Blood Analysis Experiment for Liver Disease
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Blood analysis procedures are always suggested for liver disorder disease. Four different tests were carried out to diagnose the protein, albumin, bilirubin and an electrophoresis for the given patient who had a clinical history of alcoholism. Valid assays were profound by comparing the quality control to the expected values. The total protein of the patient was 56g/L which is 4g/L lower than the reference range. The direct bilrubin levels of the patient were 11uM which should be lower than 7uM indicating a malfunction of the liver and the diseases related to the patient's diagnosis is Cirrhosis. Thus the patient was strongly recommended for LGTs and áµ§GT which would further clarify the specific disorder in the liver.
Blood is very essential medium of respiration and metabolic function. The Fluid known as blood consists of cells, cellular proteins and components. Red Blood Cells (RBC) accounts to 45% blood volume. The rest is considered as plasma.
The remainder fluid after removal of RBC can be differentiated into Plasma or Serum depending upon the technique use for extraction. Both Plasma and Serum consist of 93% of solvent water. Serum is a fluid obtained after the clot formation in the medium. Plasma is the fluid in which clots are prevented by anticoagulants. Thus the only difference in serum and plasma is that Plasma consists of proteins involved in colt formation while in serum they are absent.
Cellulose Acetate electrophoresis is a common and specific technique when proteins are separated only by charge. The advantage of using this technique is that this method is rapid and simple. Several different samples can be processed at the same time which can ensure low cost of analysis, however on the cellulose acetate strip only one sample can be applied. This method is usually applied in laboratories serum protein electrophoresis. The method works as the protein samples are placed at intervals across the top of the strip, a charge difference is induced down the column. The anode towards the top (+) and the cathode towards the bottom (-), the proteins which have more of a +ve charge are repelled more by the cathode and attracted more by the anode, thus they move faster through the gel.
The method of Electrophoresis is used to analyse the relative proportion or ratio protein fraction within the total serum proteins depending on the molecular weight of the protein. Thus the protein with higher molecular weight (MW) will have less mobility when compared protein with less molecular weight. There are several proteins with similar MW, so six clear bands are usually observed. This technique is also used to compare the quality controlled samples against the patient samples.
The Biuret test is an analysis to measure the total protein in the Serum. The peptide bond in protein is a major characteristic to differentiate with other compounds. Thus in the presence of peptide bond the cupric ion in alkaline solution will form an violet coloured complex of carbonyl and imine groups. The colour varies from pink to reddish violet product depending on tripeptides, polypeptides and proteins. Thus the intensity of colour formation is directly proportional to the number of peptide bonds of serum protein reacting with cupric ions. Thus the total protein is measured by using spectrometer.
The level of albumin in the blood can be measured using Bromocresol Green Dye also known as BCG. Albumin is an important component of blood as it maintains the level water in blood. Reduced level of albumin results in conditions like oedema in which blood fluid is retained into body tissue. The Bromocresol Green dye binds to albumin in alkaline medium which forms a complex indicating green colour. The conditions of the reaction are that the protein should be in buffer solution such that the pH is lower than the isoelectric point it has positive charge. The formation of complex shows a change in colour from yellow to green. Thus the intensity of colour change is measured by spectrophotometer at 628nm.
Jendrassik and Grof method is used to measure bilirubin level in plasma or serum. Bilirubin is a yellow coloured compound produce in the liver which characterize the plasma or serum as straw coloured fluid. It is formed as a result of catabolism of haemoglobin. As the compound is insoluble in serum or plasma, Albumin is used as a carrier to make it soluble and recycled in liver. The test for measurement of the bilirubin in patient's serum to conclude any malfunction of liver and blood destruction disorders.
The purpose of the experiment is to determine and analyse the patient blood sample and compare the with the quality control samples. The experiment is to determine a valid assay by comparing quality control samples. Valid assay is compared with the reference range and conclusion is provided whether the patient normal or abnormal.
To be able to understand the concept of clinical measurements by conducting the 4 experiments shown with the method below.
Below is a Patient Request from contains the details of the person whose Serum is used for analysis.
The provided quality control serum details are in table 2
Details of Quality Control Serum
Expected values for Quality Control Serum
- Lot no: 317UE
- Total protein: 47.5 g/L
- Expiry Date: 2012-13
- Albumin: 31.1 g/L
- Direct bilirubin: 37.7 Î¼mol/L
- Total bilirubin: 91.2 Î¼mol/L
The complete experiment consists of serum protein electrophoresis and 3 assay tests of serum. The assays were completed first and the serum protein electrophoresis was carried out last.
Serum Protein Analysis
To carry out this experiment the following equipment was required: forceps, cellulose acetate strips, barbital buffer, special applicator, electrophoresis tank, power pack, Ponceau S dye solution, acetic acid, Wicks paper.
Firstly the Vicks paper was cut according to the gel electrophoresis tank and placed on the edge of the dip of the tank. The buffer is added to almost more than half way in the dip of the tank. Using forceps the cellular acetate strips were removed from the barbital buffer where they were soaked. The cellular acetate strips where slightly blotted gently against paper towels to remove the excess buffer. Using the special applicator, the control and the patient samples were applied to the cellulose acetate strip. The cellulose acetate was then placed into the tank in between the wicks. The wicks in required and needs to be in good contact with the cellulose acetate to ensure then is a good current flow. The electrophoresis top lid was then fixed on and the power pack was set to 4mA (4mA because the there was 2 samples in the electrophoresis tank, one of my group and one of another) . The button run was then set and the strips were left to run on the electrophoresis for 80 minutes. After then was completed, power was switched off and the strip was removed with forceps from the electrophoresis tank. The strip was stained with the dye (Ponceau S dye solution). The excess dye was removed by staining to strip with 5% acetic acid for around 30 seconds. The strip was left to be dried on paper towel.
Total Protein by Biuret method
To following out this experiment, the following equipment is required: 5 test tubes, test tube rack cuvette, 0.9% NaCl, Serum (patient's sample) Biuret Reagent (dye) and the Quality control serum, spectrophotometer, timer.
There were 5 test tubes be reactions which were set up. In test tube 1 (Saline Blank) 2.5 ml of 0.9%NaCl was added. In test tube 2 (test) 2.4ml of 0.9 NaCl was added and 0.1 ml of patients sample. Test tube 2 reaction was repeated with test tube 3 as duplicate was prepared. In test tube 4, 2.4 of 0.9% NaCl was added and then 0.1ml of Quality control (Randox). Test tube 3 reaction was repeated in test tube 5 as a duplicate was prepared. Once all these test tubes are prepared the Biuret reagent (dye) is added to each test tube (3.0ml) with a staggering on time of 1 minute. This was done because it was important to keep all the test tubes were left for 30 minutes at room temperature exactly. The test tubes were mixed to ensure all samples are mixed with the reagent. The spectrophotometer was adjusted to 550nm absorbance. Test tube 1 was used to adjust the spectrometer to 0. And then after 30 minutes when colour changes had been established each test tube was added to the spectrometer according to the time specifically at 30 minutes. The absorbance of each test tube was noted down in the lab practical book.
Albumin by Bromcresol Green Binding
To following out this experiment, the following equipment is required: 5 test tubes, test tube rack cuvette, BCG solution, Serum (patient's sample) Biuret Reagent (dye) and the Quality control serum, spectrophotometer, timer.
There were 5 test tube reactions which were set up. In test tube 1 (Blank), 4.0ml of BCG solution was added. In test tube 2 (test) 4.0ml of BCG solutions was added. Test tube 2 reaction was repeated with test tube 3 as this is a duplicate. In test tube 4 (Patients serum) 4.0ml of BCG solution was added, again this was repeated with test tube 5 as it is also a duplicate of the Quality control (Patients serum). Once these were all prepared, only in the test test-tubes and the quality control (patient serum) test tubes 20ul of patient's serum was added to test tubes 2-5. When the serum was added, there was a colour change to green of the reagents. This process was done at a 1 minute interval to ensure all of the test tubes were left to incubate for the same time which was 10 minutes. Whilst waiting for incubation of the test tubes for 10 minutes in total, the spectrophotometer was set to 628nm and was adjusted to 0 with using the blank test tube (test tube 1). After 10 minutes, absorbance of each test was read and noted down.
Bilrubin - Jendrassik & Grof method
The Concentration and absorbance of 6 standard duplicate are provided. A calibration curve is plotted using the mean Absorbance and Concentration of the given 6 Standards.
Duplicates of Test Blank with 0.2ml d.H2O, 0.05ml Patient Serum were made. Duplicates of Test Direct were prepared in a test tube using 0.2ml d.H2O, 0.05ml of patient serum and 0.125ml of diazo reagent. Then duplicates of third test tube of Test total were prepared using same values of Test direct with 0.5ml caffeine. The Test tubes are mixed thoroughly and incubated in room temperature for 10 minutes. Then all three test tubes with duplicates are then incubated with 0.025ml of ascorbic acid and 0.375ml of Tartrate. Test blank and test direct are incubated with 0.5ml caffeine and only test blank is incubated with 0.125ml diazo reagent. All are mixed and reading of absorbance was measured by spectrophotometer at 607nm where water is used as 0 blank. Mean are calculated and referred to the calibration curve of 6 Standards to obtain Bilirubin concentration in ÂµM. This last step of the test was the repeated with quality control serum instead of the patient serum.
Experiment 1: Total Protein by Biuret method
Table 1 shows the total protein by Biuret, the values in table shows Absorbance 1 and 2 are duplicates were created and then mean absorbance was calculated. Saline Blank is a reagent blank which just contained the reagent with no sample added. This was used to zero the spectrophotometer and correct for any intrinsic absorbance (colour) due to the reagents at the wavelength used. The concentration (g/L) was determined from the calibration curve. The concentration was measured from the calibration curve which was absorbance of 550nm against protein g/L. Graph 1 shows a positive correlation relationship between the Absorbance (607nm) against the protein (g/L).
Experiment 2: Albumin by Bromcresol Green Binding
Table 2 shows the albumin by Bromcresol green binding test; table 2 shows Absorbance 1 and 2 as duplicates were created. Saline Blank is a reagent blank which just contained the reagent with no sample added. This was used to zero the spectrophotometer and correct for any intrinsic absorbance (colour) due to the reagents at the wavelength used. The concentration (g/L) was determined from the calibration curve using the absorbance. The concentration was measured from the curve (Graph 2) which was absorbance of 628 against protein g/L. Graph 2 provided shows positive correlation relationship between the Absorbance (628nm) and the Albumin (g/L).
Experiment 3: Bilirubin by Jendrassik & Grof
Table 3 shows the Bilirubin by Jendrassik & Grof test; the absorbance 1 and 2 were duplicates to ensure accuracy of results. The concentration (uM) was determined from the graph in the appendix. Graph 3 showed absorbance against Bilirubin (uM) and graph showed positive correlation. Concentration (uM) was determined through the mean absorbance (607nm).
As observed before starting the experiment, observations were made to ensure that patient request form was duplicate to the patient sample, unfortunately this wasn't the case as there were letter errors included on the patients sample which would mean if this was done in a lab with real life observations it would be important to ensure that is the patients sample if urgent or in other case a new sample would need to be required.
Observations from experiment 1, the total protein experiment was 43.5g/L within 10% of the expected value 47.5g/L shows that the results are valid. Comparing patient sample to the reference range 60-80g/L and the patient has a value of 56g/L, represents low protein levels in the blood.
Experiment 2 (Albumin by Bromcresol Green Binding) the albumin control 17.5g/L is not with the 10% of the expected value of 27.0g/L shows the results of the quality control is invalid. Comparing patient sample to the reference range 38-50g/L and the patient's value 37g/L are very near close enough to the normal range which indicates that the patient's albumin levels are very near to normal. The slightly low values indicate patient may be suffering from jaundice which is a pale colour of eyes or skin.
Experiment 3; Bilirubin by Jendrassik & Grof two bilirubin products were obtained, one direct and one total. Direct is conjugated and the total is the unconjugated (indirect) and the conjugated products together. The Quality control was used to check the assay is working. Compared to the expected values, Quality control value for the direct test was 47.5uM and the expected value is 32.2uM showing results are not valid and this is the same for the Quality control total test was 74uM and the expected was 91.9uM. However the difference between the Direct and Test is clear as it is with the expected results. Invalid results could be due to pipetting error or the incubation time. Accuracy is important as due to inaccuracy it can cause invalid results to the experiment. Comparing patient sample to the reference range 2-20uM of the bilrubin total is 14uM, representing that the patients total bilrubin levels are normal, whereas the bilrubin direct levels are abnormal as patients levels are higher 11uM then the reference range <7uM.
Bilirubin by Jendrassik & Goff test indicates that the patient may be suffering from Cirrhosis as the patient's clinical history indicates etOH which is abbreviated for alcoholism (etOH represents ethanol which is related to Alcoholism). Thus it can be concluded that the patient has abnormal levels of blood protein especially albumin. The requested test in the patient request form is the LFTs. The albumin levels were also low compared to the reference range indicating jaundice and this is well linked to Cirrhosis. This disease also indicates that Serum Albumin is a recommended test to indicate is patient is suffering from Cirrhosis. These tests also indicate that the patient is suffering from liver function problems.
Experiment 4, the serum protein electrophoresis technique was used, on the strip one band was identified and comparing to figure 2 it shows that the band present on the electrophoresis run was the Î²2 towards the anode.
The two tests that were requested for the patient to be tested for were a liver function test (LFTs) and Gamma Glutamyltransferase (áµ§GT) is an enzyme. The LFTs test is done to determine the state of the patient's liver. This is essential because patient is possibly suffering from liver function problems due to the consumption of alcohol which has caused the disease Cirrhosis. The other recommended test which is áµ§GT is also very essential to perform as it can detect liver disease and bile duct injury. This is an essential test for the patient as they are a regular alcohol drinker, there áµ§GT increases along which can cause a liver disease to the patient, this test could then indicate that the patient is possibly suffering from the disease indentified earlier 'Cirrhosis'.
Providing the results determined from the several tests demonstrated suggest that patient may be infected with the disease Cirrhosis, observations have been made clearly to the fact that the patient request form directs that the patient is required for 2 further test are; áµ§GT and LFTs. Patient also has a clinical history of etOH all which support the findings.
Error while conducting bilirubin test are obvious as very low volumes of serum is used in pipetting. The improper mixing may also show wrong absorbance reading in all assay test. The gel electrophoresis technique can be improved by increasing the time for voltage.
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