Clinical Biochemistry And Blood Transfusion Assignment Sample

Clinical Biochemistry And Blood Transfusion Assignment

Laboratory-1

Creatinine - Jaffe method

Introduction

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Creatinine is the type of chemical compound that is leftover from the process of producing the energy components produced in the muscles. Creatinine is excreted from our bodies during urination. It is found in the substances like creatine and creatine phosphate, which is known as the nitrogen waste product (Barganouxet al. 2018). It has to remember that creatinine is not utilized in the body while it is excreting out from the body during urination in the kidney.

The proportion at which these substances are excreted out is proportional to the body mass. It is thus referred to as the most fruitful marker of endogenous disease and thus it can be used in the treatment of kidney-related diseases. It is primarily measured as the assessment of kidney function and it has an impact on the measurement of urine or urea (Syme et al. 2020). With help of Jaffe method, creatinine can be detected in serum and urine through changing into orange colour within alkaline medium. In this method, the colour change is measured at 520 nm for identification of creatinine in body.

Creatanine levels in the body

(Source: Syme et al. 2020)

However, the level of plasma in creatinine is very much independent of intake of water, ingestion of the protein, exercises, and the rate of plasma elevation. As the production rate is constant, the plasma elevation rate of creatinine is the indication of kidney impairment and under excretion process. The decreased levels do not have any clinical significance in the measurement of plasma creatinine.

According to the UK standard, the formula to calculate “Glomerular Filtration Rate (GFR)” of “Diet Renal Disease” is “eGFR(ml/min/1.73m 2)=186 x (Sample A serum Creatinine /88.4)-1.154 x(Age)- 0.203 x (0.742 if female) x (1.210 if black)”

In this clinical procedure, the Jaffe test method is used for the measurement of creatinine levels (Kumeet al. 2018).

Materials and procedure

Principle of the Jaffe method

The creatinine present in the alkaline solution chemically reacts with picric acid, which can form a complex of colouring substances that can absorb around 500 nm. The quantity of the colouring complex is directly proportional to the concentration of the creatinine (den et al. 2018).

Creatinine + Picric acid = Creatinine picrate complex

 Jaffe’s Method Procedure

(Source: den et al. 2018)

Safety precautions and materials are required

Materials required	Precautions taken
Standard solution of the creatinine	? As picric acid is very poisonous, it has to be checked that it does not enter the mouth directly. ? Has to wear a full PPE kit. ? After the lab, take all the contaminated clothes immediately. ? When the reagent is in contact with the eyes, clean the eyes with the water very carefully for a few minutes.
Samples
R1-Picric acid
R2-NaOH
Pipettes and the useful correct
Water Bath
Spectrophotometer(Wavelength-500 nm)
POPE
Timer
Distilled water
Test tubes
Types of cuvettes
Cleaning towels
Marking pen

Procedure

• Has to ensure that the water bath or heat block is set up at 37-degree centigrade.
• Has to label the five test tubes and the five cuvettes which are Blank(B), Control (C2), Control (C1), Standard(Std), and Sample(A).
• Has to warm up the temperature to 37 degrees centigrade
• Has to set up the spectrophotometer at the wavelength range of 500 nm
• Has to add 200 micro-litres of standard, sample, and controls into the respective tubes.
• Have to add equal volumes of 2 ml of reagent working into all the five test tubes.
• Has to mix the samples by gentle shaking of the test tubes and has to keep on the water bath as soon as the timer is on.
• Has to transfer all the blank into the blank cuvette and the spectrometer has to calibrate.
• Again after 30 seconds, the absorbance (A1) has to read the defined standard, sample, and controls within the time of 1 minute (Wiles et al. 2019). The transferring of the sample has to take place of respective cuvettes.
• Record the table on the given results.
• Has to return the samples into the test tube and has to keep in the bath for about 2 minutes.
• After two minutes the sample has to be transferred, controls and the standards have to be kept in the respective cuvettes.
• Measure the second absorbance. Record the results.
• Has to throw the contents present in the cuvettes and keep the test tube down in a sink and has to rinse with the cold water. Dry with tissue paper.
• Clean all the work surfaces.

Interpretation of the results

Table 1: Standard samples (A1) and (A2)

Standards	A1	A2
Blank (B)	0	0
Std	0.129	0.16
Sample A	0.48	0.552
C1	0.18	0.193
C2	0.32	0.33

Calculator

Creatinine concentration= (0.552-0.48)/(0.16-0.129)*169=566.9677 micromol/l

Table 2

A2-A1	Sample and creatinine concentration	Result Interpretation	Calculation of eGFR for sample A	Creatinine test for normal reference =44-105 micromol/l C1=30-45micromol/l c2=87-120micromol/l
Std	0.031
Sample A	0.072	45.66	Normal		0.63
C1	0.13	237.76	High		0.7
C2	0.01	182.89	High		0.53

Graphical result

Compare and contrast

The diagnosis test like haemolysis, bilirubin, and lipid is done with the creatinine test. This method and the test are always subjected to interferences against the high levels of substances reducing the substances (Wiles et al. 2019).

Test 2

Glucose test using glucose oxidase-peroxidase (GOD-POD) method

Introduction

The blood sugar levels in the body are regulated by the hormones, glucagon, and the insulin hormone, which act oppositely. The hormones are secreted by the cells of islets present in the pancreas, and hence it is referred to as the pancreatic hormone. Thus, when the blood glucose levels increase to a certain amount, hormones then act efficiently. It triggers the glucose to form glycogen (Iwatni et al. 2020). Similarly, when the glucose level is increased the hormones trigger to secrete more glucose from the liver cells and increase the blood glucose level in the body. Thus, it proves a handy measurement to control the blood glucose concentration in the body.

Glucose concentration method

(Source: Iwatni et al. 2020)

The determination of the blood glucose is used in the clinical test used to determine the glucose for diabetes patients. Hyperglycemia, hyperthyroidism, neoplasm, and cortical hyper-function-like disorders can be checked by this process. It is one of the most frequently performed tests in the laboratory (Ichimura et al. 2021). The estimation of the plasma glucose method is treated by the GOD-POD method system. That diagnosis is very fruitful as it is very simple, single step, and done rapidly.

Principle of GOD-POD method

In this process, glucose is oxidized by an enzyme-like GOD to give D-gluconic acid and hydrogen peroxide. In the presence of enzyme peroxidase, hydrogen peroxide oxidizes to phenol which again combines with 4-Aminoantipyrine to give the result quinone imine dye which is red coloured (Bhatt et al. 2021). The colour intensity is directly proportional to the glucose concentration.

Glucose+oxygen+water=Gluconic acid+hydrogen peroxide

2H2O2+4-aminophenazone +phenol=quinone imine+4H20

Materials procedure and precaution

Materials Procedure	Precautions
Samples	? Do not insert the pipette into the mouth. ? R1 contains Sodium azide ? Do not contact directly with the substances like mucous membranes. it can cause skin inflammation and eye problems.
Pipettes
Water Bath
Maintaining the temperature at 37 degrees centigrade
PPE
Timer
Distilled water
Test tubes
Reagent 1
Reagent 2
Cuvettes
Clean towels
Glucose standard
Marker pen

Procedure

• Has to set up the water bath at 37 degrees centigrade
• The wavelength of the spectrophotometer has to be at 500 nm.
• Has to label the five test tubes as Blank (b), Control(c1), standard(Std) , Control(C2) and Sample(B).
• Warm the test tubes at 37 degrees centigrade.
• Take the pipette of 20 micro-litres of controls standard and the 20 micro-litres of water into the tube blank (Shinataroet al.2018).
• Add 2mls of reagent working into all the test tubes.
• I have to mix the contents for 10 minutes at 37 degrees centigrade.
• Calibrate the spectrophotometer.
• Has to transfer the standards, samples, and controls into the cuvettes and has to read the absorbance.
• Record the results.
• Clean all the surface area.
• Rinse all the tubes and put the cuvettes down the running water with the sink and keep them on the bench.

 Graph showing the glucose concentration

(Source: Bhatt et al. 2021)

Interpretation of the results

Table 1-Standard Concentration of glucose is 5.63mmol/L

Absorbance	Sample	Result interpretation	Glucose test normal reference ranges=3.5-5.0mmol/L(Fasting) Random=3.5-8.0mmol/L C1=2=3.5mmol/L C2=3.5-8.2mol/L
Std	0.338
Sample B	0.14	13.59		High
C1	0.22	3.19		Normal
C2	0.55	22.11		High

Graphical result

Limitations of the Tests

There should be a minimum detection limit of 5 mg/dl and the maximum range should be 500mg/dl. The presence of haemolysis, cholesterol, bilirubin, and so on samples in the patients leads to abnormal results (Garcia et al. 2019). The substances, which are reducing like creatinine, uric acid, and ascorbic acid are reacted with water, and can thus decrease the levels of glucose.

Reference

Bargnoux, A.S., Kuster, N., Cavalier, E., Piéroni, L., Souweine, J.S., Delanaye, P. and Cristol, J.P., 2018. Serum creatinine: advantages and pitfalls. J Lab Precis Med, 3(8), p.71.

Bhatt, M.P., Rai, N., Pokhrel, S., Acharya, P., Marhatta, S.B., Khanal, D.P., Nagila, A. and Chataut, P.D., 2021.Standardization of Visible Kinetic Assay for the Estimation of Plasma Glucose by Glucose Oxidase and Peroxidase Method. Journal of Manmohan Memorial Institute of Health Sciences, 7(1), pp.49-59.

den Elzen, W.P., Cobbaert, C.M., Gunnewiek, J.M.K., Bakkeren, D.L., Van Berkel, M., Frasa, M.A., Herpers, R.L., Kuypers, A.W., Ramakers, C., Roelofsen-de Beer, R.J. and Van Der Vuurst, H., 2018. Glucose and total protein: unacceptable interference on Jaffe creatinine assays in patients. Clinical Chemistry and Laboratory Medicine (CCLM), 56(8), pp.e185-e187.

García?Ponce, Á.L., Martínez?Poveda, B., Blanco?López, Á., Medina, M.Á. and Quesada, A.R., 2019. Not all has been said about glucose oxidase/peroxidase: New pedagogical uses for a classical and robust undergraduate laboratory experiment. Biochemistry and Molecular Biology Education, 47(3), pp.341-347.

Ichimura, S., Kakita, H., Asai, S., Mori, M., Takeshita, S., Ueda, H., Kondo, T., Ohashi, W., Okumura, A. and Yamada, Y., 2021. Acetaminophen elevates unbound bilirubin levels by the glucose oxidase?peroxidase method. Pediatrics International, 63(9), pp.1069-1074.

Iwatani, S., Yamana, K., Nakamura, H., Nishida, K., Morisawa, T., Mizobuchi, M., Osawa, K., Iijima, K. and Morioka, I., 2020. A novel method for measuring serum unbound bilirubin levels using glucose oxidase–peroxidase and bilirubin-inducible fluorescent protein (UnaG): No influence of direct bilirubin. International journal of molecular sciences, 21(18), p.6778.

Küme, T., Sa?lam, B., Ergon, C. and Sisman, A.R., 2018. Evaluation and comparison of Abbott Jaffe and enzymatic creatinine methods: Could the old method meet the new requirements?. Journal of clinical laboratory analysis, 32(1), p.e22168.

ShintaroIchimura, M.D., Asai, S., Mori1MD, M., Satoru Takeshita, M.D., Kondo, H.U.M.T. and Kakita, H., Acetaminophen elevates unbound bilirubin levels by the glucose oxidase-peroxidase method.

Syme, N.R., Stevens, K., Stirling, C., McMillan, D.C., and Talwar, D., 2020.Clinical and analytical impact of moving from Jaffe to enzymatic serum creatinine methodology. The Journal of Applied Laboratory Medicine, 5(4), pp.631-642.

Wiles, K., Bramham, K., Seed, P.T., Nelson-Piercy, C., Lightstone, L. and Chappell, L.C., 2019. Serum creatinine in pregnancy: a systematic review. Kidney international reports, 4(3), pp.408-419.