Blood cell lysing composition and uses thereof

Abstract

Exemplary blood cell lysis compositions include a buffer and a secondary alcohol ethoxylate at a concentration in a range of about 2.5% to about 20% weight / volume (w / v). The secondary alcohol ethoxylate can include Tergitol TM TMN-100X or Tergitol TM 15-S-9. The composition can be configured to lyse at least 90% of blood cells in a blood sample.

Description

Technical Field

[0001] This specification generally relates to exemplary blood cell lysis compositions and their uses. Technical Background

[0002] In optical diagnostics, whole blood samples can cause light scattering that can affect measurement precision. Lysing the cells in a whole blood sample can reduce this type of light scattering. Invention Overview

[0003] Exemplary blood cell lysis compositions comprise a buffer and a secondary alcohol ethoxylate in concentrations ranging from about 2.5% to about 20% by weight / volume (w / v). Blood cell lysis compositions may include one or more of the following features, alone or in combination.

[0004] Secondary alcohol ethoxylates may include at least one of the following: Tergitol TM Type 15-S-12, Tergitol TM Type 15-S-30, Tergitol TM 15-S-5 type, Tergitol TM Type 15-S-7, Tergitol TM Type 15-S-9, Tergitol TM NP-10 type, Tergitol TM NP-4 type, Tergitol TM NP-40 type ethoxylated nonylphenol, Tergitol TM NP-7 type sodium (3,9-diethyltridecane-6-sulfonate), Tergitol TM Type 8 sodium (2-ethylhexyl sulfate), Tergitol TM NP-9 type, Tergitol TM TMN-100X, TEOS Tergitol TM Sodium (7-ethyl-2-methylundecyl-4-yl) sulfate, Tergitol TM TMN-10 or Tergitol TM TMN-6. Secondary alcohol ethoxylates can be Tergitol. TM TMN-100X or Tergitol TM 15-S-9.

[0005] The buffer may include at least one of 3-(N-morpholino)propanesulfonic acid (MOPS), phosphate buffer (PBS), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]ethanesulfonic acid (TES), 2,2-bis(hydroxymethyl)-2,2',2"-nitrotriethanol or 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris).

[0006] The hematopoietic cell lysis composition may include a biocidal agent. The biocidal agent may include at least one of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), Proclin 300, 1,2-benzisothiazolin-3-one (BIT), dodecyl guanidine hydrochloride (DGH), gentamicin, levofloxacin, colistin, octylisothiazolinone (OIT), or amikacin. The biocidal agent may include CMIT and MIT. The ratio of CMIT to MIT may be approximately 3:1 (CMIT:MIT). The concentration of CMIT:MIT in the hematopoietic cell lysis composition may be 1% (w / v).

[0007] The blood cell lysis composition may have a pH of about 6 to about 8. The secondary alcohol ethoxylate may be present at a concentration ranging from about 5% to about 18% (w / v). The secondary alcohol ethoxylate may be present at a concentration ranging from about 9% to about 14% (w / v). The secondary alcohol ethoxylate may be biodegradable. The blood cell lysis composition may be formulated to lyse at least 90% of the blood cells in a sample in less than 2 seconds. The secondary alcohol ethoxylate may have a foam height of about 75 mm to about 125 mm over 5 minutes (0.1 wt% active ingredient). The secondary alcohol ethoxylate may have a foam height of about 40 mm to about 75 mm over 5 minutes (0.1 wt% active ingredient).

[0008] Exemplary methods for determining hemoglobin levels in a blood sample include (i) mixing a blood cell lysis composition with the blood sample to dissolve one or more cells in the blood sample, and (ii) determining the hemoglobin level in the blood sample. Exemplary methods may include one or more of the following features, individually or in combination.

[0009] Hemoglobin may include at least one of oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, methemoglobin, thiohemoglobin, cyanomethemoglobin, or fetal hemoglobin. The method may include measuring bilirubin levels.

[0010] An exemplary method for preparing a blood-dissolving composition includes mixing a buffer with a secondary alcohol ethoxylate at a concentration ranging from about 2.5% to about 20% by weight / volume (w / v). Exemplary methods may include one or more of the following features, alone or in combination.

[0011] Secondary alcohol ethoxylates can have a foam height of about 40 mm to about 75 mm over 5 minutes (0.1 wt% active ingredient). Secondary alcohol ethoxylates can be or include Tergitol. TM TMN-100X or Tergitol TM 15-S-9.

[0012] An exemplary blood cell lysis composition includes (i) a buffer and (ii) a secondary alcohol ethoxylate, which is or contains Tergitol. TM TMN-100X or Tergitol TM 15-S-9. Blood cell lysis compositions may include one or more of the following features, alone or in combination.

[0013] The concentration of the secondary alcohol ethoxylate can range from about 9% to about 14% (w / v). The composition can be formulated to dissolve at least 90% of the cells in a sample in less than 2 seconds.

[0014] Two or more features described in this specification (including this summary) can be combined to form embodiments not specifically described in this specification.

[0015] Details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the specification, drawings, and claims. Attached Figure Description

[0016] Figure 1A This is a block diagram showing an example optical system used for CO2 pulse oximeter measurement of blood samples.

[0017] Figure 1B It is a graph showing absorbance measurements obtained using the example optical system.

[0018] Figure 2 This is a bar graph comparing the total hemoglobin (tHb) measurements of the secondary alcohol ethoxylate hematopoietic cell lysis composition and the control hematopoietic cell lysis composition.

[0019] Figure 3 This is a dot plot showing the total hemoglobin (tHb) measurements of two example secondary alcohol ethoxylate hematopoietic cell lysis compositions relative to a control hematopoietic cell lysis composition.

[0020] Figure 4This is a dot plot showing the oxyhemoglobin (O2Hb) measurements of two example secondary alcohol ethoxylate hematopoietic cell lysis compositions relative to a control hematopoietic cell lysis composition.

[0021] Figure 5 This is a dot plot showing the carboxyhemoglobin (COHb) measurements of two example secondary alcohol ethoxylate hematopoietic cell lysis compositions relative to a control hematopoietic cell lysis composition.

[0022] Figure 6 This is a dot plot showing the methemoglobin (MetHb) measurements of two example secondary alcohol ethoxylate hematopoietic cell lysis compositions relative to a control hematopoietic cell lysis composition.

[0023] Figure 7 This is a dot plot showing the deoxyhemoglobin (HHb) measurements of two exemplary secondary alcohol ethoxylate hematopoietic cell lysis compositions relative to a control hematopoietic cell lysis composition. Invention Details

[0024] A CO-plasmometer is a device that measures the concentration of different hemoglobins and the oxygen saturation of hemoglobin in a blood sample. The oxygen saturation, or "sO2," of a blood sample corresponds to the proportion of functional hemoglobin in the blood sample that binds to oxygen.

[0025] An exemplary CO-oxygen meter includes an optical system configured to measure different forms of hemoglobin and oxygen saturation based on the optical absorbance of hemoglobin forms in a blood sample. For example, the CO-oxygen meter may be configured to measure the optical absorbance of one or more of the following: functional hemoglobin types, including oxygen-carrying oxyhemoglobin (O2Hb) and deoxyhemoglobin (HHb), or dysfunctional hemoglobin derivatives such as carboxyhemoglobin (COHb), methemoglobin (MetHb), sulfohhemoglobin (SHb), and cyanomethemoglobin (CNmetHb). Exemplary CO-oxygen meters of the type described herein may also be configured to simultaneously measure the concentrations of fetal hemoglobin, adult hemoglobin, and bilirubin in a blood sample. Bilirubin is a byproduct of hemoglobin degradation, as described below.

[0026] CO-oxygen saturation measurement can be used to diagnose a variety of medical conditions, including but not limited to anemia, suspected carbon monoxide poisoning or other non-respiratory hypoxia, blood disorders, circulatory problems, and lung diseases. CO-oxygen saturation measurement can depend on the accurate measurement of light scattering in the optical system. In this respect, whole blood cell samples scatter more light in the optical system than lysed blood cell samples. Therefore, it may be beneficial to lyse blood samples before using them to obtain CO-oxygen saturation spectroscopic measurements. Lysed blood samples reduce light scattering, which can produce more accurate and precise CO-oxygen saturation measurements.

[0027] Therefore, the example systems and processes described herein involve mixing a blood cell lysis composition with a whole blood sample prior to obtaining a CO-oxygenation spectral measurement. The blood cell lysis composition lyses the blood sample. Lysis involves disrupting the cell membranes of whole blood cells, thereby releasing lysates, including hemoglobin, from the whole blood cells. Any one or more exemplary blood cell lysis compositions described herein (collectively, “blood cell lysis compositions”) can be used to lyse whole blood samples in the context of CO-oxygenation or other processes.

[0028] As previously mentioned, during CO-oxygenation, lysed blood samples may produce less light scattering than whole blood, potentially leading to more accurate CO-oxygenation measurements. Exemplary blood cell lysis compositions include one or more surfactants, one or more buffer solutions (“buffers”), a biocide, and DI (deionized) water. Surfactants include compounds that lyse cells by disrupting cell membranes to release lysates. For example, surfactants can be synthesized via the reaction of fatty alcohols and ethylene oxide. Exemplary surfactants that can be used to lyse whole blood samples include alcohol ethoxylates. Exemplary alcohol ethoxylates have the chemical formula R(OC₂H₄). n OH. Here, R refers to the connected hydrogen or hydrocarbon side chain, and n refers to the number of units in ethylene oxide (OC2H4).

[0029] An exemplary blood cell lysis composition includes a nonionic surfactant, such as a secondary alcohol ethoxylate, in a buffer containing a preservative, possibly DI water, and a biocide. When the blood cell lysis composition is mixed with whole blood prior to CO2 oximetry, the secondary alcohol ethoxylate acts to lyse the blood sample. The secondary alcohol ethoxylate may comprise 11 to 15 ethylene oxide units, which can be marketed under the trade name Tergitol. TM Commercialization is not yet possible. Examples of secondary alcohol ethoxylates under this trade name include, but are not limited to, Tergitol. TM Type 15-S-12, Tergitol TM Type 15-S-30, Tergitol TM 15-S-5 type, Tergitol TM Type 15-S-7, Tergitol TM Type 15-S-9, Tergitol TM NP-10 type, Tergitol TM NP-4 type, Tergitol TM NP-40 type ethoxylated nonylphenol, Tergitol TM NP-7 type sodium sulfate (3,9-diethyltridecane-6-sulfonate), Tergitol TMType 8 sodium sulfate (2-ethylhexyl sulfate), Tergitol TM NP-9 type, Tergitol TM TMN-100X, TEOS Tergitol TM Sodium sulfate (7-ethyl-2-methylundecane-4-yl), Tergitol TM TMN-10 or Tergitol TM TMN-6. Other examples of secondary alcohol ethoxylates that may be included in blood cell lysis compositions include, but are not limited to, ECOSURF. TM For example, ECOSURF TM EH-9 and ECOSURF TM SA-9.

[0030] The chemical structures of exemplary secondary alcohol ethoxylates of nonionic surfactants that may be part of an exemplary blood cell lysis composition include, but are not limited to, the following:

[0031] (1) Tergitol TM NP-10, where z = 10, and its chemical structure is as follows:

[0032]

[0033] (2) Tergitol TM 15-S-7, where x = 7 and Tergitol TM 15-S-9, where x = 9, and its chemical structure is as follows:

[0034]

[0035] (3) Tergitol TM TMN-6, where n=6 and Tergitol TM TMN-10, where n = 10, has the following chemical structure.

[0036]

[0037] (4) Tergitol TM TMN-100X, which may include Tergitol TM TMN-10 and Tergitol TM The ratio of TMN-6 is 70:30.

[0038] In some embodiments, exemplary blood cell lysis compositions comprising the surfactants described herein may include one or more secondary alcohol ethoxylates at one or more concentrations of the following: between about 2.0% or 2.5% and about 20% by weight per volume (w / v), between 5% and 18% (w / v), between 9% and 14% (w / v), between 9% and 11% (w / v), between 9% and 10% (w / v), between 9.0% and 9.5% (w / v), and between 10% and 12% (w / v). Between 10% and 11% (w / v), between 10.0% and 10.5% (w / v), between 11% and 13% (w / v), between 11% and 12% (w / v), between 11.0% and 11.5% (w / v), between 12% and 14% (w / v), between 12% and 13% (w / v), between 12.0% and 12.5% ​​(w / v), between 13% and 14% (w / v), or between 13.0% and 13.5% (w / v).

[0039] Surfactants used for lysis, including secondary alcohol ethoxylates described herein, can reduce the surface tension in liquid blood samples and induce foam formation in blood samples when air is mixed in. Foam is detrimental to light absorbance measurements because it causes additional light scattering. In some embodiments, surfactants used for cell lysis in CO-oxygen assays can reduce or minimize foam formation and / or generate foam that dissipates rapidly before spectroscopic measurements. Exemplary surfactants for lysis may include secondary alcohol ethoxylates with a surface tension between 20 and 40 dynes / cm when measured at 25°C with 1% active material. Exemplary surfactants for lysis may include secondary alcohol ethoxylates having a measurable foam height of less than 110 mm in an aqueous solution of 0.1% by weight of the active material. For example, a standard measurement of foam height can be performed five minutes after the surfactant is added to the aqueous solution. The foam height generated by secondary alcohol ethoxylates can be measured, for example, using the Ross-Miles method. In an exemplary embodiment, the surfactant has a 5-minute (0.1 wt% active ingredient) foam height ranging from about 75 mm to about 125 mm. In an exemplary embodiment, the surfactant has a 5-minute (0.1 wt% active ingredient) foam height ranging from about 40 mm to about 75 mm.

[0040] In the context of CO-oxygenation, turbidity is a measure of the amount of light scattered by an optical system when light passes through a blood sample. Optical transparency can be a function of the turbidity of a blood sample; for example, the less light scattered by a blood sample, the greater the optical transparency associated with the blood sample. In some embodiments, the exemplary blood cell lysis compositions described herein lyse 90% to 100% of the cells in a whole blood sample within 1 to 3 seconds to reduce scattering effects and improve target optical clarity during CO-oxygenation. In this regard, compositions containing Tergitol... TM 15-S-9 and / or Tergitol TM TMN-100X, a surfactant with a similar structure to secondary alcohol ethoxylates, can be used to dissolve at least 90% of blood cells within 1 to 3 seconds.

[0041] In some embodiments, nonionic surfactants other than those described above can be used to lyse blood cells in a CO-oxygenation environment or elsewhere. For example, octylphenol ethoxylates can be part of a blood cell lysis composition for lysing blood samples, such as whole blood samples. Examples of octylphenol ethoxylates include ethylene oxide adducts of octylphenol. Triton X-100 is an exemplary octylphenol ethoxylate that can be used to lyse blood samples. Triton X-100 has the following chemical structure,

[0042]

[0043] In some embodiments, the exemplary blood cell lysis composition may include a buffer other than a surfactant. Exemplary buffers include aqueous solutions that resist pH changes by neutralizing small amounts of added acid or base using a weak acid and its salt or a weak base and its salt. Examples of buffers that can be used in the hemolymph lysis compositions described herein include, but are not limited to, the following: 3-(N-morpholino)propanesulfonic acid (MOPS), phosphate buffer (PBS), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]ethanesulfonic acid (TES), 2,2-bis(hydroxymethyl)-2,2',2"-nitrotriethanol or 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris). In some embodiments, the buffer may include 3-(N-morpholino)propanesulfonic acid (MOPS). In some embodiments, the buffer maintains the pH of the hemolymph lysis composition between 6 and 8. In some embodiments, the buffer is selected to reduce the effect of pH on spectral changes in hemoglobin in CO-oxygenation measurements; for example, pH changes can alter the absorption spectrum and the measurement of MetHb or other substances.

[0044] In some embodiments, the exemplary blood cell lysis compositions described herein may include biocides. Exemplary biocides include compounds designed to destroy, inhibit, render harmless, or exert control over any harmful organisms that may be present in the blood cell lysis composition. Examples of biocides that can be used in blood cell lysis compositions include, but are not limited to, the following: 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), with a CMIT:MIT ratio of approximately 3:1, and traded under the name Proclin. TM 300, 1,2-benzisothiazolin-3-one (BIT), dodecyl guanidine hydrochloride (DGH), gentamicin, levofloxacin, colistin, octylisothiazolinone (OIT), or amikacin. In some embodiments, the concentration of the CMIT:MIT biocide in the hematopoietic cell lysis composition may be 1% by weight per volume (w / v).

[0045] CO-oxygen meters may be part of a system used for measurements at point-of-care (POC) sites or central laboratories. Anemia, blood disorders, circulatory problems, and lung diseases can affect blood oxygen saturation levels and, if left untreated, can lead to organ damage or death. CO-oxygen meters can be used at POC sites and / or central laboratories to determine the oxygen saturation of hemoglobin, total hemoglobin, or functional or dysfunctional hemoglobin in blood samples, which may be evidence of such conditions. The blood cell lysis compositions described herein can be used to improve CO-oxygenation measurements by reducing scattering effects from red blood cells and by buffering the lysed blood sample to reduce or minimize the influence of pH on spectral measurements performed by the CO-oxygen meter. In this regard, in some embodiments, the CO-oxygen meter system may measure the pH, pO2, pCO2, and Na+ of blood samples in addition to CO-oxygen saturation and / or bilirubin measurements. + K + Ca 2+ Cl - glucose, lactate, and / or hematocrit.

[0046] Figure 1AAn exemplary CO-Oximeter system 5 is shown. The CO-Oximeter system 5 includes an optical absorption system configured to illuminate a blood sample and determine the spectral absorbance of the blood sample. The blood sample analyzed by the CO-Oximeter system 5 may be a whole blood sample lysed using one or more blood cell lysis compositions described herein. More specifically, in the example CO-Oximeter 5, the blood cell lysis composition and the blood sample are introduced into a mixing chamber in an optical cell 7—for example, via a mixing valve. Movement of the blood sample and the blood cell lysis composition through the mixing chamber, simultaneously driven by a solenoid, causes the blood sample and the lysis composition to mix and lyse red blood cells. The resulting mixture constitutes a lysed blood sample that can be used to obtain optical absorbance measurements via a CO-Oximeter, reducing or eliminating scattering effects caused by red blood cells.

[0047] The CO-oximeter 5 also includes a broad-spectrum white light-emitting diode (LED) light source 10 to illuminate the lysed blood in the mixing / optical chamber, and a neon lamp 11 with a high signal-to-noise ratio, which can be used as a wavelength reference for optical absorbance measurement. The exemplary CO-oximeter 5 also includes a high-resolution spectrometer 12 having a holographic diffraction grating 14 and a charge-coupled device (CCD) 19 contained in a chamber 13. The CCD 19 includes an integrated circuit that may include an array of capacitors to provide high signal-to-noise ratio digital imaging. The CCD 19 communicates with a computing system 17. This communication can be wired or wireless and is indicated by dashed lines 18. The computing system 17 is configured to perform computational analysis to resolve the sample spectrum detected by the CO-oximeter, as described below.

[0048] In the CO-oxygen meter 5, a lysed blood sample is illuminated in an optical cell 7 by light 4 emitted from a light source 10. Light 4 is reflected from mirrors 20a and 20b through the optical cell 7 containing the lysed blood sample to a spectrometer 12. In some embodiments, the optical cell 7 includes two parallel-plate optical windows 8 separated by a path length 9 through which light 4 is transmitted to the spectrometer 12. The wavelengths composed of the light passing through the lysed blood sample are separated in the spectrometer 12 by a holographic diffraction grating 14. These components are guided and struck a CCD 19. The CCD 19 generates a charge based on the incident light, which is read by an electronic device and becomes a digital copy of the light pattern falling on the CCD 19.

[0049] The light source 10 can be controllable—for example, via a computational system 17—to irradiate the lysed blood sample within a wavelength range such as 480 nm to 650 nm. The spectrometer 12 measures light passing through the lysed blood sample at different wavelengths. Measurements in different or varying wavelength ranges enable the CO-oxygen meter 5 to distinguish the absorbance spectra of different hemoglobin types or derivatives. In one example, the absorbance of a component of the blood sample, such as a hemoglobin derivative, includes the attenuation of transmitted radiant power through that component due to physical absorption processes that account for reflection, scattering, or other physical phenomena. Based on the measured spectral values, the noise-corrected absorbance of hemoglobin or hemoglobin derivatives in the blood sample can be determined using the following equation:

[0050] Abs = log 10 [IB / IS].

[0051] In the preceding equation, IB refers to the dark-corrected intensity spectrum of the process control solution (PCS) B. PCS-B is a colorless solution used to provide a zero-concentration reference for CO-oxygenation measurement. In the above equation, IS refers to the dark-corrected intensity spectrum of hemoglobin or a hemoglobin derivative.

[0052] Data on the absorbance of different hemoglobins or hemoglobin derivatives in a blood sample are transmitted from spectrometer 12 to computing system 18. The computing system 18 analyzes and processes this data and presents it to the user in a graphical user interface (GUI). For example, absorbance spectra of hemoglobin and / or hemoglobin-related products can be collected and stored in computer memory. Computing system 17 can use this data to determine the concentration of hemoglobin and / or hemoglobin-related products in the blood sample. Based on this information, measurements such as total hemoglobin (tHb) can be obtained, which indicates the total concentration of hemoglobin in the blood sample.

[0053] refer to Figure 1B The computing system 17 can also generate a GUI 25. GUI 25 plots the optical absorbance (y-axis) of various wavelengths of light (x-axis) for different hemoglobins and / or hemoglobin-related products in the lysed blood sample. In GUI 25, the different hemoglobins and / or hemoglobin-related products include O2Hb 21, HHb 22, CoHb 23, MetHb 24, and bilirubin 25.

[0054] As previously mentioned, oxygen saturation, or "sO2," corresponds to the ratio of oxygen-saturated hemoglobin to total functional hemoglobin in a blood sample. sO2 can predict the amount of oxygen available for tissue perfusion. In some implementations, the calculation system can determine the oxygen saturation of hemoglobin in a blood sample using O2Hb and HHb concentrations based on CO-oxygen saturation measurements. The equation for determining oxygen saturation in a blood sample is as follows:

[0055] sO2=100x[O2Hb / (O2Hb+HHb)]%.

[0056] The CO-pulse oximeter 5 and the calculation system 17 can be configured to determine the amount of bilirubin in a lysed blood sample. In this regard, bilirubin is a yellow compound that occurs in the normal catabolism pathway of vertebrate heme proteins (“heme”). This catabolism is likely a necessary process for the body to clear waste products generated from aging or abnormal red blood cell destruction. To measure bilirubin, its absorbance, as measured by the CO-pulse oximeter 5, can be compared with a predefined standard based on Beer Lambert's law. At this point, the calculation system can determine the absorbance of bilirubin in the blood sample using the following equation:

[0057] A = log 10 (IB / IS)=εCL

[0058] In the above, A is the value corresponding to the absorbance of the lysed blood sample, IB and IS are as described above, C is the concentration of the solution containing the lysed blood sample, ε is the molar extinction coefficient, and L is the path length of light propagation. Total bilirubin can be reported as a plasma equivalent concentration. In this respect, when analyzing whole blood, the calculation system can perform hematocrit correction to adjust for the dilution effect from red blood cells, thereby obtaining a plasma equivalent concentration. Hematocrit correction can be performed using the following equation:

[0059] Bili p =Bili b / (1-Hct)

[0060] Above, Bilibili p Corresponding to the concentration of total bilirubin in the plasma phase, Bili b Hct represents hematocrit as a fraction of the total bilirubin concentration in whole blood. Hct is determined by multiplying total hemoglobin in grams per deciliter (g / dL) by a constant of 0.03. The constant 0.03 is an example based on the average concentration of hemoglobin in red blood cells.

[0061] The blood cell lysis composition described herein can be used for optical clinical chemistry or immunoassays not specifically described herein, and in conjunction with… Figure 1AOther instruments besides those shown are used together. The following describes an experiment using a surfactant that may be included in the exemplary blood cell lysis composition.

[0062] In the first example, the surfactant is targeted to meet the following criteria: at the test concentration, the surfactant dissolves 100% of the blood cells in 1 to 2 seconds or less; the surfactant is compatible with the buffers and other chemicals used in the blood cell lysis composition; the surfactant does not interfere with the expected absorbance measurement; the surfactant does not chemically interact with hemoglobin; the surfactant does not chemically interact with bilirubin; the surfactant has relatively low foaming properties; the surfactant meets the established product requirements for CO-oxygen analyzers; and the surfactant is environmentally friendly. The above example criteria are not intended to limit the selection of other surfactants for other blood cell lysis compositions.

[0063] Several surfactants were screened based on the above criteria. Tergitol, a secondary alcohol ethoxylate... TM TMN-100X (hereinafter referred to as secondary alcohol ethoxylate 1) and Tergitol TM Type 15-S-9 (hereinafter referred to as secondary alcohol ethoxylate 2) was tested and found to meet the above standards. As described above, each blood cell lysis composition includes a secondary alcohol ethoxylate in a buffer and a preservative.

[0064] Table 1 below shows the surface tension and foam height of the blood cell lysis composition after mixing with water for 5 minutes during lysis using secondary alcohol ethoxylate 1, secondary alcohol ethoxylate 2, and Triton-X100. Triton-X100 is a control (or reference) surfactant compared to the secondary alcohol ethoxylate surfactant. As shown, the foam heights of secondary alcohol ethoxylate 1 and 2 decreased faster over time than that of surfactant Triton X-100. Similar foam heights and rapid foam dissipation may be an advantage of secondary alcohol ethoxylates, as foaming can lead to optical scattering that could interfere with or adversely affect CO-oxygen saturation measurements. In Table 1 below, "% by weight of active substance" refers to surface tension, a property of the surfactant that indicates / involves the effectiveness of the material as a surfactant.

[0065] Table 1

[0066]

[0067] Table 2 below shows the components of the exemplary blood cell lysis composition. In the exemplary test procedure, secondary alcohol ethoxylate 1 and secondary alcohol ethoxylate 2 are included in the exemplary blood cell lysis composition described with respect to Table 2 below at a concentration of 9% to 14%, and tested in a CO-oxygen saturation system of the type described herein.

[0068] 3-(N-morphorline)propanesulfonic acid (MOPS), phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]ethanesulfonic acid (TES), 2,2-bis(hydroxymethyl)-2,2',2"-nitrotriethanol or 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris)

[0069] Table 2

[0070] chemicals weight% MOPS (3-morpholino-1-sulfonic acid) 3-5％ NaOH, 2N 4-8％ <![CDATA[ProClin TM 300]]> 1-2％ Secondary alcohol ethoxylate 1 or secondary alcohol ethoxylate 2 9-14％ Deionized water 71-83％

[0071] The blood cell lysis compositions in Table 2, including secondary alcohol ethoxylate 1 or 2, meet the above criteria. For example, whole blood samples mixed with blood cell lysis compositions including secondary alcohol ethoxylate 1 or 2 exhibit approximately 100% whole blood cell lysis (e.g., 90% or greater lysis) after 1 to 2 seconds. Furthermore, the absorption spectra of the calibration solution and the lysed blood samples obtained at multiple wavelengths are substantially unaffected by either of the test surfactants.

[0072] Figure 2This is a bar graph comparing the total hemoglobin (tHb) concentration of blood samples mixed with a hemoglobin lysis composition containing secondary alcohol ethoxylate 1 28 or a hemoglobin lysis composition containing secondary alcohol ethoxylate 2 26, and a blood sample mixed with a control hemoglobin lysis composition containing Triton X-100 27. The y-axis represents the “δ(tHb-Avr Ref tHb) bias,” which refers to the difference between the total hemoglobin concentration obtained using each test hemoglobin lysis composition (26 and 28) and the mean total hemoglobin concentration, as shown by the dashed line 29 obtained using Triton X-100 27. Dashed lines 30 and 31 show the analytical range (+0.5 to -0.5) of acceptable bias around the mean 29 for hemoglobin lysis compositions based on Triton X-100 27. Blood cell lysis compositions including secondary alcohol ethoxylate 1 26 or secondary alcohol ethoxylate 2 28 showed total tHb concentrations ranging from 7 to 21 g / dL, similar to blood cell lysis compositions including Triton X-100 27, with only a small negative bias. Example data suggest that, considering the rate and integrity of lysis, relatively small or no effect on sample spectra, and the accuracy and precision of tHb measurements, secondary alcohol ethoxylate 1 and 2 may perform as well as or better than Triton X-100 27 in blood cell lysis compositions.

[0073] The following experiments were conducted in triplicate. Some experiments were conducted on different dates ("12-20" or "01-08").

[0074] Figure 3 This is a dot plot comparing the total hemoglobin (tHb) concentration of blood samples mixed with test hemoglobin lysis compositions including secondary ethoxylate 1 28 or secondary ethoxylate 2 26 and blood samples mixed with control hemoglobin lysis compositions including Triton X-100 27. The y-axis represents "δ(tHb-reference)," which refers to the difference between the total hemoglobin value obtained using the test hemoglobin lysis composition and the total hemoglobin value obtained using the control hemoglobin lysis composition including Triton X-100 27. The x-axis represents "Avg.tHbcal control (g / dL)," which refers to the average total hemoglobin value in g / dL obtained using control hemoglobin lysis composition 27. The total hemoglobin values ​​obtained using test hemoglobin lysis compositions including secondary ethoxylate 1 28, secondary ethoxylate 2 26, and the control hemoglobin lysis composition including Triton X-100 27 are plotted together. The data indicate at least partial overlap at all levels of total hemoglobin.

[0075] in this regard, Figure 3The tHb sample measurements from different types of samples are shown, aggregated according to tHb values ​​(g / dL). Figure 3 The tHb clusters shown include (i) low tHb and lipemic samples 34; (ii) normal tHb, all COHb levels, all HHb levels, and all MetHb levels, as well as Intralipid TM Sample 35; (iii) Intralipid TM Sample 36; high tHb sample 37; and high tHb sample 38. Dashed lines 32 and 33 show the range of acceptable deviations for the tested hematopoietic lysis compositions. The data suggest that, considering the rate and integrity of lysis, relatively small or no effect on the sample spectra, and the accuracy and precision of tHb measurements, secondary alcohol ethoxylates 1 and 2 may perform as well as, or better than, control hematopoietic lysis compositions including Triton X-100 27, when determining total hemoglobin using CO-oxygen saturation measurements.

[0076] Figure 4 This is a dot plot comparing the oxyhemoglobin (O2Hb) concentrations of blood samples mixed with test hematopoietic lysis compositions of the type described herein, including secondary ethoxylate 1 28 or secondary ethoxylate 2 26, and blood samples mixed with control hematopoietic lysis compositions including Triton X-100 27. The y-axis represents “δ(%O2Hb-reference)”, which refers to the difference in oxyhemoglobin obtained using each test hematopoietic lysis composition compared to the control hematopoietic lysis composition including Triton X-100 27. The x-axis represents “Avg-reference O2Hb (%)”, which refers to the percentage average of oxyhemoglobin values ​​obtained using the control hematopoietic lysis composition including Triton X-100 27. Values ​​of oxyhemoglobin obtained using hematopoietic lysis compositions including secondary ethoxylate 1 28, secondary ethoxylate 2 26, and control lysis composition 27 are plotted together to illustrate the overlap of oxyhemoglobin levels.

[0077] in this regard, Figure 4 The figure shows the O2Hb sample measurements from samples clustered according to their O2Hb values ​​(g / dL). The O2Hb clusters shown include: 60% deoxygenated Hb sample (HHb) 41; 30% HHb sample 42; 20% MetHb and 20% CoHb sample 43; 5-10% MetHb sample 44; 5-10% CoHb sample 45; 0% Intralipid TM Sample 46; and all LD (lipidemia), 1% COHb, 0.5-1% Intralipid TMAnd 0-1% MetHb sample 47. Dashed lines 39 and 40 show the analytical range of acceptable deviation for testing the blood cell lysis compositions. The data suggest that, considering the rate and integrity of lysis, relatively small or no effect on the sample spectrum, and the accuracy and precision of O2Hb measurements, secondary alcohol ethoxylate 1 and secondary alcohol ethoxylate 2 may perform as well as or better than control blood cell lysis compositions, including Triton X-100 27, when determining oxyhemoglobin using CO-oxygen saturation measurements.

[0078] Figure 5 This is a dot plot comparing (i) the concentration of dysfunctional hemoglobin carboxyhemoglobin (COHb) in blood samples mixed with test hematopoietic lysis compositions including secondary ethoxylate 1 28 or secondary ethoxylate 2 26, and (ii) the concentration in blood samples mixed with control hematopoietic lysis compositions including Triton X-100 27. The y-axis represents “δ(COHb-reference)”, which refers to the difference between carboxyhemoglobin obtained using each test hematopoietic lysis composition and carboxyhemoglobin obtained using the control hematopoietic lysis composition including Triton X-100. The x-axis represents “Avg control COHb (%)”, which refers to the percentage average of carboxyhemoglobin values ​​obtained using hematopoietic lysis composition 27. Carboxyhemoglobin values ​​obtained using hematopoietic lysis compositions including secondary ethoxylate 1 28 and secondary ethoxylate 2 26 are plotted together with those obtained using control hematopoietic lysis composition 27 and demonstrate overlap of carboxyhemoglobin at all levels.

[0079] in this regard, Figure 5 The figure shows the measured values ​​of COHb samples aggregated based on COHb values ​​(g / dL). Figure 5 The COHb cluster shown includes: all LD (hyperlipidemia), tHb, and Intralipid. TM Samples 50 (HHb and MetHb); 5% COHb sample 51; 10% COHb sample 52; and 20% COHb sample 53. Dashed lines 48 and 49 show the acceptable range of deviation for the tested hematopoietic lysis compositions. The data suggest that, considering the rate and integrity of lysis, no effect on sample spectra, and the accuracy and precision of COHb measurements, secondary alcohol ethoxylates 1 and 2 may perform as well as or better than the control hematopoietic lysis composition containing Triton X-100 27.

[0080] Figure 6This is a dot plot comparing the concentrations of dysfunctional methemoglobin (MetHb) in blood samples mixed with hematopoietic lysis compositions including secondary ethoxylate 1 28 or secondary ethoxylate 2 26 and blood samples mixed with a control hematopoietic lysis composition including Triton X-100 27. The y-axis represents "δ(MetHb-reference)," which refers to the difference in methemoglobin obtained using each hematopoietic lysis composition compared to that obtained using the control hematopoietic lysis composition including Triton X-100. The x-axis represents "Avg control MetHb (%)," which refers to the percentage average of methemoglobin values ​​obtained using control hematopoietic lysis composition 27. Methemoglobin values ​​obtained using hematopoietic lysis compositions including secondary ethoxylate 1 28, secondary ethoxylate 2 26, and control hematopoietic lysis composition 27 are plotted together, showing overlap at all methemoglobin levels.

[0081] Figure 6 The measurements are shown from donors aggregated based on their MetHb values ​​(g / dL). Figure 6 The MetHb cluster shown includes: all LD (lipemia donors), tHb, COHb, and Intralipid. TM Samples 56 (HHb and 0% MetHb); 57 (5% MetHb); 58 (10% MetHb); and 59 (20% MetHb). Dashed lines 54 and 55 show the range of acceptable deviations for testing the blood cell lysis compositions. The data suggest that, considering the rate and integrity of lysis, relatively small or no effect on the sample spectra, and the accuracy and precision of MetHb measurements, secondary alcohol ethoxylates 1 and 2 may perform as well as or better than the reference used in blood cell lysis compositions for determining methemoglobin in CO-oxygen saturation measurements.

[0082] Figure 7This is a dot plot comparing the concentrations of deoxyhemoglobin (HHb) in blood samples mixed with hematopoietic lysis compositions including tert-ethanol ethoxylate 1 28 or tert-ethanol ethoxylate 2 26 and blood samples mixed with a control hematopoietic lysis composition including Triton X-100 27. The y-axis represents "δ(HHb-reference)", which is the difference between the deoxyhemoglobin obtained using each hematopoietic lysis composition and the deoxyhemoglobin obtained using the control hematopoietic lysis composition including Triton X-100. The x-axis represents "Avg control HHb (%)", which is the percentage average of the deoxyhemoglobin values ​​obtained using control hematopoietic lysis composition 27. Deoxyhemoglobin values ​​obtained using hematopoietic lysis compositions including tert-ethanol ethoxylate 1 28, tert-ethanol ethoxylate 2 26, and the control hematopoietic lysis composition including Triton X-100 27 are plotted together. The plot of these values ​​shows the overlap of deoxyhemoglobin levels.

[0083] in this regard, Figure 7 The HHb measurements of the samples aggregated based on HHb values ​​(g / dL) are shown. Figure 7 The HHb clusters shown include: all LD (lipemia donors), tHb, COHb, and Intralipid. TM Samples 62 (MetHb and 0% HHb); 63 (30% HHb); and 64 (60% HHb). Dashed lines 60 and 61 show the acceptable range of deviation for the hematopoietic lysis compositions containing secondary alcohol ethoxylate 1 and secondary alcohol ethoxylate 2. The data suggest that, considering the rate and integrity of lysis, relatively small or no effect on the sample spectra, and the accuracy and precision of HHb measurements, secondary alcohol ethoxylate 1 and secondary alcohol ethoxylate 2 may perform as well as or better than a reference used in hematopoietic lysis compositions to determine deoxyhemoglobin based on CO-oxygen saturation measurements.

[0084] Therefore, the blood cell lysis composition produces tHb, O2Hb, COHb, MetHb and HHb results within an acceptable predetermined reference range.

[0085] In another embodiment, Multi-4 (“M4”) is a Level 3 control material used in the determination of human CO-oxygen saturation hematopoietic lysis measurements. Table 3 below shows that, for each of the Example Level 3 Multi-4 determinations (L1, L2, and L3) performed, the exemplary hematopoietic lysis compositions of secondary alcohol ethoxylate 1 (“1” in the table) and secondary alcohol ethoxylate 2 (“2” in the table) produced results of tHb, O2Hb, COHb, MetHb, and HHb within the specified reference ranges (“Range”), which are comparable to the values ​​produced using a control (“Ctrl”) hematopoietic lysis composition including Triton-X-100. Acceptable result ranges are shown, with “min” referring to the minimum acceptable result and “max” referring to the maximum acceptable result for each class L1 through L3 of M4. The mean (“ave”), standard deviation (“SD”), and delta mean (“δ”) of each secondary alcohol ethoxylate relative to the control mean are also shown. Data suggest that, considering the rate and integrity of lysis, the relatively small or no effect on sample spectra, and the accuracy and precision of tHb, O2Hb, COHb, MetHb, and HHb measurements, secondary alcohol ethoxylate 1 and secondary alcohol ethoxylate 2 may perform as well as or better than the control blood cell lysis composition used in determining oxyhemoglobin using CO-oxygen saturation measurements.

[0086] Table 3

[0087]

[0088] Referring to Table 4 below, when used to measure total hemoglobin (tHb) in highly turbid samples, hematopoietic lysis compositions including secondary alcohol ethoxylate 1 or 2 also provided results very similar to control hematopoietic lysis compositions including Triton X-100 (δ|<0.1). Such samples include naturally occurring turbid samples. In the table below, “LD” refers to the lipemia donor (patient) sample identifier (ID), as shown in (1-6), which includes up to 1% Intralipid-20. TM Table 4 below shows that the tHb values ​​produced by secondary alcohol ethoxylate hematopoietic lysis compositions 1 and 2 are close to or within the standard deviation of the tHb values ​​determined using CO-oxygen saturation measurements of oxyhemoglobin. The data in Table 4 indicate that secondary alcohol ethoxylate 1 and 2 perform as well as or better than Triton X-100 when determining total hemoglobin based on CO-oxygen saturation measurements of hyperlipidemic blood samples in the hematopoietic lysis compositions.

[0089] Table 4

[0090]

[0091] The blood cell lysis compositions described herein may have one or more of the following advantages. As described above, the blood cell lysis compositions may satisfy one or more of the following target thresholds: the efficiency and speed of blood cell lysis by the blood cell lysis composition; the degree of interference exhibited by the blood cell lysis composition in the intended optical measurements; the degree of interference exhibited by the blood cell lysis composition in blood hemoglobin chemistry; the degree of interference exhibited by the blood cell lysis composition in bilirubin chemistry; and the degree of foaming that causes scattering in optical measurements.

[0092] The blood cell lysis compositions described herein, and the surfactants contained therein, or both, may be biodegradable.

[0093] Blood cell lysis compositions and surfactants contained therein have been described for use with CO oxygen saturation measurement systems. However, the blood cell lysis compositions and surfactants contained therein can be used in any suitable amounts and are not limited to use in CO oxygen saturation measurement.

[0094] The blood cell lysis composition described herein can be mixed with blood samples from any suitable mammal, including but not limited to humans, mice, primates, dogs, or livestock for the purpose of blood cell lysis.

[0095] At least a portion of the CO-oxygen saturation measurement system described in this specification and its various modifications may be configured or controlled at least in part by one or more computers, such as computing system 17, using one or more computer programs tangibly contained in one or more information carriers, such as in one or more non-transitory machine-readable storage media. The computer programs may be written in any form of programming language, including compiled or interpreted languages, and may be in any form, including as standalone programs or as modules, parts, subroutines, or other units suitable for use in a computing environment. The computer programs may be implemented on one computer or multiple computers at one location, or distributed across multiple locations and interconnected via a network.

[0096] Actions related to configuring or controlling the test system described herein may be performed by one or more programmable processors that execute one or more computer programs to control or perform all or some of the operations described herein. All or part of the test system and processes may be configured or controlled by dedicated logic circuitry, such as FPGAs (Field-Programmable Gate Arrays) and / or ASICs (Application-Specific Integrated Circuits) or embedded microprocessors located in the instrument hardware.

[0097] Processors suitable for executing computer programs include, for example, general-purpose and special-purpose microprocessors, and any one or more processors of any type of digital computer. Typically, a processor receives instructions and data from read-only memory or random access memory, or both. The components of a computer include one or more processors for executing instructions and one or more storage area devices for storing instructions and data. Typically, a computer will also include, or be operatively coupled to, receiving data from or transferring data to one or more machine-readable storage media, or both, such as mass storage devices for storing data, such as magnetic, magneto-optical disks, or optical disks. Suitable non-transitory machine-readable storage media for embodying computer program instructions and data include all forms of non-volatile storage areas, including, for example, semiconductor storage area devices such as EPROM (Erasable Programmable Read-Only Memory), EEPROM (Electrically Erasable Programmable Read-Only Memory), and flash memory storage area devices; magnetic disks, such as internal hard disks or removable disks; magneto-optical disks; and CD-ROM (Optical Disc Read-Only Memory) and DVD-ROM (Digital Versatile Optical Disc Read-Only Memory).

[0098] Elements of the different embodiments described can be combined to form other embodiments not previously specifically described. Elements can be excluded from the systems described above without adversely affecting their operation or the general operation of the systems. Furthermore, various individual elements can be combined into one or more individual elements to perform the functions described in this specification.

[0099] Other embodiments not specifically described in this specification are also within the scope of the appended claims.

[0100] The trademarks mentioned in this application are represented in the following compositions (in order of appearance):

[0101]

[0102]

Claims

1. The use of a blood cell lysis composition for lysing blood cells for absorbance measurement, said blood cell lysis composition comprising: Buffer; and Secondary alcohol ethoxylates, in concentrations ranging from about 2.5 percent (%) to about 20% by weight / volume (w / v); wherein the secondary alcohol ethoxylate is Tergitol. TM TMN-100X or Tergitol TM 15-S-9.

2. The use according to claim 1, wherein the buffer comprises at least one selected from 3-(N-morpholino)propanesulfonic acid (MOPS), phosphate buffer (PBS), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]ethanesulfonic acid (TES), 2,2-bis(hydroxymethyl)-2,2',2"-nitrotriethanol or 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris).

3. The use according to claim 1, wherein the blood cell lysis composition further comprises a biocidal agent, wherein the biocidal agent comprises at least one selected from 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 2-methyl-4-isothiazolin-3-one (MIT), Proclin 300, 1,2-benzisothiazolin-3-one (BIT), dodecyl guanidine hydrochloride (DGH), gentamicin, levofloxacin, colistin, octylisothiazolinone (OIT), or amikacin.

4. The use according to claim 3, wherein the biocidal agent comprises CMIT and MIT.

5. The use according to claim 4, wherein the ratio of CMIT to MIT (CMIT:MIT) is approximately 3:

1.

6. The use according to claim 4, wherein the concentration of CMIT:MIT in the blood cell lysis composition is 1% (w / v).

7. The use according to claim 1, wherein the blood cell lysis composition has a pH of about 6 to about 8.

8. The use according to claim 1, wherein the secondary alcohol ethoxylate is present at a concentration in the range of about 5% to about 18% (w / v).

9. The use according to claim 1, wherein the secondary alcohol ethoxylate is present at a concentration in the range of about 9% to about 14% (w / v).

10. The use according to claim 1, wherein the secondary alcohol ethoxylate is biodegradable.

11. The use according to claim 1, wherein the composition is configured to lyse at least 90% of the blood cells in a sample in less than 2 seconds.

12. A method for determining the level of hemoglobin in a blood sample, the method comprising: (i) Mixing the blood cell lysis composition of claim 1 with a blood sample to form a mixture of the blood cell lysis composition and the blood sample, and lysing one or more cells in the blood sample; (ii) Obtain the measured values ​​of the resulting mixture, and (iii) Determine the level of hemoglobin in the blood sample, at least in part, based on the measurement results.

13. The method according to claim 12, wherein the hemoglobin comprises at least one of oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, methemoglobin, thiohemoglobin, cyanomethemoglobin, or fetal hemoglobin.

14. The method of claim 12, further comprising measuring bilirubin levels.

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