The haemolytic anaemias, congenital and acquired / by J.V. Dacie.

  • Dacie, John V. (John Vivian), Sir.
Date:
1954
Catalogue details

Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: The haemolytic anaemias, congenital and acquired / by J.V. Dacie. Source: Wellcome Collection.

    557/580 (page 507)
    Plasma Haemoglobin 507 Every effort must be made to prevent haemolysis during the collection and manipulation of the blood. A clean vene¬ puncture is essential ; a relatively wide-bore needle should be used and the syringe, first rinsed Avith sterile saline, should fill spontaneously with blood. When the required amount of blood has been withdrawn, the needle should be detached and nine parts of blood added to one part of 3-8% sodium citrate. All glassware must be scrupulously clean. Method. 0-1 ml. of plasma (or a larger volume of an appro¬ priate dilution of the plasma, see later) is added to 2 ml. of the benzidine reagent and 1 ml. of the hydrogen peroxide solution in a large test tube. A control tube, in Avhich 0-1 ml. of distilled water is substituted for the plasma, and a standard tube, contain¬ ing a known amount of haemoglobin, are also set up. The mixtures are allowed to stand at room temperature for one hour and then 20 ml. of a 20% by volume aqueous solution of glacial acetic acid are added to each tube. The colours developed are compared in a photoelectric colorimeter using the colour developed by the control tube as a blank. A blue-green (Ilford 624) filter is suitable. If the haemoglobin content of the plasma to be tested is abnor¬ mally high, the plasma should be diluted until it is just visibly tinged with haemoglobin. Normal Range. 1-4 mg. haemoglobin per 100 ml. plasma (Crosby and Dameshck, 1951). Reagents. Benzidine Solution. 0-5 g. of pure benzidine dihydroehloride (Merck) is dissolved in 15 ml. of hot (not boiling) distilled water, and then 25 ml. of 95% ethyl alcohol and 10 ml. of glacial acetic acid added. The benzidine solution will keep for several weeks in a dark bottle at 2° to 5° C, Hydrogen Peroxide. 0-6% solution, prepared by diluting a 3% (10 vols.) solution with distilled water before use. DEMONSTRATION OF HEMOSIDERIN IN URINE The urine is centrifuged and the supernatant removed and replaced by an equal volume of a freshly-made solution of 1% potassium ferrocyanide in 1% HCl (made by mixing equal volumes of 2% potassium ferrocyanide and 2% HCl). The deposit is resuspended in the acid-potassium ferrocyanide solution and alloлved to stand at room temperature for 5 to 10 minutes. The suspension is then re-centrifuged. The deposit is transferred to a slide, covered with a coverslip and examined under the micro-