.

Potential applications of the protocol

The single-cell gel electrophoresis (SCGE), also known as the Comet assay, is a rapid and highly sensitive fluorescent microscopic technique utilized to evaluate DNA damage and repair at the individual cell level (Tice et al., 2000). The alkaline (pH >13) comet assay has significantly enhanced the versatility and effectiveness of DNA damage detection (Cotelle and Ferard, 1999, Singh et al., 1988). This technique can identify various types of DNA damage, including single- and double-strand breaks, oxidative DNA base damage, DNA-DNA/protein/DNA-drug cross-linking, and assessing DNA repair processes. As a result, its application has expanded to encompass a wide range of eukaryotic cells (Cotelle and Ferard, 1999, Singh et al., 1988, Nandhakumar et al., 2011). In the Comet assay, the migration of DNA is influenced by both its size and the number of broken DNA ends. Initially, the length of the Comet tail increases with damage, reaching a maximum size determined by the electrophoretic conditions. The tail moment, a composite measure of tail length and the fraction of DNA in the Comet tail serves as the standard unit for assessment (Kumaravel and Jha, 2006, Olive and Banath, 2006, Olive et al., 1998, Anderson et al., 2003). The assay has a wide range of applications, including human biomonitoring, genotoxicology, ecological monitoring, and as a tool for investigating DNA damage and repair in various cell types in response to different DNA-damaging agents. % Tail DNA is also recommended for use in these applications (Ladeira and Smajdova, 2017, Kumaravel and Jha, 2006). The Comet assay is highly suitable for clinical applications due to its simplicity, cost-effectiveness, and minimal cell requirement, providing results within a short timeframe. This method is valuable for investigating DNA damage in cancer research and studies related to lifestyle and dietary factors. In cancer research, it proves beneficial for assessing DNA damage pre-, during, and post-therapy, including radiotherapy and chemotherapy, used for a dietary study. In that type of study, the effects of consumption of specific foods or supplements that may be protective against DNA damage are observed (Fikrova et al., 2011). Research has demonstrated that DNA damage and impaired DNA repair mechanisms play a vital role in the development and advancement of cancer. Consequently, numerous investigations have employed the Comet assay to examine the DNA damage and repair properties in a diverse array of tumour cells when exposed to various DNA-damaging agents. These studies include analyses conducted on both human tumour cell lines and tumour cells isolated from cancer patients (McKenna et al., 2008). Also, the white blood cells, known as lymphocytes, are viable surrogates for cancer cells in human studies (Dusinska and Collins, 2008, Najafzadeh et al., 2012, Anderson et al., 2014).

The novel protocol examined in the current research is efficient in terms of time and eliminates the need for two unnecessary cycles of washing and centrifugation. We are confident that this innovative protocol can be implemented across various Comet assay variations for the assessment of DNA damage and repair (e.g. treating the cells with UV or chemicals). The variation and limitations of the method have been explained by Olive and Banáth 2006 (Olive and Banath, 2006).

Glass slides from BDH (SuperfrostTM) were coated with a 1% agarose cold lysing solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, and 1% Triton X-100 at pH 10. Subsequently, low-melting point agarose (< 43°C) was applied to the slides. For electrophoresis, a cold electrophoresis buffer comprising 300 mM NaOH and 1 mM EDTA at a pH below 13 was used. The process was completed by treating the slides with a neutralizing buffer containing 400 mM Tris at pH 7.5, and 20 µg/ml ethidium bromide (or alternative DNA stains such as SYBR-green, YO-PRO-1, DAPI) was applied (Olive and Banath, 2006).

Fluorescent microscope, Komet 6 software, and horizontal electrophoresis Comet assay system.

Method

In the current investigation, a total of 1200 blood samples were analyzed, comprising 300 from healthy individuals, 300 from individuals suspected of or predisposed to cancer, and 600 from confirmed cancer patients. The age range of the cancer patient group was 35 to 72 years, while that of the healthy control group was 27 to 61 years. Blood samples from the cancer and suspected or precancerous individuals were procured from the skin, colorectal, respiratory, breast, and urology cancer clinics at St. Luke’s Hospital, Bradford. The patients had either not received treatment or had undergone surgical procedures at the relevant department of Bradford Royal Infirmary (BRI), Bradford, West Yorkshire, UK. Additionally, 300 healthy volunteers were enlisted from the School of Medical Sciences at the University of Bradford (West Yorkshire, UK). Ethical permission was obtained from both the BRI Local Ethics Committee (reference numbers 04/QI202/132, 04/Q1202/15 and12/YH/0464) and the University of Bradford’s sub-committee of Research Ethics involving Human Subjects (reference number 0405/8). Patients were selected based on their random referral to a clinic upon suspicion of disease and whether their potential cancer diagnosis was confirmed.

Each blood sample was divided into two equal volumes and placed in separate falcon tubes. The cells were then exposed to UVA+B (PUVA lamp) with an intensity of approximately 1.20 mW/cm2 for 15 minutes, and the results were compared to untreated cells using the comet assay.

This protocol was designed for simplicity and speed in preparing slides. Whole blood samples were collected using a vacutainer connected to a heparinised tube, with a minimum of 2 mL of blood. The whole blood in heparinised tubes can remain stable for up to 72 hours when stored in the dark at room temperature. Subsequently, the whole blood was transferred to a falcon tube and mixed with an equal volume of RPMI and 10% DMSO (an iron-chelating agent) to prevent the oxidative effect of iron on the white blood cells. The mixture was then aliquoted into 1.5mL Eppendorf® tubes and stored at -80ºC. To initiate the comet assay, the blood mixture is thawed by allowing the tube to reach room temperature for 15 minutes. The blood samples should not be kept for longer than 15 minutes at room temperature (the temperature should be that to avoid producing more DNA damage or starting DNA repair in the lymphocytes).

Procedure

Preparation

To prepare the slides, dust-free, frosted-end microscope slides should be coated with 1% normal melting point agarose at a temperature of approximately 100ºC and allowed to dry at room temperature overnight. Additionally, low melting point agarose (0.5%) should be prepared at a temperature of around 40 ºC.

For the chemical treatment, each 1.5mL Eppendorf® tube should have 100µL of whole blood plus the treatment compound, topped up with RPMI to a volume of 1mL. It is essential to include one tube without any treatment as a negative control and another as a positive control with a compound that induces DNA damage in cells to create a standardized assay. Following the incubation period, the Eppendorf® tubes should be centrifuged for 3 minutes at 900x g. After discarding 960µL of the supernatant, 40 µL plus the pellet should be retained. Subsequently, 100 µL of 0.5% low melting agarose should be pipetted and mixed with 40 µL of the pellet, and the 140 µL mixture should be spread on the prepared coated slide and covered with a cover slip. The slides should then be placed on a cold surface for 2-3 minutes to allow solidification of the agarose before being transferred to the lysis solution.

For the UV treatment, 40 µL of whole blood should be mixed with 100 µL of 0.5% low melting point agarose, spread on the slide, and covered with a cover slip. The remaining procedure can be carried out as described previously. The slides should be kept in the lysis solution at a temperature of 4-8ºC for a minimum of 2 hours before electrophoresis.

It is recommended that a voltmeter be used to adjust the electrophoresis tank to establish the required voltage (0.6 V/cm) within the tank should the voltage from the power system be inaccurate. Subsequent procedures can be carried out in accordance with the Olive and Banath protocol of 2006.

Running Comet assay in 4 hours.

Results and Discussion

The study conducted a comparative analysis of the comet assay results using whole blood (WB) and isolated lymphocytes (IL) from three different groups: healthy individuals, suspected precancerous, and cancer patients. A total of 1200 samples were analysed, consisting of 300 samples from healthy individuals, 300 from suspected cancer patients, and 600 from cancer patients. The results from the healthy individual samples indicated that the mean Olive Tail Moment (OTM) was 1.31 in WB and 1.04 in IL, while the mean %Tail DNA was 8.66 in WB and 8.02 in IL. For the suspected cancer group, the mean OTM in WB was 2.23 and in IL was 1.98, and the mean %Tail DNA was 2.3 in WB and 2.02 in IL. In the cancer group, prior to UV A+B exposure, the mean OTM was 2.6 in WB and 2.02 in IL, with the mean %Tail DNA recorded as 11.8 in WB and 12.01 in IL (Figures 2 and 3). Statistical analysis using the student’s t-test revealed no significant differences between the use of whole blood and isolated lymphocytes in the comet assay (p-value = 0.59 for OTM and p-value = 0.16 for %Tail DNA). However, when DNA damage was induced by treating the cells from WB and IL with UV A+B, a significant difference was observed compared to unexposed cells (p-value = 0.01). This suggests that the response to UVA+B in white blood cells (WBC) from whole blood is similar to that in isolated lymphocytes from the different study groups (Figures 2 and 3). Furthermore, the application of the Student’s t-test indicated no statistically significant differences for OTM (p-value = 0.59) and %Tail DNA (p-value = 0.16) between whole blood and isolated lymphocytes. White blood cell (WBC) use, particularly human WBC, has seen increased utilisation in genotoxicity, human monitoring and molecular epidemiology (Gajski et al., 2014). The present protocol is a possible technique to reduce the time of the whole experiment and could help in automating the Comet assay. In conclusion, using whole blood instead of isolated lymphocytes saves time, is still very sensitive and requires less than 2 mL of blood from each individual.

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We extend our sincere gratitude to Professor Badie Jacob, Professor Andrew Wright, Dr Nader Ghaderi, and the entire staff of the colorectal, dermatology, respiratory, prostate, and breast cancer clinics at St. Luke’s Hospital in Bradford. Their unwavering dedication, time, and support in the collection of blood samples were instrumental in the successful execution of this study.

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The use of isolated peripheral lymphocytes and human whole blood in the comet assay

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The use of isolated peripheral lymphocytes and human whole blood in the comet assay

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Associated Publications

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