WO2007137582A1

WO2007137582A1 - Methods and devices for rapid assessment of severity of injury and disease

Info

Publication number: WO2007137582A1
Application number: PCT/DK2007/000252
Authority: WO
Inventors: Kristian Bangert; Lars Otto Uttenthal
Original assignee: Antibodyshop A/S
Priority date: 2006-05-30
Filing date: 2007-05-30
Publication date: 2007-12-06

Abstract

Methods and devices for rapid assessment of the severity of injury and/or disease within hours of the injury or initial presentation of disease symptoms in situations in which laboratory facilities are out of reach based upon measurement of a component of the extracellular actin scavenging system are provided.

Description

METHODS AND DEVICES FOR RAPID ASSESSMENT OF SEVERITY OF

INJURY AND DISEASE

Field of the invention The present invention provides a method for rapid assessment of the severity of injury and/or disease within hours of the injury or initial presentation of disease symptoms in situations in which laboratory facilities are out of reach. In this method, a component of the extracellular actin scavenging system present in blood, preferably a blood protein Gc-globulin, is measured. The present invention is thus useful in the field of clinical medicine and surgery, in particular in the fields of traumatology, trauma surgery, emergency medicine, critical care medicine, first aid and rescue work. Devices for rapid assessment of severity of an injury and/or disease via measurement of a component of the extracellular actin scavenging system present in blood, preferably a blood protein Gc-globulin, are also provided.

Background of the Invention

The current method of assessing the severity of injury or disease in which major organ damage is suspected is by visual inspection and clinical examination by the first responder at the site of presentation of the injured or diseased subject. This is a subjective evaluation conditioned by the level of medical or first-aid training of the responder, which may vary from complete lack of training or experience to a high level of training and experience in emergency medicine and rescue work. However, it is difficult even for trained physicians to make a precise evaluation of the severity of injury of victims of mass-casualty incidents (Ashkenazi et al . Prehospital Disaster Med. 2006 21:20-23). In everyday cases of injury or medical emergency, the injured subject or patient will be brought at the slightest suspicion of non-triviality to a hospital or trauma center. In mass casualty situations such as train crashes, earthquakes or terrorist bombings, this is not feasible because of lack of immediately available resources. Thus, triage, defined as prioritizing of the injured survivors for treatment or transport to treatment facilities in order to obtain the best overall outcome in terms of survival or the avoidance of permanent disability, is applied. The more accurate the triage, the higher the survival rate and the lower the number of complications leading to prolonged hospitalization or lifelong disability.

In the case of medical emergencies not involving trauma, there are also difficulties in the initial clinical assessment of conditions associated with major organ damage. For example, in intoxication with acetaminophen (also called paracetamol) , there is a risk of acute liver failure and hepatic encephalopathy. An accurate clinical assessment relies on the clinical history and presentation, from which is it usually impossible to tell whether the patient is developing acute liver failure. Currently, the diagnosis of this condition must await the results of laboratory tests. A release of actin into the circulation that is sufficient to cause a fall in blood levels of Gc-globulin is seen in severe trauma and acute liver failure. It has been demonstrated in several studies (Dahl et al . Injury 1999 30: 275-281; Schiodt et al . Hepatology 1996 23:713-718; Lee et al. Hepatology 1995 21:101-105; Schiodt et al . Liver Transpl. 2005 11:1223-1227; Dahl et al . Crit Care Med. 1998 26:285-289; Schiodt et al . Crit Care Med. 1997 25:1366-1370; Dahl et al. Crit Care Med. 2003 31:152-156) that the Gc- globulin concentration in plasma or serum has a prognostic value in cases of trauma and acute liver failure: the lower the Gc-globulin concentration, the lower the chance of survival . The fall in Gc-globulin occurs rapidly and in cases of trauma is already apparent at 30 minutes from the insult (Dahl et al. Injury 1999 30:275-281). The prognostic value of the Gc-globulin level in cases of trauma was as high as that of the widely used injury scoring system, TRISS (trauma injury severity score) (Dahl et al . Injury 1999 30:275-281). There is a consistent difference in Gc-globulin levels between survivors and non-survivors during the first 24 hours (Dahl et al . Crit Care Med. 2003 31:152-156) and for even longer periods after the insult (Dahl et al . Crit Care Med. 1998 26:285-289).

Methods of measuring actin-free Gc-globulin, total Gc- globulin (actin-bound plus actin-free) , and gelsolin have been disclosed. However, they are all laboratory methods suffering from one or more of the following disadvantages which make them unsuitable for the purposes of the present invention: requiring the use of non-portable apparatus, requiring technical skill, and taking a long time to perform.

For example, a method of measuring actin-free Gc- globulin uses competitive ELISA to determine the concentration of total Gc-globulin in a sample, followed by crossed Immunoelectrophoresis to determine the proportion of actin-free Gc-globulin (Schiodt et al . Eur. J. Gastroenterol. Hepatol. 1995 7:635-640). This method is laborious, time-consuming and complicated requiring experienced staff and is an indirect way of measuring actin- free Gc-globulin via crossed Immunoelectrophoresis.

Another method of estimating actin-free Gc-globulin is by rocket Immunoelectrophoresis of the sample alone and in the presence of an excess of actin in a gel containing polyclonal antibody against Gc-globulin. The amount of actin-bound and actin-free Gc-globulin can then be calculated from the height of the precipitin peaks (Goldschmidt-Clermont et al. Clin. Chim. Acta 1985 148:173- 183) . This is also a laborious and time-consuming indirect method of measuring actin-free Gc-globulin, requiring technical skill and taking more than one day to perform. Another method of measuring actin-free Gc-globulin employs sandwich ELISA in which a dilution of the sample is mixed with labeled detection antibody that binds only to actin-free Gc-globulin. The labeled complexes are captured by a monoclonal antibody coated onto the wells and capable of binding Gc-globulin complexed with the detection antibody. This method requires laboratory facilities. A method of measuring total Gc-globulin using nephelometry after mixing the sample with polyclonal antibody against Gc-globulin has been disclosed (Wians et al. Liver Transpl . Surg. 1997 3:28-33). A similar method of measuring gelsolin uses nephelometry after mixing the sample with polyclonal antibody against gelsolin has been disclosed (Dahl et al. Shock 1999 12:102-104). Both of these methods are designed for use on large automated laboratory equipment .

Another method of measuring gelsolin uses the ability of gelsolin in the sample to polymerize added pyrene actin, actin polymerization in the mixture being quantified by spectrofluorimetry (Smith et al . J^". Lab Clin. Med. 1987 110:189-195). This method also requires technical skill and non-portable laboratory apparatus . The present invention provides a method and devices for assessing severe trauma and acute liver failure as well as other diagnoses involving major tissue damage to be made via measurement of a component of the extracellular actin scavenging system present in blood, preferably a blood protein Gc-globulin, in the field at the site of injury or at the first point of care without recourse to laboratory facilities .

Summary of the Invention

An aspect of the present invention relates to a method for rapidly assessing severity of an injury and/or disease in subject comprising determining a level of a component of the extracellular actin scavenging system, preferably a blood protein Gc-globulin, present in blood of a subject, wherein a decrease in blood levels of Gc-globulin is indicative of a severe injury or disease. The method of the present invention is preferably performed in the field at the site of injury or at the first point of care. An important aspect of the invention is that the measurement of the protein should be made as soon as possible after injury.

Another aspect of the present invention relates to portable devices for measuring a component of the extracellular actin scavenging system, preferably a blood protein Gc-globulin, in blood of an injured subject or a subject suffering from a disease which can be used at or near a site of injury or first point of care to assess severity of the injury and/or disease in the subject. An essential feature of the method and devices of the present invention for determining the blood levels of these biomarkers is that they must be performable by persons without laboratory training on small, portable, preferably pocket-sized and battery-powered devices under field conditions, and give results within a few minutes. Brief Description of the Figures

Figure 1 provides a diagram of the extracellular actin scavenging system in the blood. Dying cells rupture and release both filamentous F-actin and non-filamentous G-actin units into the blood. F-actin is capped and severed by gelsolin into G-actin units and the G-actin units are transferred to Gc-globulin, which binds them as tight non- covalent 1:1 complexes. Both actin-gelsolin and actin-Gc- globulin complexes are removed by scavenger cells of the reticulo-endothelial system, many of which are represented by the Kupffer cells lining the liver sinusoids. The effect of massive actin release is thus to deplete both gelsolin and Gc-globulin, but some free gelsolin can be regenerated from actin-gelsolin complexes by passing the actin to Gc- globulin. Free .Gc-globulin can only be regenerated by de novo synthesis in the liver.

Detailed Description of the Invention

The method of the present invention provides a means for improving the initial assessment of the severity of injury and/or disease involving major organ damage at the site of presentation of the injury or disease, where the injured subject or patient is found at sites in which there is no immediate access to laboratory facilities . The method of the present invention involves the quantitative and/or qualitative measurement of the level of a selected biological marker molecule, hereinafter referred to as the "biomarker", in the blood of the subject by means of a rapid test device. The device can be used at the point of care, with or without the use of a portable reading instrument that can provide a quantitative estimate of the concentration or functional activity of the biomarker. The method and devices of the present invention are thus applicable to assessing the severity of injury of victims at the scene of a mass-casualty accident, natural disaster or an act of war or terrorism, and/or in vehicles transporting injured or diseased subjects to care facilities, and/or in the emergency reception rooms of hospitals or other care facilities .

In the present invention severity of injury or major damage to muscle and liver in a subject, preferably a mammalian subject, more preferably a human subject, is assessed by measuring the level in blood of a biomarker comprising a component of the extracellular actin scavenging system, preferably a blood protein Gc-globulin in blood, by any method or device that is capable of determining such level with sufficient rapidity and accuracy and of such a nature as to be portable and usable at the site at which the injured or diseased subject is encountered, as well as in any vehicle used to transport the subject to care facilities, and in the emergency reception rooms of such facilities, such methods or devices commonly being described as being for use at the "point of care" . An important aspect of the invention is that the measurement of the biomarker is made as soon as possible after the injury or the presentation of the medical emergency has occurred, preferably within 1 hour, preferably within 45 minutes, such as within 30 minutes, more preferably within 15 minutes and most preferably within 10 minutes or even 5 minutes.

The method of the present invention in one embodiment comprises the steps of measuring the concentration of actin- free Gc-globulin in a sample of blood plasma from the individual whose injury or disease is to be assessed, and comparing the measured concentration with a selected cutoff value determined to be lower than the concentration values found in healthy, uninjured individuals. The extent to which the measured actin-free Gc-globulin concentration falls below the cutoff level is indicative of the severity of the injury or disease.

The cutoff level of actin-free Gc-globulin in blood plasma above which the concentration of this biomarker is not indicative of the severity of injury or disease, because such a level can be found in healthy, uninjured individuals, is preferably a level of 260 μg/mL or less, such a value between 260 μg/mL and 200 μg/mL, such as 250 μg/mL, or 240 μg/mL, or 230 μg/mL, or 220 μg/mL, or 210 μg/mL.

The cutoff level of actin-free Gc-globulin in blood plasma below which the concentration of this biomarker is indicative of very severe tissue damage carrying a high risk of death without radical medical intervention is preferably a level of 100 μg/mL or less, such a value between 100 μg/mL and 30 μg/mL, such as 90 μg/mL, or 80 μg/mL, or 70 μg/mL, or 60 μg/mL, or 50 μg/mL, or 40 μg/mL.

The result of the measurement of the biomarker provides objective information about the severity of injury or the severity of muscle damage or liver damage of the subject. This information enables the health care provider or first responder at the scene at which the injured or diseased subject is encountered to institute appropriate immediate measures, to prioritize the care of the subject in relation to other injured subjects at the scene of a major accident, disaster or hostile attack, and to inform the hospital or other care facility that is due to receive the injured or diseased subject. The invention can be used in any situation where this assessment of the severity of injury severity or tissue damage is wanted and where it is not feasible to obtain the same information from conventional laboratory tests. This will typically be in situations outside a hospital or in the emergency reception facilities of a hospital or other care center.

Gc-globulin (also known as vitamin D-binding protein) is a blood protein that is responsible for the last step in the extracellular scavenging of actin that is released into the blood as a consequence of trauma to tissues including skeletal muscle and of injury to the liver. Actin is the single most abundant protein in mammals, including humans. It is located within the cells where its ability to self- assemble and form fibers makes it an important component of the cytoskeleton, which is of vital importance for cell structure and function. It is especially abundant in muscle, where it forms a major component of the actin-myosin fibers responsible for muscle contraction. Lysis of cells (e.g. muscle cells in crush injury) , or the apoptotic or necrotic death or cells in major organ damage, releases actin into the circulation, where the formation of actin fibers may obstruct and damage small vessels, leading to organ failure and ultimately to death (Haddad et al . Proc. Natl. Acad. Sci. USA 1990 87:1381-1385). Actin entering the blood stream is therefore scavenged by a system of blood proteins, illustrated in Figure 1, which together disassemble actin fibers and remove actin from the circulation (Lee, W. M. and Galbraith, R. M. N. Engl. J. Med. 1992 326:1335-1341). The extracellular actin scavenging system in the blood consists essentially of two proteins present in the plasma, gelsolin and Gc-globulin. Gelsolin disassembles actin fibers, known as F-actin, and forms temporary complexes with actin that either give up the actin units to Gc-globulin or are removed from the circulation. Gc-globulin forms tightly bound 1:1 complexes with the actin units, known as G-actin, and these complexes are removed by scavenger cells of the reticuloendothelial system. The protection against vascular damage afforded by the formation of actin-Gc-globulin complexes is demonstrated by the fact that the intravenous injection into rats of Gc-globulin in complex with an otherwise lethal dose of actin left the animals unharmed (Haddad et al . Proc. Natl. Acad. Sci . USA 1990 87:1381- 1385) . Actin release into the blood, and its subsequent scavenging from the blood, thus depletes Gc-globulin from the circulation. Gc-globulin can therefore act as a biomarker of the extent of such actin release. In the present invention the biomarker that is measured is preferably actin-free Gc-globulin, representing that fraction of the Gc-globulin in the blood that is not complexed with actin and which therefore represents the actin-scavenging reserve capacity due to Gc-globulin at the time of blood sampling.

A further aspect of the present invention relates to measuring the level in blood of biomarkers comprising other components the extracellular actin scavenging system for the purpose of determining the severity of traumatic injury and liver damage; these biomarkers include, but are not limited to, actin-Gc-globulin complex, total Gc-globulin (actin- bound plus actin-free) , total gelsolin (actin-bound plus actin-free), actin-gelsolin complexes, and actin-free gelsolin; in addition to the determination of an individual component, the determination of combinations of two or more of these components may also provide useful information on the severity of injury or disease, and serve as prognostic and diagnostic indicators. Functional measurements of aspects of the actin scavenging activity present in a sample of the subject's blood may also provide similar information; this could include determining the influence of the sample on de-aggregation of F-actin or on the formation of F-actin from G-actin, provided that, for the purpose of the present invention, these determinations can be made by methods that can be used in the field or at the point of care.

Thus, by the term "level" as used herein it is meant to include concentration of the biomarker and/or functional activity of the biomarker.

An aspect of the present invention is that the level of the chosen biomarker in the sample must be measurable by a rapid method that can be performed by personnel who are not trained in laboratory techniques and which can be used in the field at the scene of major accidents, disasters or hostile acts. In this context, the term "rapid" means results are obtained within 10 minutes ,^' more preferably within 5 minutes of testing. The method and devices of the present invention therefore fall within the category of clinical analyses known as "point-of-care" or "near-patient" testing. The present invention is not limited to any particular test method or format. Instead, any method for determining the biomarker which fulfills the requirements of providing rapid test results, uses only equipment that can readily be carried to the site of testing, and can be operated by personnel without specialized training beyond a short introduction can be used.

Accordingly, devices of the present invention analyze an individual sample obtained from a subject, preferably whole blood taken with a sampling means carried separately or incorporated into the device and coated with an anticoagulant. The sample is applied to an individual test cassette of the device of the present invention, which contains the antibodies and antibody conjugates in a precisely aliquoted and stabilized form necessary for the analysis of the biomarker. Such antibodies and antibody conjugates may be bound to a solid phase, or dissolved in a solution containing preservatives and stabilizers, either case permitting a shelf life of at least 6 months at an average ambient temperature of 25 degrees centigrade. The test cassette also contains the support systems that allow the analytical process to proceed automatically once the sample is applied and the cassette inserted into a portable reading instrument carried separately or incorporated within the device itself. The test cassette is small enough to permit at least 10 such cassettes to be carried in a pocket and to fit into a pocket-sized reading instrument, for example the dimensions are smaller, and preferably much smaller, than maximum dimensions of 15 cm long, 5 cm wide and 2 cm deep. Each production batch of test cassettes is produced with a consistency such that the coefficient of variation of test results obtained with the same sample applied to different individual cassettes is less than 10%, preferably less than 5%.

These features distinguish the analytical methods and devices to be used in the present invention from the analytical methods previously used to measure the said biomarkers .

There are various clinical situations in which the rapid analysis of the biomarker in accordance with the present invention may be applied.

One exemplary embodiment is in mass-casualty situations such as major accidents, e.g. train crashes, or natural disasters, e.g. earthquakes, or acts of war or terror, e.g. bomb explosions, where there is a need for the triage of a large number of victims. The rescue worker will carry test strips or cassettes, capillary blood sampling devices, and the portable reading instrument in pockets of the uniform. The use of the invention will provide an objective assessment of injury severity which will complement the visual assessment and. improve the accuracy of triage, especially in cases of internal injury.

Another exemplary embodiment is at accidents, such as traffic accidents, involving a smaller number of casualties. The invention will enable the first responders to obtain an objective assessment of injury severity which may influence the decision on where to take the injured (i.e. to which level of trauma center) and will improve the information that they can give to the receiving hospital on what to expect.

Another exemplary embodiment is in situations where hospitals and medically trained personnel are far away. This could be in Navy or civilian ships at sea, coastguard or other patrols, or hunters, mountaineers, prospectors or explorers in remote areas . The invention will provide an objective injury assessment to the rescuer who has to take the decision whether or not to get the injured to hospital by helicopter or by making for the nearest harbor.

Another exemplary embodiment is in situations not connected with traumatic injury but with medical conditions involving a risk of major organ damage, especially acute liver failure (such as due to acetaminophen overdose or certain types of hepatitis) without nearby laboratory facilities . The method and devices of the present invention can help the first responder or ambulance team to assess hepatic injury so that the receiving hospital can be given the best information about the patient to be admitted. Another exemplary embodiment is in the situations described above in which a second or further measurement of Gc-globulin or another marker of the actin scavenging system will provide information on the patient's progress. A further fall in the Gc-globulin concentration will be associated with a poor prognosis and vice versa. This information is as important as the first reading, since these measurements reflect the current state of the patient.

The following non-limiting examples are provided to illustrate how the analysis of biomarkers in accordance with the present invention may be performed.

EXAMPLES

Example 1: Immunochromatographic or "lateral flow" device A lateral flow device comprised of a strip of porous nitrocellulose is coated near its distal end with a capture antibody, capable of binding only actin-free Gc-globulin, applied as a transverse band. A further transverse band of antibody against antibodies of the species from which the detection antibody is derived is placed distally to the capture antibody band and serves as a control of strip function. The proximal end of the strip contains the detection antibody against Gc-globulin adsorbed or linked to labeled polystyrene particles or particles of dye complex. This is overlaid by a filter that retains red blood cells in the sample. When an aliquot of blood sample (taken with an anticoagulant-coated capillary dispenser) is applied to the proximal end of the strip, the labeled particles attached to detection antibody travel along the strip by capillary attraction. When reaching the band of capture antibody, only those particles which have bound actin-free Gc-globulin in the filtered plasma will be retained, giving rise to a detectable band. Particles reaching the control band of antibody against the detection antibody will produce a detectable band whether or not any Gc-globulin has been bound. The intensity of the labeled bands can be read by eye in the case of colored particles or by means of the appropriate detection device for the label used. A positive result is indicated by color development or the accumulation of label in both bands, while a negative result is indicated by color development or other label only in the control band. Failure of color development or other label in the control band indicates inadequate strip function. The sensitivity of the test can be regulated by adjusting the proportion of labeled particles coated with detection antibody. Batches of strips can be pre-calibrated and equipped with a calibration code that can be read by the detection device, so that a quantitative or semiquantitative result can be read from the device. The device may be a battery-powered handheld device of dimensions that allow it to be carried in an appropriate jacket or leg pocket of a rescue worker's uniform. Many variations of the individual aspects of this lateral flow technology are possible, as known to those skilled in the art.

Example 2 : Minicolumn device

The minicolumn contains a frit made of compressed polyethylene particles allowing the passage of fluid and red cells. The frit is coated with capture antibody against Gc- globulin. The minicolumn is incorporated into a device, which by means of automated liquid handling allows the fixed volume of anticoagulated sample to be applied at a fixed flow rate and volume, followed by detection antibody against actin-free Gc-globulin complexed with dye. After the passage of wash solution, the color intensity of the frit is read by light diffusion photometry. The batches of frits are pre- calibrated and the minicolumns equipped with a calibration code that can be read by the device, so that a quantitative result can be displayed by the instrument without the need for prior calibration with standards. This portable instrument can also be made suitable for field use. Example 3 : Turbidimetric device

The fixed volume of anticoagulated sample is dispensed into a cassette containing a dilution of antibody against Gc-globulin. The reaction of the antibody with the Gc- globulin in the sample produces turbidity proportional to the Gc-globulin concentration, which can be read by a small, portable battery-operated photometer.

Claims

What is Claimed is:

1. A method for assessing severity of injury or disease in a subject comprising rapid measurement of a level of a biomarker comprising a component of the extracellular actin scavenging system in a sample of the subject by means of a method than can be performed independently of laboratory facilities at the site where the subject is encountered or a site of first point of care, said measurement being performed within 1 hour after the injury or the presentation of disease.

2. The method of claim 1 wherein the injury or disease is associated with the release of actin into the bloodstream and the sample is blood.

3. The method of claim 1 or 2 wherein the biomarker measured is one or more of actin-free Gc-globulin, total Gc- globulin, actin-Gc-globulin complex, actin-free gelsolin, total gelsolin and actin-gelsolin complex.

4. The method of claim 3 wherein a functional activity of one or more of the biomarkers is measured.

5. The method of any of claims 1 through 4 wherein measurement of the biomarker or its functional activity is performed by means of one or more antibodies or other binding molecules capable of binding to the biomarker or a product of its functional activity.

6. The method of any of claims 1 through 5 wherein measurement of the biomarker or its functional activity is performed on a test cassette which generates a quantitative or qualitative signal that can be read by visual inspection or by means of a portable reading device that is carried to a site of injury or first point of care of the subject.

7. The method of any of claims 1 through 6 wherein the subject is a human.

8. The method of any of claims 1 through 7 wherein the injury is traumatic in origin.

9. The method of any of claims 1 through 7 wherein the injury to tissues is due to disease or intoxication.

10. A portable device for rapid assessment of a level of a biomarker in a sample wherein said biomarker is a component of the extracellular actin scavenging system, said portable device comprising a test cassette comprising one or more antibodies or other binding molecules capable of binding to the biomarker or a product of its functional activity.

11. The portable device of claim 10 further comprising a blood sampling means .

12. The portable device of claim 10 further comprising a portable reading device for the test cassette.