CAPÍTULO II FUENTES DE INGRESOS
2.3 FACULTADES PARA ESTABLECER IMPUESTOS
1. Purpose: Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is one of the most common human enzyme deficiencies, in the world.
During G6PD deficiency, the red cells are unable to regenerate reduced Nicotinamide adenine dinucleotide phosphate (NADPH) a reaction that is normally catalyzed by the G6PD enzyme. Since the X chromosome carries the gene for G6PD enzyme, this deficiency mostly affects the males as females are protected by the other normal X chromosome. The two major conditions associated with G6PD deficiency are hemolytic anemias and neonatal jaundice, which may result in neurological complications and death. Screening and detection of G6PD deficiency helps in reducing such episodes, through appropriate selection of treatment, patient counseling and abstinence from disease-precipitating drugs such as anti-malarials like primaquine and other agents and favism.
2. Principle: G6PD in RBCs is released by a lysing agent present in the reagent. The G6PD released catalyzes the oxidation of Glucose-6 phosphate with the reduction of NADP+ to NADPH. The rate reduction of NADP+ to NADPH is measured as an increase in absorbance, which is proportional to the G6PD activity in the sample.
G6P + NADP + ---> 6-Phosphogluconic acid +NADPH + HG6PD + 3. Performance specifications:
Linearity: This method is linear for up to 20 Unit/g of Hb.
4. Primary sample:
4.1. Use only whole blood as specimen
4.2. Collect 2 mL of venous blood in a vacutainer EDTA anticoagulant tube.
4.3. Do not use lysed blood for testing as it may give very high results 4.4. Do not use contaminated/turbid samples for testing.
4.5. Sample materials: Fresh whole blood sample collected in EDTA, heparin or ACD, red cell G6PD in whole blood is reported to be stable for 7 days at 2–8°C, but is unstable in hemolysates freezing is not recommended.
74
5. Type of container and additive: Add 2.0 mL of blood in an EDTA vacutainer tube and shake well for 30 sec.
6. Reagents/Consumables:
L1: G6PDH reagent 5 × 1 mL L2: Starter reagent 10 mL
Reagent preparation: Make up G6PD reagent (L1) with distilled water as per the volume mentioned on the label. This working reagent is stable for 6 hours at RT and at least 5 days when store at 2–8°C. The starter reagent (L2) is ready to use.
Add 1.0 mL of blood to the EDTA added tube and shake well for 30 sec.
7. Instrument: Semi-autoanalyzer Chema.
8. Procedure:
8.1. Wavelength: 340 nm Temperature: 37°C Light path: 1 cm
8.2. Pipette into a clean dry test tube labeled test (T) 8.3. Addition sequence T (mL)
G6PD working reagent (L1) 1.0 Whole blood 0.01
Mix well and incubate for 5–10 min at RT and add Starter reagent 2.0
8.4. Mix well and incubate for 5 min at 37°C and read the initial absorbance A0 and repeat the absorbance reading after every 1, 2 and 3 minutes.
8.5. Calculate the mean absorbance change per minute (ΔA/min) If the G6PD activity is very low the absorbance change per minute will also be very low.
In such cases, read the initial absorbance A1 and read another absorbance A2 exactly 5 min later.
Calculate the mean absorbance change per min.
ΔA/min = A – A2 1 5
9. Reference range: G6PD Activity (U/g Hb) : 6.4 to 18.7 at 37°C 10. Critical/Alert values: Not applicable
11. Safety precautions:
11.1 Handle all samples as potentially infectious
11.2. Handle all reagents with care and avoid contact with eye, mouth and skin
11.3. Do not perform mouth pipette
11.4. Discard used reagents and sample as per disposal procedure
75
12. Potential sources of variability: Lysed blood specimens may give falsely elevated values.
AMYLASE
1. Purpose: Quantitative estimation of the activity of amylase in human serum. The clinical significance of the estimation of amylase lies almost entirely in the diagnosis of acute pancreatitis, in which the enzyme level frequently exceeds more than 10 times the normal values. Some other causes include salivary gland disorder, abdominal disturbances affecting the pancreas and intake of drugs.
2. Principle: Amylase is a hydrolytic enzyme that splits complex carbohydrates such as starch and glycogen to glucose.
Amylase
Starch ---> glucose
The iodometric method is based on the ability of iodine to form a vivid blue color in combination with starch. The byproduct of amylase action may also form colored substances with iodine but at different wavelengths from the characteristic starch-iodine complex. In this method, iodine color reagent is added to the substrate-sample mixture after an incubation period. The greater the amount of amylase activity, the lighter will be the color.
3. Performance specifications:
3.1. Linearity: This method is linear for amylase activity up to 300 Somogyi units/dL in serum.
3.2. Measurement range: This method has a measurement range of 50–300 Somogyi units/dL of amylase activity in serum.
3.3. Sensitivity: The minimum detection limit of amylase in serum by this method is 50 Somogyi units/dL.
3.4. The amylase of serum is activated by chloride ions, so the dilutions of serum must be made in physiological saline.
4. Primary sample:
4.1. Collect 4 mL of blood in a plain vacutainer tube for blood collection.
4.2. Do not use lysed serum for testing as it may give very high results 4.3. Do not use contaminated/turbid samples for testing
4.4. Process the sample on the same day within 3 hours of collection or store at –20 °C for one week.
5. Type of container and additive: Use plain vacutainer tube for blood collection.
76
6. Reagents/Consumables:
6.1. Phosphate buffer:
Na2HPO4 - 1.735 g
KH2PO4 - 1.009 g (for 1 liter)
Mix 84 mL of A and 16 mL of B solution (pH 7.4) 6.2. Starch solution: 5 g/liter
6.3. Prepare the buffer and starch solution fresh every time a sample is processed or at intervals of not more than 2 to 3 days.
6.4. Buffered substrate: 0.4 mg/mL. Weigh 20 mg starch in 50 mL of phosphate buffer.
6.5. Iodine reagent stock: Dissolve 30 g of potassium iodide with 250 mL water, weigh 13 g iodine in a closed container andtransferred quantitatively to a liter volumetric flask with the iodide solution.
Shake well to dissolve and make to the mark with water.
Standardize against thiosulphate. The iodine concentration should be 47.5 to 52.5 mmole/liter. Adjust if necessary.
6.6. Working iodine standard solution: Prepare from reagent 3 by diluting 1 to 10 with water.
6.7. Sodium chloride solution: 0.9 g in 100 mL water.
7. Instrument: DU- 640 Beckman spectrophotometer.
8. Procedure: Dilute the serum/plasma 1: 10 with saline
Test Control
Buffered substrate (mL) 1.0 1.0
Incubate 37°C for 5 minutes
Serum (1:10) (mL) 0.1 —
Incubate at 37°C for 15 minutes
Iodine, working standard (mL) 0.4 0.4
Water (mL) 8.5 8.6
Then measure the color at 660 nm using water as a blank.
9. Calculation:
Amylase activity = (Absorbance of control – Absorbance of test) × 800 Absorbance of control
1 Somogyi unit of amylase activity = 5 mg starch hydrolyzed under aforesaid conditions (Enzymatic reaction for 15 minutes at 37°C at pH 7.4).
Amount of starch present in the reaction mixture = 0.4 mg 0.4
5 = amylase units
Enzyme activity factor (amylase activity/dL serum)
77
= 0.4 5 × 100
0.1 × 10*
*Dilution factor
10. Reference range: 60–180 Somogyi units/dL (or) 95 –290 IU//L 11. Critical/Alert level value: Not applicable
12. Potential sources of variability:
12.1. Lysed serum specimens may give falsely elevated values 12.2. Amylase activity remains stable in serum for up to one week,
on refrigeration for two months
12.3. With the exception of heparin, all common anticoagulants inhibit amylase activity because they chelate Ca (II) Citrate, EDTA, and oxalate inhibit it by 15%
12.4. The amylase of serum is activated by chloride ions, so the dilutions of serum must be made in physiological saline 12.5. Misleading increases in the activities of amylase and pancreatic
amylase in the serum of a patient with macroamylasemia.
BIBLIOGRAPHY
1. Kanai L Mukherjee. Medical Laboratory Technology Iodometric Method 1988;Vol III:1037-9.
2. Method of Huggins and Ruisel. Practical Clinical Biochemistry by Harold Varley 1980;1080.