CLS 3280Clinical Chemistry

Diabetes Testing Lab Exercise

This week, you will perform quality control testing and testing for diabetes mellitus. You must work in groups of three, share equipment, and share some test results. Accuracy of results is important. Unknown Samples: Patient A45 year old woman, known diabetic. Samples provided: serum and EDTA whole blood. Patient B23 year old man, no other information available to the laboratory. Samples provided: serum and EDTA whole blood. Patient Cgirl, 5 weeks old, lethargic. Sample provided: serum only. Test Procedures: You must run tests for glucose, glycohemoglobin, and fructosamine on Patient A and Patient B. You must run glucose testing only on Patient C.. Each student in the group should do one assay, and students may share test results. Perform glucose testing on quality control material, too. Before starting, make sure the Pointe 180 Spectrophotometer is turned on and warmed up. You will be running your tests in batches. This means you will need to set up your test tubes in a batch for one type of test, and complete that work, and then move on to doing a different batch for a different test. For example, to do the glucose batch, you will label clean tubes for all of the samples that need glucose testing performed (quality control and patient samples), add reagent, prewarm the reagent, and then add the correct volume of the correct sample, and follow the rest of the instructions until the testing is completed and you have valid test results.

Glucose Procedure:
Note: If an R flag appears next to the result, the sample should be diluted appropriately with normal saline and rerun. Multiply the printed result by your dilution factor to get your final result. 1. Label one 12mm x 75mm test tube for each control and one for each patient sample (thats five tubes total). 2. You will also need a blank tube containing glucose oxidase reagent (1.0 mL). 3. Place the blank and reagent tubes in the heating block for 3-5 minutes. 4. Add 10 L of sample to the appropriate tube, cover with parafilm, mix, then incubate at 37 C for 3 minutes. 5. Press TEST, 21, ENTER 6. When USE STORED CURVE is displayed press YES, ENTER 7. When prompted, read the blank tube. 8. Read the sample tubes. The printed values are your final results. Write your results in the table below. Write the quality control results on the Levey-Jennings charts provided.

Glucose Patient Results (3 points)

Patient A Result: Glucose: (mg/dL) Patient B Result: Patient C Result:

Glucose Quality Control Results (2 points)

Level 1 Result: Glucose: (mg/dL) Level 2 Result:

CLS 3280Clinical Chemistry

Glycated Hemoglobin Procedure:

A. Hemolysate Preparation 1. Label a 12 x 75 mm tube for each patient hemolysate. 2. Dispense 500 L Lysing Reagent into the tube. 3. Place 100 L of the well-mixed EDTA whole blood sample into the appropriately labeled lysing reagent tube. Mix. 4. Allow to stand for 5 minutes or until complete lysis is evident. B. Glycated Hemoglobin Preparation 1. Label a13 x100 mm glass tube for each patient. 2. Dispense 3.0 mL of Glycohemoglobin Cation-Exchange Resin reagent into each tube. Note: Before use, mix the resin by inverting at least 10 times. 3. Add 100 L of the appropriate hemolysate (from step A3) to the resin reagent in each tube. 4. Position a filter separator in each tube so that the rubber sleeve is approximately 1cm above the liquid level. 5. Place the tubes on a rocker and mix continuously for 5 minutes. 6. Remove the tubes from the rocker. 7. Push the filter separator into the tubes until the resin is firmly packed. 8. Pour each supernatant into its appropriately-labeled 12 x 75 mm tube. 9. Run the test on the spectrophotometer: a. Press the ABS key to select Absorbance Mode b. When the display shows SELECT FILTER, select the 405 nm filter (filter #2), then press the ENTER key. c. When the display shows SELECT DIFF FILT, press the zero key (for no differential), then press ENTER d. When the display prompts for a blank, insert your blank tube. Use deionized water as your blank. e. Read and record the absorbance value for your patient test (supernatant from step #8). This reading is for glycated hemoglobin. C. Total Hemoglobin 1. Label a 12 x 75 mm tube for each patient test. 2. Dispense 5.0 mL deionized water into each tube. 2. Place 20 L of the appropriate hemolysate (from step A3) into the correct tube. Cover with parafilm and mix. 3. Run this test as shown in the box above, using the same wavelength. 4. Read and record the absorbance value for each patient test. This reading is for total hemoglobin. Absorbance Readings and Final Results for Glycated Hemoglobin (4 points) Patient A Patient B Glycated hemoglobin absorbance reading: Total hemoglobin absorbance reading: % Glycohemoglobin: (see calculation*) *Calculate each patients results this way: (Please show your calculations.)(Standard (calibrator) values will be on the board.) 1. For each patient sample, calculate the ratio (R) of glycohemoglobin absorbance to total hemoglobin absorbance. 2. Us this equation to calculate the glycohemoglobin result in percent: Patient Glycohemoglobin (%) = R patient/R standard X Standard Concentration (%)

CLS 3280Clinical Chemistry

Fructosamine Procedure:
1. Label one 12mm x 75mm test tube for each patient sample (thats two tubes total). 2. You will also need a blank tube containing fructosamine reagent (1.0 mL). 3. Place the blank and reagent tubes in the heating block for 3-5 minutes. Run the test on the spectrophotometer: 4. Press the ABS key to select Absorbance Mode 5. When the display shows SELECT FILTER, select the 550 nm filter (filter #5), then press the ENTER key. 6. When the display shows SELECT DIFF FILT, press the zero key (for no differential), then press ENTER 7. When the display prompts for a blank, insert your blank tube. 8. Add 50 L of each patient sample to its pre-labeled tube of reagent, at timed intervals. (Example: time each one and add samples 1 minute apart.) 9. Incubate each tube in the heating block for 10 minutes. 10. After exactly 10 minutes, read the absorbance of each patient sample+reagent tube and then return the tube to the heating block. 11. Incubate for another 5 minutes. 12. Read the absorbances again. 13. Calculate* each patients fructosamine value as shown below. Write your results in the table. Absorbance Readings and Final Results for Fructosamine (2 points) Patient A Patient B First absorbance reading: Second absorbance reading: Fructosamine (mmol/L) *Calculate each patients results this way: (Please show your calculations.)(Calibrator values will be on the board.) A = absorbance A2 sample - A1 sample X A2 Calibrator A1 Calibrator Calibrator Concentration = Fructosamine in sample

CLS 3280Clinical Chemistry Rewrite all results in the appropriate summary tables below.

Summary of Results for Patient A: Test Results for Patient A Glucose Glycohemoglobin Fructosamine Summary of Results for Patient B: Test Results for Patient B Glucose Glycohemoglobin Fructosamine Summary of Results for Patient C: Test Results for Patient C Glucose Questions:

Reference Intervals

Reference Intervals

Reference Intervals

1. Add your Glucose QC results to the Levey-Jennings charts posted in the lab. Next, review the QC results. Are they within +/- 2 standard deviations of the mean? (See posted values.) Can your patient test results be reported to the physician? Why or why not? (1 point)

2. List appropriate reference ranges for each analyte in the summary table above. Which values are abnormal? List them here. Remember to keep track of which values belong to which patient. (3 points)

3. List critical value limits for glucose. Do any of the patient samples have critical glucose values? If so, which ones? What would you do with the patient results that exceed critical value limits? (1 point)

CLS 3280Clinical Chemistry 4. List the most likely diagnosis (as related to carbohydrate metabolism) for each patient, explain your choices, and describe further testing necessary for each one (6 points)

5. Describe, in your own words, the test methods used (see package inserts). (3 points)