JP2012524518A - How to detect hypoxia - Google Patents

Abstract

Methods for detecting hypoxia of tissue in fetal scalp blood sampled, eg, in the liver, aorta, gastrointestinal tract, or parturition, prior to surgery or transplantation, lactate dehydrogenase (LDH) in plasma obtained from a sample ) To measure the total amount. This method may include additional measurements of K, Mg, Ca, AST, ALT, lactic acid in plasma and / or blood. An increase in one or more values of LDH, Mg, Ca, AST, ALT, and lactic acid is an indicator of hypoxia in the fetus. The use of a plasma separator in the method is also disclosed.
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Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to Swedish application No. 0602158-8 filed on Oct. 13, 2006, application No. PCT / SE2007 / 05050738, filed Oct. 12, 2007. , Both of which are incorporated herein in their entirety.

  The present invention relates to a method for detecting hypoxia. One aspect of the present invention relates to a method for detecting hypoxia in the blood of a fetal scalp during delivery that indicates a risk of damage to fetal organs. In another aspect, the present invention relates to a hypoxia detection device.

  Acute perinatal asphyxia during or at the end of delivery, ie, hypoxia (insufficient oxygen saturation of fetal blood) is an important factor in neuronal damage in the form of neonatal hypoxic-ischemic encephalopathy (HIE) The cause remains. It is found in 2-9 / 1000 term infants, followed by cerebral palsy (CP) and death in severe cases. From a global perspective, approximately 4 million newborns die each year and approximately 23% are attributed to acute perinatal asphyxia. Due to lack of funding, developing countries are in trouble and this is a serious problem as many people understand in Western countries. Sweden is seen as a representative country in Western Europe, and asphyxia will occur at about 7/1000 term birth, and 2/1000 children will be born with HIE. In order to prevent persistent damage caused by perinatal asphyxia, it is important to detect hypoxia in the fetus as soon as possible with respect to its onset. Early detection makes it possible to decide whether to intervene at a stage where persistent failure does not occur. Intervention essentially consists of removing the infant as quickly as possible by beneficial delivery, especially by caesarean section.

  The detection of acute perinatal asphyxia currently involves monitoring the fetal heart rate, followed by pH measurement, fetal scalp blood lactate sampled from the vagina, if a fetal heartbeat pattern is seen as a bad sign It is carried out by measuring.

  pH and lactic acid are indicators of metabolic acidosis that occurs by switching to anaerobic metabolism in the context of inadequate oxygen supply. In fetuses lacking oxygen, pyruvate is metabolized to lactic acid and energy. At present, measuring pH is a great standard. However, rapid measurement of pH requires 35 μL of scalp blood, which is not easily obtained. As some studies show, the first measurement failure is very common (20%). Since only 5 μL of blood is required and analysis can be performed at the bedside, lactic acid is easy to measure. The analysis of lactic acid can be performed within 1 minute and is consequently fast enough.

  Lactic acid and pH are also indicators of acute asphyxia. As such, it provides an indication of an overall healthy fetus exposed to sudden sudden onset of hypoxic ischemia during labor. A significant proportion of all infants who develop hypoxic-ischemic encephalopathy have symptoms of hypoxic ischemia before entering the delivery phase. They are vulnerable to hypoxic ischemia during parturition and do not respond in the same way as healthy fetuses, so that currently used methods are not sufficient for this group of patients.

  A method of monitoring labor, including the measurement of lactic acid in fluids such as vaginal fluid, is disclosed in WO2005 / 034762A1. Preliminary results from a recent Swedish study that arbitrarily extracted fetal blood pH and lactic acid at delivery, lactic acid as an indicator of acidosis is as good as pH. Neither lactic acid nor pH is an ideal predictor of moderate / severe HIE; the sensitivity is only 67% for lactic acid and 50% for pH, but the specificity is about the same, 76 for lactic acid. %, And pH is 73%. Also, the sensitivity and specificity for predicting acidosis in newborns is less than 70% for lactic acid and pH. A recent Swedish report detects certain perinatal asphyxia that can lead to brain damage even in fetuses monitored by a combination of heart rate recording (CTG) and STAN (analysis of heart rate recording ST segments) It is reported that there is a risk that it cannot be done (SBU Alert-rapport nr 2006-04).

  Enzymes known to be elevated in newborns exposed to asphyxia during labor are LDH (lactate dehydrogenase), also known as liver enzymes, ALT (alanine aminotransferase) and AST (aspartate aminotransferase). ). LDH is found in most cells in the body and is considered a non-specific enzyme. It is therefore rarely used in clinical practice. LDH was previously used as a marker for myocardial injury, but has now been replaced by more specific tests. In particular, AST and ALT are more specific for liver damage. In studies of plasma and liver enzymes working and delivery effects in low-risk Chinese female newborns, LDH (lactate dehydrogenase), ALT (alanine transaminase), AST (aspartate transaminase), GGT (γ -Glutamyltransaminase) was measured and showed a correlation between maternal and newborn (Mongrelli M et al., J Obstet Gyneecol Res, 26 (1): 61-63, 2000).

During or at the end of parturition, when the fetus is exposed to hypoxia, its blood flow is restored from the “less important organs” (kidney, liver, fat and intestine) in support of the brain, heart and adrenal glands. Will be distributed. This tends to damage cells in the lowest organs. Cell damage results in leakage of enzymes that enter the blood circulation. If hypoxia is severe, the cells will die and even the enzyme concentration in the blood will rise. The rate of decrease of LDH, AST and ATL (12-36 hours) detects cases that are more vulnerable to hypoxic ischemia during delivery for previous hypoxia that begins before delivery Make it possible. Hypoxia also affects the balance of electrolytes in the fetus. One example is the influx of calcium from intracellular blood during hypoxia. It has been shown that serum ionized calcium levels increase during hypoxia in a neonatal animal model. Calcium also predicts outcome in human infants (regardless of brain damage). Other enzymes and electrolytes of interest that change during hypoxia in neonatal mammals are potassium (K ⁺ ), magnesium (Mg ²⁺ ), sodium (Na ⁺ ), glucose, creatinine kinase (CK) and GGT. It is.

  From a global perspective, about 4 million children die each year, and about 23% of these deaths result from acute perinatal asphyxia. The pathological mechanism of hypoxic ischemia that induces neuronal damage in the brain is biphasic, starting with the main phase immediately after birth. If the infant is successfully revived, this major phase will be followed by a free interval that lasts for several hours. In 2 of 7 infants choking, this free interval includes secondary energy depletion leading to late cell death in the child's brain, and seizures (also known as HIE) ) Will continue with the clinical picture. Free intervals offer the possibility of minimizing delayed cell death from hypothermic treatment (cooling the child's brain to 34.5 ° C). However, there is currently no reliable means to predict that a child can develop HIE and, as a result, enjoy the benefits of hypothermia treatment.

  Hypoxia is a significant concern in many other medical conditions during or at the end of parturition. For example, colorectal cancer is the most common tumor in both men and women and its incidence is increasing year by year. The latest treatment is surgery, which removes the radical part of the intestine. In most of this case, the distal and proximal ends of the intestine are then reconfigured. This is called an anastomosis. During this progression, arterial blood supplied to the part of the intestine where the tumor is located is blocked when the arterial vessel is severed. In 7-10% of all surgeries, post-surgical complications due to anastomotic leakage can be predicted. In such cases, intestinal contents leak into the abdomen, causing inflammation, peritonitis, sepsis and potential death. The main reason for this complication is an inadequate supply of blood to the area for anastomosis as a result of the removal of blood vessels. The latest solution is a simple reoperation. Undesirably, it is not currently possible during surgery to predict whether an anastomosis will leak or not.

  Other areas where hypoxia is a major concern include vascular surgery and liver transplant surgery. For example, the main factor determining morbidity and mortality in patients after liver transplantation therapy is preservation of liver graft damage (Leemaster 1997). Leakage of LDH, AST and ALT into the perfusate is a degree of loss of liver cell membrane integrity (Kevis 2007).

  One major drawback of previous devices and methods for detecting hypoxia is that only LDH can be analyzed in plasma or serum. In addition, they require a minimum of 150 μL of whole blood for such measurements. Such a volume of blood is difficult to obtain from small animals, certain tissues or prenatal children during labor. Another problem is that LDH is also present in red blood cells, and hemolysis (red blood cell rupture) will give incorrect high value (beyond detection limits).

  Therefore, there is a need for a method that requires only a small amount of blood to detect LDH alone or together with electrolytes and liver enzymes to detect hypoxic ischemia. Furthermore, there is a need for improvements in the detection of fetal oxygen supply during delivery.

  The present invention provides at least a number of devices and methods that can analyze LDH and optionally AST, ALT, Mg and lactic acid within 10 minutes of whole blood in 10 microliters of whole blood. Satisfy that request. These analyzes can measure free hemoglobin together to confirm the absence of hemolysis, which results in incorrect LDH increases.

  Embodiments of the present invention include a method for detecting hypoxia, particularly hypoxia in fetal scalp blood sampled during labor. In such an embodiment, the method may comprise measuring LDH (lactate dehydrogenase) in plasma. In addition to LDH, it is preferable to measure one or several markers selected from the group consisting of K, Mg, Ca, AST, ALT and lactic acid in the blood of the fetal scalp. Particularly preferred is a combination of any of K, Mg, Ca, AST, ALT and lactic acid with LDH. Further, a combination with LDH, lactic acid, Mg and AST and / or ALT is preferable.

  As used throughout this specification, “LDH” and “lactate dehydrogenase” should be understood to mean all dehydrogenases, ie, not their isozymes.

  In the method of the present invention, a limited amount (preferably about 5-25 μL) of scalp blood is sampled from the fetus through the vagina during delivery in the environment near the patient. Samples are subjected to separation of blood cells, especially plasma (serum) from blood cells, which are analyzed for LDH, possibly K, Mg, Ca, AST, ALT, lactic acid within minutes to find symptoms. The As a result, the medical team can conclude directly that it indicates whether the child is suffering from acute asphyxia, ie whether a cesarean section is required. Thanks to the present invention, many sufferings associated with hypoxia can be eliminated and many remedies can also be achieved.

  In a study conducted by the inventors, LDH, AST and ALT in blood samples from children were analyzed within the first 24 hours. It was found by 193 tested children. Nineteen of these children showed signs of asphyxia in the form of an Apgar score <7 at 5 minutes of age. According to the ROC curve, a sensitivity and specificity of 96% for LDH was observed at a cutoff value of 1000 U / L (see FIG. 1). For HIE, the sensitivity is 100% and the specificity is 96% at the same cutoff value. In other words, this is because the main marker according to the method of the present invention is that 95% (or more) of all children with true hypoxia are healthy and 95% of all healthy children who are healthy at the same time. It means to decide. AST and ALT represent 86% sensitivity and 90% specificity at cutoff values of 55 U / L and 18 U / L, respectively. Despite the fact that this study is preliminary, it represents an unexpected improvement over the methods that exist today.

From a cost perspective, the results are surprising. Using today's method, approximately 10 unnecessary caesarean sections are performed to find one child with HIE, ie a total of 11 cesarean sections to find one child with HIE. Of which 10 are unnecessary. The sensitivity and specificity of the present invention indicates that the results are opposite, i.e., only one out of 10 cesarean sections was unnecessary. Thus, society can save millions and at least bring more children to use natural labor.
Described below. A table showing significant improvements by the method of the present invention compared to known methods is shown below.

  In healthy humans, red blood cells are known to contain about 150 times more LDH than serum. Therefore, since it is passed through the plasma, it is important to perform sampling and separation in a way that does not damage the red blood cells to prevent the LDH content from passing into the plasma. It is also important to consider a small amount of scalp blood sample that can be used for analysis. In the method of the present invention, plasma is separated into plasma and blood cells by passing it through a porous matrix, referred to in this application as a “membrane” on a solid phase that can hold blood cells. Plasma is analyzed for LDH by conventional methods. Analyzing K, Mg, Ca, AST, ALT and / or lactic acid in plasma or blood; when analyzing in blood, a scalp blood sample is derived from the plasma obtained for the determination of LDH; Separate into one or more parts for analysis of other hypoxic markers. The use of other methods used in the art, such as microcentrifugation, microfluidic compact disc technology and magnetophoresis, to separate plasma from blood cells in the scalp blood sample of the present invention is of the present invention. Within range.

  Methods for analyzing LDH in fluids such as plasma are known in the art and can be applied to measure LDH in serum obtained from fetal blood; see, eg, Pinto PV C et al. , Clin Chem 15: 339-349, 1969. Commercial equipment used in the last-mentioned method is commercially available (Vitros® DT60 II Chemistry System; Ortho-Clinical Diagnostics, Inc., USA). Methods for the analysis of blood K, Mg, Ca, ALT, AST and lactic acid are routinely used in clinical chemistry and are therefore within the reach of those skilled in the art.

  According to the present invention, when the fetus is hypoxic, the levels of LDH, K, Mg, Ca, ALT, AST and lactic acid in scalp blood sampled from the fetus through the vagina during parturition are evaluated and standardized. (Baseline) is the corresponding level in the fetus during and / or immediately after parturition in the context of a problem-free birth. In particular, in fetuses in labor that suffer from severe hypoxia, ie hypoxia that places the fetus at high risk of obtaining HIE, the levels of LDH, ALT and AST are increased by a factor of two, especially more than three times.

  According to a preferred embodiment of the present invention, a characteristic care device is provided that uses at least one, preferably a number of markers, to determine whether it is in acute hypoxia within minutes. A method for detecting hypoxia in a scalp blood sample during labor is disclosed.

  According to a further preferred variant, the instrument is further connected to one or several analytical means of K, Mg, Ca, AST, ALT, lactic acid.

  According to a further preferred embodiment, the invention relates to a plasma separation device which is used simply and directly in the device / method of the invention.

  In another aspect, the invention includes a method for assessing hypoxia in a mammalian tissue site. In such an embodiment, the method of the invention includes collecting a blood sample from a tissue site, the blood sample comprising plasma and blood cells, and separating plasma derived from the blood cells. The amount of LDH in plasma is measured, and the presence of hypoxia in the tissue site is evaluated from the amount of LDH in plasma. Methods for evaluating hypoxia at a tissue site include analysis of blood samples derived from mammalian digestive tract (eg, mammalian intestine), brain collected by specific organ (eg, mammalian aorta) or microcatheter. Analysis of samples derived from spinal fluid, analysis of urine or peritoneal fluid, and analysis of samples derived from organs transplanted into mammals in need of transplantation. The method of assessing hypoxia according to embodiments of the present invention predicts the likelihood of brain damage after prenatal hypoxia and to the limbs of a mammal before, during and after a medical procedure or surgery. Allows assessment of blood flow.

  In the following, the present invention will be described in more detail by means of more or less schematic illustrations of preferred but non-limiting embodiments of the invention.

  Accordingly, while describing the invention in general terms, the symbols are implemented with the accompanying drawings that are not necessarily drawn to scale.

FIG. 1 shows ROC curves for sensitivity and specificity. FIG. 2 shows a block diagram schematically illustrating an embodiment of the method of the invention. FIG. 3 shows a cross-sectional view of a first embodiment of the separation device in sequential steps used in the method of the invention. FIG. 4 shows a cross-sectional view of a first embodiment of the separation device in sequential steps used in the method of the invention. FIG. 5 shows a cross-sectional view of a first embodiment of the separation device in sequential steps used in the method of the invention. FIG. 6 shows an embodiment of the capillary device of the present invention intended to be used to collect test scalp blood. FIG. 7 illustrates a rapid test analysis system according to another embodiment of the present invention. FIG. 8 shows an embodiment of a disposable card device for rapid testing of the present invention. FIG. 8 shows a schematic diagram of the action normally linked to the present invention.

  The present invention will now be described in more detail below with reference to some accompanying drawings, which do not show all embodiments of the present invention. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are statutes to which the present disclosure is applicable. Is provided to meet the necessary requirements.

  The present invention provides an instrument and method capable of analyzing LDH, optionally AST, ALT, Mg and lactic acid within minutes (or seconds) of 10 microliters of whole blood. Analyzes can be measured together with free hemoglobin to confirm the absence of hemolysis, which results in incorrect LDH increases.

  In a position aspect, the present invention includes a method for detecting hypoxia. According to certain embodiments, the method may include collecting a blood sample; measuring the total amount of LDH in the blood. The blood sample can be collected from any mammal or an alternative derived from any organ transplanted into the mammal in need of transplant.

  In another embodiment, the method of detecting hypoxia comprises collecting a blood sample, the blood sample comprising plasma and blood cells. Preferably, the plasma is separated from the blood cells so that the plasma can be analyzed in the absence of blood cells. The separated plasma can be analyzed for the total amount of LDH in the plasma. The detection of LDH itself can be achieved. Based on the LDH amount or a combination with other prognostic markers, hypoxia can be easily detected.

  In a preferred embodiment, the detection of hypoxia comprises analyzing a blood sample to determine the content or amount of a plurality of prognostic markers. In one such embodiment, the method measures the amount of LDH in a blood sample, or preferably plasma, and is essentially selected from the group consisting of K, Mg, Ca, AST, ALT and lactic acid. Measuring the amount of at least one additional prognostic marker.

  Preferably, the blood sample for analysis is manipulated to separate plasma from blood cells. In one embodiment, this separation can be achieved by using a semi-permeable membrane or centrifugation. When utilizing a semipermeable membrane, plasma preferably passes through the membrane and blood cells are retained in the membrane. Thus, plasma is separated from the blood cell bulk for all practical purposes. The plasma is then sampled and tested in the absence of blood cells.

  According to various embodiments, the sample volume for analysis is greatly reduced over previous methods. In one embodiment, the volume of blood for detection of hypoxia is 5 μL to 60 μL, or 5 μL to 25 μL, or preferably about 5 to 15 μL, especially 10 μL. In certain embodiments, the volume of blood for detection of hypoxia comprises about 5 μL to 150 μL, or 10 μL to 120 μL, or 10 μL to 100 μL, or 10 μL to 80 μL.

Advantageously, embodiments of the present invention allow detection of hypoxia in a variety of environments. For example, embodiments of the invention may include blood from fetal scalp, gastrointestinal tract (eg, colon anastomosis), certain organs (eg, liver and aorta), cerebrospinal fluid from lumbar drain, and organs to be transplanted. Including, but not limited to, detecting hypoxia in the medium. Furthermore, embodiments of the present invention provide:
Evaluation and / or monitoring of liver (eg, of mammals potentially suffering from multi-organ dysfunction), peripheral tissues (eg, trauma, sepsis, bleeding or extensive surgery), prediction of brain damage after prenatal asphyxia, As well as monitoring of peripheral blood flow in mammals.

  The plasma separation device 1 of the present invention in a continuous process of use in the present invention is shown in FIGS. Device 1 typically includes an annular housing in a hydrophobic polymer material such as poly (tetrafluoroethylene). The plasma collection compartment 2 in the housing, like that of the housing, has an upper opening closed by a plasma separation membrane 11 supported by a grid 12 of the same or similar material. The partition bottom 13 is inclined toward the center in the form of a wide-angle pyramid. Extending from the side wall of the compartment 14 is a tubular conduit 15 with a valve 16. The conduit 15 extends to a negative pressure source such as a vacuum pump (not shown). In FIG. 3, 40 μL of scalp blood sample 7 is placed on the outer surface of the membrane 11 and the valve is closed. By opening the valve 16, the compartment 14 is placed under negative pressure (FIG. 4); thereby, the plasma 7 ′ is sucked into the compartment 14 and accumulates in its inclined bottom 13, and the blood cell 7 ″ becomes the membrane 11. Retained. After closing the valve 6, the state shown in FIG. 5 is reached, and the pressure in the compartment becomes equal to the atmospheric pressure by the air drawn into the membrane 11. Other pressure equalization in the compartment 14 can be achieved through the conduit 15 after stopping the formation of negative pressure or by other suitable means. FIG. 5 shows the removal of the plasma sample 7 from the compartment 14 by means of a suction syringe, the suction syringe cannula 10 being inserted through the membrane 11. In order to avoid contamination of the plasma sample 7 with blood cells on the membrane, a separation insertion port (not shown), for example, a rubber septum is provided which is arranged in the separation opening in the upper part or side wall of the instrument 1. The block diagram of FIG. 2 illustrates the principle of the method of the present invention.

The compartment further comprises a second conduit arranged to connect to the bottom of the compartment and second valve means for controlling the discharge of liquid (plasma) that accumulates at the bottom of the compartment. The compartment also includes means for detecting accumulated LDH and other markers. ^Film, 5mm ^{2 ~1000m 2,} it is particularly preferable to have an area of 20 mm ² to 300 mm ^2.

Analysis of scalp blood The analysis was successfully repeated using a scalp blood sample from a healthy adult male using standard equipment used in clinical routines for pH or lactate measurements. Samples use and do not use vaginal tubes (to avoid amniotic fluid contamination in real life situations). The average time of application of blood samples to the plasma separation card to show the results was 7 minutes. By using the instrument applied to the method of the present invention, in particular, the negative pressure applied to the vacuum side of the membrane is well controlled by using electrical means, and by measuring time, The time required for this was considerably shortened, for example 3-4 minutes or less. Thus, the use of the method of the invention in the detection of fetal scalp blood at the bedside in an emergency situation is completely feasible.

  Measurements showed that LDH in scalp blood was significantly higher than finger capillaries; it was 400 U / L in finger blood, with or without an amniotic fluid tube 1044 U / L and 1127 U / L. Differences between scalp and umbilical cord blood similar to those seen for LDH were obtained for glucose and hemoglobin in the fetus. The measurement of LDH in fetal scalp blood is unknown in the art.

  FIG. 6 includes a disposable card 2 and an analysis means 3 for performing a point of care test, i.e. within 7 minutes, preferably within 2 minutes, more preferably within a few seconds in the environment near the patient. Fig. 2 shows a system of the present invention that makes it possible to show. The disposable card 2 is preferably arranged with a number of different detection cells 20A-20E, which are described in detail in FIG. 7, in fact only LDH (or, for example, two cells for LDH + AST). A card that only tests can be sufficient in some applications.

  FIG. 6 shows two continuous steps according to the method of the present invention. On the upper left side, a new card 2 is supplied so that the test blood 7 is supplied using the glass capillary device 4, and the first step of filling the blood with an amount of about 10 μL, for example, is shown. In a continuous process, the glass capillary device 4 is inserted into the compartment 21 of the card 2 in order to connect the blood sample 7 with the card 2 and the disposable card 2 arranged in the device 3, and the blood sample 7 directly Analysis is performed and will be described in detail in connection with FIG. The card comprises plasma 7 'derived from blood sample 7 in at least one detection cell 20A-E, preferably blood cells in at least one other cell, more preferably whole blood in at least one other cell. It is equipped with a device that allows it to enter (eg, essentially known as a microfluidic chamber for plasma / serum distribution). Prior to entering the detection cell, plasma / serum / whole blood enters the reaction chamber (26A-26E) in which the reagent (preferably dried) is deposited. Thus, the reaction will occur before detection. Optical measurements (eg, spectrophotometry known per se and described in US Pat. No. 4,803,159, which is incorporated herein by reference) are processed directly by the processor 31 of the instrument 3 and shown on the display 32. And / or provided as data output 33 on, for example, printed paper. Furthermore, the analytical instrument 3 preferably comprises a barcode reading arrangement that can read and process a barcode 22 with a unique code for each disposable card 2. Preferably, the analytical instrument 3 is surrounded by a casing 35 that is portable. Connections are provided to supply the necessary supplies, for example, power (if not battery powered). In other embodiments, it is made smaller by using an external processor, for example, a laptop that manufactures equipment that can be connected to the laptop via a USB cable.

In FIG. 7, the aspect of the specific disposable card 2 of this invention is shown. The card 2 has five detection cells 20A to 20E, all of which are optical detection cells. The first detection cell 20A is for ALT. The second detection cell 20B is for AST. The third detection cell 20C is for total LDH. The fourth and fifth detection cells 20D, 20E are for lactic acid and Mg ²⁺ , respectively. The card shown in the embodiment has a circular planar shaped body 23 having a diameter that facilitates processing, for example in the range of 20-120 mm, preferably 40-100 mm. The material of the body 23 can be selected from a wide range, for example, poly (tetrafluoro) ethylene, polyethylene, polypropylene, polystyrene, and the like. As shown schematically in FIG. 7, the card 2 comprises a chamber 21 adapted to mount a glass capillary device 4 for supplying a blood sample 7. Interface 23 in a manner known per se, which relates to the chamber 21 and protects further transport of the blood sample at its bottom (compare the membrane 11 shown in FIGS. 3-5, including the membrane and the plasma collection compartment) There is. A sample splitter 24 between the plasma separation membrane 25 and the interface 23, possibly allowing the supply of whole blood to several detection cells, for example by printing reagents 26A, B for detecting ALT and AST, respectively. Next, a large volume of whole blood sample (shown by dotted lines) is supplied to the plasma separator 25 by a printing reagent that detects, for example, total LDH and lactic acid in the plasma 7 ', respectively. The blood cell 7 ″ obtained after the separation is carried out after mixing the optical reagent 20D with the printing reagent 26D in order to detect Mg ²⁺ .

  Test results using various marker combinations in a specific disposable card of the present invention will be shown below.

  Lactate dehydrogenase increases during hypoxia and is present in all body cells. LDH present in the bloodstream exhibits hypoxia that is severe enough to reduce blood flow to peripheral organs. This is the onset of leakage of LDH from these cells. It is not possible to detect which organs suffer from hypoxia by detecting LDH. If hemolysis is occurring (even if it is not hypoxic, erythrocyte rupture also causes an increase in LDH), hemolysis is divided into two groups: (1) Do you obtain samples in vitro in vitro? Refers to hemolysis that occurs when stored, and (2) refers to red blood cells that rupture in vivo due to disease in vivo. Ongoing hemolysis will then give unreasonably high levels of LDH, not just because of hypoxia. The half-life (T1 / 2) of LDH depends on which of the five isomers of LDH is released into the blood. LDH1, which is mainly present in the heart, brain and blood cells, has a T1 / 2 of 120 hours, while LDH5, which is mainly found in the liver and muscle, has a T1 / 2 of 10 hours.

  In our study in neonates, patients suffering from HIE will be found by using LDH as a marker as well as 178 of 184 healthy patients. This means that there is no case for hypoxia and that 6 infants are unnecessarily delivered by caesarean section or by instrumental delivery.

  High LDH indicates the onset or recent onset of hypoxia or hemolysis somewhere in the body.

  AST is an enzyme that exists in many organs in the body but is more organ specific than LDH. AST is present in the liver, muscle and erythrocytes. AST and LDH are sensitive to hemolysis but not to the same extent.

  The T1 / 2 for AST is 12-15 hours for newborns.

  In our study, patients suffering from HIE will be found by using AST as a marker as well as 210 of 236 healthy patients. This means that there is no case for hypoxia and that 26 infants are unnecessarily delivered by caesarean section or by instrument delivery.

  High levels of AST indicate the onset or recent onset of hypoxia or hemolysis in the liver, muscle.

  ALT is an enzyme specific to the liver and is very slightly affected by hemolysis.

  The T1 / 2 for ALT is 36 hours.

  In our study, patients suffering from brain injury will be found by using AST as a marker as well as 28 of 240 healthy patients. This has been delivered unnecessarily by caesarean section or by instrument delivery.

  Elevated levels of ALT indicate the onset or development of hypoxia in the liver.

  Magnesium in the body is located 50% in the skeleton and 50% in the cells. For example, if acidosis occurs during hypoxia (decrease in pH), hydrogen ions will migrate into the cell. At the same time, Mg moves into the cell and raises the Mg level in the blood. For newborns with HIE, Mg levels are lower than healthy newborns. Mg is a better marker for acidosis than cell damage.

  Elevated Mg levels are an indicator of acidosis, a symptom of hypoxia. Low Mg levels are an indication of the development of hypoxia that induces brain damage.

  At this stage we do not have data on Mg during labor. However, we trust that Mg levels are elevated in animal studies in newly born piglets.

  This finding indicates that all healthy infants have low levels of AST and / or LDH. All of the 10 infants suffering from HIE have high levels of AST and / or LDH and have LDH. AST adds the effect of using LDH to determine whether an infant is healthy.

  High levels of LDH and AST indicate hypoxia and hemolysis.

  The table below shows the results based on the use of LDH + ALT as a marker.

  The above table shows that healthy infants have low levels of ALT and / or LDH when analyzing LDH and ALT simultaneously. Of the 10 infants suffering from HIE, all infants had elevated ALT and LDH. One infant with elevated levels of AST and LDH did not suffer from HIE. ALT adds the effect of using LDH to determine whether an infant is healthy.

  ALT can also theoretically tell if an infant is suffering from hypoxia early in the uterus because ALT remains in the blood for a long time (T1 / 2 is 36 hours). ,be interested.

  Elevated levels of LDH and ALT indicate hypoxia that affects the liver (among other organs).

  The table below shows the results based on the use of LDH + ALT + AST as markers.

  When LDH is analyzed together with AST and ALT, all enzyme levels are low and infants suffering from HIE cannot be found. In all three marker elevations, all infants suffering from HIE are found and only one infant is delivered unnecessarily without HIE.

  High levels of LDH + ALT + AST indicate the development of hypoxia or hypoxia in the previous time. The fact that the half-life of ALT is 24 hours longer than AST and LDH allows this combination to also provide a time phase of hypoxia. The LDH + ALT + AST examples are all high and remain increased after 24 hours of infant birth, indicating that hypoxia develops near the end of labor.

  In the materials used for these calculations, 23 cesarean sections and 22 instrument deliveries were actually performed in groups that did not show an index in hypoxia (HIE, acidosis or low Apgar score). This is the only cesarean section that includes infants performed for suspicious harm.

  In the above example, using LDH in combination with ALT showed that one infant was unnecessarily delivered instead of 45. This does not include aspiration delivery and only saves 880000 SEK in the cost for cesarean section. Aspiration delivery, combined with increased patient suffering, generates costs for healthcare and increases the risk of injury for both mothers and infants.

  As mentioned above, AST does not give much to the method when determining whether an infant is suffering from hypoxia. The AST remains informed for a half-life of 12 hours. Along with ALT and after birth, samples can be taken repeatedly and these two enzymes can provide information about whether hypoxia is occurring. Today's obstetricians have problems to prove whether women are hypoxic when they arrive at the hospital for delivery, or if the infant is already suffering from hypoxia, This has great legal utility.

  Conclusions based on information from the scientific literature indicate that magnesium provides knowledge about whether hypoxia exists or is a previous manifestation.

  High levels of LDH + ALT + AST + Mg affect peripheral organs and indicate the development of hypoxia that is severe enough to show acidosis. High levels of LDH + ALT + AST, low levels of Mg result in organ damage and possibly indicate a recent onset of hypoxia affecting the brain. Mg is not sensitive to hemolysis and, together with ALT, emphasizes that elevated levels of LDH are due to organ damage and not due to hemolysis.

  In the following, the examples describe the actual use of the method and apparatus of the present invention and assume that a patient named Anna has arrived at the delivery room for delivery. The staff begins to control the infant's condition by examining the heart rate using a small piece on the woman's stomach (fetal heart rate pattern, CTG). Control with CTG is performed to observe possible changes at intervals. 8 hours after parturition, Anna is opened 8 cm. It can be seen that the heart rate of the child rises with the new CTG control. The midwife calls on the obstetrician who wishes to continue taking samples from the infant's scalp if there are other signs of illness.

  Anna lying on her side seeks to move to the obstetrician's position, and the obstetrician passes a metal tube through the vagina and pushes it into the infant's scalp. She washed the amniotic fluid and cut the infant's scalp into small pieces (scalp sample). When she sees a drop of blood 7, she takes a capillary tube and collects about 10 μL of blood. (If it is necessary to clean and work quickly, it is not easy to collect blood from a small tube at the same time. 30-40 μL is a remarkably large volume in this setting, and it is useful for scalp samples to measure pH. is necessary.)

  Next, the midwife provides the obstetrician with the disposable card 2 inserted by the doctor into the capillary tube 4. Next, the card 2 containing blood is inserted into a nearby detection device 3, and the levels of LDH, AST, ALT and magnesium are analyzed by a spectrophotometer. After separation of red blood cells 7 ", analysis is performed on card 2, and the remaining plasma 7 'is reacted with reagents 20A-D on the card. Within a few minutes (eg 2 minutes) the result is shown on the instrument display. Shown as “normal-normal-normal-normal”, which tells the obstetrician that the level of any marker of LDH, AST, ALT and magnesium is not elevated. Anna continues to deliver in a natural way and later gives birth to a healthy baby.

  Later that night, the obstetrician visits room 3 where Helena gives birth to the first child. Here, similarly, CTG is not good, and a scalp sample is collected. At this time, the display shows “high-high-high-high”, which tells the obstetrician that the levels of all markers are rising. This simply means that the infant is suffering from hypoxia and decides to make an emergency caesarean section. An alarm for the operation sounds and Helena rushes to her bed room. Rapid anesthesia is performed, and the obstetrician cleans her hands and changes to a surgical gown. She then cuts her skin and uterus and gives birth to an infant just minutes after the alarm.

  As mentioned above, some markers are sensitive to hemolysis. The effect of hemolysis in this method has not been fully investigated at this stage, but will not change the results dramatically in at least some aspects. However, if hemolysis frequently appears during sampling, it is a foursome for integrating markers for hemolysis in the card. The most likely scenario is that we will use free hemoglobin (HB) as a marker choice. By measuring Hb in plasma, information about hemolysis can be obtained and how severe it is. In view of this, it is also possible to review the enzyme level depending on the degree of hemolysis.

  In some situations, if such an analysis can be colorimetric (reagent color change), LDH alone or LDH in combination with, for example, lactic acid, ALT, AST, or magnesium will determine what an infant should do. Enough.

  In FIG. 8, another test configuration embodiment is shown that uses a dry compound to perform the analysis. The advantage of using the test device 5 as shown in FIG. 8 is that no power is required in all wounds. The test device 5 is arranged with an elongated tube forming a body / case 50. Within the elongated body 50 is a channel 51 that extends into the front chamber 52 at a location adjacent to the front end of the housing 50. A pump device 53 is supplied at the other end of the channel 51 near the back end of the housing. The pump device 53 is in the form of a resilient hollow body arranged at its outlet end with a check valve 54. The outlet (through check valve 54) extends into processing device 55. At the front end of the housing 50 is a collar device 56 having a centrally located hole 57 connected to the front chamber 52 by a filter 58 to filter out blood cells. At a distance from the front end forming part of the channel 51, there is an additional chamber 59 in which the drying science means 8 is placed. An additional chamber 501 is also provided as part of the channel 51 adjacent to the pumping device 53 to form a buffer and also provide an indication regarding the volume of blood 7 in the channel 51.

  The test device shown in FIG. 8 is intended to be used in connection with labor. As shown in FIG. 8, a small amount of blood 7 is obtained through the scalp of the child's head 6. Thereafter, the test device 5 is inserted through the vagina and the collar 56 is positioned around a small amount of blood to contact the blood sample 7. In the next step, the pump mechanism 53 is activated, where the check valve 54 will spread into the air escaping from the hollow interior of the resilient pump device 53. When the pump device 53 is released, a vacuum is created (for elasticity) and connects with a small amount of blood 7 through the channel 51, whereby blood is drawn into the channel 51 through the filter 58. Thus, plasma 7 ′ enters analysis chamber 59. The pump mechanism may apply a lot of time to protect enough blood for testing, which can be measured once blood is observed in the buffer chamber 501. Thereafter, the test apparatus 5 is removed and by observing the color of the chemical means in the analysis chamber 59, it can be determined whether the child is suffering from hypoxia. As is known per se, the dry chemistry means 8 can be used to indicate various means depending on which color is displayed. For example, the following colors are used to indicate which means to take. If green is indicated, the measure should not be implemented. If red is indicated, the child should be removed as quickly as possible. If yellow is shown, a new sample should be taken within 20 minutes.

  Clinically, the test device 5 will give the same kind of indication as described above. The use of a capillary tube to collect blood from the infant's scalp can also be combined with a slightly modified test device 5, but preferably a closed system is formed in the device 5 when collecting blood. It can also be designed. This can be accomplished in many ways, for example by pushing the top with a silicone collar (not shown) against the infant's scalp or by removing the device 5 and attaching a tightly sealed cap on the top.

  We also foresee that the lactic acid test used today can be analyzed in combination with the present invention.

  In FIG. 9, there is shown a flowchart, preferably using the method and apparatus of the present invention. As shown in the flowchart, the CTG should preferably be used as an initial indicator. If the CTG is normal, there is usually no need to undertake any means. However, if the CTG is abnormal, a blood sample derived from a child's scalp may be analyzed directly according to the present invention, or optionally an ST-analysis of the fetal heart may be initiated.

  The present invention is not limited by what has been described above, but may vary within the scope of the appended claims. For example, it will be apparent to those skilled in the art that for the definition of “scalp sample” and blood samples collected from other parts of the body function at various occasions to obtain the benefits of the present invention. Samples used in the present invention include whole blood, plasma and serum.

  In other aspects, embodiments of the present invention can be advantageously used in other medical situations. Blood samples are collected from locations of interest prior to medical treatment and analyzed for prognostic markers.

  In various embodiments, a plurality of prognostic markers are analyzed. Such an embodiment provides a total amount of LDH and at least one additional prognostic marker selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactate in the plasma of both blood samples. Including measurements. Thus, the amount of each prognostic marker in the first and second samples can be compared to confirm the exact location of the anastomosis. In one embodiment, the medical procedure includes gastrointestinal anastomosis.

  For example, colorectal cancer is one of the most common tumors in both men and women. Current treatments include surgery to remove the tumor along with the root part of the intestine. In most of these cases, the distal and proximal ends of the intestine are then reconfigured. This is called an anastomosis.

  As such, certain embodiments include a method of detecting hypoxia from a blood sample collected from the mammalian digestive tract (eg, intestine). As such, certain embodiments include a point-of-care (POC) method for rapid (eg, within minutes or seconds as discussed above) detection of hypoxic ischemia from a small amount of blood. . Thus, these embodiments make it possible to determine whether the selected site of the intestine is suitable for anastomosis.

  LDH, AST, magnesium and lactic acid are markers that increase in the blood during hypoxic ischemia due to cell damage and anaerobic metabolism. Prior to excision of the blood vessel, blood from the intestine can be collected using a scalpel (or similar) and sterile capillary. Capillaries can be inserted into the aforementioned analysis card and analyzed. The result can be used as a unique patient reference value (eg, baseline). After removing the tumor (and surrounding intestine), a new test can be performed to see if the marker is elevated. If so, the region of the anastomosis can be moved close to the blood supply still present to minimize the risk of hypoxic ischemia that induces lack of anastomosis and leakage.

  In addition to sampling blood derived from the gastrointestinal tract, embodiments of the present invention advantageously include collecting a blood sample from the organ of interest or cerebrospinal fluid of the mammal during surgery.

  For example, the main cause of surgery in the thoracoabdominal aorta is aneurysm and dissection. The extensive surgery required is associated with significant morbidity and mortality. Some of the most common complications are nerve injury. The cause of most of these complications is a lack of oxygen and energy supply due to ischemia. As a specific example, such treatment causes permanent paralysis as a post-operative complication in 1-10% of patients depending on the site of aortic surgery.

  However, an embodiment of the present invention provides a method that may be a POC method for hypoxic ischemia in blood derived from specific organs or cerebrospinal fluid (CSF) through the lumbar drain during surgery. Including. As such, samples from a particular organ of interest are obtained and analyzed for LDH as detailed above. In a preferred embodiment, the method of detecting hypoxia comprises at least one additional prognosis selected from the group consisting of LDH and essentially K, Mg, Ca, AST, ALT and lactic acid in a sample. Detecting the marker. Preferably, one of the additional prognostic markers detected includes lactic acid. As a specific example, the particular organ of interest includes the mammalian aorta.

  In other aspects, embodiments of the invention can improve patient morbidity and mortality after transplantation therapy. One of the key factors affecting morbidity and mortality, especially after transplantation, is related to the preservation damage of the graft, such as a liver graft in a liver transplant. For example, leakage of LDH, AST and ALT into the perfusate is an indicator of liver cell membrane integrity loss.

  In one such embodiment, a method for detecting the presence of hypoxia in an organ transplanted into a mammal in need of detection comprises a blood sample as described above for prognostic markers prior to transplantation surgery. Collecting and analyzing the sample. In one embodiment, the sample is analyzed to determine the total amount of LDH and at least one additional prognostic marker selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactic acid in the sample. . In a preferred embodiment, the organ for transplantation includes the liver.

  Certain embodiments of the present invention may satisfy hospital demand for identification of patients at risk of developing a multi-organ dysfunction crisis. Currently, a country from the Intensive Care Unit (ICU) aims to provide a mobile team that can appear in a room (not an intensive care unit) for the evaluation of patients at risk for multi-organ dysfunction. An individual team of doctors and nurses is being developed. If confirmed to be at risk, the patient can be treated and transferred to the ICU. In Australia, the introduction of these mobile teams has reduced cardiac arrest, sudden death from cardiac arrest, the occurrence of postoperative complications and the number of days in ICU by 50%.

  These mobile teams conduct lung, kidney and blood circulation clinical tests to assess the risk of multi-organ dysfunction. However, there is no quick and reliable method for assessment of liver or metabolism. The liver is often affected by severe disease, and liver enzymes and metabolic acidosis are predictors of mortality.

  Accordingly, the apparatus of embodiments of the present invention is well suited for use by such teams. Such devices are usually small and quick enough to be taken by a mobile team (eg, in a bag) when examining in a hospital room. Since LDH, AST and ALT are present in liver cells and lactate increases during metabolic acidosis, these teams are currently evaluating the liver for other parameters to be examined by utilizing the device of the present invention. Can be added. Thus, the devices and methods of the present invention potentially identify even patients at risk for severe illness, thus saving both humanitarian and economic costs.

  In other aspects, the devices and methods of certain embodiments are well suited for use by staff within an intensive care unit (ICU). The ICU is responsible for most critically ill patients in the healthcare system. Independently, when a patient is delivered to an ICU for trauma, cardiac diagnosis, sepsis, bleeding or extensive surgery, insufficient blood circulation and saturation is a significant risk of hypoxia. Even with careful monitoring of typical parameters and treatment of abnormal conditions with fluids and medications, there is still no bedside method that can provide ICU staff with information about therapeutic effects in peripheral tissues. .

  It is known that significant increases in serum LDH, AST, ALT and lactate in patients who received severe shock are indicators of bad outcome (eg death) (Hardaway 1981). AST, ALT (formerly LDH) is measured as a clinical disorder in many ICUs as a marker of liver damage, and lactate is used as a marker for anaerobic metabolism and intestinal ischemia (Juel 2007). However, there are still no devices that can be operated by the bed or give results in a matter of minutes.

  Embodiments of the present invention include an apparatus and method for detection of hypoxia at the bedside where the results can be utilized within a few minutes at most. Such an embodiment includes obtaining a sample for analysis and detection of LDH. In a preferred embodiment, the method comprises measuring the amount of at least one additional prognostic marker selected from the group consisting essentially of AST, ALT and lactic acid in plasma.

  As discussed above, acute perinatal asphyxia, or hypoxia (insufficient oxygen saturation of fetal blood) during or at the end of parturition is a form of hypoxic ischemic encephalopathy (HIE) in newborn infants. Leaving an important cause of nerve damage. The pathological mechanism of hypoxic ischemia that induces neuronal damage in the brain is biphasic, starting with the main phase immediately after birth. If the infant is successfully revived, this major phase will be followed by a free interval that lasts for several hours. In 2 of 7 infants choking, this free interval includes secondary energy depletion leading to late cell death in the child's brain, and seizures (also known as HIE) ) Will continue with the clinical picture. Free intervals offer the possibility of minimizing delayed cell death from hypothermic treatment (cooling the child's brain to 34.5 ° C). . However, there is currently no reliable means to predict that a child can develop HIE and, as a result, enjoy the benefits of hypothermia treatment.

  Since it is important to start hypoxic treatment as soon as possible after birth (neuroprotective effect is reduced most after 5.5 hours of respiratory arrest), it is a quick point in the care system for prognostic marker detection There is clinical value. Data were collected from neonates by measuring the amount of LDH along with ALT, indicating that it provides 100% sensitivity for the prediction of HIE with> 96% specificity.

  Certain embodiments of the present invention provide a diagnostic tool for physicians in clinical settings and in research related to the treatment of hypoxia in neonates suffering from perinatal asphyxia. Furthermore, embodiments of the present invention include a method of detecting hypoxia that allows an individual to predict brain damage after perinatal asphyxia. Such a method involves obtaining a sample for analysis of LDH, as discussed in detail above. In a preferred embodiment, the relative amount of multiple prognostic markers can be measured. Preferably, the blood sample is processed such that the plasma is separated from the blood cells and the measurement of the level of various prognostic markers is based on an analysis of the plasma alone. In a preferred embodiment, the method comprises measuring the amount of LDH and at least one additional prognostic marker selected from the group consisting essentially of AST, ALT and lactic acid in plasma. Most preferably, the amount of ALT is one of the prognostic markers to be measured.

  In one method of an embodiment of the invention, the method is directed to a mammal in need, wherein measurement of plasma LDH (alone or together with other prognostic markers) indicates delayed cell death in the mammalian brain. Providing treatment for hypoxia.

  In other aspects, embodiments of the present invention can be used to assess the condition of a mammal's limb before, during and after a medical or surgical procedure. For example, trauma, fractures and vascular occlusion can affect peripheral limbs and muscle circulation (eg, compartment syndrome). There is a significant correlation between oxygen and lactate levels in ischemic muscle, LDH is observed (Yamamoto 1988), and in the blood of the femur of patients suffering from peripheral arterial occlusive disease, Lactic acid is elevated compared to the value (Rexroth 1988). The device of embodiments of the present invention makes it possible to use enzymes and lactate levels to diagnose specific limb ischemia and to assess the effectiveness of most treatments.

  Furthermore, embodiments of the invention include a method of detecting hypoxic ischemia by analyzing a sample derived from a limb of interest and measuring the total amount of LDH in plasma. Additional diagnostic markers can be quantified simultaneously with the measurement of LDH. This makes it possible to assess the blood circulation to the mammal's limb before, after and after the medical or surgical procedure.

  One skilled in the art can implement a wide variety of modifications without departing from the above description without inventive skills, such as by using glass or some other suitable material instead of plastic. .

  Many modifications and other embodiments of the invention disclosed herein will occur to those skilled in the art so that these inventions have the benefit of the teachings presented in the foregoing description and the associated drawings. Accordingly, the invention is not limited to the specific embodiments disclosed, but other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used herein, they are used for general and descriptive purposes, not for purposes of limitation.

Claims (54)

A method for evaluating hypoxia in a mammalian tissue site, comprising:
a. Collecting a blood sample from a tissue site (the blood sample comprising plasma and blood cells);
b. Separating plasma from blood cells;
c. Measuring the amount of LDH in the plasma; and d. A method comprising a step of evaluating hypoxia in a tissue site from the amount of LDH in plasma. The method of claim 1, further comprising measuring the amount of at least one additional prognostic marker in plasma selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactic acid. The method of claim 1, wherein the volume of the blood sample is 5 μL to 150 μL. The method of claim 1, wherein the volume of the blood sample is 5 μL to 25 μL. The method of claim 1, wherein the volume of the blood sample is 5 μL to 15 μL. The method of claim 1, wherein the plasma is separated from the blood cells using a semipermeable membrane such that the plasma passes through the membrane and the blood cells are retained on the membrane. The method of claim 1, wherein the blood sample is collected from a mammalian intestine. A blood sample is collected prior to the medical procedure and further includes the following steps:
a. Collecting a second blood sample after the medical procedure (the blood sample comprising plasma and blood cells);
b. Separating plasma from blood cells of a second blood sample; and c. The method according to claim 7, comprising measuring the total amount of LDH in the plasma of the second blood sample. Further comprising measuring the amount of at least one additional prognostic marker in the plasma of both blood samples selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactate. 8. The method according to 8. 10. The method of claim 9, further comprising comparing the amount of prognostic marker in the blood sample. 9. The method of claim 8, wherein the medical procedure comprises gastrointestinal anastomosis. The method of claim 1, wherein the blood sample is collected from one or more of a particular mammalian organ, cerebrospinal fluid, urine, or intraperitoneal fluid during surgery. 13. The method of claim 12, wherein the organ comprises a mammalian aorta. 13. The method of claim 12, further comprising measuring the amount of at least one additional prognostic marker in the plasma of the blood sample, selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactic acid. the method of. 15. The method of claim 14, wherein the at least one prognostic marker comprises lactic acid. The method of claim 1, wherein a blood sample is collected from an organ transplanted into a mammal in need of the organ. The method of claim 17, wherein the blood sample is collected prior to transplantation surgery. The method of claim 17, wherein the organ comprises a liver. 18. The method of claim 17, further comprising measuring the amount of at least one additional prognostic marker in plasma selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactic acid. 3. The method of claim 2, further comprising measuring the amount of at least one additional prognostic marker in plasma selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactic acid. 21. The method of claim 20, wherein the measurement of a prognostic marker allows an individual to assess the health of a mammal's liver. The method of claim 1, wherein detection of hypoxia enables prediction of brain damage after prenatal asphyxia. 23. The method of claim 22, further comprising measuring the amount of at least one additional prognostic marker in plasma selected from the group consisting essentially of AST, ALT and lactic acid. 23. The method of claim 22, further comprising measuring the amount of ALT in plasma. 23. The method of claim 22, further comprising providing hypothermia for the animal in need of treatment, wherein the measurement of LDH in plasma indicates delayed cell death in the mammalian brain. The method of claim 1, wherein the measurement of the total amount of LDH in the plasma allows assessment of blood circulation to the mammal's limb before, during or after treatment or surgery. A method for detecting acute hypoxia in fetal scalp blood, comprising collecting a blood sample during labor and processing the blood sample to look for at least one indicator for detecting hypoxia. And measuring the total amount of lactate dehydrogenase (LDH) in the plasma obtained from the sample. 28. The method of claim 27, wherein the sample is between 5 [mu] L and 60 [mu] L. 28. The method of claim 27, wherein the sample is 5 [mu] L to 25 [mu] L. 28. The method of claim 27, wherein the sample is 5 [mu] L to 15 [mu] L. 28. The method of claim 27, comprising separating plasma from blood cells using a semipermeable membrane and / or filter. ^Film, 5mm ^{2 ~1000mm 2,} preferably has an area of less than 100 mm ^2, The method of claim 31. 32. The method of claim 31, comprising attaching one surface of the membrane containing the sample, applying negative pressure to the other surface of the membrane, and collecting plasma in a compartment facing the other surface. 34. The method of claim 33, comprising transferring plasma from the compartment to an apparatus and / or instrument for measuring LDH. 34. The method of claim 33, comprising measuring LDH in plasma disposed within the compartment. 32. The method of claim 31, wherein the membrane is disposed on or integrated on a porous matrix, and plasma is drawn and moved by capillary forces. 38. The method of claim 36, wherein after the time for measuring LDH in the matrix is given, the membrane is removed from the matrix. 28. The method of claim 27, comprising separating plasma from blood cells in the sample and transferring the supernatant plasma to a device for LDH measurement. 28. The method of claim 27, further comprising comparing the measured LDH with measured LDH in plasma obtained from fetal scalp blood sampled during delivery from a fetus in non-hypoxic conditions. 40. The method of claim 39, wherein a statistically significantly increased LDH level is an indicator of hypoxia. 40. The method of claim 39, wherein LDH levels that are statistically significantly increased by three or more factors are indicative of the risk of hypoxic ischemic encephalopathy. 28. The method according to claim 27, further comprising measuring one or several members of the group consisting of K, Mg, Ca, AST, ALT and lactic acid in the scalp blood and / or plasma. A combination of a statistically significantly increased level of any of K, Mg, Ca, AST, ALT and lactic acid with a statistically significantly increased level of LDH is an indicator of hypoxia Item 43. The method according to Item 42. 28. A method according to claim 27, characterized in that the sample is collected on a disposable device comprising means for separating the plasma and a compartment arranged for performing the LDH measurement. 28. The use of a device for the separation of plasma from blood cells according to the method of claim 27, wherein the device is a housing, a compartment in the housing, a first in a housing covered with a semipermeable membrane. Use of a device comprising an opening, a second opening in the housing connected to the compartment by negative pressure or capillary force through the conduit, and valve means for controlling said connection in case of negative pressure. The valve means is disposed within the conduit, and preferably the compartment further includes a second conduit disposed to connect to the bottom portion thereof, to control the evacuation of plasma accumulated at the bottom of the compartment. 46. Use according to claim 45, comprising second valve means for. 46. Use according to claim 45, wherein the compartment housing is connected to means for measuring LDH in plasma accumulated in the housing. 28. A test system for performing the method of claim 27, wherein the system indirectly or directly receives a disposable device having a blood sample collection portion, a plasma separator, and total lactate dehydrogenase (LDH) in plasma. A system that includes compartments arranged for automatic measurement. 49. The test system of claim 48, wherein the disposable device is a flat form object and the receiving portion defines a compartment and is coupled to a capillary sample collection device. 50. The test system of claim 49, wherein there are a number of measurement compartments disposed on the card, preferably each of the compartments is connected to an upstream compartment with a reagent. 49. The test system of claim 48, wherein a sorter is disposed between the junction and the plasma separation device. 50. The test system of claim 49, wherein the compartment is in the form of an optical detection cell and the body is configured to fit within an instrument having at least one device for optical spectrum. . 49. A test system according to claim 48, characterized in that the main body is arranged with a unique code, preferably the device is equipped with a reader. 49. The test system of claim 48, wherein the disposable device includes a compartment having a dry chemical means for visual detection.

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