Method for treating red blood cells to effect sphering and reagent therefor

A method is disclosed of blood serum sample preparation for improved, more accurate and precise, electro-optical method for measuring erythrocyte volumes, individually and as an average.

BACKGROUND OF THE INVENTION 
This invention relates generally to a method for sphering or sphering and 
fixing whole blood erythrocytes without volume change for accurate and 
precise cell volume measurement. More particularly, the method employs a 
series of dilution steps whereby a protein, externally provided or 
endogenously supplied and a sphering agent are added in a protein/sphering 
agent weight ratio of from about 20:1 to about 70:1, based on total sample 
volume, and the concentration of detergent in the final sample is from 
about 2 mg./100 ml. to about 10 mg./100 ml. 
Methods which utilize the measured amount of light scattered from 
individual red cells (erythrocytes) to determine the individual and mean 
volumes of red cells, suffer from two kinds of error: 
1. The native human red cell is a biconcave disc and the amount of light 
scattered within a particular solid angle varies with the orientation of 
the cell with respect to the incident light beam; 
2. During handling, i.e. dilution and pumping, the shape of the cells can 
change depending in part on the time between the drawing of the blood and 
the time of measurement and in part on the composition of the diluted 
blood sample. 
For a discussion of the above, see Hemolysis and Related Phenomena, Chapter 
II, pp 10-49 by Eric Ponder (1948) and Transformation and Restoration of 
Biconcave Shape of Human Erythrocytes Induced by Amphiphilic Agents and 
Changes of Ionic Environment, Biochemica Et. Biophy. Acta, Bernard 
Deuticke, pp 494-500 (1968). 
The present invention eliminates both of these sources of error and permits 
vastly improved methods for determination of human red blood cell volumes. 
It is well known, see for example Ponder supra, that it is possible to 
sphere red blood cells in isotonic solution without changing their 
volumes. Since the light scattering from a perfectly sphered cell is 
invariant with orientation in a light beam, the first kind of error is 
eliminated. However, such preparations are notoriously unstable and red 
cell lysis occurs at various times after sphering, depending on the choice 
of sphering agent and the properties of the individual blood samples. 
It has now been discovered that prolonged stability of the sphered state 
can be achieved by controlling the absolute concentration of the sphering 
agent (typically a material with detergent properties) and the weight 
ratio of sphering agent to protein, either added or endogenous at any 
desired dilution in isotonic solution. This helps to assure shape 
consistency during processing and minimizes the second kind of error. 
The method of this invention can be carried out generally in two ways: 
A. A blood serum sample is diluted, typically about 1/1000, in an isotonic 
solution containing sphering agent (detergent) and albumin at the required 
concentrations; or 
B. The blood serum sample is diluted with an amount of isotonic solution 
containing the sphering agent at a concentration which is just sufficient 
to cause sphering when the dilution provides the correct ratio of sphering 
agent to the endogenous serum albumin (plasma protein) from the blood 
sample itself. The resulting sample is then simultaneously and/or 
successively fixed and further diluted by adding an isotonic solution of a 
fixing agent to harden the sphered cells and make them completely 
insensitive to processes which could otherwise cause them to change their 
shape or size or lyse and lose their contained hemoglobin. 
SUMMARY OF THE INVENTION 
In accordance with this invention, there is claimed a method for treating 
mammalian red blood cells in a sample to provide a sample which can be 
effectively measured electrooptically for determination of red blood cell 
volumes which comprises combining an anticoagulated whole blood sample 
with an isotonic solution containing sphering agent, and diluting an 
aliquot of the resulting sample with an isotonic solution containing 
protein and sphering agent. The weight ratio in the final sample of 
protein/sphering agent is from about 20:1 to about 70:1, preferably about 
50:1 and the concentration of sphering agent is from about 2 mg./100 ml. 
to about 10 mg./100 ml., preferably about 3 mg./100 ml. 
Preferably, whole blood sample is prediluted with saline, as diluent, 
resulting in about a 50% by volume dilution of sample to reduce viscosity 
and therefore assure reduction of volumetric pumping errors which stem 
from variations in blood sample viscosities. The subsequent dilution steps 
results in a final dilution of sample of about 1:1000 by volume to produce 
a dilution such that the probability of more than one cell passing through 
the incident light beam of the electrooptical detector during the 
detector's measuring time window is very low. 
The detergent used in this method is preferably an alkali metal salt of an 
alkyl sulfate, said alkyl group containing from 10 to 16 carbon atoms. 
Sodium lauryl sulfate is most preferred. 
The protein used in this method is preferably serum albumin, which is added 
externally. 
Another preferred method of this invention is similar to the 
above-described method except that in lieu of the protein/sphering agent 
dilution step, the aliquot sample is treated with a fixing agent solution, 
preferably an isotonic glutaraldehyde-containing saline solution. In this 
method, the protein required is endogenously provided in the sample as 
plasma protein. 
In another preferred embodiment of this invention, there is claimed a 
reagent for sphering red blood cells in a sample comprising a 
protein-sphering agent mixture whereby the weight ratio of protein to 
sphering agent is from about 20:1 to about 70:1 and the total 
concentration of sphering agent in the composite sample is from about 2 
mg./100 ml. to about 10 mg./100 ml. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to a method of sphering mammalian red 
blood cells in an anticoagulated whole blood sample. The method involves 
in the employment of a protein and a sphering agent in a specified weight 
ratio and a certain final sphering agent concentration. 
In the absence of protein, after addition of a sphering agent, the amounts 
of free sphering agent in solution is dependent upon the concentration of 
red cells (see Ponder above). Therefore, with a reagent with fixed optimal 
sphering agent concentration for a normal blood count, the degree of 
sphering could be either incomplete, with a blood with high red cell count 
per unit volume of solution, or could lead to lysis with a very low red 
blood cell count. Proteins, such as serum albumins, bind sphering agent 
reversibly and can therefore be used to buffer the effective concentration 
of sphering agent in the optimal range, independent of red cell count. 
The preferred concentration of sphering agent is that amount which is just 
sufficient to cause sphering when buffered with a protein such as albumin 
or plasma protein at any particular dilution of sample. The protein 
albumin can be provided in either of two ways: by outside addition or 
endogenously as plasma protein in the serum sample. 
In a preferred embodiment of this invention, the method involves combining 
a prediluted blood sample with an isotonic sphering agent-saline solution 
and then treating an aliquot thereof with a protein-sphering agent saline 
soluton. 
Preferably, the predilution step is carried out by diluting the serum 
sample about 50% by volume with a suitable isotonic diluent such as a 
saline solution. The resulting prediluted sample is combined with an 
isotonic solution containing a sphering agent (sometimes referred to 
herein as detergent). A typical first dilution results in a 50:1 dilution 
of sample. A further dilution is effected by treating an aliquot of the 
above sample with a protein-sphering agent solution to provide a dilution 
of sample of about 1000:1. The resulting sample contains sphered and 
stabilized erythrocytes at a feasible concentration for light scattering 
measurement. When such light scattering measurement is conducted employing 
a flow cell cytometer, the individual cell volumes can be determined as 
well as the number of cells. The mean volume can therefore also be 
calculated. 
A critical feature of this method involves the weight ratio of 
protein/sphering agent and the concentration of sphering agent. By 
regulating these parameters within certain limits, the sphering process is 
effectively accomplished and the analytical results highly probative. 
It has been found that a weight ratio of protein/sphering agent in the 
herein disclosed method is preferably from about 20:1 to about 70:1, with 
a ratio of 50:1 most preferred. For the final concentration of sphering 
agent, a concentration of from about 2 mg./100 ml. to about 10 mg./100 ml. 
is highly suitable, with a concentration fo 3 mg./100 ml. most preferred. 
The protein, externally supplied, is preferably a serum albumin. Other 
employable proteins include bovine, human and egg albumin. 
In a second method of this invention, the protein/sphering agent second 
dilution step is replaced by treatment with an isotonic fixing agent 
solution. In this system, the protein for the first dilution is provided 
in endogenous form in the serum sample as plasma protein. An isotonic 
solution of a sphering agent is added in a volume sufficient to bring the 
endogenous plasma protein/sphering agent ratio and also the concentration 
of sphering agent within the preferred ranges. The preferred fixing agent 
is glutaraldehyde, used in an amount to provide a final glutaraldehyde 
concentration of from 0.1% to 0.4% by weight. The isotonic fixing agent 
solution is suitably formulated with saline or a saline-sphering agent 
mixture. 
Because glutaraldehyde fixes red cells very rapidly, optimal buffering of 
the sphering agent concentration beyond the fixing agent addition step is 
considered less critical. As soon as the red cell count has been fixed, it 
becomes completely noncritical. 
The sphering agent employed in either method is suitably an alkali metal 
(sodium, potassium, lithium, cesium or rubidium) salt of an alkyl sulfate 
wherein said alkyl contains from 10 to 16 carbons. Alkali metal lauryl 
sulfates are preferred, and sodium lauryl sulfate most preferred. Other 
suitable sphering agents which may be employed in these methods include 
fatty acids, phospholipids, etc. It is to be noted that some nominal 
"sphering agents" such as crude egg lecithin (see Ponder above) actually 
contain a sphering agent as a minor impurity. For example, pure lecithin 
is not a sphering agent. It is to be understood that the weight 
concentrations discussed are of the active principle in any impure 
"sphering agent" and not the crude weight concentration. 
Both methods can be effected either continuously as in an automated system 
or in a discontinuous or discrete manner.

DETAILED DESCRIPTION OF THE DRAWING 
Referring to the sole FIGURE, a system is illustrated for measuring the 
volume of individual red cells in a discrete anticoagulated blood sample 
treated in accordance with the present invention. However, it is within 
the contemplation of the present invention that the measurement of the 
volume of red cells in successive anticoagulated blood samples may be 
effected on a continuous basis, for example, as described in U.S. Pat. No. 
3,740,143, assigned to a common assignee. 
Such system comprises a peristaltic pump 1 including pump tubes 3, 5, 7, 9 
and 10. As is understood, the relationship of the internal diameters of 
such pump tubes determines the proportioning of the sample and reactants 
introduced into the system. An aspirating probe 13 is connected along 
conduit 14, to the inlet of pump tube 5, whose outlet is connected to a 
junction 15. Probe 13 is adapted to be immersed into an anticoagulated 
blood sample 17 contained in a sample receptacle 19. It will be appeciated 
that probe 13 may be adapted, as described in U.S. Pat. No. 3,740,143, to 
be immersed, in turn, into successive sample receptacles, so as to effect 
the measurement of the red cell volumes of successive samples on a 
continuous basis. 
Also, the inlet end of pump tube 3 is connected to a source 21 of 
appropriate diluent for effecting the first dilution of the sample 17. 
Upon operation of pump 1, diluent is passed along pump tube 3 to junction 
23 in conduit 14, so as to be mixed with and dilute the sample being 
pressed from the probe 13. Also, an air line 25 from an "air-bar" 
structure 26, as described in U.S. Pat. No. 3,306,229, assigned to a 
common assignee, whose operation is phased to that of the pump 1 as 
indicated by the dashed connector, operates periodically to introduce 
occluding air segments into conduit 14. The presence of such 
"intra-sample" air segments insures proper proportioning of the sample and 
reactants into the system (and effective wash between successive samples) 
as described in the referenced patent. Concurrently, an isotonic solution 
containing the sphering agent is passed from source 27 along pump tube 7 
to junction 15, whereat it is mixed with the diluted sample passed along 
pump tube 5, to effect the second dilution of sample 17. The sample is 
flowed from junction 15 and through mixing coil 29, to effect a thorough 
mixing thereof, and subsequently along conduit 31 to a resampling fitting 
33. Fitting 33 includes a waste outlet 35 and a resampling outlet 37 
connected to the inlet of pump tube 9. The sample passes from outlet 37 
and to junction 39 along pump tube 9, excess sample and "intra-sample" air 
segments introduced into fitting 33 being passed to waste along waste 
outlet 35. A second "air-bar" structure 38 reintroduces "intra-sample" air 
segments along air line 36 into the diluted sample stream. 
The inlet of pump tube 10 is connected to a source 41 of fixing agent. The 
outlet of pump tube 10 is connected to junction 39, whereat the fixing 
agent and the twice-diluted sample are mixed and passed to mixing coil 43, 
to insure mixing of the time. The outlet of mixed coil 43 is passed to a 
resampling fitting 45, which includes a waste outlet 47 and a resampling 
outlet 49, the latter being connected to the inlet of the single pump tube 
of a secondary peristaltic pump 51. The sample is passed from the outlet 
49 and through pump 51 to a sheath-stream particle counter 53, of the type 
described in U.S. Pat. No. 3,740,143, supra. Again, excess sample and the 
"intra-sample" air segments are passed to waste along waste outlet 47. In 
counter 45, the red cells in the treated blood sample are confined to flow 
serially, so as to be individually counted and their volumes measured. The 
treated blood sample is thereafter passed to waste. The sphering of the 
red cells, according to the present invention, insures that the measured 
volume is independent of the orientation of the red cells as they progress 
through counter 53. In prior art, where the red cells were not properly 
sphered, the random orientation of the red cells proceeding through the 
particle counter often resulted in inaccurate volume determinations. 
EXAMPLE 1 
A sample (0.38 ml.) of anticoagulated whole blood is prediluted with 
isotonic saline (0.23 ml.). An aliquot (0.16 ml.) of the resulting sample 
is combined with 4.2 ml. of an isotonic saline solution containing sodium 
lauryl sulfate (3 mg./100 ml.). An aliquot (0.16 ml.) of the resulting 
diluted sample is then treated with 4.0 ml. of an isotonic saline solution 
containing bovine serum albumin (0.1%) and sodium lauryl sulfate (3 
mg./100 ml.). The final sample is placed in a flow cell and 
electrooptically measured. The red blood cell count and red blood cell 
volume were recorded. 
EXAMPLE II 
A sample (0.37 ml.) of anticoagulated whole blood is prediluted with 
isotonic saline (0.23 ml.). An aliquot (0.16 ml.) of the resulting sample 
is combined with 4.2 ml. of an isotonic saline solution containing sodium 
lauryl sulfate (3 mg./100 ml.). An aliquot (0.16 ml.) of the resulting 
diluted sample is then treated with 4.0 ml. of an isotonic saline solution 
containing glutaraldehyde (0.2%) and sodium lauryl sulfate (1 mg./100 
ml.). The final sample is placed in a flow cell and electrooptically 
measured. The red blood cell count and red blood cell volume were 
recorded. 
It should be understood by those skilled in the art that various 
modifications may be made in the present invention without departing from 
the spirit and scope thereof as described in the specification and defined 
in the appended claims.