334 THE BLOOD. 



than the latter. Its spectrum, however, is the same as that of the 

 completely reduced haemoglobin. 



In sufficiently dilute solution oxyhaemoglobin shows two bands 

 of absorption between D and E. The one to the left, which is not 

 so wide as the other, but darker and more sharply defined, borders 

 on D, while the second lies at E. 



The Quantitative Estimation of Oxyhaemoglobin. This is best 

 accomplished by Hoppe-Seyler's method, which is based upon 

 the comparison of a given amount of diluted blood with a stand- 

 ard solution of crystallized oxy haemoglobin. This solution is 

 prepared by dissolving 2 grammes of the pure coloring-matter in 

 50 c.c. of distilled water. The oxyhaemoglobin is then transformed 

 into carbon monoxide haemoglobin by passing a current of the gas 

 through the solution to saturation. It is then stored in drawn- 

 out and sealed glass tubes, such that each tube contains about 

 6 c.c. The contents of each tube, when diluted with ten times its 

 volume of water, will then represent a 0.2 per cent, solution of the 

 oxyhaemoglobin. 



A carefully measured or weighed amount of blood, not exceed- 

 ing 0.5 c.c., is now diluted with water that has been saturated with 

 carbon monoxide to exactly 5 c.c. A small drop of a very dilute 

 solution of sodium hydrate is added if necessary to remove any 

 turbidity that may exist. This solution is further saturated with car- 

 bon monoxide, and freed from fibrin by filtration. The filtrate should 

 measure exactly 4 c.c. The comparison of the two solutions, and 

 the further dilution of the blood with carbon monoxide water then 

 takes place in the so-called double pipette of Hoppe-Seyler. The 

 color of the two solutions is here equalized, and the amount of 

 haemoglobin present in the specimen of blood calculated from the 

 degree of dilution. 



Example. Suppose that we started with 0.5 gramme of blood, and 

 that the standard solution contained 0.002 gramme of oxyhaemoglo- 

 bin in the cubic centimeter. The 4 c.c. of the diluted and filtered blood 

 are further diluted in the pipette to 22 c.c., which corresponds to a 

 total solution of 27.5 c.c. for the total 5 c.c. of the first dilution. In 

 these 27.5 c.c., which represent the original 0.5 gramme of blood, 

 there will consequently be 27.5 X 0.002 = 0.0550 gramme of 

 haemoglobin. The percentage will accordingly be 11 per cent. 



In the clinical laboratory other forms of apparatus are in use, 

 such as the hcemometer of Fleischl and the hcpmoglolinometer of 

 G-owers. In the first the color of the diluted blood is compared 

 with that of a glass wedge that has been stained with the golden 

 purple of Cassius. In the second a standard solution of carmin 

 and picric acid is employed. But as these colors do not represent 

 the exact shade of oxyhaemoglobin, the results must of necessity be 

 less accurate. For clinical purposes, however, these methods are 

 sufficiently exact. 



The spectro-photometric determination of the blood coloring- 



