Patent Description:
Point-of-care testing refers generally to medical testing at or near the site of patient care, such as in an emergency room. A desired outcome of such tests is often to obtain rapid and accurate lab results to determine a next course of action in patient care. A number of such point of care tests involve taking or otherwise using a blood sample from a patient. The ideal blood sample is pure plasma separated from the source whole blood sample. Hemolysis (hemolysis), however, is a common problem which may introduce undesirable components into the plasma fraction.

<CIT> discloses an apparatus for detecting haemolysis from a sample of blood, which comprises a sealed chamber having a fixed volume and an internal pressure resulting from a quantity of air within the chamber. While receiving fluid that includes the sample, the internal pressure of the sealed chamber is raised and, as a result thereof, the plasma portion of the blood sample permeates a membrane that forms a side of the chamber. The haemolysis condition of the plasma portion is detected in accordance with a hue associated with the test volume received into the haemolysis detection means which is positioned outside the chamber. The haemolysis detector is an indicator paper that shows a visible color change in the presence of haemoglobin when treated with a developer solution, e.g. a hydrogen peroxide solution. <CIT> teaches a multi-layered test strip device for measuring an analyte in a whole blood sample. The device comprises a spreading screen, a separating layer which removes red blood cells, and a membrane reagent layer. The layers are in close and contiguous contact to each other. The membrane reagent layer contains a reagent that generates a color when it reacts with a specific analyte that is present in the blood plasma leaving the separating layer. The reading of the color of a glucose test strip can be done by the naked eye and compared to color charts of varied and defined color intensities. A reflectance reading may also be used, in which the color is compared in a computer analysis to standard reflectances. <CIT> teaches another device for visual detection of haemolysis in a whole blood sample. The device comprises a separation filter that separates the red blood cells from the blood plasma and a detection filter. Haemolysis is determined by observing through a detection window the hue of the plasma portion that has been absorbed by the detection filter. <CIT>, corresponding to <CIT> discloses a dry reagent test strip from which an analyte presence and/or concentration in a body fluid is determined. It comprises a filtering layer which provides capillary action to draw the sample through a capillary and to disperse it onto an absorbent reagent layer membrane. The reagent layer comprises a reagent selected for the analyte of interest. A preferred analyte is glucose.

<CIT> discloses a device for determining the total hemoglobin content of a sample by reflectance measurements.

<CIT> discloses a device for detecting hemolysis by taking a digital image of separated plasma.

In particular, hemolysis refers to the rupturing of erythrocytes (red blood cells) and the release of their contents, including hemoglobin and potassium, into surrounding fluid (e.g., blood plasma). Hemolysis may occur in vivo or in vitro (inside or outside the body). During any of the collection, transportation, and handling of patient whole blood samples, there is the possibility of hemolysis. If hemolysis occurs, the resulting components in the sample may cause interference in a number of tests, thereby leading to a signal reduction, reduced measurement accuracy and precision, or to false positive results at the other end of the spectrum. Further, if hemolysis occurs, it has been found that the potassium concentration in a corresponding sample may increase significantly and cause a high risk of misdiagnosis in a diagnostic test for potassium levels. Current methods do not adequately and quickly determine whether an unacceptable level of hemolysis has occurred in a blood sample.

The invention is explained in the following description in view of the drawings that show:.

The present invention is directed to a device as claimed in claim1 that enables the quick and reliable detection of hemolysis in a sample such that a sample which exhibits an unacceptable level of hemolysis can be immediately flagged or disregarded in an associated diagnostic test. In this way, via the device described herein, a sample may be rapidly tested to determine whether hemolysis has occurred and a false positive result may be avoided, for example, a false positive result in an assay for potassium levels in a blood sample.

In one aspect, a device is provided to which a sample may be added and prepared for hemolysis analysis. In an embodiment, the device is removably insertable into a detection system such as a spectrometer to determine a degree of hemolysis. If a degree of hemolysis in the sample is above a predetermined threshold value, an indication of the same may be provided and proper action may be taken in response to an analytical test with the same sample or a second (other) sample from the same sample source. If the sample indicates that no hemolysis exists above a predetermined threshold level, further testing of the sample may be performed with greater confidence in the results, or the results of previously run diagnostic tests may be interpreted with greater confidence.

Now referring to <FIG>, there is shown an exemplary device <NUM> for hemolysis detection in a sample introduced into the device <NUM>. According to the invention the device <NUM> comprises a body <NUM> having an upper portion <NUM> and a lower portion <NUM>. The body <NUM> defines an opening <NUM> therein to allow for introduction of a sample into the device <NUM>. The sample may be any sample suspected of having an unacceptable amount of hemolysis therein.

The opening <NUM> is disposed in the upper portion <NUM> of the device <NUM>. Further, in the embodiment shown, a plasma separation element <NUM> is disposed within or carried by the body <NUM> of the device <NUM>. The plasma separation element <NUM> is structured to isolate plasma and components from hemolysis (broken cell fragment, hemoglobin, etc.) from whole cells in a blood sample. A membrane <NUM> is disposed adjacent the plasma separation element <NUM> for receiving a plasma sample suspected of comprising an amount of hemolysis therein from the plasma separation element <NUM>. A slit <NUM> is defined in the body, such as in the lower portion <NUM>, for defining a pathway for energy to be applied to the membrane <NUM> from an energy source <NUM> as will be discussed below in order to detect an amount of hemolysis in a sample.

The body <NUM> of the device <NUM> may be formed from any suitable liquid impermeable material that is also inert to at least hemoglobin. For example, without limitation, the body <NUM> may be formed from a material comprising polystyrene, polyethylene, polycarbonate, polypropylene, fluoropolymer, polyester, glass, metals, ceramics, suitable composite materials, and combinations thereof as would be appreciated by those skilled in art. As noted above, one or more openings <NUM> may be defined in the device for introduction of a sample to the device <NUM>. The location, number, and size (e.g., width and depth) of the one or more openings <NUM> may be optimized for the desired application of the device <NUM>.

The sample for introduction into the device <NUM> may be one that potentially has undergone an unacceptable amount of hemolysis therein. For example, the sample may be one which could potentially create a false positive result in an assay for potassium levels by providing potassium levels which are significantly larger than the associated subject's actual potassium levels in vivo in the absence of hemolysis. For example, potassium levels inside red blood cells may be <NUM> times higher than in plasma. Thus, if hemolysis occurs, the potassium value of the sample in question may be increased significantly. When a subject's potassium levels are not actually as high as indicated, a false positive result may in turn result in misdiagnosis and mistreatment of a disorder characterized by elevated potassium levels. For example, as a result of hemolysis, a subject might be misdiagnosed with having hyperkalemia or any other disorder or condition characterized by elevated potassium levels, e.g., Addison's disease or hemolytic anemia. Further, the subject may be misdiagnosed as having elevated potassium levels as a side effect of taking medications such as water pills (diuretics) or blood pressure drugs and unnecessarily instructed to cease taking such medications to the subject's detriment. In addition, a false positive result could inadvertently lead to one unnecessarily being provided with agents to remove potassium from the intestines before potassium is absorbed or other unnecessary treatments. Thus, advantageously, the device <NUM> may be utilized in a screening process for an unacceptable level of hemolysis prior to analysis of the sample for potassium levels or for confirming the integrity of test results already run.

In certain embodiments, the sample is a whole blood sample which includes a quantity of whole blood cells, including red blood cells, white cells, and platelets. Within the sample, the extent of hemolysis may correlate to an amount of hemoglobin therein. As used herein, it is understood that the term "hemoglobin" refers to any and all hemoglobin molecules obtained either from drawn blood or by recombinant procedures in their oxygenated, deoxygenated, dimeric, tetrameric, or various polymerized forms. Hemoglobin is commonly known as the oxygen-carrying pigment and predominant protein of red blood cells. Hemoglobin is composed of four protein chains, two alpha chains and two beta chains, each with a ring-like heme group containing an iron atom. Oxygen binds reversibly to these iron atoms. In its oxygenated state, hemoglobin may be referred to as oxyhemoglobin and is characterized by a bright red. In the reduced state, hemoglobin may be referred to as deoxyhemoglobin and is characterized by a purple-blue color.

In an embodiment, a presence of hemolysis in the sample is determined by analyzing at least for an amount of oxygenated hemoglobin in the sample. In a particular embodiment, the presence of hemolysis in a sample may be determined by a degree of discoloration of the sample or brought about by contact with the sample. As such, the more red the separated plasma sample, generally, the greater the amount of hemolysis that may have taken place.

It is appreciated that prior to analysis for an extent of hemolysis, it may be desirable to prepare the plasma sample by substantially filtering out whole blood cells from the sample, particularly in the case of a whole blood sample. It is further appreciated that this may be accomplished by a number of different methods, including but not limited to centrifugation, filtration, or the like. In an embodiment, a whole blood sample is separated to produce a plasma sample comprising hemolysed cells and the contents thereof (including hemoglobin) when hemolysis has occurred in the sample.

In an aspect of the present invention, a component for separating plasma and products of hemolysis (if present) is conveniently provided in the device <NUM>. For example, in the embodiment shown in <FIG>, the plasma separation element <NUM> is included which may isolate plasma and hemolysis products, e.g., hemoglobin, from whole blood cells in a sample such as a whole blood sample. In an embodiment, the plasma separation element <NUM> comprises a plasma separation membrane as is commercially available in the art. In certain embodiments, the plasma separation membrane comprises an asymmetric material, which is able to retain a plurality of whole blood cells thereon while allowing plasma and small molecules/complexes to travel therethrough. A number of different plasma separation membranes are commercially available and may be suitable for use in the device <NUM>. For example, the plasma separation membrane may comprise an asymmetric polysulfone material as is commercially available from Pall Corporation (currently under the trademark Vivid™). Alternatively, the plasma separation element <NUM> may comprise any other suitable plasma separation membrane.

In operation, a sample <NUM> may be introduced onto the plasma separation element <NUM>. After an effective amount of time, which may be two minutes or less, a retentate may be formed on the plasma separation element <NUM> which comprises whole blood cells, such as red blood cells, white blood cells, and platelets, while a filtrate passes through the plasma separation element <NUM> and onto the membrane <NUM>. In other embodiments (not according to the invention), a plasma portion may be formed by centrifugation or filtration remote from the device <NUM> and the resulting plasma portion may be introduced directly onto the membrane <NUM>. In this instance, the device <NUM> need not include plasma separation element <NUM> and the device <NUM> may be further modified accordingly.

The membrane <NUM> may be any suitable porous or non-porous material which is inert to at least hemoglobin. For example, the membrane <NUM> may comprise a cellulosic material, a glass fiber material, a porous polymeric material, or combinations thereof. In one embodiment, the membrane <NUM> comprises a filter paper having a thickness sufficient to absorb and/or maintain a sample, such as the filtrate, thereon without adding any discoloration to the sample. For example, the membrane <NUM> may be any suitable commercial filter paper having sufficient degree of thickness to maintain the sample thereon such as a Whatman Grade <NUM>: <NUM> pore size; <NUM> thickness; medium flow filter paper.

According to the invention, the body <NUM> of the device <NUM> comprises a slit <NUM> as referred to above. In an embodiment, the slit <NUM> is defined in the lower portion <NUM> of the device as shown in <FIG>. The slit <NUM> defines a pathway for energy to be applied to the membrane <NUM> for the detection of hemolysis such as by spectroscopic methods. As such, the slit <NUM> will have a dimension, including a length and a width, suitable for a selected detection method as would be appreciated by one skilled in the art.

Referring now to <FIG>, the device <NUM> is a component of a system <NUM> also comprising a detection system <NUM> for producing and recording an output indicative of the absence, presence, and, in certain embodiments, an extent of hemolysis in a sample introduced into the device <NUM>. As such, the device <NUM> may be in a form suitable for removable insertion into or placement within the detection system <NUM> for analysis of the sample therein. For example, the device <NUM> may be in the form of a cartridge, cassette, or the like. Further, the device <NUM> may be formed from relatively inexpensive materials and may be readily disposable after a single use. In certain embodiments, the device <NUM> may be removably inserted into a corresponding receiving space <NUM> defined in a body of the detection system <NUM>.

The detection system <NUM> may comprise any suitable apparatus for generating a qualitative, semi-quantitative, and/or quantitative result corresponding to an amount of hemolysis in the sample on the membrane <NUM>. Thus, a detection system <NUM> that merely detects whether hemolysis in a sample is above a predetermined threshold is also considered to be included within the scope of the present invention. In a particular embodiment, the detection system <NUM> comprises a spectrometer as is well-known in the art comprising an energy source, a monochromator, the device <NUM> (when inserted), and a detector. The spectrometer is configured for scanning at least over a suitable range or at selected wavelength(s) for the detection of hemoglobin.

The amount of hemoglobin determined in a given sample is understood to be useful as a measurement of hemolysis. Thus, a threshold value for an acceptable amount of hemolysis will have an associated hemoglobin threshold value, which is readily determinable by one skilled in the art. If a sample has a hemoglobin content over the hemoglobin threshold value, it is understood that the sample may be said to have an unacceptable amount of hemolysis therein or also exhibits an amount of hemolysis over a hemolysis threshold value.

In an embodiment, as shown in <FIG>, the detection system <NUM> is configured to transmit energy from the energy source <NUM> to the membrane <NUM> through the slit <NUM> in the device <NUM> once the device <NUM> is inserted or otherwise properly associated with the detection system <NUM>. Thereafter, a detector <NUM> is provided which is configured to receive and measure energy transmitted from the membrane <NUM> or energy absorbed by the membrane <NUM>. The detector <NUM> may comprise a photomultiplier tube (PMT), a photodiode, or a charge coupled device (CCD) as are known in the art.

The generation of a spectrometric result on the system <NUM> may be carried out by known methods in the art such as those described in <CIT>, for example. In any case, the generated spectrum may be representative of the extent of hemolysis in the sample or on the membrane <NUM>. In an embodiment, the extent of hemolysis may correspond empirically with a degree of coloration of the plasma fraction on the membrane <NUM>. In a particular embodiment, an amount of oxidized hemoglobin in the sample corresponds with a degree of red coloration in the sample. The extent of hemolysis in a sample may thus be determined qualitatively, semi-quantitatively, or quantitatively through the use of known standards and controls as would be well understood by persons skilled in the art. For example, results may be compared to values of a calibration curve created from a plurality of standard samples having predetermined concentrations as is well-known in the art. In certain embodiments, any results on the system <NUM> may be compared to a predetermined threshold value to determine whether hemolysis in a subject sample is greater than an acceptable limit.

For system control and data management, the detection system <NUM> comprises a computing unit <NUM> comprising one or more modules configured to receive data from the detector <NUM> and determine an amount of hemolysis from the data. The computing unit may comprise, for example, a special purpose computer comprising a microprocessor, a microcomputer, an industrial controller, a programmable logic controller, a discrete logic circuit or other suitable controlling device. In an embodiment, the computing unit <NUM> may further comprise one or more input channels, a memory, and output channel(s). The memory may include a computer-readable medium or a storage device, e.g., floppy disk, a compact disc read only memory (CD-ROM), or the like. In an embodiment, the computing unit <NUM> may comprise computer readable instructions for performing any aspect of the methods or for controlling any aspect of the components described herein.

In an embodiment, the computing unit <NUM> is programmed with instructions to provide an indication when determined that a degree of hemolysis in a subject sample is greater than a predetermined threshold value for hemolysis. In a particular embodiment, the predetermined threshold value is one over which would provide a potentially misleading result in a test for an analyte such as potassium. By "misleading," it is meant that the extent of hemolysis may be sufficient to alter interpretation of the result in a subsequent test for an analyte of interest. In such cases, an amount of hemolysis over the predetermined threshold may be sufficient cause a test to inaccurately reflect an amount of an analyte in question in vivo. The indication of hemolysis over a predetermined threshold may be conveyed to the test operator of the system <NUM> by any suitable method, such as an audio or visual alarm, or by providing the indication on a display or in a message, locally or remotely from the detection system <NUM>. The predetermined threshold value may be, for example, an amount sufficient to provide a misleading potassium result in a subsequent test for potassium content within the same sample or a second sample which is/was taken from the same source. In a particular embodiment, the predetermined threshold value is one indicative of at least <NUM> % hemolysis in a sample. In another embodiment, the predetermined threshold value represents at least <NUM> % hemolysis. A <NUM>% level of hemolysis, for example, may correspond to a <NUM> mmol/L increase in a potassium reading, which may lead to misdiagnosis or mistreatment.

In certain embodiments, the detection system <NUM> is configured for communication with an existing instrument which will test for potassium levels in a blood sample, such as a blood gas analyzer <NUM> (<FIG>). Exemplary blood gas analyzers are available from Siemens Healthcare Diagnostics, Inc. and are currently sold under the trademarks: RAPIDLab <NUM>, RapidLab 348EX, RAPIDPoint <NUM>, RAPIDLab <NUM>/<NUM>, RAPIDPoint <NUM>/<NUM>, RAPIDChem <NUM>/<NUM>, and RAPIDPoint <NUM>/<NUM> Systems. In this way, the detection system <NUM> can act as a highly beneficial peripheral device to blood gas analyzer.

To accomplish this, the detection system <NUM> and blood gas analyzer <NUM> may comprise suitable wired or wireless connection(s) to facilitate communication therein such as a plug in connection as shown by exemplary USB connection <NUM>. In an embodiment, a known sample from the same source (e.g., a sample taken from the same person at or about the same time) could be added to both the blood gas analyzer <NUM> and the detection system <NUM> comprising the device <NUM>. By utilizing the detection system <NUM> as described herein, the detection system <NUM> may provide an indication that the sample introduced to the blood gas analyzer <NUM> is one that would likely or would be susceptible to providing misleading or less than optimal results by having a degree of hemolysis greater a predetermined threshold value. The indication may be an audio, visual, or written indication and may be output by any suitable method to any suitable location, component, report, or display. In this way, for example, samples which could potentially provide a misleading or false positive result for elevated potassium levels may be avoided or otherwise treated with appropriate caution.

Aspects of the present invention are demonstrated by the following examples, which are not intended to be limiting in any manner.

An experiment was carried out to confirm that hemolysis significantly decreases detector output (mV) as the degree of hemolysis increases. Whole blood samples with a range of <NUM> to <NUM> % hemolysis were used and detection was measured at <NUM>. Tables <NUM> and <NUM> and FIG. <NUM>, for example, shows a substantially linear decrease in detector output (mV) (measured at <NUM>) from <NUM> % hemolysis to <NUM>% hemolysis with a substantially notable plateau in the decrease of detector output from <NUM>-<NUM>% hemolysis.

Claim 1:
A device (<NUM>) for determining the amount of hemolysis in a plasma fraction wherein the device comprises:
a body (<NUM>) with an upper portion (<NUM>) and lower portion (<NUM>);
an opening (<NUM>) defined in the upper portion of the body for introduction of a blood sample;
a plasma separation membrane (<NUM>) disposed between the upper portion and the lower portion;
a second membrane (<NUM>) positioned adjacent and below the plasma separation membrane (<NUM>) so as to receive a plasma fraction that was separated from the blood sample by the plasma separation membrane;
a slit (<NUM>) defining a pathway for energy to be applied to the second membrane (<NUM>) from a detection system (<NUM>), characterized in that the detection system is configured to transmit energy from an energy source (<NUM>) to the membrane (<NUM>) through the slit (<NUM>) and comprises a detector (<NUM>) which is configured to receive and measure energy transmitted from the membrane (<NUM>) or energy absorbed by the membrane (<NUM>), the detection system further comprising a computing unit (<NUM>) comprising:
one or more modules configured to receive data from the detector (<NUM>) and determine a degree of hemolysis from the data.