THE REFRACTOMETRIC DETERMINATION OF HEMOGLOBIN.

BY JAMES L. STODDARD AND GILBERT S. ADAIR.

(From the Chemical LabOTatOTy of the Massachusetts General Hospital, Boston.)

(Received for publication, July 10, 1923.)

INTRODUCTION.

The standard method of determining hemoglobin concentration in blood is based on a measurement of oxygen capacity and Htif- ner’s factor, 1.34 cc. of oxygen per gm. of hemoglobin. Unfor- tunately, the Van Slyke and the Haldane machines do not give the same results for the oxygen capacity. The difference is over 10 per cent of the total, and there is no agreement as to which method is correct. The calorimetric methods are based on the oxygen capacity, so that they can offer no help as to the absolute value.

The refractometer appeared to offer the possibility of making a determination of the absolute quantity of hemoglobin by an inde- pendent method, entirely unconnected with the oxygen capacity, or even with any chemical standards. There was also the possi- bility that a rapid and convenient method might be worked out for determining hemoglobin, which could be used by those having refractometers at their disposal.

After the work was started it was found that Howard 3 years ago1 had investigated the refractive constant of hemoglobin and had suggested the possibility of a refractometric method for blood. No such method has been published, however, and the amount of elapsed time since that suggestion seemed to the writers to justify a new attack on the problem.

The principal objects in the present work were: (1) to redeter- mine the refractive constant “a” of hemoglobin; (2) to test the constancy of the relation a X c = n solution - n water; (3) to

1 Howard, F. H., J. Biol. Chem., 1920, xii, 537.

437

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438 Refractometric Determination of Hb

devise a method for the determination of hemoglobin in blood which should first of all be accurate, and give the absolute quantity of hemoglobin in terms of dry weight of the substance; (4) to com- pare the concentration of hemoglobin with oxygen capacity; and (5) to devise a quicker method that could be used with convenience.

Note.-In all oases in this paper where figures are given for the refractive index of a solution minus the refractive index of water at the same tempera- ture, it is to be understood that the last integer is in the fifth decimal place; e.g., 194 represents 0.00194.

In all the experiments the temperature was controlled by immersing the usual refractometer bath in a large (370 liter) water bath. The tempera- ture did not vary faster than O.l”C. per hour, and corrections were made for temperature when necessary.

A. Determination of “a” of Hemoglobin.

A redetermination of this value was deemed advisable for the following reasons: (1) Howard’s values were not based on human blood with the exception of one experiment where the concentra- tion was standardized only by the oxygen capacity. (2) The weight of hemoglobin thoroughly dried in an oven at 110°C. was considered preferable to the weight of crystalline hemoglobin (used by Howard). Not that it is theoretically preferable, but merely that until the ease of loss of water of crystallization and the possibility of more than one degree of hydration of the crystals are carefully investigated, it seems probable that a more constant value will be obtained from a thoroughly dried preparation. The writers do not believe their method of drying is unexceptionable. It ought, however, to give minimal values, and this is especially important in view of our later findings with relation to the oxygen capacity.

1. Preparation of the Hemoglobin.-The hemoglobin was prepared from human blood by the method of Adair. This essentially consists in laking the washed red cells with ether, adding salt to separate the stromata, and dialyzing the hemoglobin solution. All operations are done at 0°C.

Bloods from two normal human individuals were investigated.

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J. L. Stoddard and G. S. Adair

Instead of measuring the hemoglobin solution taken for the dry weight volumetrically, it was considered more accurate to get the specific gravity of the blood, and then weigh the sample taken.

2% Determination of the Concentrations of the Solutions in terms of Dry Weight.

Specimen 1.

Density (by 5 cc. pycnometer) at 19.5”C. Weights. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) 5.1338 gm.

(2) 5.1854 “ Average . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1346 (‘

Capacity of pycnometer. . . . . . . . . . . . . . . . . . . . . . . 4.9916 cc. Density. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.03336

Hemoglobin solution for dry weight (weighed in crucible). . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . 5.1942 gm.

Hemoglobin solution for dry weight in cc., calculated from density.................................... 5.001~~.

Dry weight (dried at 110°C. for 3 days}. . . . . . . . . . . 0.8173 gm. Concentration of original solution. . . . . . . . . . . . . . . . 16.34 per cent.

The ash was 0.0044 gm. The ash calculated from the hemoglobin was 0.0040 gm.

Specimen 8.

Density at 20.5”C. Weight. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2353 gm. Capacity of pycnometer. . . . . . . . . . . . . , . . . , . . . . . . . . 4.9916 CC.

Density. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0488

Hemoglobin solution for dry weight (weighed in crucible). . . . . . . . . . . . . . .

Hemoglobin solution for dry weight in cc., calculated from density.. . . . . .

Dry weight (dried to constant weight). Concentration of original solution. . . . . Ash found.............................

“‘ calculated. . . . . . . . . . . . . . . . . . . . . . . , -

1

5.2282 gm. 5.2044 gm.

4.9885 cc. 4.9624 cc. 0.9993 gm. 0.9916 gm.

29.043percent 19.933 per cent. 0.0064 gm. 0.0068 gm. 0.0050 “ 0.0050 “

2

-

_ -

3. Determination of the “n” of the Solution and of the Value of “a.”

In each case the refractive index of water at the same tempera- ture as the solution is subtracted from the refractive index of the

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440 Refractometric Determination of Hb

solution. The pipettes were calibrated, and the factors omitted in the statement of the dilutions, but, of course, used in the calculations.

Dilutions. n Solution - n water

Dilution

1:25 3,184 2~25 3,185 1:2 3,162

Average. 3,177

Specimen 1.

The maximum variation in the values is 3,185- 3,162 = 23. This is equivalent to 23+194.4= 0.12 per cent Hb.

n Solution - n water 3,177 a=

Concentration = 16.34 = 194.4

Specimen 2.

Dilutions. I

n Solution - n water Dilution ’

1:4 3,883 1:8 3,908 1:16 3,904 1:25 3,851

Average......................... 3,886.5

n Solution - n water a=

Concentration = (1) 3886.5 = 193 85

20.048 . (2) 3886.5

- = 194 45 19.988 ’

Average..................................1’94.15

The average of the three values of the factor is 194.2. Using the weighted average method of Robertsoq2 i.e. dividing the sum of the original refractive indices by the sum of the dilutions, the second specimen gives the average of 194.17. The maximum variation in the factor calculated in the various instances is 194.45- 193.85 = 0.6 or 0.3 per cent of the original factor. Applying the factor to hemoglobin concentration in blood would give an uncertainty due to the factor of 0.3 per cent of the total hemoglo- bin, or if the hemoglobin is 15 per cent, of 0.045 per cent of hemoglo- bin. This is certainly accurate enough for all practical purposes.

* Robertson, T. B., J. BioZ. Chem., 1912, xi, 179.

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J. L. Stoddard and G. S. Adair 441

The maximum variation in the factors from our average factor is 194.2-193.85 = 0.35 or 0.2 per cent of the total. This would represent, applied to blood, an uncertainty of 0.030 per cent of hemoglobin.

The difference from Howard’s factor of 183 we think is to be explained by the fact that his preparation contained water of crys-

tallization. If so, there would be 194.2 - 183

194.2 = 5.7 per cent of

water of crystallization. Gamgee (quoted in Schafer’s Text- book of physiology) found the hemoglobins of various animals to contain from 4 to 8 per cent of water. The crystals were dried in N-NUO at low temperature, and then at 110%.

B. Determination of the Constancy of the Relation a X c = n Solution -n Water.

The best way to determine whether this relation is valid is to calculate the value for the more dilute solutions from the value for the more concentrated, and note whether the observed value for the diluted solutions differs from the calculated value by more than the errors of observation.

Proportionality of the Refractive Index of Hemoglobin.

Specimen No.

1 1 1 (new dilution).

2:25 1:25 1:2

1 ( “ “ ) 1:4 1 ( “ “ ) 1:8 2 1:8 2 1:16 2 1:16 2 I:25

Dilu- tion.

--- 1:2 256.7 258.6 1.9 1:2 128.4 129.0 0.6

Original solution, a dilution 31 .O 29.4 1.6 of 1, having an index of 62.

“ ‘I ‘I 15.5 15.3 0.2 ‘I “ “ 7.8 7.7 0.1

1:4 485.7 487.7 2.0 1:4 242.8 244.0 1.2 1:s 243.9 244.0 0.1 1:s 155.0 156.0 1.0

The error of observation might be as high as 3. All deviations are within this value. There is therefore proportionality in the range covered, which is that from an 8.17 per cent solution to a 0.04 per cent solution. More concentrated solutions than 8.17

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442 Refractometric Determination of Hb

per cent are too difhcult to read. This conclusion is in complete accord with the observations of Howard on hemoglobin and of Robertson on various other protein solutions.

C. A Refractometric Method for Hemoglobin in Blood.

1. The Method.-The method in brief is: (1) Wash the red cells from a measured quantity of blood. (2) Hemolyze with dis- tilled water and saponin, add salt to throw down the stromata, and make up to volume. Centrifuge. (3) Obtain the refractive index of the solution. (4) Obt ain the refractive index of the fil- trate obtained by the heat coagulation of the hemoglobin. (5) Subtract (4) from (3) and divide by “a” for hemoglobin.

Thus the method is essentially a separation of the refractive index of hemoglobin from the refractive indices of the other blood constituents. After the proof which follows of the admissability of the various procedures, will be given a detailed statement of the steps in the method.

d. Separation from Plasma.-The amount of necessary washing with 0.8 per cent NaCl was determined repeatedly by getting the refractive index of the wash water and comparing it with that of the NaCl. The washings were done on 2 cc. of blood in a 16 to 20 cc. centrifuge tube. To the blood was added 0.8 per cent NaCl up to 15 cc. The mixture was centrifuged 5 minutes at low speed and the diluted plasma pipetted off. NaCl solution was then added as before, the tube inverted with a rubber stopper in, and thoroughly shaken, then again centrifuged. Invariably the third wash water (counting the original dilution of the blood as the fust) was not appreciably different from the NaCl solution. That this is reasonable is evident from the following calculation. If 1.2 cc. of plasma are present in the blood, the first dilution gives a plasma concentration of approximately 1.2 + 13.8 = 0.087. The next wash gives, if 0.2 cc. diluted plasma is left with the red cells, 0.087 f 71 = 0.0012, and the third, 0.0012 + 71 = 0.0000172. If the refractive index of the plasma - that of water were 1,650, then that due to the traces of plasma in the third dilution would be 1,650 X 0.0000172 = 0.028. A difference of 2.0 is all that can be read with any certainty. Of course there are traces of plasma protein that would give protein tests such as the nitric acid test,

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J. L. Stoddard and G. S. Adair 443

but they have no significance in this connection. In the method the cells after washing are made up to 10 cc. This final dilution would then correspond to the third washing above, and only two washings are necessary.

3. Methods of Bemolysis.-Hemolysis with distilled water alone was found to be incomplete under the conditions of this method. That is, using 2 cc. of blood, the washed red cells have about 7 cc. of distilled water added, then 1 cc. of 10 per cent NaCl, and then, after making up to 10 cc., are centrifuged out. The stromata appeared deep red, and careful determinations of the hemoglobin concentration in the stromata by repeated extractions, showed that it was about twice as great in the stromata as in the fluid surrounding them.

With saponin hemolysis there is hemoglobin in the solution left between the centrifugalized stromata, and also hemoglobin in solution in the stromata. Therefore, the solution cannot be pipetted from the stromata and made up to volume. If, on the other hand, the stromata are left in while the solution is made up to volume, the question is important whether the concentration of hemoglobin in the stromata is the same as that in the solution surrounding them. To settle this point a considerable number of duplicate determinations were made on 2 cc. samples of blood. In one case to the washed red cells were added about 7 cc. of dis- tilled water and about 1 mg. of saponin. Then, after thorough mixing, the solution was made up to 10 cc. with distilled water and 1 cc. of 10 per cent NaCl. The solution was centrifuged and the refractive index read. Thehemoglobin was then determined from the heat filtrate according to the method described later.

In the other case, the washed red cells were hemolyzed as in the first example, and then 1 cc. of 10 per cent NaCl was added and the solution centrifuged. The solution was then pipetted off and the stromata repeatedly hemolyzed and precipitated in the same manner until they were perfectly white and the extract was colorless. The hemoglobin was then determined in the combined extracts. In the course of the extractions, especially after the first two or three, the stromata tended to dissolve. In order to be sure that they were not adding to the refractive index of the solu- tion, the hemoglobin in these later extracts was also determined

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444 Refractometric Determination of Hh

calorimetrically, using as a standard one of the first extracts which had no dissolved stromata. Since the stromata are color- less, any increase in the refractive index should throw the, deter- mination of hemoglobin by this method out of harmony with that determined calorimetrically. No difference, not within the errors of reading the calorimeter, was found. If anything, thecolor- imetric figures were higher, probably on account of the turbidity of the solution. In fact, the difficulties in the calorimetric work from this cause made very exact determinations impossible. All that could be concluded was that either the stromata added very little to the refractive index, or else that they went through into the heat filtrate and were deducted.

Concentration of Hemoglobin in Stromata Laked with Distilled Water and

Average 2,893 2,898

A Leaving

stromate, in the solution.

SC

&xl hemoglobin,

extracting stromats.

2,873 2,852 2,849 2,866 2,798 2,812 3,130 3,123 2,816 2,836

>onin. *

Remarks.

Slight loss in pipetting A.

Here much saponin was used in A.

+ Concentration is given in terms of refractive index excess over that of water at the same temperature.

It is evident that there is no constant or significant difference between the two methods. The divergencies are probably due to the numerous manipulations in the repeated extractions. It appears, therefore, safe to rely upon the concentration of hemo- globin within the stromata being essentially the same as in the surrounding fluid when the red cells are hemolyzed with distilled water and saponin under the conditions of these experiments.

4. Possible E$ects of Saponin on the Refractive Index.-In this method about 1 mg. of saponin is used to 10 cc. of solution. A solution of 70 mg. of saponin in 100 cc. of salt solution increases the refractive index 10.5. 1 mg. in 10 cc. therefore would increase

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J. L. Stoddard and G. S. Adair

it only 10.5 X 1: = 1.5. This is within the error of reading the

refractometer, and would correspond to an amount of hemoglobin 1.5 x 5

in the original blood of 194 = 0.038 per cent. (The “5” in the

numerator allows for the dilution of the 2 cc. of blood to the 10 cc. of final solution.) Further, tests of the filtrates (obtained by heat coagulation of the hemoglobin as described later) showed that even when less than 1 mg. of saponin was used, the filtrate was hemolytic, and that the hemolytic power increased with the amount of saponin in the original solution. There seems no doubt, then, that the saponin goes through into the filtrate as do the other soluble non-coagulable constituents, and is allowed for when the heat filtrate is subtracted. Variations in the amount of saponin therefore do not introduce a source of error so far as the effect of the refractive index of saponin is concerned.

5. Possible E$ects of Solution of the Stromata.-When about 1 mg. of saponin is used there is usually little or no solution of the stromata. Occasionally the stromata dissolve somewhat, how- ever, without any apparent reason. With large amounts of sa-’ ponin they dissolve completely. It appeared logical that if the irregular slight solution made much difference duplicate deter- minations would not check. Very careful experiments showed a maximum variation in duplicates of 0.15 per cent that apparently could be accounted for in no other way. The higher values always occurred when the stromata were completely dissolved. Attempts to prepare stromata free from hemoglobin and salts were unsuccess- ful. Although the error due to this factor is small, because only small amounts of saponin are used, it was nevertheless considered advisable to investigate the refractive index of the stromata, especially as it is an element in the explanation of the short method for hemoglobin described later:

The method adopted for the solution of this problem was to add to a series of 2 cc. samples of blood increasing amounts of saponin, and estimate the concentration of hemoglobin. The following tabulation gives the results.

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446 Refractometric Determination of Hb

E$ect of Increased Amounts of Saponin with’ Solution of the Stromata.

B

“0 .J

q 8

0

% 4 ; j 2 4

8 B

ol k?

B

‘6

4

E 8

3

B & 4 4

1 f $

4 d

1

$ !3 - -----

ml. per cent mQ. Per Ce?VZt ,,SQ. Pe7 Cent mQ. pc?,’ Cd

1 Little (1 mg.). 15.74* Much (10mg.). 15,93t 2 0.6 15.80$ 3.2 15.460 8.0 16.061 22.8 15.95t 3 2.5 15.456 5.4 15.64125.9 16.24t 4 1.0 12.720 1.0 12.881 4.813.04$ 5 1.3 12.43$ 2.6 12.300 15.4 12.67t 26.0 12.73t

0.9 12.41$ 6 1.6 10.31* 2.8 10.235 16.8 10.65t 25.4 10.73t

1.0 10.31*

* Slight solution of the stromata. t Complete solution of the stromata. $ Partial solution of the stromata. $ No solution.

It will be noted that the percentage of hemogIobin varies with the degree of solution of the stromata rather than with the amount of saponin. Thus in Specimen 2 there was 0.34 per cent less hemo- globin in the case where 3.2 mg. of saponin were used than where 0.6 mg. was used. In Specimens 5 and 6 there were respectively 0.12 and 0.08 per cent less hemoglobin when about 2.5 mg. of saponin were used than when about 1 mg. was used. These last two experiments were done with the very greatest care, and it is believed represent the maximum errors due to the variation in solution of the stromata when amounts of saponin up to 3 mg. are used because the visible variation in stroma residue was as great as in any of the other similar experiments. When about 15 mg. of saponin are used the solution of the stromata becomes complete, or practically so. The apparent increase in hemoglobin due to this solution of the stromata, obtained by subtracting the figure in each series where there was none or slight solution of the atromata from the figure where there was the most complete solution gives:

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J. L. Stoddard and G. S. Adair 447

Specimen No.

per cent

0.19 0.6 0.79 0.16 0.43 0.50

Average........................................... 0.45 “ ofSpecimens5and6 . . . . . . . . . . . . . . . . . . . . 0.46

-

The refractive index of the stromata in a given amount of blood is equal to the refractive index of 0.45 per cent of hemoglobin. The error due to variations in solution of the stromata when not over 3 mg. of saponin are used is not over 0.15 per cent of hemoglobin.

6. Possible E$ects of Dissolved Leucocytes.-The leucocytes apparently do not dissolve appreciably. Even if they did, and added to the refractive index as do the stromata. their effect would

10,000 ’ be of the order of magnitude of 5,000,0co X 9 X 0.45 = 0.0081 per

cent in terms of hemoglobin concentration, which is negligible. 7. Separation of Hemoglobin frm Non-Protein Constituents by

Heat Coagulation.-At this point in the method the hemoglobin has been separated effectively as far as refractometer readings are concerned from the plasma proteins and the red cell proteins. There remains the separation from the non-protein constituents of the red cells, and from the NaCl added. These might be removed by dialysis, but the process is slow, and hemoglobin is apt to be lost by deposition on the membrane. Ultrafiltration was selected as the test method, and heat coagulation of the hemoglobin tried as a possible easy method, to be checked by ultrafiltration.

Numerous experiments on pure hemoglobin solutions showed that the hemoglobin would coagulate at 100°C. in 1 to 2 minutes, giving a clear filtrate, provided a certain amount of salt were present and the pH of the solution were close to the isoelectric point of 6.8. These conditions are fulfilled when washed red cells are hemolyaed with distilled water. The principle constituent here is hemoglobin, and the other constituents have comparatively little effect on the

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448 Refractometric Determination of Hb

PH. It is natural, therefore, that the pH of the solution should be approximately that of the isoelectric point of hemoglobin. Salts are present in sufhcient amount, for 1 cc. of 10 per cent NaCl is added in the method. Invariably in numerous experiments coagulation of the hemolyzed red cell solution has occurred in 3 minutes immersion of the test-tube containing the hemoglobin solution in boiling water, and the filtrate has been clear and prac- tically colorless. There are probably changes in the water or salt relations of the hemoglobin during the process of coagulation, but they are too slight to affect the refractometer, as the following parallel experiments on the heat filtrates and ultraflltrates show.

Comparison of Ultrafiltrates with Heat Filtrates.

n Heat fi1trste - ?I water.

218.0 216.7

4.0 4.5 4.0 6.0

98.0 95.0 202.0 200.8

n Ultm- filtrate -

n water. Solution.

- .-__.

Hemolyzed red blood cells from 1 cc. blood made up td25 cc. with N&l solution.

Same as No. 1 but no NaCl. “ “ “ 1 ‘I ‘I ‘I “ “ ‘I 1 “ with less NaCl. “ “ I‘ 1 “ “ 3 volume KHzPO~

and less NaCl.

The two methods do not differ by more than the error of read- ing the refractometer (2-3), although the filtrates represent widely varying salt content.

The only remaining question in this connection is whether the heat filtrate represents the salt concentration in the original solu- tion. It may be stated that it does not exactly, of course, but experiments on pure hemoglobin solutions show that under the conditions of this method the correction would not amount to over l/15 to l/Z0 per cent hemoglobin in the fiiid ToSEE; The authors do not care to present their results here as they are of more significance in other directions and will be published else- where.

Detailed Directions for the Method.

1. Measure 2 cc: of blood into a 17 cc. centrifuge tube, add 6.3 per cent NaCl up to about 15 cc., invert twice with rubber stopper in, rinse stopper into tube, centrifuge at moderate speed 5 minutes.

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J. L. Stoddard and G. S. Adair 449

2. Pipette off the diluted plasma, replace with 0.8 per cent N&l, shake vigorously with rubber stopper, rinse, and centrifuge as before.

3. Pipette off the supernatant fluid, add about 1 mg. of saponin, then distilled water up to 7 cc., mix thoroughly with a fine glass rod. Pipette into a 10 cc. volumetric flask, rinse the rod, pipette, and tube with 1 cc. of 10 per cent N&l, and successive small portions of distilled water until the flask is full to the mark. Mix thoroughly. Centrifuge.

4. Obtain the refractive index of the solution. 5. Put the hemolyzed blood into a test-tube loosely stoppered with a

rubber stopper, and place in boiling water for about 3 minutes. Cool, invert twice, filter through a small filter paper.

6. Obtain the refractive index of the filtrate. 7. Subtract (6) from (4), multiply by (5), and divide by 194.2. This gives

the concentration of hemoglobin in terms of dry weight in gm. per 100 ca. of blood.

The temperature at which the solution and the filtrate are read should be the same within 0.15%. or else correction must be made for the difference in temperature.

In some cases the blood may give after a time in contact with the prism a fuzzy line. This is apparently due to a deposition of material on the face of the prism, and all that is necessary is to take the prism out quickly, wipe the face with moist lens paper and then with dry, and replace it.

The dipping refractometer is the best for this work as it is more accurate.

Accuracy of the Method.-When the immersion refractometer is used the results should check within 0.1 to 0.2 per cent of hemo- globin, and the writers believe that the absolute amount is not in error by an appreciably greater amount.

The greatest sources of error are: (1) Volumetric technique; (2) temperature control; and (3) adjustment of the compensating prism. In hemoglobin solutions the border-line appears to move with adjustment of the prism, instead of merely showing colors as happens in colorless solutions. Sharpness of the line is the criterion here. No reading should be made unless the line is perfectly clear and sharp. In case adjustment is difficult wipe the prism as described above.

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450 Refractometric Determination of Hb

D. Relation of Hemoglobin Concentration to Oxygen Capacity.

Specimen No.

Oxygen capacity by

“Et!%

cc. 02 per 100 cc. blood

18.7 18.2 18.2 18.2 19.95 20.14 19.21 19.21

AVl?rage.

20.04

19.21

Hemoglobin by refrac- tometer.

gm. Hb per 100 cc. blood

14.79 14.67 14.41 14.50 15.53 15.58 15.76 15.74

Average.

15.55

15.75

Rstio.

1.264 1.25 1.26 1.255

1.29

1.22

Our method of drying would tend to give, if anything, low values for the hemoglobin. This would tend to make a high factor “a” and this in turn would tend to give a high ratio between hemoglobin and oxygen capacity. The ratio as determined by our method definitely supports the Van Slyke as opposed to the Haldane method for determining oxygen capacity.

E. A Short Method for the Refractometric Determination of Hemoglobin.

The principle of this method is to measure two equal samples of blood. To one is added a certain number of volumes of salt solution (0.8 per cent NaCl) and to the other is added the same number of volumes of salt solution containing a sufficient concen- tration of saponin to hemolyze the red cells. Subtracting the refractive indices and allowing for the refractive index of the saponin should give approximately at least the hemoglobin con- centration in the diluted blood. The points of doubt were the liberation of other constituents than hemoglobin from the red cells, and other minor points. It seemed probable that these corrections would be insignificant. The method is checked for absolute amounts with the former method.

The method in detail is:

1. Measure two 1 cc. samples of blood into two medium size test-tubes. 2. To one sample add 5 cc. of 0.8 per cent NaCl. 3. To the other sample add 5 cc. of 0.8 per cent NaCl containing about

70 mg. of saponin per 100 cc.

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J. L. Stoddard and G. S. Adair 451

4. Centrifuge (2) and (3) and read the clear solutions in the refrac- tometer. Read also the two salt solutions.

Calculations.-Subtract the refractive indices of the two salt solutions.

This gives the added refraction due to the saponin. Multiply by F6 to get

the dilution of the saponin refraction by the added 0.6 cc. (approximately) of plasma. Subtract this saponin correction from (3) and then subtract (2) from the result. This gives the increase in refraction due to the hemoglo- bin. Divide by the hemoglobin factor, or 194.2, and multiply by 6 (the dilution) to get the concentration of hemoglobin in the original blood.

Of course, the refractive index of the salt and the saponin solutions need not be determined for every hemoglobin determination. The saponin tends to precipitate somewhat with age, so that it is advisable to check it up from time to time.

Attention needs to be directed to keeping the face of the prism clean as described in the other method, as there is a greater tend- ency here for the line to become blurred. The prism should be left in the solution about 3 minutes to allow it to attain the tem- perature of the solution, then quickly cleaned if necessary, which will not alter the temperature of the whole prism, then read in half a minute.

The blood should be taken with paraffin oil to avoid hemolysis, but great care should be taken not to get the oil mixed with the blood in the operation of shaking previous to measuring the sam- ples. The best way to mix is to put the blood in a test-tube, let the oil rise to the top, pipette it off, then mix by introducing a large bored pipette so that the tip is near the bottom of the test- tube and draw the blood up and down rapidly.

It is possible that some samples of saponin may require more than 70 mg. per 100 cc. to effect hemolysis.

In the following tabulation are given the results obtained by the two methods :

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-

.-

Pl

-

Refractometric Determination of Hb

Comparison of the Long and the Short Methods.

12.30 12.56 0.26

12.49

10.23 10.45 0.24

10.49 I I I-

Average of dif- ferences. . . . . . . 0.12

Remarks.

Saponin concentration in short method, 214 mg. per 100 cc.

Saponin concentration in short method, 60 mg. per 100 cc.

Saponin concentration in short method, 70 mg. per 100 cc.

Saponin concentration in short method, 480 mg. per 100 cc.

Saponin concentration in short method, 70 mg. per 100 cc.

Saponin concentration in short method, 436 mg. per 100 cc.

Saponin concentration in short method, 70 mg. per 100 cc.

Saponin concentration in short method, 430 mg. per 100 cc.

Saponin concentration in short method, 70 mg. per 100 cc.

Saponin concentration in short method, 450 mg. per 100 cc.

In order to get the greatest accuracy, the figure for the old method in each case represents an experiment where there was no solution of the stromata. Duplicates of 3, 4, and 5, may be found opposite 3, 5, and 6, respectively, in the table in Section 5 of Part C.

The close checking of the duplicates in the short method in spite of the widely varying amounts of saponin is noteworthy.

The short method evidently gives slightly higher results than the long method, the average being 0.12 per cent. The average

of the last two experiments is believed to represent a more exact

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J. L. Stoddard and G. S. Adair 453

figure, since they were done last, with the greatest care. This means a difference of 0.25 per cent of hemoglobin. It is an inter- esting question why the short met,hod does not give higher values on account of the complete solution of the stromata which takes place. It was found (Section 5 of Part C) that the stromata added to the refractive index an amount equivalent to 0.45 per cent in terms of hemoglobin concentration. The unaccounted for difference is 0.45 - 0.25 = 0.20 per cent. As a possible explana- tion is advanced the theory that when the red cells dissolve there is no longer the inequality in the distribution of inorganic constit- uents between cells and plasma that there was before. The refractive index of the inorganic constituents of red cells and plasma was calculated from Schmidt’s tables, and found to be (in terms of excess refraction over that of water at the same temperature) :

Plasma ....................... 107.2 Red cells. .................... 73.30

Difference . . . . . . . . . . . . . . . . . . 23.9

The uncertainty of the analyses makes an estimation of the differences in non-protein organic constituents unsatisfactory.

This difference in terms of hemoglobin percent’age would be 23.9 - = 0.12 per cent and would tend to make the hemoglobin 194.2 estimated by the short method low by that amount. There remains then only 0.2 - 0.12 = 0.08 per cent to be accounted for, and this may easily be the experimental error.

In conclusion it may be stated that the close checks in the methods tabulated above are attainable only by the greatest care in all operations.

CONCLUSIONS.

The short method gives even closer checks than the long method. The results by it are 0.25 per cent of hemoglobin too high. It is advised that it be used with this correction, in preference to the long method, because there is no variation in the degree of solution of the stromata.

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454 Refractometric Determination of Hb

SUMMARY.

The refractive constant “a” for human hemoglobin, determined on pure hemoglobin solutions, the concentration of which was obtained by getting the dry weight at llO”C., was found to be 194.2. This compares with 183 found by Howard on crystal- line horse hemoglobin, and, if the substances are comparable, would indicate 5.7 per cent of water of crystallization.

The subtraction of the refractive index of water at the tempera- ture of the hemoglobin solution from the refractive index of the hemoglobin solution gives a figure proportional to the hemoglobin concentration over a very wide range.

Hemoglobin in the presence of NaCl and at a pH close to the iso- electric point of 6.8 can be coagulated in 3 minutes at lOO”C., giving a clear filtrate.

The refractive indices of the filtrates from the heat coagulation of hemoglobin correspond very closely to the ultrafiltrates from the same solutions. This gives an easy way to separate the refractive effect of hemoglobin from that of non-protein constit- uents under the proper conditions.

A method is described for obtai.ning the concentration of hemo- globin in terms of dry weight of the substance, applicable to blod.

This method compared with the oxygen capacity of the same blood obtained by the Van Slyke method, gives a ratio of 1.26, as compared with Hiifner’s of 1.34. If the oxygen capacity ‘were obtained by the Haldane method the ratio would be about 1.15.

A short method of obtaining the hemoglobin concentration by the refractometer is described. This method checks closely with the longer and more conclusive method.

We wish to express our indebtedness to Dr. Arlie V. Bock and Dr. Henry Field, Jr., for the determinations of the oxygen capacity.

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James L. Stoddard and Gilbert S. AdairDETERMINATION OF HEMOGLOBIN

THE REFRACTOMETRIC

1923, 57:437-454.J. Biol. Chem. 

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