Clinical chemistry: HPMT 353
Title and introduction: Stanbio direct Creatinine liquid color
The purpose of the experiment is quantitative enzymatic determination of creatinine in serum or urine.
Principle of the procedure: Creatinine is a chemical waste molecule that is generated from muscle metabolism. Creatinine is produced from creatine, which is a molecule of major importance for energy production in muscles. Approximately 2% of the body’s creatine is converted to creatinine every day. Creatinine is transported through the bloodstream to the kidneys. The kidneys filter out most of the creatinine and dispose of it in the urine. Because the muscle mass in the body is relatively constant from day to day, the creatinine level in the blood normally remains essentially unchanged. As the kidneys become impaired for any reason, the creatinine level in the serum or urine will rise due to poor clearance by the kidneys. Abnormally high levels of creatinine thus warn of possible malfunction or failure of the kidneys. Serum creatinine can also transiently rise after ingestion of large amount of dietary meat. Enzymatic methodology is the accurate measurement of creatinine, especially for neonates, pediatrics, and hematology units. The enzymatic assay for creatinine involves a series of coupled enzymatic reactions including creatininase enzymatic conversion of creatinine into the product creatine which itself is converted to sarcosine by creatine amidinohydrolase (creatinase), followed by oxidation of sarcosine by sarcosine oxidase (SOD) producing hydrogen peroxide. In the presence of peroxidase (POD) the hydrogen peroxide is quantified at 550 nm by the formation of a colored dye.
Material and method
1. Reagent 1 (R1) lot # 18243 Exp date 2015/12
2. Reagent 2 (R2) lot # 18244 Exp date 2015/12
3. SER-T-Fy1 (control 1) lot # 23844 expiration date 2017/06
4. SER-T-Fy2 (control 2) lot # 07332 expiration date 2015/01
5. Sample # 4
5 tubes were labeled as blank, standard, sample, control 1, and control 2.
540ul of reagent one was added to all test tubes.
12ul of standard, sample, water was added to tubes label standard, sample, and water, respectively.
Tubes mixed well and incubate for 5 min at 37C
After incubation first absorbance of spectrophotometer was recorded
180ul of reagent 2 was added to all test tubes, mix well and incubate for 5 min at 37C.
After incubation second absorbance of the spectrophotometer was recorded.
Standard (Std) Sample (Au) Control 1 Control 2
Reading 1 0.001 0.015 0.031 0.043
Reading 2 0.102 0.035 0.055 0.100
K= 0.754Standard concentration= 5 mg/dL
Creatinine (mg/dL)= ((Au2-Au1)* K/(Std 2-Std1)*K) *Std concentration
= ((0.035-0.015)* 0.754/(0.102-0.001)*0.754)* 5 — = 0.99 mg/dL
Control 1 =((0.055-0.031)*0.754/(0.102-0.001)*0.754)*5–= 1.19 mg/dL
Control 2 =((0.100-0.043)*0.754/(0.102-0.001)*0.754)*5–= 2.82 mg/dL
The patient creatinine level was 0.99 mg/dL, which is within the normal range for male and female. Normal range for men (0.9-1.5) mg/dL, female normal range (0.7-1.4) mg/dL. My control 1 was 1.19 mg/dL, which is within the control limit (0.9-1.5) mg/dL, so my control is in and I will accept it. Control 2 was (2.82) mg/dL, which is not within the control limit (3.6-6.6) mg/dL, so I have to repeat control 2 and repeat the patient sample as well, because I can’t accept the patient result if one or both of my controls is out.
Stuff that can interfere with the results is the use of fluoride or ammonium heparin sample, or clotted sample. Ascorbic acid over 200 mg/dL, hemoglobin over 500 mg/dL, bilirubin-conjugate over 32 mg/dL, some drugs can interfere with creatinine methodologies level as well. Stuffs that’s did go wrong, pipetting the correct and exact amount of the material solutions, zero the spectrophotometer at over/lower than the 540nm, all may cause falsely decrease/ increase results. The addition of the second reagent was important step; any neglected of adding it may cause false results.
The test didn’t work because control 2 is out of range, so I can’t accept the patient result. Control 2 was out of range, and this due to the fact that control 2 was expired, and definitely affecting my result. This method of measuring creatinine is linear to 30 mg/dL; any sample over 30 mg should be diluted 2 fold. The sensitivity of this procedure was (0.04 mg/dL) which is very good, this is mean that this test is detected really small amount of analyte up to 0.04 mg. this method is specific for detection of ceatinine and have no interference was observed for ascorbic acid up to 200 mg/dL, hemoglobin up to 500 mg/dL, bilirubin-conjugate up to 32 mg/dL. Creatinine test may be used to diagnose or monitor the statue of some diseases and conditions like, kidney pain, kidney failure of infection, kidney dysplasia, hemolytic uremic syndrome, and hypertension kidney disease. Increase creatinine level suggest abnormal kidney function, these include, damage to or swelling of blood vessels in kidney, bacterial infection, prostate disease, urinary track infection, kidney stone, and complications of diabetes. Some of the technical problems that occurred during testing was pipetting the correct amount of analyte, transferring of the solution from the tubes to the cuvette, and misplaced the cuvette in the spectrophotometer that leads to wrong reading of the absorbance.