ESTIMATION OF HEMOGLOBIN. 1 9 



12. PREPARATION OF HEMOGLOBIN CRYSTALS. Method of Rollett. Put defibrinated 

 blood in a platinum capsule placed on a freezing mixture, freeze the blood, and then thaw it ; 

 pour the lake-coloured blood into a plate, until it forms a stratum not more than 1^ mm. in 

 thickness, and allow it to evaporate slowly in a cool place, when crystals will separate. 



Method of Hoppe-Seyler. Mix defibrinated blood with 10 volumes of a 20 per cent, salt 

 solution, and allow it to stand for two days. Remove the clear upper fluid with a pipette, 

 wash the thick deposit of blood-corpuscles with water, and afterwards shake it for a long time 

 with an equal volume of ether, which dissolves the blood-corpuscles. Remove the ether, filter 

 the lake-coloured blood, add to it J of its volume of cold alcohol (0), and allow the mixture to 

 stand in the cold for several days. The numerous crystals can be collected on a filter and pressed 

 between folds of blotting-paper. 



Method of Gscheidlen. Take defibrinated blood, which has been exposed for twenty-four 

 hours to the air, and keep it in a closed tube of narrow calibre for several days at 37 C. When 

 the blood is spread on glass, the crystals form rapidly. [Vaccine tubes answer very well.] 



[Method of Stirling and Brito. It is in many cases sufficient to mix a drop of blood with a 

 few drops of water on a glass slide, and to seal up the preparation. After a few days beautiful 

 crystals are developed. The addition of water to the blood of some animals, such as the rat 

 and the guinea-pig, is rapidly followed by the formation of crystals of hemoglobin. Very 

 large crystals may be obtained from the stomach of the leech several days after it has sucked 

 blood.] 



13. QUANTITATIVE ESTIMATION OF HEMOGLOBIN. (a) From the Amount of Iron. 



As dry (100 C. ) hemoglobin contains 0*42 per cent, of iron, the amount of haemoglobin maybe 

 calculated from the amount of iron. If m represents the percentage amount of metallic iron, 



then the percentage of hemoglobin in blood is -.-- -, The procedure is the following: 



Calcine a weighed quantity of blood, and exhaust the ash with HC1 to obtain ferric chloride, 

 which is transformed into ferrous chloride. The solution is then titrated with potassio 

 permanganate. 



(b) Colorimetric Method. Prepare a dilute watery solution of hemoglobin crystals of a 

 known strength. With this compare an aqueous dilution of the blood to be investigated, by 

 adding water to it until the colour of the test solution is obtained. Of course, the solutions 

 must be compared in vessels with parallel sides and of exactly the same width, so as to give the 

 same thickness of fluid (Hoppe-Seylcr). [In the vessel with parallel sides, or haematinometer, 

 the sides are exactly 1 centimetre apart. Instead of using a standard solution of oxyhemoglobin , 

 a solution of picro-carminate of ammonia may be used (liajewsky, Malassez).] 



(c) By the Spectroscope. Preyer found that a 0*8 per cent, watery solution (1 cm. thick), 

 allowed the red, the yellow, and the first strip of green to be seen (fig. 17, 1). Take the blood 

 to be investigated (about 0"5 c.cm.), and dilute it with water until it shows exactly the same 

 optical effects in the spectroscope. If 1c is the percentage of Hb, which allows green to pass 

 through (0*8 per cent.), b, the volume of blood investigated (about 0'5 c.cm.), w, the necessary 

 amount of water added to dilute it, then a? = the percentage of Hb in the blood to be investi- 

 gated 



_ Jc(<w+b) 

 X ~b~ ' 



It is very convenient to add a drop of caustic potash to blood and then to saturate it with 

 CO. 



[(d) The Haemoglobinometer of Gowers is used for the clinical estimation of hemoglobin 

 (fig. 14). " The tint of the dilution of a given volume of blood with distilled water is taken 

 as the index of the amount of hemoglobin. The distilled water rapidly dissolves out all the 

 hemoglobin, as is shown by the fact that the tint of the dilution undergoes no change on 

 standing. The colour of a dilution of average normal blood one hundred times is taken as the 

 standard. The quantity of hemoglobin is indicated by the amount of distilled water needed 

 to obtain the tint with the same volume of blood under examination as was taken of the 

 standard. On account of the instability of a standard dilution of blood, tinted glycerine-jelly 

 is employed instead. This is perfectly stable, and by means of carmine and picro-carmine the 

 exact tint of diluted blood can be obtained. The apparatus consists of two glass tubes of 

 exactly the same size. One contains (D) a standard of the tint of a dilution of 20 cubic mm. 

 of blood, in 2 cubic centimetres of water (1 in 100). The second tube (C) is graduated, 100 

 degrees = 2 centimetres (100 times 20 cubic millimetres). The 20 cubic millimetres of blood 

 are measured by a capillary pipette (B). This quantity of the blood to be tested is ejected 

 into the bottom of the tube, a few drops of distilled water being first placed in the latter. The 

 mixture is rapidly agitated to prevent the coagulation of the blood. The distilled water is 

 then added drop by drop (from the pipette stopper of a bottle (A) supplied for that purpose), 

 until the tint of the dilution is the same as that of the standard, and the amount of water 

 which has been added (i.e., the degree of dilution) indicates the amount of hemoglobin." 



