WO2009126110A1 - Procédé de détermination d'une hypoxie - Google Patents

Procédé de détermination d'une hypoxie Download PDF

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Publication number
WO2009126110A1
WO2009126110A1 PCT/SE2009/050426 SE2009050426W WO2009126110A1 WO 2009126110 A1 WO2009126110 A1 WO 2009126110A1 SE 2009050426 W SE2009050426 W SE 2009050426W WO 2009126110 A1 WO2009126110 A1 WO 2009126110A1
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WO
WIPO (PCT)
Prior art keywords
plasma
ldh
blood
sample
hypoxia
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PCT/SE2009/050426
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English (en)
Inventor
Mathias Karlsson
Sofia Hiort Af Ornäs
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Calmark Sweden Aktiebolag
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Filing date
Publication date
Priority claimed from US12/101,470 external-priority patent/US8945824B2/en
Application filed by Calmark Sweden Aktiebolag filed Critical Calmark Sweden Aktiebolag
Priority to EP09729478A priority Critical patent/EP2396419A4/fr
Priority to CN200980112713.4A priority patent/CN102131938A/zh
Priority to BRPI0910870A priority patent/BRPI0910870A2/pt
Priority to JP2011503943A priority patent/JP2012524518A/ja
Publication of WO2009126110A1 publication Critical patent/WO2009126110A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Definitions

  • the present invention relates to a method of determining hypoxia.
  • One aspect of the present invention relates to methods of determining hypoxia in fetal scalp blood during labor indicative of a risk of fetal organ dysfunction.
  • the present invention relates to a device for determining hypoxia.
  • Acute perinatal asphyxia that is, hypoxia (insufficient oxygen saturation of fetal blood) during or close to birth, remains an important cause of neurological damage in form of hypoxic-ischemic encephalopathy (HIE) in newborn infants. It is seen in 2-9/1000 term infants and is followed by cerebral palsy (CP) and death in the severe cases. In a global perspective about 4 million newborn children die each year and about 23% are caused by acute perinatal asphyxia. Due to a lack of resources undeveloped countries are worse off, but as understood by the large numbers also in the western world this is a serious problem.
  • hypoxia insufficient oxygen saturation of fetal blood
  • CP cerebral palsy
  • Detection of acute perinatal asphyxia is presently done by monitoring of fetal heart rate followed by measurement of pH or lactate measurement in fetal scalp blood sampled through the vagina if an ominous fetal heart rate pattern is seen.
  • pH and lactate are indicators of metabolic acidosis caused by a switch to anaerobic metabolism in situation of insufficient oxygen supply. In an oxygen starved fetus pyruvate is metabolised to lactate and energy.
  • pH is the golden standard. The fast determination of pH however requires about 35 ⁇ l of scalp blood, which is not easily obtained. Failure in the first determination is quite common (20 %) as some studies indicate. Lactate is easier to measure since only 5 ⁇ l of blood is needed and the analysis can be carried out at bed side. Lactate analysis can be carried out within one minute and thus is sufficiently quick.
  • Lactate and pH are also indicators of acute asphyxia. As such Lactate and pH can provide an indication of a totally healthy fetus subjected to a sudden acute onset of hypoxia- ischemia during birth. A significant proportion of all infants developing hypoxic ischemic encephalopathy have had episodes of hypoxia-ischemia before entering the delivery phase. They are more vulnerable to hypoxia-ischemia during birth and do not respond in the same way as healthy foetuses and therefore the currently used methods are not sufficient for this group of patients.
  • a method for monitoring childbirth comprising the measurement of lactate in fluids, such as vaginal fluids, is disclosed in WO 2005/034762 Al .
  • lactate as an indicator of acidosis, is as good as pH.
  • lactate nor pH are ideal predictors of moderate/severe HIE: the sensitivity is only 67% for lactate and 50% for pH, whereas the specificity is about the same, 76% for lactate and 73% for pH.
  • the sensitivity and specificity in predicting acidosis in newborns are less than 70 per cent for both lactate and pH.
  • Enzymes known to be elevated in newborn infants subjected to asphyxia during labor are LDH (lactate dehydrogenase), ALT (alanine aminotransferase) and AST (aspartate aminotransferase) also known as liver enzymes.
  • LDH lactate dehydrogenase
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • liver enzymes LDH is found in most of the cells in the body, and it is considered an unspecific enzyme. Therefore it is infrequently used in clinical work.
  • Previously LDH was used as a marker of myocardial damage but has now been replaced by more specific tests.
  • AST and, in particular, ALT are more specific to liver damage.
  • hypoxia is also a serious concern in many other medical conditions.
  • colorectal cancer is one of the most common tumors in both genders, the incidence of which is increasing every year.
  • the current treatment involves a surgical procedure whereby the tumor is removed together with a radical part of the bowel. In the majority of these cases the distal and the proximal ends of the bowel are thereafter put together again. This is referred to as an anastomosis. During this procedure, arterial blood supply to the part of the bowel where the tumor is located is interrupted when the arterial vessels are cut.
  • hypoxia is a major concern
  • Other areas where hypoxia is a major concern include vascular surgery and liver transplant surgery.
  • vascular surgery and liver transplant surgery a major factor determining morbidity and mortality in patients after liver transplantation therapy is preservation injury of the hepatic grafts (Leemaster 1997).
  • LDH, AST and ALT leakage into the perfusate is a measure of loss of the membrane integrity of the liver cells (Kebis 2007).
  • LDH can only be analyzed in plasma or serum. Further, they require a minimum of 150 microliters of whole blood for such a measurement. Such volume of blood is difficult to receive from small animals, specific tissue or the unborn child during birth. Another problem is that LDH is also present in red blood cells and haemolysis (rupture of red blood cells) will lead to false high values (beyond detection limit).
  • the present invention satisfies at least some of the needs by providing a device and method where LDH, and optionally also AST 3 ALT, Mg, and lactate, can be analyzed within minutes (or alternatively in seconds) in 10 microliters of whole blood. These analyses can be measured together with free haemoglobin to make sure that haemolysis is not present with false increased values of LDH as the result.
  • Embodiments of the present invention include a method of determining hypoxia, especially hypoxia in fetal scalp blood sampled during labor.
  • the method can comprise the determination of LDH (lactate dehydrogenase) in plasma. It is preferred to determine, in addition to LDH, one or several markers selected from the group consisting of K, Mg, Ca, AST, ALT 5 and lactate in said fetal scalp blood. Particularly preferred are the combinations of LDH with any of K, Mg, Ca, AST, ALT, lactate. Also preferred is the combination of LDH, lactate, Mg, and AST and/or ALT.
  • LDH lactate dehydrogenase
  • LDH and lactate dehydrogenase refer to total lactate dehydrogenase, that is, not to isoenzymes thereof.
  • scalp blood in limited amount (preferably about 5 — 25 ⁇ L) is sampled from a fetus through the vagina during partum in an environment close to the patient.
  • the sample is subjected to separation of plasma (blood serum) from blood cells, in particular from erythrocytes, and the plasma is analyzed for LDH and, optionally, for one or more of K, Mg, Ca, AST, ALT, lactate, to present a result within minutes.
  • the medical team may directly conclude if the child suffers from acute asphyxia, i.e. indicating if caesarean section is needed or not. Thanks to the invention a large amount of the suffering related to hypoxia may be eliminated and also a large amount of savings may be achieved.
  • AST and ALT presented a sensitivity of 86 % and a specificity of 90 % at a cut off value of 55 U/L and 18 U/L respectively. Despite the fact that this study is preliminary it indicates a surprising improvement compared to existing methods of today.
  • erythrocytes are known to contain about 150 times more LDH than serum. Thus it is important to carry out sampling and separation in a manner that does not damage erythrocytes to prevent their LDH content from passing into the plasma. It is also important to take into account the small volumes of scalp blood available for analysis.
  • a sample of scalp blood is separated into plasma and blood cells by a making the plasma pass through a porous matrix, in this application termed "membrane" on a solid support capable of retaining the blood cells.
  • the plasma is analysed for LDH in a conventional manner.
  • K, Mg, Ca, AST, ALT, and/or lactate can be analyzed in plasma or in blood; if analyzed in blood, the scalp blood sample has to be divided into a portion from which plasma is obtained for the measurement of LDH, and one or more portions for the analysis of the other hypoxia marker(s). It is within the ambit of the invention to use other methods known in the art, such as micro-centeifugation, microfluidic compact disc technology and magnetophoresis, for separating plasma from blood cells in the scalp blood sample of the invention.
  • Patent No. 4,803,159 which is herewith incorporated in this application.
  • a commercial apparatus for use in the last mentioned method is on the market (Vitros® DT60 II Chemistry
  • AST, and lactate in blood are routinely used in clinical chemistry and thus within the easy reach of a person skilled in the art.
  • levels of LDH, K, Mg, Ca, ALT, AST, and lactate are elevated in scalp blood sampled from a fetus through the vagina during partum if the fetus is in a hypoxic state, the standard (baseline) being corresponding levels in a fetus during partum and/or immediately after partum in the context of an unproblematic birth.
  • levels of LDH, ALT and AST are increased by a factor of two, in particular three or more, in a fetus during partum suffering from severe asphyxia, that is, asphyxia which puts the fetus at a higher risk of acquiring HIE.
  • a method of determining hypoxia in a sample of scalp blood during labor comprising providing a point of care device, using at least one, preferably numerous, markers to within minutes determine if acute hypoxia is at hand.
  • the device is additionally in communication with analyzing means for one or several of K, Mg, Ca, AST, ALT, lactate.
  • a plasma separation device for simple and direct use in a device/method of the invention.
  • the present invention includes a method of assessing hypoxia at a tissue site of a mammal.
  • the method can comprise the collecting a blood sample from the tissue site, wherein the blood sample comprises plasma and blood cells followed by separating the plasma from the blood cells. The amount of LDH in the plasma is determined and the presence of hypoxia at the tissue site is assessed from the amount of LDH in the plasma.
  • Methods of assessing hypoxia at a tissue site can include analysis of a blood sample from a mammal's gastrointestinal tract (e.g., a mammal's bowel), analysis of a sample from a specific organ (e.g., a mammal's aorta) or cerebrospinal fluid collected for example by microcatheter, analysis of urine or intraperitoneal fluid, and analysis of a sample from an organ to be transplanted into a mammal in need thereof.
  • Methods of assessing hypoxia can allow one to predict the likelihood of brain injury after prenatal asphyxia and the assessment of blood circulation to a mammal's limbs before, during, and after a medical or surgical procedure.
  • FIG. 1 illustrates a ROC curve for sensitivity and specificity
  • FIG. 2 depicts a block diagram schematically presenting an embodiment of the method of the invention
  • FIGs. 3 - 5 depict sectional views of a first embodiment of a separation device in consecutive stages of use in the method of the invention
  • Fig. 6 presents an embodiment of capillary device according to the invention, intended to be used for collecting and testing scalp blood;
  • Fig.7 presents a quick test analyzing system according to another embodiment of the invention.
  • Fig.8 presents an embodiment of disposable card device for a quick test according to the invention.
  • Fig. 9 presents a diagram of actions taken normally in conjunction with the invention.
  • the present invention provides a device and method where LDH, and optionally also AST, ALT, Mg, and lactate, can be analyzed within minutes (or alternatively in seconds) in 10 microliters of whole blood. These analyses can be measured together with free haemoglobin to make sure that haemolysis is not present with false increased values of LDH as the result
  • the invention comprises a method of determining hypoxia.
  • the method can comprise collecting a blood sample; and determining the total amount of LDH in the blood.
  • the blood sample can be collected from any mammal or alternatively from any organ to be transplanted into a mammal in need thereof.
  • the method of determining hypoxia comprises collecting a blood sample, wherein the blood sample includes plasma and blood cells.
  • the plasma is separated from the blood cells such that the plasma can be analyzed without the presence of blood cells.
  • the isolated plasma can be analyzed for the total amount of LDH in the plasma. As such, a determination of LDH can be achieved. Based on the LDH quantity, or alternatively in conjunction with other prognostic markers, a determination of hypoxia can be readily realized.
  • the determination of hypoxia includes analyzing a blood sample to determine the quantity or amount of multiple prognostic markers.
  • the method comprises determining the amount of LDH in a blood sample, or preferably the plasma thereof, and determining the amount of at least one additional prognostic marker selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactate.
  • the blood sample for analysis is manipulated such that the plasma is separated from the blood cells.
  • this separation can be achieved by use of a semi-permeable membrane or centrifuge.
  • the plasma preferably passes through the membrane and the blood cells are retained on the membrane. Accordingly, the plasma is for all practical purposes isolated from the bulk of the blood cells. The plasma can then be sampled and tested in the absence of blood cells.
  • the sample volume for analysis can be greatly reduced from prior methods.
  • the volume of blood for determining hypoxia comprises from 5 ⁇ L to 60 ⁇ L, or from 5 ⁇ L to 25 ⁇ L, or preferably from about 5 tol5 ⁇ L, and in particular 10 ⁇ L. In certain embodiments, the volume of blood for determining hypoxia comprises from about 5 ⁇ L to 150 ⁇ L, or from 10 ⁇ L to 120 ⁇ L, or from 10 ⁇ L to 100 ⁇ L, or from 10 ⁇ L to 80 ⁇ L.
  • embodiments of the present invention allow for the determination of hypoxia in a wide variety of circumstances.
  • embodiments of the present invention include, but not limited to, the determination of hypoxia in blood from a fetal scalp, a gastrointestinal tract (e.g., colon anastomosis), specific organs (e.g., liver and aorta), cerebrospinal fluid from a lumbar drain, and organs to be transplanted.
  • a plasma separation device 1 according to the invention is shown in Figs. 3 - 5 in consecutive stages of use in the method.
  • the device 1 comprises a generally circular housing in a hydrophobic polymer material such as poly(tetrafluoroethylene).
  • a plasma collection compartment 2 in the housing has a top opening closed by a plasma separation membrane 11 supported by a grid 12 of same or similar material as that of the housing.
  • the compartment bottom 13 slants towards its center in the form of a wide-angled cone.
  • a tubular conduit 15 provided with a valve 16.
  • the conduit 15 extends to a source of negative pressure such as a vacuum pump (not shown).
  • a source of negative pressure such as a vacuum pump (not shown).
  • a 40 ⁇ L sample 7 of scalp blood has been deposited on the exterior face of the membrane 11, the valve 16 being in a closed position.
  • the compartment 14 is put under negative pressure (Fig. 4); thereby plasma T is sucked into the compartment 14 accumulating on its slanting bottom 13, the blood cells 7" being retained by the membrane 11.
  • Fig. 5 illustrates the removal of the plasma sample 7 from the compartment 14 by means of an aspirating syringe, the cannula 10 of which has been inserted through the membrane 11.
  • a separate insertion port (not shown) can be provided such as, for instance, a rubber septum disposed in a separate opening in a top or side wall of the device 1.
  • the block diagram of Fig. 2 illustrates the principles of the method of the invention.
  • the compartment may additionally be provided with a second conduit arranged to communicate with a bottom section thereof provided with a second valve means for controlled emptying of fluid (plasma) accumulated at the bottom of the compartment.
  • the compartment may comprise means for determining LDH and other markers in the accumulated plasma. It is preferred for the membrane to have an area of from 5 mm 2 to 1000 mm 2 , in particular from 20 mm 2 to 300 mm 2 .
  • a system including a disposable card 2 and an analyzing instrument 3 for performing point of care testing, i.e. in an environment nearby the patient, to allow for presenting a test result within 7 minutes, preferably within 2 minutes and more preferred within seconds.
  • the disposable card 2 is preferably arranged with a number of different detection cells 2OA - 2OE, as will be explained more in detail in connection with Fig. 7, but indeed a card merely testing LDH (or e.g. two cells for LDH+AST) may in some applications be sufficient.
  • Fig. 6 two consecutive steps according to the method of the invention are presented.
  • the first step where new card 2 is provided to be supplied with test blood 7 by means of a glass capillary device 4, being filled with whole blood amounting to, e.g. about 10 ⁇ L.
  • the glass capillary device 4 has been inserted into a compartment 21 of the card 2 to interface the blood sample 7 with the card 2 and the disposable card 2 placed into the instrument 3, whereby a directly analysis of the blood sample 7 may be performed as will be explained more in detail in connection with Fig. 7.
  • the card is provided with devices (as known per se, e.g.
  • micro fluidic channels for distribution of plasma/serum to allow for plasma T from the blood sample 7 to enter into at least one of the detection cell 2OA - 2OE, and preferably blood cells in at least one other cell and more preferred hole blood to at least one other cell.
  • a reaction chamber 26A-26E
  • reagents preferably dried
  • reaction will take place prior to the detection.
  • optical devices as known per se, and for example described in US 4935346 (which is hereby incorporated by reference) performing optical measurements. The optical measurement (e.g.
  • the processor 31 of the instrument 3 will directly be handled by the processor 31 of the instrument 3 and shown in display 32 and/or provided as a data output 33, e.g. on a printed paper.
  • the analyzing instrument 3 is preferably provided with bar code reading arrangement 34 enabling reading and processing a bar code 22 with a unique code for each disposable card 2.
  • the analyzing instrument 3 is enclosed within a housing 35 to make it portable. Connections (not shown) are provided to supply the instrument 3 with necessary supplies, e.g. power (if not battery operated).
  • the instrument may be made smaller by using an external processor, e.g. of a laptop making the instrument connectable to laptop, e.g.
  • FIG. 7 there is shown an embodiment of an exemplary disposable card 2 according to the invention.
  • the card 2 is arranged with five detection cells 2OA - 2OE, all of them being optical detection cells.
  • the first detection cell 20A is for ALT.
  • the second detection cell 20B is for AST.
  • the third detection cell 2OC is for LDH total.
  • the fourth and fifth detection cells 2OD, 20E are for lactate and Mg 2+ respectively.
  • the card according to the shown embodiment has a circular flat shaped body 23, having a diameter which makes it easy to handle, e.g. a diameter within the range of 20 - 120 mm, preferably 40 - 100 mm.
  • the material of the body 23 may be chosen from a wide range, for example poly(tetrafluoro)ethylene 3 polyethylene, polypropylene, polystyrene and similar.
  • the card 2 is provided with a chamber 21 adapted to be fitted with a glass capillary device 4 supplying a blood sample 7.
  • a glass capillary device 4 supplying a blood sample 7.
  • the plasma separation device 25 including a membrane and a plasma collection compartment, please compare with the membrane 11 shown in Figs. 3-5).
  • a sample splitter 24 (as indicated by dotted lines) to provide the possibility to supply whole blood to some detection cells, e.g. via printed reagents 26 A, B, to detect ALT and AST respectively .
  • a larger amount of the sample of whole blood may then possibly be supplied to the plasma separation device 25, e.g. to via printed reagents 26 C, D detect LDH total and lactate respectively in the plasma T .
  • Blood cells T ' obtained after separation may be conducted after mixing with a printed reagent 26 D to optical cell 2OE to detect Mg 2+ .
  • Lactate dehydrogenase is increasing during hypoxia and is present in all of the body's cells. LDH present in the blood stream indicates hypoxia severe enough to reduce the blood flow to peripheral organs. This is the onset on leakage of LDH from these cells. By detecting LDH it is not possible to determine which organ that is suffering from hypoxia. If haemolysis has occurred (rupture of red blood cells will also cause an increase in LDH, even if it is not by hypoxia).
  • Haemolysis is divided into two groups: (1) In vitro, which means that the haemolysis occur when taking the sample or at storage in test tubes, and (2) In vivo which means that the red blood cells within the patient have ruptured due to illness. An ongoing haemolysis will then give falsely high levels of LDH, not due to only hypoxia.
  • the half-life (T 1 A) for LDH is dependent on which type of the five isomers of LDH that has leaked out into the blood.
  • LDHl which is mainly present in the heart, brain and red blood cells has T 1 A 12O h while LDH5 mainly appears in the liver and muscles have T 1 A at 10 h.
  • LDH as a marker as well as 178 of 184 healthy patients. This means that no case of hypoxia would be missed and 6 babies would be unnecessarily delivered by caesarean section or by instrumental delivery.
  • High LDH indicates ongoing or a recent episode of hypoxia somewhere in the body or haemolysis.
  • AST is an enzyme that is present in many of the organs in the body but is more organ specific than LDH. AST is mainly present in the liver, muscles and red blood cells. AST is, as well as LDH, sensitive towards haemolysis but not to the same extent.
  • TA for AST is 12-15 h for newborns.
  • AST as a marker as well as 210 of 236 healthy patients. This means that no case of hypoxia would be missed and 26 babies would be unnecessarily delivered by caesarean section or instrumental delivery. [0058] High level of AST indicates ongoing or a recent episode of hypoxia in the liver or muscles or haemolysis.
  • ALT is a specific enzyme for the liver and is very little affected by haemolysis.
  • TA for ALT is 36h.
  • An elevated level of ALT indicates an ongoing or an episode of hypoxia in the liver.
  • the magnesium in the body is to 50 % located in the skeleton and 50 % inside cells. If an acidosis occurs (lowered pH), for example during hypoxia, hydrogen ions will move in to the cells. Mg will at the same time be transported out of the cells into the blood which leads to elevated Mg levels in the blood. For newborns with HIE the Mg levels are lower than for healthy newborns. Mg is more of a marker for acidosis than cell damage.
  • An elevated Mg level is an indication of acidosis which is a symptom for hypoxia.
  • a low Mg level is an indication of an episode of hypoxia that led to brain damage.
  • ALT is also of interest since it theoretically would be able to tell if the baby suffered from hypoxia earlier when still in uterus since ALT will remain in the blood for a long time
  • Elevated levels of LDH and ALT indicate hypoxia that has affected the liver (amongst other organs).
  • the midwife is next to the obstetrician providing a disposable card 2 where the doctor is inserting the capillary tube 4.
  • the card 2 with the blood 7 is then inserted into a detection instrument 3 positioned nearby where levels of LDH, AST 5 ALT and magnesium are analyzed by a spectrophotometer 4.
  • the analyses takes place after the separation of red blood cells 7" on the card 2 and the remaining plasma T reacts with reagents 20 A-D on the card.
  • Within minutes e.g. about two minutes
  • the result shows on a display on the instrument. It shows "normal-normal-normal-normal" which tells the obstetrician that none of the markers LDH, AST, ALT and magnesium are at elevated levels.
  • Anna is continuing the labor by natural means and is later giving birth to a healthy child.
  • LDH alone or LDH in combination with for example lactate, ALT, AST, or magnesium may be enough to judge how the baby is doing, if so the analysis can be colorimetric (change of colour of the reagents).
  • FIG. 8 there is shown an embodiment of alternative testing arrangement, wherein dry chemistry is used to perform the analysis.
  • An advantage with testing device 5 as shown in Fig. 8 is that no power supply at all would be needed.
  • the testing device 5 is arranged with elongated tube formed body/casing 50.
  • Within the elongated body 50 there is a channel 51 opening up into a front chamber 52 positioned adjacent the front end of the housing 50.
  • a pumping device 53 At the other end of the channel 51 there is, near the rear end of the housing 50, there is provided a pumping device 53.
  • the pumping device 53 is in the form of a resilient hollow body arranged with a check valve 54 at the outlet end thereof. The outlet (via the check valve 54) opens up into a disposal compartment 55.
  • a collar device 56 having a centrally positioned hole 57 communicating with the front chamber 52 via a filter 58, that filters out blood cells.
  • a further chamber 59 arranged with dry chemistry means 8.
  • a further chamber 501 creating a buffer and also providing indication regarding volume of blood 7 in the channel 51.
  • the testing device 5 is introduced via the vagina and put onto the blood sample 7, by positioning the collar 56 around the blood drop 7.
  • the pumping mechanism 53 is activated wherein the check valve 54 will open up to let air escape from the hollow inner of the resilient pumping device 53.
  • the pumping device 53 is released a vacuum will be created (thanks to the resiliency), which is communicated to the blood drop 7 via the channel 51, whereby blood will be sucked into the channel 51 via the filter 58. Accordingly blood plasma T will enter into the analyzing chamber 59.
  • the pumping mechanism may be applied a number of times to safeguard sufficient blood for the testing, which may be determined once blood is observed in the buffer chamber 501.
  • the testing device 5 is removed and by observing the colour of the chemical means in the analyzing chamber 59 it may be determined if the child is suffering from hypoxia.
  • the dry chemical means 8 may be used to indicate different measures to be taken depending on which colour is presented. For instance the following colours could be used to indicate which measure is to be taken. If it shows a green colour no measure should be taken. If a red colour is presented the child should be taken out as quickly as possible. If yellow colour is presented a new sample should be taken within less than 20 minutes.
  • testing device 5 would give the same kind of indications as above. It is evident that the use of a capillary tube to collect the blood from the baby's scalp, could also be combined with a slightly modified testing device 5, but preferably also designed such that when the blood is collected a closed system is created within the device 5. This can be achieved in many ways, e.g. by pushing the top with a silicon collar (as shown) against the scalp of the baby or by removing the device 5 and attach a tight sealed cap on the top (not shown).
  • Fig. 9 there is shown a flow chart on when to preferably use the method and devices respectively according to the invention.
  • CTQ should preferably be used as a primary indicator. If the CTG is normal no measure would normally be necessary to undertake. However if the CTG is abnormal either directly a blood sample from the child's scalp should be analysed according to the invention or possibly it may be proceeded by a ST-analysis of the fetal heart.
  • the invention is not limited by what has been described above but may be varied within the scope of the appending claims. For instance it is evident for the skilled person that the definition "scalp blood" also blood samples collected from other parts of the body may at occasions function to obtain the advantages according to the invention.
  • Samples to be used with the invention include whole blood, blood plasma and blood serum.
  • a blood sample is collected from a location of interest prior to a medical procedure and analyzed for prognostic markers.
  • the plasma and blood cells are separated from each other prior to determining the respective amounts of prognostic markers in the plasma (e.g., total amount of LDH in the plasma).
  • a second blood sample can be obtained from the point of interest and analyzed in the same manner as the initial sample. The determination of prognostic markers in each sample can determined and compared to assess the presence of hypoxia.
  • multiple prognostic markers are analyzed. Such embodiments comprise determining total amount of LDH and 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. Accordingly, the respective amounts of each prognostic marker in the first and second samples can be compared to identify a proper location for an anastomosis.
  • the medical procedure comprises anastomosis of the gastrointestinal tract.
  • colorectal cancer is one of the most common tumors in both genders.
  • the current treatment involves a surgical procedure where the tumor is removed together with a radical part of the bowel. In the majority of these cases the distal and the proximal ends of the bowel are thereafter put together again. This is referred to as an anastomosis.
  • certain embodiments comprise a method of determining hypoxia from a blood sample collected from a mammal's gastrointestinal tract (e.g. bowel).
  • certain embodiments comprise a point-of-care (POC) method for a quick (e.g. within minutes or seconds as discussed above) determination of hypoxia-ischemia from a small blood volume.
  • POC point-of-care
  • LDH, AST, magnesium and lactate are markers that increase in blood due to cellular damage and anaerobic metabolism during hypoxia-ischemia.
  • blood from the bowel can be collected using a scalpel (or the like) and a sterile capillary.
  • the capillary can be inserted into an analysis card described above and analyzed.
  • the result can preferably used as a unique patient reference value (e.g., a baseline).
  • a new test can be performed to see if the markers have increased. If so, the area for anastomosis can be relocated closer to the still existing blood supply to minimize the risk for hypoxia-ischemia induced anastomosis insufficiency and leakage.
  • embodiments of the present invention can beneficially comprise collecting blood samples from a specific organ of interest or cerebrospinal fluid of a mammal during a surgical procedure.
  • a specific organ of interest or cerebrospinal fluid of a mammal For instance, the main reasons for surgery in the thoracoabdominal aorta are aneurysms and dissection. The extensive operation required is associated with significant morbidity and mortality. Some of the most common complications are neurological injuries. The reason for the majority of these complications is insufficient oxygen and energy supply due to ischemia. As an example, such operations cause permanent paralysis as a post operative complication in 1-10% of the patients depending on the location for surgery on the aorta.
  • embodiments of the present invention comprise a method, which can be a POC method, for hypoxia-ischemia detection during surgery in blood from specific organs or cerebrospinal fluid (CSF) through a lumbar drain.
  • a sample from a specific organ of interest is obtained and analyzed for LDH as described in detail above.
  • the method for detecting hypoxia includes determining LDH and at least one additional prognostic in the sample selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactate.
  • one of the additional prognostic markers determined includes lactate.
  • a specific organ of interest can include a mammal's aorta.
  • embodiments of the invention can improve the morbidity and mortality rates in patients after transplantation therapy.
  • One of the key factors impacting morbidity and mortality rates in patients after transplantation is related to preservation injury of grafts, such as the hepatic grafts in a liver transplant.
  • LDH, AST and ALT leakage into the perfusate is an indication of loss of the membrane integrity of the liver cells.
  • the method for determining the presence of hypoxia in an organ to be transplanted into a mammal in need thereof can comprise collecting a blood sample and analyzing the sample, as described above, for prognostic markers prior to the transplantation surgery.
  • the sample is analyzed to determing the total amount of LDH and at least one additional prognostic marker in the sample selected from the group consisting essentially of K 5 Mg, Ca, AST 3 ALT and lactate.
  • the organ for transplant comprises a liver.
  • Certain embodiments of the present invention can satisfy a need at clinics for identify patients with a risk of developing the critical condition multi organ dysfunction.
  • ICU intensive care unit
  • a patient can be treated and transferred to the ICU.
  • the introduction of these mobile teams has reduced the incidence of cardiac arrest, sudden death due to cardiac arrest, post operative complications and days at ICU by as much as 50%.
  • devices and methods according to embodiments of the present invention can potentiality identify even more patients in risk of severe illness and therefore save both humanitarian and economical costs.
  • devices and methods according to certain embodiments are well suited for use by staff in the intensive care unit (ICU).
  • the ICU is responsible for the most critically ill patients in the health care system. Independently, if the patient is taken to the ICU because of trauma, cardiac diagnosis, sepsis, haemorrhage or extensive surgery there is always a substantial risk of hypoxia if the circulation and saturation are insufficient. Even if the typical parameters are carefully monitored and abnormalities treated with fluids and drugs, there remains no bedside method capable of providing the ICU staff information about the effects of a treatment in the peripheral tissue.
  • Embodiments of the present invention include a device and a method for determining hypoxia bedside, wherein the results are available within a matter of a few minutes at most. Such embodiments include obtaining a sample for analysis and determination of LDH. In preferred embodiments, the methods include determining the amount of at least one additional prognostic marker in the plasma selected from the group consisting essentially of AST, ALT and lactate.
  • hypoxia insufficient oxygen saturation of fetal blood
  • HJE hypoxic-ischemic encephalopathy
  • the free interval offers a possibility to minimize the delayed cell death by hypothermia treatment (cooling the child's brain to 34.5°C).
  • hypothermia treatment cooling the child's brain to 34.5°C.
  • Certain embodiments of the present invention provide a diagnostic tool for the physician in the clinical workplace and in research around hypothermia treatment in neonates subjected to perinatal asphyxia. Further, embodiments of the present invention include a method for the determination of hypoxia, in which an individual is capable of predicting brain injury after prenatal asphyxia.
  • Such methods include obtaining a sample for analysis of LDH as discussed in detail above.
  • the relative amounts of multiple prognostic markers can be determined.
  • the blood sample is treated such that the plasma is separated from the blood cells and the determination of the levels of the various prognostic markers is based on an analysis of the plasma alone.
  • the method comprises determining the amount of LDH and at least one additional prognostic marker in the plasma selected from the group consisting essentially of AST, ALT and lactate. Most preferably, the amount of ALT is one of the prognostic markers determined.
  • the method includes providing hypothermia treatment to a mammal in need thereof, wherein the determination of LDH (alone or with other prognostic markers) in the plasma indicate delayed cell death in a mammal's brain.
  • embodiments of the present invention can by used to assess the status of a mammal's limbs before, during, and after medical or surgical treatment. For instance, trauma, fractures and vessel occlusions can affect the circulation to peripheral limbs and muscles (e.g compartment syndrome). There exists a significant correlation between oxygen in ischemic muscle and levels of lactate and LDH is observed (Yamamoto 1988) and lactate is elevated in femoral blood in patients with peripheral arterial occlusive disease compared to control values (Rexroth 1988). Devices according to embodiments of the present invention make it possible to use enzyme and lactate levels to diagnose ischemia of a specific limb and also to assess the effects of most treatments.
  • embodiments of the present invention comprise a method for determining hypoxia-ischemic by analyzing a sample from a limb of interest and determining the total amount of LDH in the plasma. Additional prognostic markers can be quantified at the same time as the determination of LDH. This allows an assessment of blood circulation to a mammal's limbs before, during, and after a medical or surgical treatment.

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Abstract

L'invention porte sur un procédé de détermination d'une hypoxie d'un tissu, par exemple dans le foie, l'aorte ou le tractus gastro-intestinal, avant une chirurgie ou une transplantation, ou dans un prélèvement de sang du cuir chevelu du fœtus prélevé au cours du travail. Le procédé comprend le dosage de la lactate déshydrogénase (LDH) totale dans le plasma obtenu à partir de l'échantillon. Le procédé peut comprendre le dosage supplémentaire de K, Mg, Ca, AST, ALT, lactate dans le plasma et/ou le sang. Des valeurs accrues d'un ou plusieurs parmi LDH, Mg, Ca, AST, ALT, lactate sont indicatives d'une hypoxie dans le fœtus. L'invention porte également sur l'utilisation d'un appareil de séparation de plasma dans le procédé.
PCT/SE2009/050426 2008-04-11 2009-04-23 Procédé de détermination d'une hypoxie WO2009126110A1 (fr)

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EP09729478A EP2396419A4 (fr) 2008-04-11 2009-04-23 Procédé de détermination d'une hypoxie
CN200980112713.4A CN102131938A (zh) 2008-04-11 2009-04-23 确定缺氧的方法
BRPI0910870A BRPI0910870A2 (pt) 2008-04-11 2009-04-23 método de determinação de hipoxia
JP2011503943A JP2012524518A (ja) 2008-04-11 2009-04-23 低酸素状態の検出方法

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US12/101,470 US8945824B2 (en) 2006-10-13 2008-04-11 Method of determining hypoxia
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013057604A (ja) * 2011-09-08 2013-03-28 Yamasa Shoyu Co Ltd 肝臓におけるハイポキシアの検出法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023426A2 (fr) * 1999-09-30 2001-04-05 Varian Associates, Inc. Genes humains et proteines relatifs a l'hypoxie et leurs utilisations
US20030232396A1 (en) * 2002-02-22 2003-12-18 Biolife Solutions, Inc. Method and use of protein microarray technology and proteomic analysis to determine efficacy of human and xenographic cell, tissue and organ transplant
WO2008054303A2 (fr) * 2006-10-13 2008-05-08 Mathias Karlsson Procédé de détermination d'une hypoxie dans le sang du cuir chevelu pendant le travail

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023426A2 (fr) * 1999-09-30 2001-04-05 Varian Associates, Inc. Genes humains et proteines relatifs a l'hypoxie et leurs utilisations
US20030232396A1 (en) * 2002-02-22 2003-12-18 Biolife Solutions, Inc. Method and use of protein microarray technology and proteomic analysis to determine efficacy of human and xenographic cell, tissue and organ transplant
WO2008054303A2 (fr) * 2006-10-13 2008-05-08 Mathias Karlsson Procédé de détermination d'une hypoxie dans le sang du cuir chevelu pendant le travail
US20080213744A1 (en) * 2006-10-13 2008-09-04 Mathias Karlsson Method of Determining Hypoxia

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MONGELLI, M ET AL.: "Effect of Labour and Delivery on Plasma Hepatic Enzymes in the Newborn", J. OBSTET. GYNAECOL. RES., vol. 26, no. 1, 2000, pages 61 - 63, XP008108988 *
SANDERS, R. D. ET AL.: "Perinatal neuroprotection", CURR ANAST, vol. 18, 2007, pages 215 - 224, XP022320755 *
SAUGSTAD, OD: "Is lactate a reliable indicator of tissue hypoxia in the neonatal period?", ACTA PAEDIATRICA, vol. 91, 2002, pages 17 - 19, XP008139118 *
See also references of EP2396419A4 *
VAGHOLKAR, K. R.: "Healing of anastomosis in the gastrointestinal tract: Retrospective study of 35 cases", BOMBAY HOSPITAL JOURNAL, vol. 43, no. 2, 2001, pages 269 - 280, XP008139121 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013057604A (ja) * 2011-09-08 2013-03-28 Yamasa Shoyu Co Ltd 肝臓におけるハイポキシアの検出法

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EP2396419A1 (fr) 2011-12-21
RU2010140243A (ru) 2012-05-20

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