17-OH CORTICOSTEROIDS TROUBLESHOOTING GUIDE
(b1) Thorough mixing in color development step (before and after incubation) is essential.
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17-OH CORTICOSTEROIDS TROUBLESHOOTING GUIDE | ||
| PROBLEM | CAUSE | POSSIBLE REMEDY |
| 1. Low Standard Curve) | (a) Improper aspiration | (a) See General Problems 3a and 7a |
| (b) Low incubation temp | (b) The water bath must be at 60°C. Check it with a good thermometer. | |
| (c) Inaccurate pipetting | (c) See General Problems 3c | |
| (d) Alcohol wash concentration greater than 5% | (d) Make sure that the alcohol wash is properly formulated. | |
| (e) Elution Solvent alcohol content too low | (e) Left over water due to improper aspiration might lower the Elution Solvent alcohol concentration. Make sure that the aspiration step is not skipped. When Elution Solvent is made in your lab, check the percentage by G.C. or density (refer to USP). | |
| 2. Low Control Value | (a) No salt is added | (a) Urine and control recoveries are affected significantly by the salt content. Make sure approx. 2.2 gm of salt is added in all the tubes. |
| (b) Alcohol wash concentration greater than 5% | (b) See Step 1d | |
| (c) Elution solvent Alcohol content too high | (c) When Elution Solvent is made in your lab, check the percentage by G.C. or density (refer to USP). | |
| (d) Decomposition of corticosteroids | (d) Higher Sodium Hydroxide concentration (more than 0.1N) or improper cooling of the sample (during Reagent Blank and color reagent addition) may be suspected. | |
| 3. High Control Value | (a) Improper aspiration | (a) Low concentration of alcohol in Elution Solvent affects desorption of "free" steroids more than conjugated steroids. |
| (b) Slow flow rate | (b) If column takes more than 5 minutes for urine flow, the resultant values may be on the high side. To correct the problem see Step 2 in general Problems section. | |
| 4. Erratic Standard Curve and Control Value | (a) Erroneous pipetting | (a) All pipetting steps must be carefully done.See General Problems 3c |
| (b) Inconsistent mixing | (b) Thorough mixing of eluate prior to pipetting the 2.0 mL
aliquot is essential. See General
Problems 3d. (b1) Thorough mixing in color development step (before and after incubation) is essential. |
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| (c) Improper reading | (c) Make sure the cuvette is thoroughly cleaned. Wait until the reading is stable before recording. (Air bubbles tend to cause erratic readings, therefore, wait until the bubbles have dissipated before recording. | |
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