Sharq Elneil College 4th Year General Clinical Chemistry-I Practical Manual 2022-2023 PDF

SHARQ ELNEIL COLLEGE SCHOOL OF MEDICAL LABORATORY SCINCES DEPARTMENT OF CLINICAL CHEMISTRY 4th Year Practical Manual Dr. ZUHAIR YAGOUB ABDELKARIM Medical laboratory Science, Clinical Chemistry Department. Gener...

SHARQ ELNEIL COLLEGE SCHOOL OF MEDICAL LABORATORY SCINCES DEPARTMENT OF CLINICAL CHEMISTRY 4th Year Practical Manual Dr. ZUHAIR YAGOUB ABDELKARIM Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 1  Student Name……………………………………..  University No:………………………… Third Edition by: Dr. Zuhair Yagoub Abdelkarim Contents  COLORIMETER & SPECTROPHOTOMETER (Calibrations)  COLORIMETER & SPECTROPHOTOMETER (Selection of Filters)  COLORIMETER & SPECTROPHOTOMETER (Wave length Accuracy)  COLORIMETER & SPECTROPHOTOMETER (Base line Stability & Stray Light Detection)  USES & MAINTANCE OF BLANCES  AUTOMATIC PIPITES CALIBRATION  PREPARTION OF WORKING STDs FROM STOCK STD.  FLAME PHOTOMETER (Preparation of STDs)  METHOD OF MEASURING SP.G (Weighting Technique)  PREPARTION OF WORKING STDs FROM STOCK STD.  TURBIDIMETRY MESURING  PROTEIN STANDARD CURVE (Number of points formula)  GLUCOSE STANDARD CURVE (Number of points formula) Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 2 Practical NO (1) Colorimeter & spectrophotometer Date-------------------------------------- CALIBRATION OF COLORIMETER AIM:  To know how to: prepare low concentration from high concentration.  To: apply Beer and Lambert low. REAGENT Methyl red solution of conc. ( g/dl) PROCEDURE:  Make several solutions of known concentration including one of just water to use as blank using the formula RV/O where: R: Required Concentration V: Required Volume O: Original Concentration  Put each one in the cuvette and, insert it in the colorimeter and take reading as following" Concentration Absorbance ( con) (O.D) Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 3  If the method follows, beer& Lambert low the absorbance proportional to concentration.  On a graph paper, make graph of concentration (con) VS absorbance (O.D). __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 4  Practical NO ( 2 ) Date----------------------------------- COLORIMETER (Selection of Filters) AIM: To: verify the complementary relationship between the wavelength absorbed by colored solution and the wavelength of the filters. Reagent: Methyl red solution, Potassium permanganate solution Principle: When light path through color solution characteristic portion of mixed wavelength is absorbed, the reaming light will then assume the complementary to the wave length absorbance. Procedures 1. Use the filter (wavelength430nm) and distilled water to adjust colorimeter zero. 2. Pour in the colored solution into the cuvette. 3. Insert the cuvette in the colorimeter. 4. Compare the color of the solution with the color of the filter, which gives the highest absorbance. 5.. Repeat the previous steps with the rest of the filters. 6. Repeat the previous steps with all the colored solution to an end. Filter wavelength Filter color Solution color (nm) 430 490 520 540 580 600 Conclusions: Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 5 __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ _________________________________________________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 6  Practical NO ( 3 ) Date----------------------------------- COLORIMETER (Wave length Accuracy) _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ ___________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 7  Practical NO ( 4 ) Date----------------------------------- COLORIMETER & SPECTROPHOTOMETER (Base line Stability & Stray Light Detection) _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ ___________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 8  Practical NO ( 5 ) Date----------------------- Uses and Maintenance of Balances AIM To obtain accurate and reliable mass measurements with a single-pan analytical balance, at the same time, prevent damage to the balance. RULES FOR USES ANALYTICAL BALANCES: 1. Close the balance door, while weighing an object, in order to prevent air currents from disturbing the reading. When finished, the operator should close the balance door to prevent dust and dirt from entering the balance. 2. Only glass, ceramic, metal or plastic objects and containers shall been place in direct contact with the balance pan. 3. Do not handle objects to be weigh with bare hands. Moisture, grease and dirt on your fingers will affect the weight of the objects. 4. To be weigh accurately, all objects must be at room temperature. A warm object sets up convection currents inside the balance field, which will make an object appear lighter than it really is. In addition, warm air inside the field is less dense than the air that it displaces and this leads to a negative determinate error. 5.Never weigh chemicals directly in contact with the balance pan. Use containers such as beakers, flasks and weighing bottles. 6. All objects and materials that have removed from desiccators will absorb moisture and thereby gain weight. It is therefore good practice to record weights after identical time intervals. By using this technique, it is possible to minimize the effect of moisture absorption. 7. The use of weighing paper must been avoid when using an analytical balance. 8. Do not spill chemicals inside the balance enclosure. If a spill occurs, clean it up immediately. Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 9  Practical No ( 6 ) Date......................... Automatic pipits Calibration Pipettes are a type of lab equipment used to measure and transfer very small volumes of liquid. Accuracy and precision in pipette measurement are necessary as any discrepancy in volumes dispensed can affect the results of an experiment. To ensure accuracy, it is necessary to check the pipette calibration every few months. The calibration process helps to check whether or not the equipment is dispensing the proper volumes so it can be fixed if it is not. 1- Gather the necessary materials. To check the calibration of a pipette, you will need the pipette, pipette tips, distilled water, a beaker, a thermometer, a balance, and weigh boats. The balance needs to be specific to micrograms to calibrate micropipettes with a maximum of 1 µL.  You won’t need more than 5 mL of water. Fill the beaker with the water.  Make sure the pipette tips are the correct tips that properly fit the pipette. 2- Measure the temperature of the distilled water. Place the thermometer in the water and leave it for at least one minute. If the red line on the thermometer is still moving, leave it for another minute. After a minute, record the temperature in a notebook. Remove the thermometer and dry it off when you are finished.  Knowing the temperature of the water is important for the calculations that will be done to check the calibration. Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 10  3- Place the weigh boat on the balance and zero it out. Ideally, you will use a balance that has doors on it and is an isolated chamber. Place the weigh boat in the chamber and close the doors. If you don’t have doors, simply place the weigh boat on the balance. Press the “Zero” or “Tare” button and wait until the balance reads zero.  Zeroing the scale subtracts the weight of the plastic boat and allows you to measure just the weight of whatever you put into the boat. 4- Prepare the pipette for calibration. Wipe down the pipette with ethanol to kill any contaminants before beginning and make sure nothing is clogging the tip end of the pipette. Place the appropriate tip on the end and set the volume that you want to test.  For calibration, test both the smallest volume and the largest volume the pipette can dispense. 5- Pre-rinse the tip before calibration. Press the button to the first stop and stick the tip into the distilled water so that about 2 mm is immersed in the liquid. Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 11  Release the button to aspirate some liquid and then dispense the liquid again by pushing the button down. Repeat this three times to pre-rinse the tip.  Push the button to the second stop to dispense any liquid that remains in the tip and remove the pipette from the water. 6- Aspirate the calibration volume. With the tip out of the liquid, push the button down to the first stop. Place about 2 mm of the tip into the distilled water and release the button to aspirate the liquid into the tip. Wait about 1 second before removing the tip from the water.  Make sure the tip is completely immersed during the whole aspiration process. You don’t want any bubbles in the tip or the results will be wrong. 7- Dispense the liquid into the weigh boat on the scale. Place the tip against the bottom of the weigh boat and push the button down to the first stop. Move to another spot slightly away from the water and push the button to the second stop. Keeping the button pressed, lift the tip away from the weigh boat.  Keep the tip on the pipette as you will use it again to weigh out more calibration tests. MAke sure the tip does not touch anything.  Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 12  8- Record the weight on the balance. Close the door if you are using a chamber balance. Wait until the numbers stop changing. Record this number in your notebook.  It’s important to wait until the number has stabilized before recording it. Your readings will be incorrect if you don’t wait. 9- Repeat this process to take at least 10 readings. Zero the scale, pre-rinse the tip, aspirate the same volume, dispense the volume, and then record the weight. You want to have multiple recordings of the same volume and then you can average them all together.  You can repeat this same process with different volumes as long as you take multiple readings per volume.  Part2 Calculating the Results 10- Define the formula for calculated volume. The formula for calculating the volume dispensed by the pipette is V = w * Z where w is the weight of the water, Z is the conversion factor based on the density of the water, and V is the calculated volume of how much water was dispensed.  You can find the Z variable by looking up the density of water using the temperature recorded at the beginning of the experiment. Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 13   For example: If the temperature of the water was 23°C, then you will use a Z value of 1.0035 µg/mg. 11- Average the replicates together. You should have weighed the volume of water dispensed by the pipette at least 10 times. In order to average all of these values together, add them all up and divide by 10. If you did more or fewer trials, add up each trial and divide by the total number of trials.  For example: The ten weights you recorded for a pipette volume of 10µL are as follows: 9.89, 10.01, 10.02, 9.99, 9.95, 10.04, 9.96, 10.01, 9.99, and 9.98.  The average is: (9.89 + 10.01 + 10.02 + 9.99 + 9.95+ 10.04 + 9.96 + 10.01 + 9.99 + 9.98)/10 = 99.84/10 = 9.984 12- Plug the variables into the equation and solve. Once you have determined the proper numbers to use for each variable, you can plug them into the equation and solve for the calculated volume. To solve, simply multiply the average weight of all the trials by the Z value.  For example: V = w * Z = 9.984 * 1.0035 = 10.019 Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 14  13- Calculate the accuracy of the pipette. To calculate the accuracy you can use the equation A = 100 x Vavg/V0, where A is the accuracy of the pipette, Vavg is the average calculated volume and V0 is the value you set the pipette to dispense. Accuracy should be between 99-101%.  If the pipette is properly calibrated, the calculated value should be very close to the actual value you set on the pipette.  For example: A = 100 x Vavg/V0 = 100 x 10.019/10 = 100 x 1.0019 = 100.19%  This particular pipette is properly calibrated. 14- Send the pipette for calibration, if necessary. If your pipette does not pass the calibration test, stop using it for experiments immediately. Pipettes are very delicate and expensive pieces of lab equipment. You cannot fix the calibration yourself so you will need to send them in for proper servicing. Alternatively, some companies will come to your lab and calibrate your pipettes there.  Contact the company that makes your particular brand of pipette for servicing. Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 15 __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 16  Practical No ( 7) Date…………………………. PREPARTION OF WORKING STDs FROM STOCK STD. _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 17  Practical No ( 8) Date…………………………. ESTIMATION OF ELECTROLYTES ESTIMATION OF SODIUM & POTASSIUM BY FLAME PHOTOMETER Aim To determine Na+ and K+ by flame photometer Specimen: Blood collected on heparin. STD: Na+ & K+ STD are prepared together from NaCL & KCl as follow: 1) Na=140m.mol\l. 2) K=5m.mol\l. Preparation of sample & STD: Dilute both the sample, STD 1:100 AS follow: a) Add 0.1ml from the sample to 9.9 ml of D.W. b) Add 0.1 ml from the STD to 9.9ml of D.W. Theory: The purpose of the flame is twofold, chemical bonds are broken to produce atom, and then atoms absorb energy from the flame and enter an excited electronic state. The excited atoms return to the ground state by emitting light energy that is characteristic for that atomic species. Principle: Using compressed air, diluted serum or plasma is sprayed as affine mist of droplets (nebulizer) into anon luminous gas flame which become colored by characteristic emission of sodium or potassium metallic ions in the sample. Light of wavelength corresponding to the metal being measured which selected by alight filter or prism system and allowed to fall on photosensitive detector system. Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 18 Compositions: 1. Nebulizer (atomizer): In this, the sample mixed with air and sprayed to the burner at constant and reproducible rate. Compressed air is used to provide stream of air to draw in and nebulize the sample. 2. Mixing chamber with baffles: In the mixing chamber the atomized sample and fuel are mixed. The baffle deflects any large droplets as waste and allowing only the small droplets to enter the flame. 3. Burner: This converts the metallic ions to uncharged atoms and excited them to emit light 4. Lens and Filter System: Lens focuses the emitted light from the flame and narrow band filter select the wavelength of the metal being measured (Na transmit yellow light of 589nm, K transmit at 767nm). 5. Photosensitive detector system: Is used to convert the emitted light into electric current. Reference Value: 1. Na : 135--------145 mmol\l 2. K: 3.5--------5 mmol\l. Con K _____________ Con of Na_____________ Comment ___________________________________________________________ ___________________________________________________________ ___________________________________________________________ ___________________________________________________________ ___________________________________________________________ ___________________________________________________________ ___________________________________________________________ ___________________________________________________________ ____________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 19  Practical No ( 9) Date…………………………. PREPARTION OF WORKING STDs FROM STOCK STD. _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ _______________________________________ ___________________________ Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 20  Practical No ( 10 ) Date…………………………. Method of measuring S.G r Weighing technique The technique requires the use of an analytical balance capable of weighing to 3dicimal i.e 1mg sensitivity. Procedure: 1) Place on each pan of analytical balance a small beaker containing a few ml of tape water. 2) Using pasture pipette transfer water from one beaker to other until the weight is equal on both sides. 3) -using a dry volumetric pipette adds 10ml of distilled water to left hand beaker. 4) Using a dry volumetric pipette adds 10ml of urine to right hand beaker. 5) Add weight to the left hand pan until once again the weight is equal on both sides. - Total the number of milligram used. - calculate the mass density of urine from the following formula Urine density = 10000+ number of gram used 10000 Example number of gram used =270gramUrine density = 10000+ 270 1000 =1.027g/ml Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 21  Practical No ( 11) Date…………………………. Measurement urine protein By Turbidimetry Method Reagents  3% T.C.A  Protein standard 100mg/dl.  Principle: Trichloroacetic acid(T.C.A)denaturise the urine protein as strong acid and so caused turbidity directly proportional with conc. of protein present in urine sample and can be measured using blue filter(430 nm). Sample: Spot urine sample collected under certain construction (not diluted). Procedure: REAGENT BLANK STD TEST 3% T.C.A 1.5ml 1.5 ml 1.5 ml D.W 0.5 ml ------ ------ W.STD (100 mg/dl.) ------ 0.5ml ----- Urine sample ------ ------- 0.5 ml Mix well; incubate for 5 min at R.T. Read the absorption of TEST and STD against BLANK. At 430 nm Calculation: 𝑂.𝐷 𝑇 Urine protein mg/dl=: × 𝐷𝑓 × 𝑠𝑡𝑑 𝑐𝑜 (Df = 10) 𝑂.𝐷 𝑠𝑡𝑑 Interference factors: Amorphous urate, epithelial cell and casts, for calculation Medical laboratory Science, Clinical Chemistry Department. General Clinical Chemistry-I (4th Year) Page 22 Sensitivity: