Pointe 180 QT
Mineral Determination
Skills = 10 points
Objectives:
Upon completion of this exercise, the student will be able to:
1. Determine magnesium and calcium concentrations by colorimetric methods, as available.
2. Determine phosphorus concentrations by absorption methods, as available.
3. Explain the clinical significance of abnormal magnesium, calcium, and phosphorus values.
4. Discuss commonly employed automated and manual methods for determining magnesium, calcium, and phosphorus and be able to explain the principle of each method.
Materials:
a. Magnesium (Xylidyl Blue) reagent 1 and 2 ( Reorder # HM929-120)
b. Calcium (o-Cresolphthalein) reagent 1 and 2 (Reorder # C7503-120)
c. Inorganic Phosphorus reagent (Reorder # P7516-120)
d. Test tubes
e. Rack
f. Timer
g. Pointe 180 QT instrument
h. Pointe 180 Chemistry Calibrator (Reorder # C7506-50)
i. Controls
References:
1. Pointe 180 QT Magnesium- XB package insert
2. Pointe 180 QT Calcium- package insert
3. Pointe 180 QT Inorganic Phosphorus- package insert
Discussion:
By definition, electrolytes are ions capable of carrying an electric charge. Mineral ions such as calcium, phosphorus, magnesium, copper, zinc, etc., are also capable of carrying an electric charge and are therefore also considered to be electrolytes.
Magnesium
Magnesium is a co-factor in reactions using ATP, such as carbohydrate metabolism, muscle contraction, and blood coagulation. Magnesium also assists in neuromuscular transmission, and in maintenance of the intracellular potassium concentration.
Increased serum magnesium
a. Renal failure
b. Diabetic acidosis
c. Addison’s Disease
d. Vitamin D intoxication
Decreased serum magnesium
a. Reduced intake
b. Decreased absorption
c. Increased excretion due to renal, endocrine, or drug induced conditions
Expected Values
1.6-2.6 mg/ dl
Methods of Magnesium Determination:
a. Atomic Absorption Spectrophotometry – most accurate and reliable assay.
b. Colorimetry – The three most common methods for measuring magnesium are colorimetric, either using methylthymol blue, formazan dye, Eriochrome Black T, calmagite, or Xylidyl Blue (Magon).
Principle of Reaction:
Serum magnesium ions react with Xylidyl Blue in an alkaline solution to produce a red complex that is measured spectrophotometrically. The intensity of the color produced is directly proportional to magnesium concentration.
Specimen Collection and Handling:
1. Use fresh, unhemolyzed serum or heparinized plasma.
Procedure:
1. Turn the Pointe 180 ON.
2. Prepare the controls by following the package insert.
3. Reconstitute the Pointe 180 chemistry calibrator by adding 5.0 ml of diluent to the lyophilized serum. Gently invert the vial intermittently over a period of 20 minutes. The calibrator is stable for seven days when stored at 2-8 0C.
4. Prior to use, gently invert the chemistry calibrator 5-10 times.
5. From the Pointe 180 main menu, select “Manage Test” then select “Edit Test”.
6. Highlight the test to edit- “Magnesium”, then select test. If you do not see the test you need to edit, use the > to forward to next page.
7. Confirm that “Magnesium” appears at the top of the screen, then review the “Standard Conc” box to confirm the value matches the value on the test package insert and /or lab report sheet.
8. If the value matches, hit “Exit”, then “Exit” again.
9. If the value does not agree with the calibrator (standard) value, touch the box with the red color.
10. Select “Clear”, then type in the new value based off the test package insert or the lab report sheet, then hit “Enter”.
11. Hit “Exit”, then hit “Exit” again.
12. Prepare the Magnesium working reagent (Magnesium reagent) by adding equal volumes of R1 and R2 reagents. Once prepared, the working reagent is stable for 48 hours at 18- 25 OC. The solution should be blue.
13. Label test tubes as “Blank”, “CS” (CalibratorStandard), “C1”(Control 1),” and “C2”(Control 2). Label the tubes around the lip of the test tube. Do not label in a vertical fashion.
14. Pipet 1.0 ml of magnesium working reagent to each tube.
15. Pipette 0.01ml (10 uL) chemistry calibrator to the “C” (Calibrator) tube. Mix. The solution should be a fushia color.
16. Pipette 0.01ml (10 uL) chemistry control 1 to the “C1” tube. Mix. The solution should be a fushia color.
17. Pipette 0.01ml (10 uL) chemistry control 2 to the “C2” tube. Mix. The solution should be a fushia color.
18. Place the “Blank,” “C”, “C1”, and “C2” tubes in the heat block to incubate for 3 minutes.
19. From the main menu, select “Run Test”, select “magnesium” by using the > button to scroll down to locate test 35 (magnesium). Note- you can select tests by pressing with either your finger or the tip of a pen or pencil.
20. Screen will display magnesium 545/630 “Referencing air”, then “Print full header”, select “YES”
21. When the header prints, confirm that the calibrator/standard value matches the test package insert and/or the lab result sheet.
22. Next prompt will ask whether to “Use stored blank”, select “No”.
23. “Referencing air” will be displayed again, then
24. P place the “Blank’ into the Pointe 180 measurement well, making sure to insert the tube all the way down in the well.
25. incubator for 3 minutes.
26. Select Magnesium test parameters by pressing RUN TEST
a. Then use > button to scroll down to locate test 35 (magnesium)
b. Use the appropriate arrows to highlight magnesium, then select.
27. The Pointe 180 QT will then reference air and display “USE STORED BLANK”. If “NO” is selected the blank tube should be read containing reagent.The absorbance of the blank will be displayed- note on lab report sheet. An audible beep will sound once the Pointe 180 has read the tube. At “Remove tube” prompt, remove the blank. At the “Use stored curve” prompt, hit “Discard”.
28. The Pointe 180 will now prompt for the standard/calibrator. Insert the tube labeled as “C”. The screen will display the absorbance and concentration, note on lab report sheet. Then remove “C” tube.
29. A curve will be displayed- verify the curve is linear, then select “Accept”.
30. The Pointe 180 will now prompt for sample 1- this will be the first control- “C1”. Insert C1, then record results. Repeat with C2. If controls are in range, continue with patient samples.
31. To enter the calibration mode press “DISCARD” in response to the “USE STORED CURVE” prompt.
32. Verify that the stored standard value matches the value of the standard being used. This value is found on the lab report sheet.
33. Pipette 0.01ml (10 uL) calibrator to the “S” (Standard) tube. Mix.
34. Allow tube to incubate at 37oC for 3 minutes. Make sure to push the tube solidly down in the well. Following the 3 minute incubation, follow the screen prompt.
35. Read and record absorbance of all tubes. An audible beep will sound once the Pointe 180 has read the tube.
36. To store factor in the Pointe 180 QT, press ACCEPT.
37. Pipette 0.01ml (10 uL) of patient sample into 1.0 ml of magnesium working reagent control (C1) and control (C2) to the tubes with the working reagent. Mix.
38. Allow tubes to incubate at 37oC for 3 minutes. Following the 3 minute incubation, insert the sample in the measurement well. follow the screen prompt.
39. Read and record absorbance and concentration of patient sample.
40.
41. Read and record absorbance of all tubes.Once done with the analysis of all specimens, hit “QUIT”.
Procedure Notes
1. Hemolized samples should not be used because the red cells contain twice the magnesium concentration as serum. Hemolized samples would falsely elevate the results.
2. Grossly icteric or lipemic samples should be avoided.
3. Certain drugs and other substances are known to influence circulating magnesium levels.
Calcium
Calcium is the most abundant mineral in the human body, most of which (98%) is present in the
skeleton. One half of the remaining calcium is found in extracellular fluid and the rest in a variety
of tissues. Calcium plays a very important role in skeletal mineralization, and it is also vital for
basic physiologic processes such as blood coagulation, neuromuscular conduction, and for the
maintenance of normal muscle tone.
Increased serum calcium
a. Hyperparathyroidism
b. Bone metastases/ malignancies
c. Multiple myeloma
d. Vitamin D intoxication
e. Paget’s disease
Decreased serum calcium
d. Tetany/Convulsions
e. Hypoparathyroidism
f. Vitamin D deficiency/ malabsorption of vitamin D
g. Chronic renal disease
Expected Values
8.5- 10.5 mg/ dl
Methods of Calcium Determination:
a. Atomic Absorption Spectrophotometry – most accurate and reliable assay.
b. Colorimetry – The three most common methods for measuring calcium are colorimetric, either using o-cresolphthalein, or Arsenzo III dye.
Principle of Reaction:
Calcium reacts with o-Cresolphthalein complex in an alkaline solution to produce a purple color that is measured spectrophotometrically. The purple color absorbs at 570 nm (550-580nm). The intensity of the color produced is directly proportional to calcium concentration.
Specimen Collection and Handling:
1. Use fasting, fresh, non-hemolyzed serum.
2. Remove serum from clot as soon as possible as red cells can absorb calcium.
Procedure:
1. From the Pointe 180 main menu, select “Manage Test” then select “Edit Test”.
2. Highlight the test to edit- “Calcium”, then select test.
3. Confirm that “Calcium” appears at the top of the screen, then review the “Standard Conc” box to confirm the value matches the value on the test package insert and /or lab report sheet.
a. If the value matches, hit “Exit”, then “Exit” again.
b. If the value does not agree with the calibrator (standard) value, touch the box with the red color.
i. Select “Clear”, then type in the new value based off the test package insert or the lab report sheet, then hit “Enter”.
ii. Hit “Exit”, then hit “Exit” again.
4. Prepare the Calcium working reagent by adding equal volumes of R1 and R1 reagents. The solution should be a yellow-orange color. Let stand for twenty minutes at room temperature before use. Once prepared, the working reagent is stable for 2 weeks refrigerated.
5. Label test tubes as “Blank”, “C” (Calibrator), “C1”(Control 1), and “C2”(Control 2). Label the tubes around the lip of the test tube. Do not label in a vertical fashion.
6. Pipet 1.0 ml of calcium working reagent to each tube.
7. Pipette 0.02ml (20 uL) chemistry calibrator to the “C” (Calibrator) tube. Mix. The solution should be a fushia color.
8. Pipette 0.02ml (20 uL) chemistry control 1 to the “C1” tube. Mix. The solution should be a fushia color.
9. Pipette 0.02ml (20 uL) chemistry control 2 to the “C2” tube. Mix. The solution should be a fushia color.
10. Allow the “Blank,” “C”, “C1”, and “C2” tubes to sit at room temperature for 3 minutes.
11. From the main menu, select “Run Test”, select “calcium”- test 11.
12. Screen will display calcium 570 “Referencing air”, then “Print full header”, select “YES”
13. When the header prints, confirm that the calibrator/standard value matches the test package insert and/or the lab result sheet.
14. Next prompt will ask whether to “Use stored blank”, select “No”.
15. “Referencing air” will be displayed again, then place the “Blank’ into the Pointe 180 measurement well, making sure to insert the tube all the way down in the well.
16. The absorbance of the blank will be displayed- note on lab report sheet. An audible beep will sound once the Pointe 180 has read the tube. At “Remove tube” prompt, remove the blank. At the “Use stored curve” prompt, hit “Discard”.
17. The Pointe 180 will now prompt for the standard/calibrator. Insert the tube labeled as “C”. The screen will display the absorbance and concentration, note on lab report sheet. Then remove “C” tube.
18. A curve will be displayed- verify the curve is linear, then select “Accept”.
19. The Pointe 180 will now prompt for sample 1- this will be the first control- “C1”. Insert C1, then record results. Repeat with C2. If controls are in range, continue with patient samples.
20. Pipette 0.02ml (20 uL) of patient sample into 1.0 ml of magnesium working reagent Allow tubes to incubate at room temperature for 3 minutes. Following the 3 minute incubation, insert the sample in the measurement well.
21. Read and record absorbance and concentration of patient sample.
22. Once done with the analysis of all specimens, hit “QUIT”.
1. Using plastic vessels, prepare the working reagent (Calcium reagent) by adding equal volumes of R1 (Color) and R2 (Buffer) reagents. Label this as the “working reagent.” Let stand for twenty minutes at room temperature before use. Once prepared, the working reagent is stable for 2 weeks refrigerated.
2. Label test tubes as “Blank”, “S” (Standard), “C1” (Control 1), and “C2” (Control 2). Label the tubes around the lip of the test tube. Do not label in a vertical fashion.
3. Pipet 1.0 ml of working reagent to each tube.
4. Select Calcium test parameters by pressing RUN TEST,
a. Then use > button to scroll down to locate test 11 (calcium)
b. Use the appropriate arrows to highlight calcium, then select.
5. The Pointe 180 QT will then reference air and display “USE STORED BLANK”. If “NO” is selected the blank tube should be read containing reagent. Insert Blank.
6. To enter the calibration mode press “DISCARD” in response to the “USE STORED CURVE” prompt.
7. Verify that the stored standard value matches the value of the standard being used. This value is found on the lab report sheet.
8. Pipette 0.02ml (20 uL) calibrator to the “S” (Standard) tube. Mix.
9. Allow tube to incubate at room temperature for 3 minutes. Following the 3 minute incubation, follow the screen prompt.
10. Read and record absorbance of standard tube.
11. To store factor in the Pointe 180 QT, press ACCEPT.
12. Pipette 0.02ml (20 uL) of control (C1) and control (C2) to the tubes with the working reagent. Mix.
13. Allow tubes to incubate at room temperature for 3 minutes. Make sure to push the tube solidly down in the well. Following the 3 minute incubation, follow the screen prompt.