CA1233096A

CA1233096A - One step method for sphering and fixing whole blood erythrocytes

Info

Publication number: CA1233096A
Application number: CA000475939A
Authority: CA
Inventors: Leonard Ornstein; Young R. Kim
Original assignee: Technicon Instruments Corp
Current assignee: Bayer Corp
Priority date: 1985-03-07
Filing date: 1985-03-07
Publication date: 1988-02-23

Abstract

UNITED STATES PATENT APPLICATION
OF: LEONARD ORNSTEIN
YOUNC RAN KIM
FOR: ONE STEP METHOD FOR SPHERING AND
FIXING WHOLE BLOOD ERYTHROCYTES

Abstract of the Disclosure A rapid one step, one-reagent method is disclosed for the isovolumecric sphering and fixing of whole blood erythrocytes.

Description

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Background to the Invention This invention relates generally to a rapid one-reagent method for sample preparation for improved electron optical methods or measuring human and animal erythrocyte volumes and cell hemoglobin contents. More particularly, it relates to a one step method for treating mammalian red blood cells in a sample which can be effectively measured electrooptically for determination of red blood cell volumes whereby the one-step, single reagent treatment provides isovolumetric sphering of erythrocytes in the sample indepen-dent of erythrocyte concentration in the original blood sample and without inducing louses.
Prior art methods utilizing the measured amount of light scattered from individual red cells (erythrocytes) to determine the individual and mean volumes of red cells were subject to error for two reasons: since the native human red cell is a biconcave disc, the amount of light scattered will vary with the orientation of the cell with respect to the incident light beam; and, the shape of the cells can change during dilution and pumping steps.
For a discussion of the above, see Hemolysis and Related Phenomena, Chapter II, pup 10-49 by Eric Ponder (1948) and Transformation and Restoration of Biconcave Shape of Human Erythrocytes Induced by Amphilic Agents and Changes of Ionic Environment, Biochemical et Biopsy. Act, Bernard Deuticke, pup 494-500 (1968).
In Ornstein et at U.S. 4,412,004, commonly assigned, a method is disclosed and claimed which eliminates both of the above-described causes of error and permits a vastly improved method for determination of human red blood cell volume. In Thea patent, the inventive method involves a two-step procedure of first sphering erthrocytes isovolumetrically and then fixing isotonically. The method was devised as a two-step procedure in order to be assured that sphering had reached completion before the fixing process began, otherwise, cells might become 'tstiffened'l by cross-link-in which occurs during fixation and thus becomes "frozen in ~33~6 some intermediate shape between the native biconcave shape and the desired spherical shape. Fixing immediately after sphering permitted the use of higher sphering agenttprotein ratios in the fixing solution because cells which might otherwise lyre, were fixed before that could happen. This leads to a more convenient implementation for automated clinical use.
It has now been found, quite unexpectedly, that a single reagent containing appropriate and limited ranges of so -2-~L23;3~36 concentrations of both sphering agent and fixing agent can achieve complete isovolumetric sphering of all erythrocytes of a large range of clinical samples of blood, independently of the concentration of erythrocytes in the whole blood sample, and without inducing louses of any of the erythrocytes in any of the diluted blood samples. It was not obvious to one of ordinary skill in the art that the employment of particular parameters in a one-step method as disclosed and claimed herein could allow for a procedure in which the speed of the sphering step remains sufficiently greater than the fixing process so that the cells are completely sphered before they have appreciably stiffened. This leads to a faster and still moxie convenient implementation for automated clinical use.
SUMMARY OF THE INVENTION
In accordance with this invention, there is claimed a method for treating mammalian red blood cells in a whole blood sample which can be subsequently effectively measured electrooptically for determination of red blood cell volumes which comprises treating in a single step an anti coagulated whole blood sample with an isotonic reagent solution containing both a sphering agent and a fixing agent at a temperature in the range of 4C to 37C over a reaction period of up to 180 seconds employing a whole blood dilution factor of from 1:300 to 1:4,000, wherein the sphering agent and the fixing agent are present in the reagent at concentrations and at a ratio which provides isovolumetric sphering of the erythrocytes in the sample independent of the concentration of the erythrocytes in the whole blood sample and without inducing louses of the erythrocytes in the resulting diluted sample.

In one preferred embodiment, the concentration of sphering agent in the reagent is from about 0.5 my % to about :;

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3.0 my %, preferably where it does not exceed 2.0 my %, and most preferred is a concentration of about 1 my I.
In another embodiment and in accordance with this invention, the sphering agent employed is an alkali metal salt of an alkyd sulfate, e.g. sodium dodecyl sulfate. It is understood however, that any suitable sphering agent known in this technology is applicable.
In another embodiment and in accordance with this invention, the fixing agent, e.g. glutaraldehyde, is provided at a concentration of from about 0.05% to about 0.3% preferably at about 0.2%. For the purposes of this invention glutaraldehyde is preferred; however, other aldehydes, such as formaldehyde and other fixing agents known in this technology are employable.
In another embodiment and in accordance with this invention, a phosphate buffer is included in the aqueous reagent, preferably at a concentration adjusted to provide a reagent pi of from 6.0 to 7.5, and more preferably, at a concentration of about 0.035 M to provide a reagent pi of about 7.3.
In another embodiment and in accordance with this invention, sodium chloride is added to the aqueous reagent, preferably at a concentration adjusted to provide a reagent osmolarity to about 290 mOs/kg.

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l In another embodiment and in accordance with this

2 invention, the diagnostic method is effected at about 25~C

3 over a period of about 60 seconds and at a whole blood dilution

4 factor in the range 1:600 to 1:700.

6 In further accordance with this invention, there 7 is claimed a composition for sphering red blood cells in 8 an anti coagulated whole blood sample in a single step to 9 ` provide isovolumetric sphering il1dependent of the red blood lo cell concentration in the whole blood sample and without if louses inducement which comprises an aqueous sphering agent/
12 fixing agent reagent comprising from about 0.5 my % to about 13 3-0 my % (mg/lO0 ml) of a sphering agent and from about 0.5% to about 14 0.3% (wow) of a fixing agent.

Detailed Description of the Invention . . .. _ 17 The herein disclosed and claimed invention 18 relates Jo a one step method for the isovolumetric sphering 19 and fixing of whole blood erythrocytes. It is a significant improvement over prior art methods with regard to its 21 kinetics and its ability to be adapted to an automated 22 system. It allows for the production of single formulations 23 of reagents which completely and isovolumetrically sphere 24 all erythrocytes of a large range of clinical samples of blood, independently of the concentration of erythrocytes 26 in the blood samples, and without inducing louses of any of 27 the erythrocytes in any of the diluted blood samples.

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, ~13~6 It has been found, unexpectedly, that conditions can be employed, such that, when whole blood is added to, and rapidly and thoroughly mixed with a single reagent containing appropriate arid limited ranges of concentrations of both sphering agent and fixing agent, complete isovolumetric sphering of all erythrocytes of a large range of clinical samples of blood can be achieved without inducing louses of any of the erythrocytes in any of the diluted blood samples. That such is achievable resides in the utilization of concentrations and temperatures which permit a particular reaction kinetics -the rate of sphering remains sufficiently greater than the rate of fixing so that the cells are completely sphered before they have stiffened appreciably.
A preferred set of conditions for the instant invention is set forth hereinbelow:
Temperature: room temperature (25C + 8C) Whole blood dilution factor: 600 _ 100 Reaction time: 60 seconds + 40 seconds Reagent formulation:
sodium dodecyl sulfate: 1 my % + 0.2 my %
glutaraldehyde: 0.2% + 0.1%
phosphate buffer: 0.035 + 0.015 M at pi 7.3 + 0.2 sodium chloride: to adjust reagent osmolarity to 290 +

5 mOs/kg 1 j When a whole blood sample is mixed with the 2 above reagent, red cells are isovolumetrically sphered 3 ' for more accurate and precise cell volume and hemoglobin 4 , measurements by a light scattering flow cytometry. A
preferred flow cytometry for the measurement uses a Hone

6 red laser (wavelength 632.8 no) as the light source,

7 I' and the illuminated field is defined as a 20 em x 100 em

8 , slit imaged in the center of the sheathed flow cell. Two

9 optical light scatter measurements are taken, the low

10 , (2 to 3) angle signals and high (5 to 15) angle

11 signals.

12 1

13 The low angle signals and the high angle signals

14 1 are paired to form a 50 x 50 erythrocyte volume and hero-glob in cytogram (the other high angle signals can be used 16 , to generate the platelet histogram). The cytogram's 17 data are processed by coincidence trimming followed by a 18 non-linear two-dimensional transformation. A computer 19 , program transforms the scatter amplitude signals into j I erythrocyte volume and hemoglobin concentration histograms 21 as disclosed in commonly assigned 22 ' Canadian patent application Serial No. 461,953, filed 23 August 28, 1984.

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The following parameters can be generated from the cell preparation:
1. Red blood cell count (RBC) 2. Mean corpuscular volume (MCV) 3. Cell hemoglobin concentration mean (CHUM) 4. Hematocrit (HOT) 5. Red cell volume distribution width (ROW) 6. Hemoglobin distribution width (HOW) 7. Platelets (PBC) 8. Mean platelet volume (MPV) 9. Plateletcrit (POT) 10. Platelet distribution width (POW) Broader parameters applicable to the present invent lion are provided hereinbelow:
Reaction Temperatures: can be varied from 4C to 37C
without risking any significant change in results. OWE
at 4C and -0.62% at 37C in Noel blood MCV were observed.
No change occurred in RBC and Pit counts at either temperature.

Whole Blood Dilution Factor: can be varied from 1:300 to 1:4,000. Without risking any significant change in results.
Less than +1% at 1:300 and less than -1% at 1:4,000 in fresh whole blood MCV has been observed.

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Sphering Agent Concentration: can be varied from 0.5 my % to 3.0 my % when all test samples are fresh whole bloods. Aged blood samples are more prone to louses at concentrations above 2.0 my %.
Fixing Agent Concentration: can be reduced to 0.05%. Above 0.3% interface with uniform shoring and excessive cross-linking takes place before sphering of erythrocytes is complete.
Pi of the Regent can range from 6.0 to 7.5. One pi unit beyond these limits produces -1.5% change in MCV and +1.5%
in CHUM to whole blood.
Reaction Times: at 180 seconds results in less than 1% change in whole blood MCV and CHUM. Up to 300 seconds result in MCV's with less than -2% error and Shakeups with less than +2% error.
EXAMPLE
Reagent Formulation Sodium Dodecyl Sulfate 3 my %
Glutaraldehyde 0.1 %
phosphate buffer 0.02 M
Nail To bring up 290 miasmal ~33q~6 Instrument 1) Light Source: Hone red laser (I = 632.8 no) 2) Illumination Field: 20 em x 100 em slit 3) Sheathed Flow cell (Sheath fluid = phosphate buffered saline, 290 miasmal, pi 7.4) 4) Two Optical Light Scatter Measurements Angle:
Low 2 to 3 High 5 to 15 5) Sample Stream Width: 15U

10 Procedure 1) 0.01 ml whole blood (ETA anti-coagulated) sample is added to 6.0 ml of the reagent and mixed immediately.
The red cells will be isovolumetrically sphered.

2) Aspirate the sample of sphered and fixed red cells through the sample stream and take 40 second reading.

The low angle signals and the high angle signals at low gain are paired to form a 50 x 50 RBC cytogram. The other high angle signals (lo low gain) is used to generate the platelet histogram. The cytogram's data is processed by 20 coincidence trimming followed by a non-linear two dimensional transformation, A computer program transforms the scatter amplitude signals into RBC volume and a hemoglobin concentration histogram.

The system must be recalibrated using 10 normal bloods whose parameters are predetermined by reference methods.

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, . .
_ I

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for treating mammalian red blood cells in a whole blood sample which can be subsequently effectively measured electrooptically for determination of red blood cell volumes which comprises treating in a single step an anticoagulated whole blood sample with an isotonic reagent solution containing both a sphering agent and a fixing agent at a temperature in the range of 4°C to 37°C over a reaction period of up to 180 seconds employing a whole blood dilution factor of from 1:300 to 1:4,000, wherein said sphering agent and said fixing agent are present in the reagent at concentra-tions and at a ratio which provides isovolumetric sphering of the erythrocytes in the sample independent of the concentra-tion of said erythrocytes in the whole blood sample and without inducing lysis of said erythrocytes in the resulting diluted sample.

2. The method of claim 1 wherein the concentration of said sphering agent in the reagent is from about 0.5 mg/100 ml to about 3.0 mg/100 ml.

3. The method of claim 2 wherein said concentration does not exceed 2.0 ml/100 ml.

4. The method of claim 3 wherein said concentration is about 1 mg/100 ml.

5. The method of claim 1 wherein said sphering agent is an alkali metal salt of an alkyl sulfate.

6. The method of claim 5 wherein said alkyl sulfate salt is sodium dodecyl sulfate.

7. The method of claim 1 wherein the concentration of said fixing agent is from about 0.05 w/w% to about 0.3 w/w%.

8. The method of claim 7 wherein said concentration is about 0.2 w/w%.

9. The method of claim 1 wherein said fixing agent is glutaraldehyde.

10. The method of claim 1 wherein said aqueous reagent includes a phosphate buffer.

11. The method of claim 10 wherein the phosphate buffer is adjusted to provide a reagent pH of from 6.0 to 7.5.

12. The method of claim 11 wherein said phosphate buffer concentration is about 0.035M and provides a reagent pH
of about 7.3.

13. The method of claim 1 wherein said aqueous reagent includes sodium chloride.

14. The method of claim 13 wherein the sodium chloride concentration is adjusted to provide a reagent osmolarity to about 290 Os/kg.

15. The method of claim 1 wherein said temperature is maintained at about 25°C (room temperature).

16. The method of claim 1 wherein said reaction period is about 60 seconds.

17. The method of claim 1 wherein said whole blood dilution factor is about 1:600 to 1:700.

18. The method of claim 1 wherein said aqueous reagent comprises sodium dodecyl sulfate at a concentration of about 1 mg %, glutaraldehyde at a concentration of about 0.2%, about pH 7.3 phosphate buffer at a concentration of about 0.035M and sodium chloride in an amount to provide a reagent osmolarity to about 290 mOs/kg.

19. The method of claim 1 wherein the resulting, diluted sample is subjected to light scattering measurement for determination of at least one of red blood cell volumes and cell hemoglobin content.

20. The method of claim 1 wherein the resulting, diluted sample is measured with a flow cytometer.

21. A composition for sphering red blood cells in an anticoagulated whole blood sample in a single step to provide isovolumetric sphering independent of the red blood cell concen-tration in the whole blood sample and without lysis inducement which comprises an aqueous sphering agent/fixing agent reagent comprising from about 0.5 mg % to about 3.0 mg % of a sphering agent and from about 0.05% to about 0.3% of a fixing agent.

22. The composition of claim 21 wherein said aqueous reagent includes a phosphate buffer to provide a reagent pH of from 6.0 to 7.5.

23. The composition of claim 22 wherein said phosphate buffer concentration is about 0.035 M and provides a reagent pH
of about 7.3.

24. The composition of claim 21 wherein said aqueous reagent includes sodium chloride in an amount to provide a reagent osmolarity of from 280 mOs/kg to about 300 mOs/kg.

25. The composition of claim 24 wherein said sodium chloride concentration provides a reagent osmolarity to about 290 mOs/kg.

26. The composition of claim 21 wherein said sphering agent is an alkali metal salt of an alkyl sulfate.

27. The composition of claim 26 wherein said alkyl sulfate salt is sodium dodecyl sulfate.

28. The composition of claim 21 wherein said fixing agent is glutaraldehyde.

29. A composition as claimed in claim 21 wherein said aqueous reagent comprises about 1 mg % sodium dodecyl sulfate, about 0.2% glutaraldehyde, about 0.035 M phosphate buffer at about pH 7.3 and sodium chloride in an amount to provide a reagent osmolarity of about 290 mOs/kg.