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→Mechanism of Acute Inflammation
Interleukin (IL)-6 is a pleiotropic cytokine with a wide range of biological activities. It induces pro- and anti-inflammatory reactions and is rapidly induced in the course of acute inflammatory reactions. IL-6 is produced by lymphoid and nonlymphoid cells and helps regulate immune reactivity, the acute phase response, inflammation, oncogenesis, and hematopoiesis. Studies have shown that IL-6 appears to be both a marker and mediator of sepsis and persists in the plasma much longer than other proinflammatory cytokines.<ref name="Arti5">Interleukin-6. Critical Care Medicine, 33(12 Suppl), S463-5, 2005, Song, M., & Kellum, J. A. https://doi.org/10.1097/01.ccm.0000186784.62662.a1 </ref> Therefore IL-6 serves as a valuable biomarker for the early detection of sepsis.
== History of Sepsis ==
The oldest report that associates sepsis with wounds goes all the way back to a discovery by Edwin Smith. In 1826 he found a papyrus in Luxor, Egypt, which was written around 1600 BC. This papyrus seemed to be a copy of an even older manuscript written around 3000 BC. In this manuscript, 48 cases of traumatic lesions between wounds, fractures, and dislocations are mentioned. Clear references to fever as a secondary phenomenon in the wound – with emphasis on the fever as a part of the monitoring of the patients’ evolution - are found in five out of the forty-eight references. Thus, without being familiar with the concept of infection or inflammation, these Egyptian physicians were able to identify some clear signs of what we know nowadays as local suppuration and systemic infection.<ref name="Arti6">The History of Sepsis from Ancient Egypt to the XIX Century, (M. C. F. P. E.-L. Azevedo (Ed.); p. Ch. 1). IntechOpen, 2012, Botero, J. S. H., https://doi.org/10.5772/51484 </ref><ref name="Arti7"> The last 100 years of sepsis. American Journal of Respiratory and Critical Care Medicine, 173(3), 256–263, 2006, Vincent, J.-L., & Abraham, E., https://doi.org/10.1164/rccm.200510-1604OE </ref><ref name="Arti8"> Sepsis History, https://www.news-medical.net/health/Sepsis-History.aspx, 2018, Ryding, S. </ref>
The first reported use of the word sepsis (σηψις) is in a poem of Homer in the Iliad, where it is a derivative of the word sepo (σηπω), which translates to “I rot”. Yet, the first use of sepsis in a medical context can be found in the Hippocratic corpus, written around 400 BC. The use of this word was related to the phenomenon discovered by the Egyptians. Hippocrates described sepsis as a dangerous odiferous biological decay that could occur in the body. Furthermore, it was believed that this decay took place in the colon and from there “dangerous principles” were released, which could cause “auto-intoxication”. Hippocrates was the first one to try and find antisepsis properties and potential medicinal compounds.<ref name="Arti6">The History of Sepsis from Ancient Egypt to the XIX Century, (M. C. F. P. E.-L. Azevedo (Ed.); p. Ch. 1). IntechOpen, 2012, Botero, J. S. H., https://doi.org/10.5772/51484 </ref><ref name="Arti9">Sepsis and septic shock: a history. Critical Care Clinics, 25(1), 83–101, viii, 2009, Funk, D. J., Parrillo, J. E., & Kumar, A., https://doi.org/10.1016/j.ccc.2008.12.003</ref><ref name="Arti8"> Sepsis History, https://www.news-medical.net/health/Sepsis-History.aspx, 2018, Ryding, S. </ref>
In the nineteenth century, large growth in the knowledge on the origin and transmission of infectious diseases occurred. One of the physicians who contributed significantly to this development was Ignaz Semmelweiss (1818-1865). He was a physician in Vienna, Austria. In 1841, while working on a maternity ward in a hospital he noticed that there was a high rate of death from childbed fever. Nowadays this is also known as puerperal sepsis. He made the observation that women whose deliveries were assisted by midwives had a significantly lower percentage of infection (2%) than deliveries assisted by medical students (16%). Back then, the medical students practiced both autopsies and childbirth deliveries on the same day without washing their hands. When one of Semmelweis’ colleagues died of an infection, Semmelweis made the connection between the medical students, the deliveries, the autopsies, and puerperal sepsis. Semmelweis’ comment on this situation was “The fingers and hands of students and doctors, soiled by recent dissections, carry those death-dealing cadaver’s poisons into the genital organs of women in childbirth”. When a handwashing policy was implemented, the rates of puerperal sepsis dropped to under 3%.<ref name="Arti9">Sepsis and septic shock: a history. Critical Care Clinics, 25(1), 83–101, viii, 2009, Funk, D. J., Parrillo, J. E., & Kumar, A., https://doi.org/10.1016/j.ccc.2008.12.003</ref><ref name="Arti7"> The last 100 years of sepsis. American Journal of Respiratory and Critical Care Medicine, 173(3), 256–263, 2006, Vincent, J.-L., & Abraham, E., https://doi.org/10.1164/rccm.200510-1604OE </ref><ref name="Arti8"> Sepsis History, https://www.news-medical.net/health/Sepsis-History.aspx, 2018, Ryding, S. </ref>
In 1964 Dr. Edward Frank from Boston published a management strategy for septic shock. This strategy consisted of continuous monitoring of systemic arterial pressure, central venous pressure, cardiac output, urinary output, blood volume, blood chemistries, gases, pH and electrolytes. Some of these are still used nowadays, such as blood monitoring and urinary output. Aided by the discovery of antibiotics by Alexander Fleming, it was also recommended to find the cause of the infection.<ref name="Arti10">The History of Sepsis Management Over the Last 30 Years. Elsevier, 15(2), 116–117, 2014, Zehava L., N., https://daneshyari.com/article/preview/3235901.pdf </ref>
== Mechanism of Acute Inflammation ==
When the human body is subjected to harmful stimuli, an inflammatory response is activated to remove these stimuli and, if necessary, initiate a healing process. Cellular and molecular events take place to minimize injury and infection. Common characteristics of inflammation on tissue level are redness, heat, pain and loss of tissue function, which all result from local immune, vascular and inflammatory cell responses to infection or injury.<ref name="Arti11">Inflammatory responses and inflammation-associated diseases in organs, Oncotarget, 9(6), 7204–7218, 2018, Chen, L., Deng, H., Cui, H., Fang, J., Zuo, Z., Deng, J., Li, Y., Wang, X., & Zhao, L., https://doi.org/10.18632/oncotarget.23208 </ref> The mentioned events are a consequence of specific complex molecular pathways involving different types of receptors, transcription factors, leukocytes and eventually cytokines that induce inflammatory responses. Even though different stimuli may evoke different inflammation pathways in the human body, in general, a common mechanism is applied which can be summarized in four steps as stated in research done by Chen et al., 2018 <ref name="Arti11">Inflammatory responses and inflammation-associated diseases in organs, Oncotarget, 9(6), 7204–7218, 2018, Chen, L., Deng, H., Cui, H., Fang, J., Zuo, Z., Deng, J., Li, Y., Wang, X., & Zhao, L., https://doi.org/10.18632/oncotarget.23208 </ref>:
1. Cell surface pattern receptors recognize harmful stimuli
2. Inflammatory pathways are activated
3. Inflammatory markers are released
4. Inflammatory cells are recruited
Pathogen-associated molecular patterns (PAMPs) trigger inflammatory responses through activation of specific pattern recognition receptors. As a result, the production of proinflammatory cytokines is induced. Proinflammatory cytokines are produced predominantly by activated macrophages and are involved in the upregulation of inflammatory reactions.<ref name="Arti12">Cytokines, inflammation, and pain. International Anesthesiology Clinics, 45(2), 27–37, 2007, Zhang, J.-M., & An, J., https://doi.org/10.1097/AIA.0b013e318034194e </ref> Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are cytokines that mediate receptor activation in order to trigger crucial intracellular signaling pathways that may start the healing process.
However, in the case of acute inflammation, the response to an infection is dysregulated and often disproportional to the severity of the infection. The response gets overheated, overactivated, and can damage the body from within. Potential consequences of this overly strong reaction include infections, organ dysfunction (severe sepsis), or septic shock which is a state of circulatory failure where circulatory, cellular and metabolic abnormalities are associated with an increased risk of death. These reactions are often caused by coagulation (i.e. formation of blood clots) dysregulation. The hypercoagulability of sepsis is thought to be driven by the release of tissue factor from disrupted endothelial cells.<ref name="Arti13">Sepsis: The evolution in definition, pathophysiology, and management. SAGE Open Medicine, 7, 2050312119835043–2050312119835043, 2019, Gyawali, B., Ramakrishna, K., & Dhamoon, A. S., https://doi.org/10.1177/2050312119835043 </ref> When the human body suffers from severe sepsis, activated monocytes and endothelial cells, along with circulating microvesicles, become sources of tissue factor<ref name="Arti14">Role of extracellular vesicles in the development of sepsis-induced coagulopathy. Journal of Intensive Care, 6, 68, 2018, Iba, T., & Ogura, H., https://doi.org/10.1186/s40560-018-0340-6 </ref>.
This factor then causes the systemic activation of the coagulation cascade resulting in the production of thrombin, activation of platelets, and formation of platelet–fibrin clots. These structures can result in local perfusion defects leading to tissue hypoxia and organ dysfunction <ref name="Arti13">Sepsis: The evolution in definition, pathophysiology, and management. SAGE Open Medicine, 7, 2050312119835043–2050312119835043, 2019, Gyawali, B., Ramakrishna, K., & Dhamoon, A. S., https://doi.org/10.1177/2050312119835043 </ref>. Moreover, research has shown that dysregulated apoptotic immune cell-death plays a crucial part in immune dysfunction and mortality of sepsis. Apoptosis is a “programmed cell death” to limit damage of surrounding tissue during the immune response<ref name="Arti15">Apoptosis: a review of programmed cell death. Toxicologic Pathology, 35(4), 495–516, 2007, Elmore, S., https://doi.org/10.1080/01926230701320337 </ref>. It is a vital component of many processes in the human body such as cell turnover, proper development and functioning of the immune system <ref name="Arti15">Apoptosis: a review of programmed cell death. Toxicologic Pathology, 35(4), 495–516, 2007, Elmore, S., https://doi.org/10.1080/01926230701320337 </ref> Most cells that undergo enhanced apoptosis in sepsis are of lymphoid origin, hence less immune cells are left to fight off the infection itself <ref name="Arti16">Host–pathogen interactions in sepsis. The Lancet Infectious Diseases, 8(1), 32–43, 2008, van der Poll, T., & Opal, S. M., https://doi.org/https://doi.org/10.1016/S1473-3099(07)70265-7 </ref>. Since no effective treatment for sepsis exists yet, early diagnosis and recognition is crucial.<ref name="Arti17">Sepsis and septic shock: current approaches to management. Internal Medicine Journal, 49(2), 160–170., 2019, Thompson, K., Venkatesh, B., & Finfer, S., https://doi.org/10.1111/imj.14199 </ref>
This is where IL-6 plays an important part. As mentioned, IL-6 is a cytokine that functions as a crucial mediator during the acute phase of response to inflammation in sepsis.<ref name="Arti18">Diagnostic and prognostic value of interleukin-6, pentraxin 3, and procalcitonin levels among sepsis and septic shock patients: a prospective controlled study according to the Sepsis-3 definitions. BMC Infectious Diseases, 19(1), 968, 2019, Song, J., Park, D. W., Moon, S., Cho, H.-J., Park, J. H., Seok, H., & Choi, W. S., https://doi.org/10.1186/s12879-019-4618-7 </ref> Research on the clinical value of IL-6 in patients with sepsis and septic shock describes that IL-6 is considered controversial regarding its diagnostic and prognostic values, where meta-analysis of diagnostic value of IL-6 has shown that IL-6 only offers moderate success in differentiating sepsis from non-infectious systemic inflammatory response syndrome (SIRS).<ref name="Arti19">Role of interleukin-6 to differentiate sepsis from non-infectious systemic inflammatory response syndrome. Cytokine, 88, 126–135, 2016, Ma, L., Zhang, H., Yin, Y.-L., Guo, W.-Z., Ma, Y.-Q., Wang, Y.-B., Shu, C., & Dong, L.-Q., https://doi.org/10.1016/j.cyto.2016.08.033 </ref> Hence it is recommended that IL-6 is used as a biomarker to confirm infection rather than differentiate between sepsis and SIRS<ref name="Arti19">Role of interleukin-6 to differentiate sepsis from non-infectious systemic inflammatory response syndrome. Cytokine, 88, 126–135, 2016, Ma, L., Zhang, H., Yin, Y.-L., Guo, W.-Z., Ma, Y.-Q., Wang, Y.-B., Shu, C., & Dong, L.-Q., https://doi.org/10.1016/j.cyto.2016.08.033 </ref>.
== Medical Application & Relevance ==
As mentioned, sepsis causes each year more than 11 million deaths while affecting approximately 50 million people worldwide.<ref name="Arti2">WHO - Health topics - Sepsis, WHO, 2021, https://www.who.int/health-topics/sepsis#tab=tab_1 </ref> Mortality rates from sepsis, according to data from the Surviving Sepsis Campaign 2012, were approximately 41% in Europe versus approximately 28.3% in the United States.<ref name="Arti20">Surviving Sepsis Campaign: association between performance metrics and outcomes in a 7.5-year study. Critical Care Medicine, 43(1), 3–12, 2015, Levy, M. M., Rhodes, A., Phillips, G. S., Townsend, S. R., Schorr, C. A., Beale, R., Osborn, T., Lemeshow, S., Chiche, J.-D., Artigas, A., & Dellinger, R. P., https://doi.org/10.1097/CCM.0000000000000723 </ref> Severe sepsis causes dysfunction of multiple organs with as a consequence a state of chronic critical illness involving severe immune dysfunction and catabolism.<ref name="Arti21">Sepsis: pathophysiology and clinical management. BMJ, 353, i1585, 2016, Gotts, J. E., & Matthay, M. A., https://doi.org/10.1136/bmj.i1585 </ref> Despite efforts of experimental and clinical research during the last thirty years, the ability to positively influence the course and outcome of the disease continues to be inadequate. Options of successful and specific interventions are limited and often unsuccessful. If sepsis is detected early and has not affected vital organs yet, administration of different antibiotics have shown to increase chances of survival.<ref name="Arti22">Sepsis—Pathophysiology and Therapeutic Concepts. Frontiers in Medicine, 8, 609, 2021, Jarczak, D., Kluge, S., & Nierhaus, A., https://doi.org/10.3389/fmed.2021.628302 </ref><ref name="Arti20">Surviving Sepsis Campaign: association between performance metrics and outcomes in a 7.5-year study. Critical Care Medicine, 43(1), 3–12, 2015, Levy, M. M., Rhodes, A., Phillips, G. S., Townsend, S. R., Schorr, C. A., Beale, R., Osborn, T., Lemeshow, S., Chiche, J.-D., Artigas, A., & Dellinger, R. P., https://doi.org/10.1097/CCM.0000000000000723 </ref>
Furthermore, early symptoms of sepsis are rather generic, e.g. fever, chills, feeling dizzy or nausea, and thus are not often attributed to sepsis in the early stages.<ref name="Arti23">NHS inform Scotland - Sepsis, https://www.nhsinform.scot/illnesses-and-conditions/blood-and-lymph/sepsis, 2020 </ref> When sepsis goes undetected for too long, or if effective treatment is withheld, sepsis can rapidly progress to septic shock where risk of mortality increases by 7.6% each hour . Hence, a patient suffering from sepsis must be diagnosed correctly in time to prevent worsening of the disease with death as a potential consequence.<ref name="Arti22">Sepsis—Pathophysiology and Therapeutic Concepts. Frontiers in Medicine, 8, 609, 2021, Jarczak, D., Kluge, S., & Nierhaus, A., https://doi.org/10.3389/fmed.2021.628302 </ref>
Currently, sepsis is diagnosed with standard laboratory techniques, which takes 12-72 hours before results are obtained. These tests are based on blood cultures looking for bacterial or viral infections.<ref name="Arti24">How is sepsis diagnosed and treated?, Centers for Disease Control and Prevention. (n.d.). Retrieved September 28, 2021, from https://www.cdc.gov/sepsis/diagnosis/ </ref> However, research shows that sepsis cannot always be attributed to only the infectious agent and the immune response that follows, but also to more complex and indirect factors such as significant alterations in coagulation, immunosuppression and organ dysfunction.<ref name="Arti13">Sepsis: The evolution in definition, pathophysiology, and management. SAGE Open Medicine, 7, 2050312119835043–2050312119835043, 2019, Gyawali, B., Ramakrishna, K., & Dhamoon, A. S., https://doi.org/10.1177/2050312119835043 </ref> Hence, it is often difficult to diagnose sepsis in a patient on time since multiple causes might be at hand. Early diagnosis is therefore key to battle sepsis. With a biosensor that detects IL-6 in the early stages, symptoms can be classified as belonging to sepsis and consequently, the mortality and severity of sepsis can be decreased. Sepsis is treatable if it is identified and treated quickly which in most cases leads to full recovery with no lasting problems<ref name="Arti23">NHS inform Scotland - Sepsis, https://www.nhsinform.scot/illnesses-and-conditions/blood-and-lymph/sepsis, 2020 </ref>.
== State of the Art ==
Currently, the concentration of unbound IL-6 is typically measured in serum, i.e. blood samples are clotted and refrigerated-centrifuged within 25-40 minutes after collection. The serum is then suited for use in several immunoassays, e.g., ELISA, CLIA, ECLIA, and RIA. Alternatively, the serum can be frozen and stored at -20 ℃ or lower for later use.<ref name="Arti25">Plasma and Serum Preparation, ThermoFisher Scientific, 2007, https://www.thermofisher.com/nl/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-preparation-protocols/plasma-and-serum-preparation.html </ref><ref name="Arti26">Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions. Journal of Immunological Methods, 153(1–2), 115–124, 1992, Thavasu, P. W., Longhurst, S., Joel, S. P., Slevin, M. L., & Balkwill, F. R., https://doi.org/10.1016/0022-1759(92)90313-i </ref> In addition, commercial assays also allow for measurement in plasma. Whole blood is collected into anticoagulant-treated tubes e.g., EDTA-treated or citrate-treated. Cells are removed from plasma by centrifugation for 10 minutes at 1000-2000 x g using a refrigerated centrifuge. Centrifugation for 15 minutes at 2000 x g depletes platelets in the plasma sample.
{| class="wikitable" style="margin-bottom:0"
!Company
!Product
!Test name
!Sample volume (μL)
!Sample matrix
!Range (pg/mL)
!Sensitivity (pg/mL)
!Precision (CV%)
!Incubation time (min)
!Measuring Technique
!Assay type
|-
|Beckman Coulter
|A16369
|Access IL-6 Assay
|110
|Serum or plasma
|0.5 - 1500
|0.5
|N/A
|35
|CLIA
|One-step immunoenzymatic sandwich assay
|-
|Roche COBAS
|5109442190
|Elecsys IL-6
|30
|Serum or plasma
|1.5 - 5000
|1.5
|N/A
|18
|ECLIA
|Sandwich
|-
|ThermoFisher Scientific
|MAN0014630
|Human IL-6 ELISA Kit
|100
|Serum, plasma, buffered solution, or cell culture supernatants
|7.8 - 2500
|< 2.0
|Intra-assay: CV = 6.2%, Inter-assay CV = 7.9%
|180
|ELISA
|Solid-phase sandwich
|-
|R&D Systems Inc.
|D6050
|Quantikine® Human IL-6 Immunoassay
|100
|Serum, plasma, or cell culture supernatants
|3.1 - 300
|0.7
|Intra-assay: CV = 2.0% - 4.2%, Inter-assay: CV = 3.8% - 6.4%
|270
|ELISA
|Sandwich
|-
|abcam
|ab178013
|Human IL-6 ELISA Kit
|N/A
|Serum, EDTA plasma, cit plasma, cell culture supernatants
|7.8 - 500
|1.6
|Intra-assay CV = 2.1%, Inter-assay CV = 2.4%
|90
|ELISA
|Sandwich
|-
|RayBio
|ELH-IL6
|RayBio® Human IL-6 ELISA Kit
|100
|Serum, plasma, cell culture supernatants
|3.0 - 1000
|3.0
|Intra-assay CV =< 10%, Inter-assay CV =< 12%
|150
|ELISA
|Sandwich
|}<div style="margin-bottom:1em"><sub>''Table 1: Commercially available IL-6 well-plate assays.</sub><br />
Immunoassays, in general, provide a relatively slow method for measuring specific biomarkers like IL-6. Therefore, faster methods are the subject of intensive research. A commercially available rapid test is the Milenia® QuickLine IL-6 test: Milenia Biotec.<ref name="Arti27">Milenia Biotec, 2016, https://www.milenia-biotec.com/en/product/il6/#nav-overview </ref> This lateral flow immunoassay is designed for the semi-quantitative evaluation of human Interleukin-6 in serum, plasma, cell culture supernatant, amniotic fluid or cerebrospinal fluid.<ref name="Arti28">Rapid and sensitive detection of interleukin-6 in serum via time-resolved lateral flow immunoassay, Analytical Biochemistry, 588, 113468, 2020, Huang, D., Ying, H., Jiang, D., Liu, F., Tian, Y., Du, C., Zhang, L., & Pu, X., https://doi.org/10.1016/j.ab.2019.113468</ref><ref name="Arti29">Development of quantum dot-based fluorescence lateral flow immunoassay strip for rapid and quantitative detection of serum interleukin-6, Journal of Clinical Laboratory Analysis, 35(5), e23752, 2021, Tang, J., Wu, L., Lin, J., Zhang, E., & Luo, Y., https://doi.org/10.1002/jcla.23752 </ref> In the Milenia® QuickLine IL-6 test, the sample is pipetted in the sample application port. IL-6 of the patient's sample binds to a first monoclonal anti-IL-6 antibody conjugated to gold particles. The IL-6-loaded gold particles diffuse through the membrane and overflow the test line (T). There, a second monoclonal antibody specific for IL-6 is coated on the membrane; so the gold particles are bound specifically and become visible as a colored line. The color intensity is proportional to the concentration of IL-6 in the sample and intensifies during the incubation time. The surplus of gold particles continues to diffuse over the test strip. The conjugate specific antibodies printed as a control line on the membrane (control line, C) capture the gold conjugate and therefore a visible line develops during the incubation time.<ref name="Arti30">A Point of Care Lateral Flow Assay for Rapid and Colorimetric Detection of Interleukin 6 and Perspectives in Bedside Diagnostics, J Clin Med Res, 2(2), 1–16, 2020, de Souza Sene, I., Costa, V., Bras, D. C., de Oliveira Farias, E. A., Nunes, G. E., & Bechtold, I. H., https://doi.org/https://doi.org/10.37191/Mapsci-2582-4333-2(2)-032 </ref><ref name="Arti31">Duplex Shiny app quantification of the sepsis biomarkers C-reactive protein and interleukin-6 in a fast quantum dot labeled lateral flow assay, Journal of Nanobiotechnology, 18(1), 130, 2020, Ruppert, C., Kaiser, L., Jacob, L. J., Laufer, S., Kohl, M., & Deigner, H.-P., https://doi.org/10.1186/s12951-020-00688-1 </ref>
{| class="wikitable" style="margin-bottom:0"
!Company
!Product
!Test name
!Sample volume (μL)
!Sample matrix
!Range (pg/mL)
!Sensitivity (pg/mL)
!Incubation time (min)
!Measuring Technique
|-
|Milenia Biotec
|MQL6 1
|Milenia® QuickLine IL-6 test
|100
|Serum, plasma, cell culture supernatants, amniotic fluid, cerebrospinal fluid
|50 - 10000
|50
|20
|LFIA
|-
|}<div style="margin-bottom:1em"><sub>''Table 2: Commercially available rapid test, Milenia® Quickline IL-6 test.</sub><br />
== Safety & Lab protocols ==
<b>Safety</b>
Interleukin-6 is present in blood plasma, thus the sample preparation in the lab will also take place with human blood plasma as matrix. It is of crucial importance to handle human blood plasma with care due to potential pathogens that can be transmitted, such as Hepatitis viruses and HIV.<ref name="Arti32">Recommendations for the production, control and regulation of human plasma for fractionation, World Health Organization, 2007, https://www.who.int/bloodproducts/publications/TRS941Annex4blood.pdf </ref> Sigma Aldrich has screened the blood plasma in advance on HIV, Hepatitis B and Hepatitis C, however caution is recommended all the same.<ref name="Arti33">Plasma from human, Sigma Aldrich, Retrieved November 12, 2021 from https://www.sigmaaldrich.com/NL/en/product/sigma/p9523, n.d. </ref> Specific lab protocols and rules will be listed below.
== References ==
<references />
