Patent Application
20150029492
The disclosure relates the direct determination of total hemoglobin concentration in whole blood without the need for transforming the hemoglobin through secondary reactions.
Traditional total hemoglobin measurements have generally been done indirectly, requiring significant sample quantities from a venous draw, extensive sample preparation and the use of toxic and dangerous chemicals. The assay reagents are typically used to lyse the blood cells to release the hemoglobin molecules to which accompanying molecules react with and bind unto the hemoglobin molecules to form a stable compound that is optically detectable, and whose concentrations are proportional to the amount of hemoglobin in the sample. The need for reagents adds to the cost of the assay and makes it less accessible in the developing world. Certain reagents also require specific storage conditions which makes them less useful in areas where climate control is rare. Thus there is a need for a method to determine total hemoglobin content in modified or unmodified forms even in very small blood samples.
Unless otherwise stated, the following terms used in this application, including the specification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
Described herein is a convenient and inexpensive method for quantifying total hemoglobin directly and rapidly in a system designed for use at the point of care using a very small blood sample. The sample requires no addition of reagents to accomplish the measurement. The sample is exposed to light at a plurality of wavelengths. Absorbance, transmission or reflectance measurements are made and the ratio of the measurements at two wavelengths indicate the amount of total hemoglobin.
A small amount of blood is drawn into a sample holder. Only a few drops are needed. The sample holder can be any holder normally used to collect blood samples provided that the holder material allows transmission of the wavelengths of light used for analysis. Example sample holders include glass capillaries (open on both ends) as well as test strips incorporating a capillary channel that is open to the atmosphere at both ends of the channel. When one end of the capillary is touched to a drop of blood, the blood is drawn into the channel. The sample holder may be uncoated and need not contain any reagents. No reagents (such as lysing agents) need to be added to the sample. It should be noted however that the disclosed method can be used with samples where a lysing agent has been added to the blood sample. The sample is then analyzed spectrophotometrically.
Example sample holders of various shapes are shown in
The sample is exposed to light at at least two wavelengths. The selected wavelengths should be sufficiently separated on the spectrum but this does not mean that the edges of the ranges of the two wavelengths cannot overlap. While not wishing to be bound by any particular theory, it is believed that the use of multiple wavelengths avoids error due to hemoglobin variants and side reactions. In one embodiment, the sample is exposed to both blue light as well as red light. In some embodiments the blue light has wavelengths spanning 390 to 520 nm, or 390 to 495 nm, or any subrange of any of the foregoing. In some embodiments, the blue light has a wavelength of between 390 nm and 520 nm or a plurality of wavelengths selected from wavelengths of 390 nm to 520 nm. In some embodiments, the red light has wavelengths spanning 500 to 700 nm, or 570 to 750 nm, or 620 to 750 nm or any subrange of any of the foregoing. In some embodiments, the red light has a wavelength of between 500 nm and 700 nm or a plurality of wavelengths selected from wavelengths of 500 nm to 700 nm. As shown in
The reflectance of the light off of the sample is detected or alternatively, the transmission of light through the sample of light is detected. In yet another alternative, the absorbance of light by the sample is determined. Standard spectrophotometric techniques can be used to make the spectrophotometric measurements. For ease of detection of the multiple wavelengths, the reflected or transmitted light is detected by a detector array such as a charge coupled device (CCD) or photodiode array. The sample is exposed to light for an extended period of time during which several measurements are taken. The sample may be exposed from 5-20 seconds, from 8-15 seconds or about 10 seconds. Measurements are taken every second, every half second or every quarter second. In some embodiments, the measurements are taken at several positions along the capillary channel.
The measurements for each color (or wavelength) of light are averaged and then the ratio is taken of the average of one color (wavelength) to the average of the other color (wavelength). If measurements are taken at multiple positions along the capillary channel, all measurements, regardless of position, for each color (wavelength) are averaged. For example if the measurements are transmission measurements and the light is blue light and red light, the ratio is determined as follows: (average of all transmission measurements of blue light)/(average of all transmission measurements of red light). This ratio correlates to the total hemoglobin concentration in the sample. After creation of a calibration curve, the ratio of optical measurements for a sample is used to determine the total hemoglobin concentration for the sample.
When larger samples are available, this method can be used with larger sample holders and spectrophotometers that hold larger volumes.
In some embodiments, the sample is exposed to many wavelengths of light and the two wavelengths used for analysis are those that are measured spectrophotometrically.
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Haemoglobincyanide (HiCN) references were prepared at 6.8, 9.7, 11.9, 16.3, 21.0 and 25.2 g/dL. The standards were prepared and the calibration curve calculated using the standard World Health Organization (WHO) method (“Haemoglobinometry” Chap 7 in the Blood and Safety and Clinical Technology, Guidelines on Standard Operating Procedures for Haematology published by WHO). The transmission of red light (from 500 to 700 nm) and blue light (from 390 to 520 nm) through the sample was measured. For each sample the transmission of each color was measured 20 times at ½ second intervals. The ratio of blue to red was determined for each measurement and then the average ratio was determined. The last 5 measurements for each sample are shown below in Table 1. Spectrophotometric analysis was performed by the Avie™ Alc available from MEC Dynamics in San Jose, Calif.
The resulting calibration curve is shown as
HiCN references from 5 to 23 g/dL were prepared by serial dilution and their concentration determined using the measurement procedure described in Example 1.
Fifty whole blood samples were tested using both the procedure in Example 1 as well as using a Hemocue™ Hb 201 available from HemoCue AB of Angelholm Sweden.
To assess the precision of the disclosed method, samples having low, mid and high hemoglobin concentrations were tested 20 times each using the method in Example 1. The results in Table 2 illustrate the precision of the disclosed method.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the invention as defined in the appended claims.
This application claims the benefit of the earlier filing date of U.S. Provisional Application 61/686,670 filed Apr. 9, 2012 which is hereby incorporated by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/035849 | 4/9/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61686670 | Apr 2012 | US |
