Investigation of Complement Function in Vitro Results:  The first part of this practical involved measuring the CH100 total haemolytic complement classical pathway using a kit based upon the principle of radial immunodiffusion.The principle of this kit was based on serum which consisted of active complement components radially diffusing from a cylindrical well into an agarose gel which contained sheep red blood cells sensitised with rabbit anti-sheep red blood cell antibodies. While diffusing the C1 is bound by the sheep red blood cells. It is then incubated and the complement cascade continues resulting in lysis of the sheep red blood cells producing a clear zone, where the size relies on the classical complement activity of the serum at the start.Fig.1: The Total Haemolytic complement plate (Lot number: 444827 and Expiry date: 30/08/2018) after incubation. The zones of lysis of various sizes were measured to determine the activity of the classical complement pathway in the patient’s samples.Table 1: The diameters of the zones of lysis were measured and recorded in the table below:Title: The Classical complement pathway zone of lysisSampleZone of lysis (mm)% Activity CH100 units/mlCalibrator (Neat)9.0100991Calibrator (1/2 dilution)8.550495.5Calibrator (1/4 dilution)7.025247.75Control9.0??Patient one9.0??Patient two9.0??Patient three9.0??By measuring the zones of lysis produced by the sera of known complement activity, which was the calibrators of various dilutions used in this practical, a calibration curve was constructed by plotting the diameters of the zones of lysis of the calibrator against the complement activity of the calibrators on semi log graph paper.From the curve of the Classical complement pathway in figure 3, obtained the percentage activity for the control and patient samples.Table 2: Percentage activity of the Classical complement activity in the samples are recorded in the table below:Title: Percentage activity of the Classical complement activitySampleZone of Lysis (mm)% Activity CH100 units/mlControl9.0100991Patient one9.0100991Patient two9.0100991Patient three9.0100991 The interpretation of the results is that the control value should be 505 CH100 units /ml but in this practical it was 991 CH100 units/ml. Therefore, the control did not work and so the method used to measure the Classical complement activity is invalid.The second part of this practical involved measuring the alternative haemolytic pathway using a kit based upon the principles of radial immunodiffusion.The principle of this kit was based on serum which consisted of active complement components radially diffusing from a cylindrical well into an agarose gel which this time contained chicken red blood cells. While diffusing the C3b molecules coated on the chicken cells bind to membrane surfaces which allows the creation of the C5 convertase. It is then incubated and the complement cascade continues resulting in lysis of the chicken red blood cells producing a clear zone, where the size relies on the alternative complement activity of the serum at the start.Fig.2: The Total Haemolytic Complement plate (Lot number: 444827 and expiry date: 30/08/2018) after incubation and the zones of lysis of various sizes were measured to determine the activity of the alternative complement pathway in the patient’s samples.Table 3: The diameters of the zones of lysis were measured and recorded in the table below:Title: The alternative complement pathway zone of lysisSampleZone of lysis (mm)% Activity Calibrator (Neat)8.5133Calibrator (1/2 dilution)7.066.5Calibrator (1/4 dilution)3.533.25Control7.0?Patient one8.0?Patient two8.5?Patient three8.0?By measuring the zones of lysis produced by the sera of known complement activity, which was the calibrators of various dilutions used in this practical, a calibration curve was constructed by plotting the diameters of the zones of lysis of the calibrator against the complement activity of the calibrators on semi log graph paper.From the curve of the Alternative complement pathway, obtained the percentage activity for the control and patient samples.Table 4: The percentage activity of the Alternative complement pathway in the samples are recorded in the table below:Title: The percentage activity of the Alternative complement pathway activitySampleZone of lysis (mm)% ActivityControl7.066.5Patient one8.0100Patient two8.5133Patient three8.0100The interpretation of the results is that the control value should be 78% activity but in this practical it was 66.5% activity. Therefore, the control did not work and so the method used to measure the Alternative complement activity is invalid.The third part of this practical involved measuring the C1 esterase inhibitor using a functional assay.The principle of this kit was based on the C1 Esterase inhibitor inhibiting the activated forms of the first component of complement which is the C1 esterase. Any remaining C1 esterase not bonded to the C1 inhibitor is then reacted with C1 esterase substrate and pNA is released. The amount of the chromogenic substrate reacted with C1 esterase is proportional to the absorbance measured at the wavelength 405nm.Table 5: The absorbance values at wavelength 405nm are recorded in the table below.Title: The absorbance values form the C1 Esterase Inhibitor Functional assay Sample Absorbance at 405nm% Activity Coagulation Reference (Neat)0.616  125Coagulation Reference (1/2 dilution)0.77662.5Coagulation Reference (1/4 dilution)1.623 31.25Coagulation Control Normal0.641?Coagulation Control Abnormal0.938?Patient Three0.7052?Patient Four0.742?By measuring the absorbance of each of the calibrators of various dilutions used in this practical, a calibration curve was constructed by plotting the absorbance values of the calibrators against the percentage of the C1 inhibitor in the calibrators on graph paper.From the curve of the C1 Esterase Inhibitor in figure 4, obtained the percentage activity for the controls and patient samples.Table 6: The percentage activity of the C1 Esterase Inhibitor for the samples were recorded in the table below:Title: The percentage activity of the C1 Esterase InhibitorSample % Activity Control Normal122Control Abnormal92Patient three116Patient four112The Coagulation control Abnormal for C1-Inhibitor Lot Number: 3P73BC1KExpiry date: 2020-01-31 Range: 41.7 – 69.5% Mean: 55.6%The Coagulation control Normal for C1-Inhibitor Lot Number: 1P72BC1KExpiry date: 2020-01-31 Range: 88.1 – 146.9% Mean: 117.5%The interpretation of the results is that the normal control is within range but the abnormal control is out of range. Therefore the test method used is invalid.Discussion: In this practical the classical and alternative complement pathway haemolytic activities in patient serum samples were measured. One assay measured the classical pathway and the other assay measured the alternative pathway. The initial step of complement system differs in various pathways. Nevertheless, each of the pathways develop enzyme complexes. The C3 convertase, which splits C3 into C3a and C3b; and the C5 convertase, which splits C5 into C5a and C5b.C3b, therefore created, connects C3 convertase to develop C5 convertase. C5 convertase, made by the alternative, classical, or lectin pathway, starts the initiation of final elements of the complement system to develop membrane attack complex (MAC) and eventually destroys the pathogen. This happens across three pathways; Classical pathway, triggered with binding of antibody and antigen, Alternative pathway, triggered at bacterial cell surfaces, and Mannose binding Lectin pathway, triggered by a plasma lectin that attaches to mannose residues on microorganisms.The classical pathway begins with the formation of antigen-antibody complex which provokes conformational alterations in the Fc fraction of the antibody which uncovers a site for binding with the C1 protein. Therefore, the antibody only triggers the complement system once combined to an antigen. C1 is comprised of two molecules each of C1r and C1s subunits and one molecule of C1q. C1q attaches to the antibody bound antigen. Proteases C1r and C1s assist to split C4 and C2.When C1 is bound to the immune complex it recruits an additional protein C4 which is split into C4a and C4b. C4a leaves while stimulates C4b binds to the target surface close to C1q. Then, C4b draws C2 which is too split into C2a and C2b. C2a binds C4b developing the C4b2a complex while C2b leaves. C4bC2a then triggers C3. The C4b2a complex is also called the C3 convertase as this transforms C3 into an active structure by splitting C3a and C3b. One molecule of C4b2a can split a great amount of C3 molecules. C3b binds to the bacterial surface or to the convertase itself. C3b once bound to C3 convertase develops C4bC2aC3b (C5 convertase) which triggers C5.In contrast to the classical pathway, in the alternative pathway, it is activated when a bacterium arrives in the body, due to inflammation, complements reaches the area, where C3 molecules directly bind with the antigen and become initiated. In the alternative pathway, serum C3 comprising an unsteady thioester bond experiences gradual unprompted hydrolysis to produce C3a and C3b. C3b binds the surface of pathogen and next binds to an additional serum protein known as factor B. Next factor B uncovers the position which functions as the substrate for enzymatically active serum protein D. Next factor D splits B into Ba and Bb creating the C3 convertase (C3bBb). C3 convertase then produces C5 convertase which eventually creates a MAC as in classical pathway (Ross, 2014).In the measurement of the classical pathway activity of the patients, controls were used to ensure that the method used and the calibration curve obtained from the different dilutions of calibrators gave correct results. The control was the first results looked at before the patient results were examined. The control result in table 2 should be 505 CH100 units but in this practical it was 991 CH100 units. Therefore, the control did not work and so the method used to measure the Classical complement activity is invalid. A possible reason of the method not being valid is that the total haemolytic complement plate used was out of date.However, in a clinical setting these results would have been disregarded for the patients and the method performed again or change until the control values were in range. So assuming that the control values were in range and looking at the results for all three patients, they had 991 CH100 units/ml classical complement pathway activity. The normal adult serum classical complement reference range is 392 – 1029 CH100 units/ml. Therefore all these patients have normal classical complement activity.In the measurement of the alternative complement activity of the patients, again controls were used in order to ensure that the method employed gave correct results. The control results again were looked at first. The results of the control value should be 78% activity but in this practical it was 66.5% activity. Therefore, the control did not work and so the method used to measure the Alternative complement activity is invalid. Again in a clinical setting the patient’s results would have been disregarded and the method performed again or changes done to it until the control results were in the target range. Again a possible reason that the control was out of range is that the total haemolytic complement plate was out of date.However, again assuming that the control results were in range and the method employed was valid, the patient results were then analysed. Patients one and three had 100% activity and patient two had 133% activity. Assuming again that all these patients are adults, the normal adult serum alternative pathway complement reference range is 55 – 178%. Therefore all these patients are in the normal alternative complement activity.In the third part of this practical a C1 Inhibitor functional assay was used to measure the C1 esterase inhibitor functional activity in patient samples.The complement cascade is rapidly activated and highly amplified by the generation of C3 and C5 convertases. There are three main levels of control that limit the potential harm that uncontrolled complement activation might cause: (1) the initiation step in the Classical and Lectin pathways; (2) the C3 and C5 convertases of all three pathways; and (3) the assembly of the MAC. Both soluble and membrane-bound regulatory proteins serve these functions, which help terminate complement activation and direct it to appropriate targets.A plasma serine proteinase inhibitor (serpin) is the C1 esterase inhibitor (C1-INH). C1-INH binds covalently to C1r and C1s when they are activated, irreversibly their activity is inhibited and so reducing Classical pathway initiation. C1-INH preventing activation of C1r and C1s in addition eliminates them from the C1 complex, revealing positions on the collagen-like area of C1q. Similarly, C1-INH stops MASP-1 and MASP-2, kallikrein, factor XIa, factor XIIa, and the Lectin pathway plasmin and the contact, coagulation, and fibrinolytic systems (Rich et al., 2018).The C1 esterase inhibitor functional assay was carried out on two patient samples along with two controls, one abnormal and one normal. Controls again were used in order to ensure that the method used gave valid results. The control results were looked at first before going onto look at the patient results. The control results were that the normal control was within the target range, however the Abnormal control was outside the target range. Therefore, the method used wasn’t valid and the patient results should be discarded.In a clinical setting these results would be disregarded for the patients and the method would be repeated or altered until the controls were within range. Assuming that for this practical that both controls were within range, will then go onto interpret the patient results.The biological reference range for normal serum is 70 – 130% and anything less than 30% is considered deficient for C1 inhibitor activity. Applying this information to the patient results in table 6, both patients had normal C1 inhibitor activity.The C1 inhibitor is an important regulatory protein of the complement system. Deficiencies or abnormalities in the C1 inhibitor can occur individuals. The C1 inhibitor as the name suggests plays an inhibiting role in the complement system, kinin generating system, clotting system and the fibrinolytic system. Hereditary Angioedema is a common illness in patients with C1 inhibitor deficiency. Angioedema is the swelling of tissues under the skin or in mucus membranes which do not cause itchiness. Swelling can occur in places such as the feet, hands, mouth, bowel and in the airways. When there is swelling present in the skin it is generally localised, not red or itchy. Severe abdominal pain can occur when swelling occurs in the bowel wall. A patient’s ability to breathe can be extremely compromised if swelling occurs in the airways. Angioedema usually lasts for about 3 days.The majority of the proteins and regulators of the complement system are inherited as autosomal recessive genes. However, there are two exceptions to this rule. The properdin deficiency is inherited as a X linked recessive trait and the deficiency of the C1 inhibitor is inherited as autosomal dominant.Conclusion:From carrying out this practical in the investigation of complement function in vitro, if the controls worked in all these three tests, it is possible to measure the complement activity in patients. It is also possible to identify the specific complement pathway and whether the complement regulatory proteins are present or reduced. This practical also highlighted the importance of controls in order to stand over the results produced for the patients in order for the patient to be properly diagnosed and treated. As detecting complement abnormalities in patients early and vigorously is vital in order for them to lead productive lives.References: Rich, R., Fleisher, T., Shearer, W., Schroeder, H., Frew, A. and Weyand, C. (2018). Clinical immunology. 5th ed. [Miejsce nieznane]: Elsevier, pp.299 305.Ross, G. (2014). Immunobiology of the Complement System. 1st ed. Burlington: Elsevier Science.