==> Sodium hydroxide (NaOH) …………… 4 g(Role=Formation of Alkaline Hematin D) ==> Triton X-100 (or equivalent) ………… 25 g(Role=Detergent to dissolve lipid of the RBCs membrane to rupture it and release Hb) ==> Distilled water ………………………………. 1000ml(Role=solvent for preparation of the reagent)
Why detergents dissolve the red cell memebrane???CLICK HERE
First the detergent dissolve the lipids embedded in the RBCs membrane leading to formation of pores in the membrane …… this leads to leaks of hemoglobin from these pores (i.e. hemolysis) ….. we do this, because we can’t measure the hemoglobin concentration while it is contained within the RBCs
Then, NaOH react with the released hemoglobin to form the Alkaline Hematin D-575 which is stable colored compound that can be measured photo-metrically by the spectrophotometer of colorimeter and whose concentration equals the real hemoglobin concentration
==> Dissolve the sodium hydroxide in the distilled water in a clean conical flask.
==> Stir using a glass rod until the crystals have completely dissolved.
==> Add the Triton X-100 (or equivalent) and mix well.
==> Filter the solution into a clean glass-stoppered reagent bottle, using Whatman No. 1 (or equivalent) filter-paper.
==> Label the bottle “ALKALINE HAEMATIN D REAGENT” and write the date.
==> Store at room temperature (20–25°C).
==> Check the quality of the solution(see the quality control below).
==> Alkaline haematin D (AHD) reagent will keep for several months at 20–25°C. If a precipitate forms during storage, the reagent should be filtered before use.
==> Use filtered rainwater if distilled water is not available.
Quality control of alkaline hematin D reagent
An alkaline haematin D standard solution (AHD standard) supplied by the central laboratory is used to test the quality of new batches of AHD reagent in peripheral level laboratories.
1) Fill a clean cuvette with distilled water. Place the cuvette in the cuvette chamber and adjust the haemoglobinometer or colorimeter to read zero at 540nm wavelength.
2) Replace the distilled water with AHD reagent. The haemoglobinometer or colorimeter should read zero.
3) Pipette 20ml of AHD standard into a test-tube containing 3 ml of the freshly prepared AHD reagent (1:150 dilution).
5) Repeat the procedure using the previous batch of AHD reagent. Compare the results.
6) If the haemoglobin values differ by more than 5 g/l, discard the freshly preparedAHD reagent and prepare a new batch, paying attention to accurate measurementof the constituents and the cleanliness of the glassware.
The AHD standard stock solution will keep for 8 months at 4–8°C.