WO2016090134A1 - Haptoglobine en tant que biomarqueur pour l'infirmité motrice cérébrale chez les nouveau-nés - Google Patents

Haptoglobine en tant que biomarqueur pour l'infirmité motrice cérébrale chez les nouveau-nés Download PDF

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WO2016090134A1
WO2016090134A1 PCT/US2015/063723 US2015063723W WO2016090134A1 WO 2016090134 A1 WO2016090134 A1 WO 2016090134A1 US 2015063723 W US2015063723 W US 2015063723W WO 2016090134 A1 WO2016090134 A1 WO 2016090134A1
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newborn
level
hprp
sample
biological sample
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Catalin Buhimschi
Irina Buhimschi
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Research Institute At Nationwide Children's Hosptial
Ohio State Innovation Foundation
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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
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • 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
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/805—Haemoglobins; Myoglobins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics

Definitions

  • Prematurity represents a key risk factor for short and long term adverse neonatal outcomes.
  • infection-induced preterm birth represents a unique clinical entity that raises considerably the risks.
  • Acting synergistically with prematurity, intra-amniotic infection, histological chorioamnionitis and Early Onset Neonatal Sepsis (EONS) increase the risk of neonatal intraventricular hemorrhage (IVH), and long term consequences such as neuro-developmental delay, and cerebral palsy (CP).
  • placental histological markers of inflammation/infection The major disadvantage of placental histological markers of inflammation/infection is that first they are becoming available relatively late after birth. For this reason their significance is limited to late counseling and research purposes. Second, placental histologic examination, like any tests that relies on interpretation of visual inspection, has its limitations. A significant heterogeneity in assessment of tissue inflammatory responses to infection and poor to moderate intra- and inter-operator variability have been reported. Redline et al. Pediatr Dev Pathol 6:435-48 (2003). Third, lesions consistent with mild acute chorioamnionitis are also found in uncomplicated pregnancies and more often associated with labor at term.
  • Hp switch-on pattern at birth can serve as a "biomarker” that can improve diagnosis of EONS over that achieved using clinical criteria alone.
  • the obstacle of an absent "gold standard” diagnostic test for EONS was circumvented by using latent class analysis (LCA), a statistical method that has gained increased acceptance for situations when a perfect gold standard diagnostic test does not exist. From all the variables entered in the analysis, the Hp switch pattern had the highest discriminative power while gestational age (GA) at birth, sex, preterm premature rupture of membranes (PPROM) and Apgar scores were excluded.
  • GA gestational age
  • PPROM preterm premature rupture of membranes
  • MFMU Maternal-Fetal Medicine Units
  • CP cerebral palsy
  • Haptoglobin is absent at birth in healthy term newborns. Antenatal exposure to intra-uterine inflammation results in the precocious "switch-on" of Hp with levels dependent on the major fetal phenotypes; Hpl-1, Hpl-2, Hp2-2.
  • the inventors investigated the relationships between fetal Hp switch-on and phenotypes on immediate and late childhood outcomes independently of exposure to magnesium in-utero. The source of data for the analysis was a secondary analysis of a multicenter clinical trial of magnesium sulfate for prevention of cerebral palsy. Rouse et al, N. Eng. J. Med. 359(9), 895-905 (2008).
  • Laboratory analyses and cluster assignments were done by investigators blinded to clinical data. Primary outcome was occurrence of intra- ventricular IVH gr3-4 and/or death.
  • Figures l(A-C) provide images and a graph showing cord blood haptoglobin (Hp) and haptoglobin-related protein (HpRp) immuno-reactivity revealed by Western blot (A&B) and ELISA (C).
  • Hp cord blood haptoglobin
  • HpRp haptoglobin-related protein
  • C ELISA
  • Figures 2A and 2B provide: (A) Clustering flowchart based on probability of "antenatal exposure to infection/inflammation”. Posterior probabilities for inclusion in the "likely exposed” cluster were calculated for all 8 possible combinations of indicators (cord blood Hp and HpRP switch pattern, IL-6, suspected EONS) and their modal characteristics (yes/no). The number of newborns presenting each combination in the Yale cohort is included in parentheses. All newborns with cluster-2 probability levels >50% (black boxes) were considered “likely exposed”. Alternatively, a diagnosis of "likely non-exposed” was assigned to newborns with probability levels ⁇ 50% (grey boxes).
  • Figure 3 provides a scheme representing the results of BEAM50 classification. Based on Hp and HpRP levels, whether the levels are weak or strong, and the level of IL-6, there are 6 possible combinations which can help determine the level of risk for a poor pregnancy outcome. Analysis of the data indicates that cluster ⁇ , in which IL-6 levels are high and Hp and/or HpRP levels are low includes newborns that have the highest risk of developing cerebral palsy
  • Figure 4 provides a set of graphs providing a model of the relationships among the timing of the infectious trigger, cord blood IL-6 and Hp levels which generate the 3 newborns clusters described herein.
  • the present invention provides a method of diagnosing a newborn as having an increased risk of developing cerebral palsy is described.
  • the method includes obtaining a biological sample from the newborn; determining the level of Hp and/or HpRP in the biological sample and comparing it to a control value; determining the level of IL-6 in the biological sample and comparing it to a control value; and diagnosing the newborn as having an increased risk of developing cerebral palsy if the level of Hp and/or HpRP is lower than the control value, and the level of IL-6 is greater than the control value.
  • the present invention also provides a method of diagnosing a newborn as having an increased risk of developing retinopathy of prematurity, that includes obtaining a biological sample from the newborn; determining the level of Hpl-2 and/or Hp2-2 in the biological sample and comparing it to a control value, and diagnosing the newborn as having an increased risk of developing retinopathy of prematurity if the level of Hpl-2 and/or Hp2-2 is higher than the control value.
  • diagnosis can encompass determining the likelihood that a subject will develop a disease, or the existence or nature of disease in a subject.
  • diagnosis also encompasses determining the severity and probable outcome of disease or episode of disease or prospect of recovery, which is generally referred to as prognosis).
  • treatment refers to obtaining a desired pharmacologic or physiologic effect.
  • the effect may be therapeutic in terms of a partial or complete cure for a disease or an adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and can include inhibiting the disease or condition, i.e., arresting its development; and relieving the disease, i.e., causing regression of the disease.
  • therapy encompasses activity carried out to treat a disease.
  • the specific activity carried out to conduct therapy can include use of surgery, radiotherapy, hormonal therapy, chemotherapy, or the use of one or more therapeutic agents (e.g., anticancer agents).
  • therapeutic agents e.g., anticancer agents.
  • pharmaceutically effective are intended to qualify the amount of an agent which will achieve the goal of improvement in disease severity and the frequency of incidence over treatment of each agent by itself, while avoiding adverse side effects typically associated with alternative therapies. The effectiveness of treatment may be measured by evaluating a reduction in symptoms.
  • One aspect of the invention provides a method of diagnosing a newborn as having an increased risk of developing cerebral palsy.
  • the method includes the steps of obtaining a biological sample from the newborn or newborn umbilical cord; determining the level of Hp and/or HpRP in the biological sample and comparing it to a control value; determining the level of IL-6 in the biological sample and comparing it to a control value; and diagnosing the newborn as having an increased risk of developing cerebral palsy if the level of Hp and/or HpRP is lower than the control value, and the level of IL-6 is greater than the control value.
  • Hp is an acute phase glycoprotein with a great variety of important biological functions, of which the most recognized is hemoglobin binding.
  • Hp is a tetrameric protein of two a and two ⁇ chains derived from a single mRNA that encodes a precursor that is cleaved post-translationally to generate the two chains.
  • the liver is the main site of Hp synthesis, although Hp synthesis has been demonstrated many extrahepatic sites such as adipose tissue, lung, ovary, testis, arteries and placenta.
  • Hp is an immunomodulatory protein linked to human susceptibility, as opposed to resistance, to infection.
  • Two allelic variants (Hpl and Hp2) in humans code proteins with different oc-chains.
  • Hp is essentially absent at birth and the adult phenotype (Hp 1-1; Hp 1-2 or Hp2-2) emerges within the first year of life.
  • the HpO-0 phenotype lacks both alleles.
  • HpRP shares greater than 90% homology with Hp.
  • Hp-related (HpRP) gene which also contains elements of retroviral insertion.
  • the transcription of the HpRP gene generates 21 kDa protein, but is silenced in the adult liver through an unknown mechanism.
  • human serum, breast carcinoma, decidua and placenta are four compartments where the HpRP protein product appears expressed.
  • HpRP is 90% identical with Hp, which explains cross-reactivity in Western blots and ELISA assays with most available antibodies. Bueler M R, Bersinger N A., Br J Obstet Gynaecol., 96(7), 867-9. (1989).
  • the levels of other biomarkers for neonatal sepsis can also be determined using the methods described herein.
  • the method also includes determining the level of C-reactive protein (CRP) and comparing it to a control value.
  • CRP C-reactive protein
  • PCT procalcitonin
  • Cerebral palsy is a group of permanent movement disorders that appear in early childhood. While the central feature of CP is a disorder with movement, difficulties with thinking, learning, feeling, communication and behavior often occur along with CP. Cerebral palsy is caused by abnormal development or damage to the parts of the brain that control movement, balance, and posture. CP is classified by the types of motor impairment of the limbs or organs, and by restrictions to the activities an affected person may perform. There are three main CP classifications by motor impairment: spastic, ataxic, and athetoid/dyskinetic CP.
  • Cerebral palsy is characterized by abnormal muscle tone, reflexes, or motor development and coordination. In some cases, there are joint and bone deformities and contractures (permanently fixed, tight muscles and joints). The classical symptoms are spasticity, spasms, other involuntary movements (e.g., facial gestures), unsteady gait, problems with balance, and/or soft tissue findings consisting largely of decreased muscle mass. Scissor walking (where the knees come in and cross) and toe walking (which can contribute to a gait reminiscent of a marionette) are common among people with CP who are able to walk, but taken on the whole, CP symptomatology is very diverse. The effects of cerebral palsy fall on a continuum of motor dysfunction, which may range from slight clumsiness at the mild end of the spectrum to impairments so severe that they render coordinated movement virtually impossible at the other end of the spectrum.
  • Cerebral palsy is typically diagnosed on the child's and mother's history and a physical examination.
  • a general movement assessment which involves measuring movements that occur spontaneously among those less than four months of age, appears to be most accurate.
  • further diagnostic tests can be carried out such as neuroimaging with computed tomography (CT) or magnetic resonance imaging (MRI), with MRI being preferred over CT due to diagnostic yield and safety.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • Another aspect of the invention provides a method of diagnosing a newborn as having an increased risk of developing retinopathy of prematurity.
  • the method includes obtaining a biological sample from the newborn; determining the level of Hp 1-2 and/or Hp2-2 in the biological sample and comparing it to a control value; and diagnosing the newborn as having an increased risk of developing retinopathy of prematurity if the level of Hp 1-2 and/or Hp2-2 is higher than the control value.
  • the present invention also provides a method of diagnosing a newborn as having an increased risk of developing retinopathy of prematurity.
  • Retinopathy of prematurity is a disease of the eye affecting prematurely-born babies generally having received intensive neonatal care, in which oxygen therapy is used on them due to the premature development of their lungs. It is also known as Terry syndrome or retrolental fibroplasias. ROP involves disorganized growth of retinal blood vessels which may result in scarring and retinal detachment, and can lead to blindness in serious cases.
  • Risk factors for ROP include prematurity, high exposure to oxygen, low birth weight, antenatal infection, and cardiac defects. Almost all infants with ROP have a gestational age of 31 weeks or less (regardless of birth weight) or a birth weight of 1250 g (2.76 lbs) or less; these indications are generally used to decide whether a baby should be screened for ROP.
  • the traditional screening procedure involves pupillary dilation using eye drops, after which the retina is examined using a special lighted instrument (an indirect ophthalmoscope). The peripheral portions of the retina are sometimes pushed into view using scleral depression during the examination.
  • the methods involve providing or obtaining a biological sample from the subject, which can be obtained by any known means including needle stick, needle biopsy, swab, and the like.
  • the biological sample is a blood sample, which may be obtained for example by venipuncture.
  • the biological sample can be obtained before birth, e.g., by cordocentesis, or at any time after birth, preferably within the first 24 hours, 48 hours or 72 hours (within 1 day, 2 days or 3 days) after birth.
  • Biomarkers of the invention can be measured in different types of biological samples, preferably biological fluid samples such as blood.
  • biological samples that may be used in methods of the invention, although not intended to be limiting, include blood, such as umbilical cord blood or neonatal blood, decidua (the thick layer of modified mucous membrane that lines the uterus during pregnancy), or placental material.
  • a sample can be prepared to enhance detectability of the biomarkers. For example, a sample from the subject can be fractionated. Any method that enriches for a biomarker of interest can be used.
  • Sample preparations are optional and may not be necessary to enhance detectability of biomarkers depending on the methods of detection used. For example, sample preparation may be unnecessary if an antibody that specifically binds a biomarker is used to detect the presence of the biomarker in a sample. Sample preparation may involve fractionation of a sample and collection of fractions determined to contain the biomarkers. Methods of prefractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis and liquid chromatography.
  • a sample is pre-fractionated by anion exchange chromatography.
  • Anion exchange chromatography allows pre-fractionation of the proteins in a sample roughly according to their charge characteristics.
  • a Q anion-exchange resin can be used, and a sample can be sequentially eluted with eluants having different pHs.
  • Anion exchange chromatography allows separation of biomolecules in a sample that are more negatively charged from other types of biomolecules. Proteins that are eluted with an eluant having a high pH is likely to be weakly negatively charged, and a fraction that is eluted with an eluant having a low pH is likely to be strongly negatively charged.
  • anion exchange chromatography separates proteins according to their binding characteristics.
  • biomolecules in a sample can be separated by high-resolution electrophoresis, e.g., one or two-dimensional gel electrophoresis.
  • a fraction containing a biomarker can be isolated and further analyzed by gas phase ion spectrometry.
  • two-dimensional gel electrophoresis is used to generate two-dimensional array of spots of biomolecules, including one or more biomarkers. See for example Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1997).
  • the two-dimensional gel electrophoresis can be performed using methods known in the art. See for example Deutscher ed., Methods In Enzymology vol. 182.
  • biomolecules in a sample are separated by, e.g., isoelectric focusing, during which biomolecules in a sample are separated in a pH gradient until they reach a spot where their net charge is zero (isoelectric point).
  • This first separation step results in one- dimensional array of biomolecules.
  • the biomolecules in one-dimensional array is further separated using a technique generally distinct from that used in the first separation step.
  • two-dimensional gel electrophoresis can separate chemically different biomolecules in the molecular mass range from 1000-200,000 Da within complex mixtures. The pi range of these gels is about 3-10 (wide range gels).
  • HPLC high performance liquid chromatography
  • HPLC instruments typically consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Biomolecules in a sample are separated by injecting an aliquot of the sample onto the column. Different biomolecules in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. A fraction that corresponds to the molecular weight and/or physical properties of one or more biomarkers can be collected. The fraction can then be analyzed by methods of detection described herein.
  • samples containing biomarkers can be treated with one or more stabilizing agent and the container used for collection of the sample(s) may be pretreated with one or more stabilizing agent prior to measuring the levels of biomarkers.
  • stabilizing agent refers to one or more molecules, such as polypeptides or nucleic acids, that can be used to prevent the degradation of the biomarkers.
  • the stabilizing agent is a protease inhibitor, including any of 4-(2-Aminoethyl) benzenesulphonyl fluoride (AEBSF) and Pefabloc SC, Antipain and Antipain-dihydrochloride, Aprotinin, Benzamidine and Benzamidine hydrochloride, Bestatin, Chymostatin, E-64 (L-trans- epoxysuccinyl-leucylamide-(4-guanido)-butane or N-[N-(L-trans-carboxyoxiran-2-carbonyl)- L-leucyl]-agmatine), Ethylenediaminetetraacetic acid and its sodium salt (EDTA-Na2), Leupeptin, Ethylmaleimide, Pepstatin and Pepstatin A, Phosphoramidon, Sodium azide, Trypsin inhibitor or E-aminocaproic acid.
  • AEBSF 4-(2-Aminoethy
  • a biological sample may be fresh or stored.
  • Biological samples may be or have been stored or banked under suitable tissue storage conditions.
  • the biological sample may be a tissue sample expressly obtained for the assays of this invention or a tissue sample obtained for another purpose which can be subsampled for the assays of this invention.
  • tissue samples are either chilled or frozen shortly after collection if they are being stored to prevent deterioration of the sample.
  • the methods of the present invention are directed towards diagnosing whether a newborn has an increased risk of developing cerebral palsy or retinopathy of prematurity.
  • a newborn having an increased risk is a newborn that is more likely to develop a condition (e.g., cerebral palsy) than an average newborn. Since about 2.1 per 1,000 live births have cerebral palsy, an increased risk for a newborn developing cerebral palsy is a probability higher than 0.21% that the newborn will develop cerebral palsy.
  • the increased risk is from 1% to 5%, from 5% to 10%, from 11% to 25%, from 26% to 50%, from 51% to 75%, from 76% to 90%, or 91% or greater.
  • a newborn i.e., neonate
  • Newborns are defined as human children in the period immediately succeeding birth and continuing through the first 28 days of extrauterine life.
  • the newborn can either have been full term, or have been premature.
  • the newborn is premature.
  • a newborn human can be considered to be premature if it is premature born before the end of the full term of gestation. In some embodiments, the newborn human is premature if it was born 1 week, 2 weeks, 3 weeks, or 4 weeks or more before the end of full term.
  • the newborn can already have been diagnosed as having an increased risk of developing cerebral palsy (CP) or retinopathy of prematurity (ROP) as a result of other factors.
  • the present method is directed towards determining if the risk of developing cerebral palsy or retinopathy of prematurity is heightened relative to what it would be in view of the existing risk factors.
  • a subject at increased risk can be at a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% level of increased risk as compared with a typical subject.
  • cerebral palsy between 40% and 50% of all children who develop cerebral palsy are born prematurely.
  • Multiple-birth infants are also more likely than single-birth infants to have CP. They are also more likely to be born with a low birth weight. In those who are born with a weight between 1 kg and 1.5 kg CP occurs in 6%. Among those born before 28 weeks of gestation it occurs in 11%. Genetic factors are believed to play an important role in prematurity and cerebral palsy generally. While in those who are born between 34 and 37 weeks the risk is 0.4%.
  • risk factors for cerebral palsy include: problems with the placenta, birth defects, low birthweight, breathing meconium into the lungs, a delivery requiring either the use of instruments or an emergency Caesarean section, birth asphyxia, seizures just after birth, respiratory distress syndrome, low blood sugar, genetics, and infections in the baby.
  • About 2% of all CP cases are inherited, with glutamate decarboxylase- 1 being one of the possible enzymes involved.
  • Most inherited cases are autosomal recessive, meaning both parents must be carriers for the disorder in order to have a child with the disorder.
  • the newborn has an increased risk of developing cerebral palsy and/or retinopathy of prematurity as a result of being diagnosed as having been exposed to infection in utero. Infections in the mother, even infections that are not easily detected, may substantially increase the risk of a newborn developing the disorder. O'Shea, T.M., Ment Retard Dev Disabil Res Rev. 8(3): 135-45 (2002). Infections of the fetal membranes known as chorioamnionitis also increases the risk.
  • Levels of biomarkers such as Hp, allelic variants of Hp, HpRP, and IL-6 can be determined using a variety of different methods.
  • the levels of biomarkers are determined using an immunoassay.
  • the levels of the biomarker are determined using a method other than an immunoassay.
  • biomarkers such as Hp, HpRP, and IL-6 can be detected using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF).
  • MALDI-TOF matrix-assisted laser desorption-ionization time-of-flight mass spectrometry
  • the polypeptide of interest may be further purified and/or quantified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).
  • Analytical methods particularly suited to the preparation of pure proteins are immunohistochemistry, ion- exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
  • a particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • Hp and/or HpRP levels can be assessed, for example by western blot, with the appearance of -40 kDa Hp and/or HpRP bands (corresponding to the beta-chain) and optionally accompanied by one or two additional bands that are Hp and/or HpRP specific.
  • Full term newborns have essentially no Hp and normal levels of Hp and/or HpRP in newborns are considered undetectable and below clinical assay limits.
  • a determination of switched Hp could be made when a signal (or assay value) is measured that is above a pre- established cut-off.
  • the cut-off for the signal (or assay value) upon which a determination can be made may vary with method sensitivity depending on the assay.
  • a determination of switched Hp and/or HpRP obtained from visual inspection of a western blot may, under the conditions used, correspond to an immunoreactivity level above 3,370 ng/mL in ELISA immunoreactivity (>3.37 micrograms/mL).
  • minimum detection levels can be around 2 mg/dl of protein and an analyte (e.g. a protein) is considered undetectable if the concentration is below the detection limit.
  • Normal levels of newborns at term are at undetectable levels. In newborns with uncomplicated pregnancy and delivery the switch to adult levels of about 100-150 mg/dL occurs within the first year of life.
  • antibodies are provided that are specific for Hp and/or HpRP or cytokines, such as IL-6 found in fluid samples of a fetus or newborn.
  • Antibodies provided herein include polyclonal and monoclonal antibodies, as well as antibody fragments that contain the relevant antigen binding domain of the antibodies.
  • the term "antibody” as used herein refers to immunoglobulin molecules or other molecules which comprise at least one antigen-binding domain.
  • antibody as used herein is intended to include whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, multi-specific antibodies, single chain antibodies, epitope- binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), disulfide- linked Fvs (sdFv), fragments comprising either a VL or VH domain, and totally synthetic and recombinant antibodies.
  • the antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • type e.g., IgG, IgE, IgM, IgD, IgA, and IgY
  • class e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass of immunoglobulin molecule e.g., immunoglobulin molecule.
  • Monoclonal antibodies may be produced in animals such as mice and rats by immunization.
  • B cells can be isolated from the immunized animal, for example from the spleen.
  • the isolated B cells can be fused, for example with a myeloma cell line, to produce hybridomas, that can be maintained indefinitely in in vitro cultures.
  • hybridomas can be isolated by dilution (single cell cloning) and grown into colonies. Individual colonies can be screened for the production of antibodies of uniform affinity and specificity.
  • Hybridoma cells may be grown in tissue culture and antibodies may be isolated from the culture medium.
  • Hybridoma cells may also be injected into an animal, such as a mouse, to form tumors in vivo (such as peritoneal tumors) that produce antibodies that can be harvested as intraperitoneal fluid (ascites).
  • tumors in vivo such as peritoneal tumors
  • the lytic complement activity of serum may be optionally inactivated, for example by heating.
  • Biomarker polypeptides or effective fragments thereof may be used to generate antibodies.
  • the amount of polypeptides used for immunization will vary based on a number of factors, including the animal which is immunized, the antigenicity of the polypeptide selected, and the site of injection.
  • the polypeptides used as an immunogen may be modified as appropriate or administered in an adjuvant in order to increase the peptide antigenicity.
  • polypeptides, peptides, haptens, and small compounds may be conjugated to a carrier protein to elicit an immune response or may be administered with and adjuvant, e.g. incomplete Freund's adjuvant.
  • Protocols for generating antibodies including preparing immunogens, immunization of animals, and collection of antiserum may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y., 1988) pp. 55-120 and A. M. Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984).
  • antibody fragment as used herein is intended to include any appropriate antibody fragment which comprises an antigen-binding domain that displays antigen binding function.
  • Antibodies can be fragmented using conventional techniques. For example, F(ab') 2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab') 2 fragment can be treated to reduce disulfide bridges to produce Fab 1 fragments. Papain digestion can lead to the formation of Fab fragments.
  • Antibody fragments including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains.
  • An immunoassay can be used to detect and analyze biomarkers in a biological sample.
  • An immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g., a biomarker).
  • An immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to a biomarker from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically reactive with that biomarker and not with other proteins, except for polymorphic variants and alleles of the biomarker. This selection may be achieved by subtracting out antibodies that cross-react with the biomarker molecules from other species.
  • a sample obtained from a subject can be contacted with the antibody that specifically binds the biomarker.
  • the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
  • solid supports include glass or plastic in the form of, e.g., a microliter plate, a stick, a bead, or a microbead.
  • Antibodies can also be attached to a probe substrate or a protein chip.
  • the mixture is washed and the antibody- marker complex formed can be detected.
  • This detection reagent may be, e.g., a second antibody which is labeled with a detectable label.
  • detectable labels include magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads.
  • the biomarker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound biomarker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the biomarker is incubated simultaneously with the mixture.
  • an indirect assay wherein, for example, a second, labeled antibody is used to detect bound biomarker-specific antibody
  • a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the biomarker is incubated simultaneously with the mixture.
  • Methods for measuring the amount or presence of an antibody-marker complex include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a gating coupler waveguide method or interferometry).
  • Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods.
  • Electrochemical methods include voltammetry and amperometry methods.
  • Radio frequency methods include multipolar resonance spectroscopy.
  • EIA enzyme immune assay
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmune assay
  • Western blot assay a Western blot assay
  • slot blot assay a slot blot assay
  • Immunoassays can be used to determine presence or absence of a biomarker in a sample as well as the quantity of a biomarker in a sample.
  • the amount of an antibody-marker complex can be determined by comparing to a standard.
  • a standard can be, e.g., a known compound or another protein known to be present in a sample. It is understood that the test amount of biomarker need not be measured in absolute units, as long as the unit of measurement can be compared to a control.
  • the immunoassay can include SERS sensors.
  • SERS surface-enhanced Raman scattering
  • the immunoassay can be included in a lateral flow device.
  • biosensors alone are applied to whole blood samples, blood samples need to be pre- treated to remove cells prior to detection. Interference of sample matrix generally reduces the sensitivity of electrochemical, colorimetric and visible-light fluorescent biosensors.
  • One of effective solutions is to integrate a biosensor with microfluidic modules to form a microfluidic device. Compared to biosensors alone, a single microfluidic device is capable of handling sample pretreatment, concentration, transport and detection. This results in a highly automated operational workflow, allowing operation by a layperson.
  • microfluidic devices require less volume of sample and reagent; have better portability and capability of multiplexing. Hence microfluidic devices have been widely used for biomarker detection. Yetisen et al., Lab Chip, 13: 2210-2251 (2013).
  • a microfluidic device the flow in microfluidic channels is driven either by an active flow with a pump or by passive control with the capillary force.
  • the capillary force- based microfluidics such as later flow strips is very attractive because these devices are simple and inexpensive.
  • disposable paper-based lateral flow strips utilize low- cost materials and do not require expensive fabrication equipment such as clean-room lithography.
  • Parolo C Merkoci A., Chem. Soc. Rev., 42: 450-457 (2013).
  • a plastic-base lateral flow strip can be used to conduct an immunoassay.
  • the biomarker levels can be determined by mass spectrometry.
  • a preferred mass spectrometric technique for use in the invention is Surface Enhanced Laser Desorption and Ionization (SELDI), as described, for example, in U.S. Pat. No. 5,719,060 and U.S. Pat. No. 6,225,047, in which the surface of a probe that presents the analyte to the energy source plays an active role in desorption/ionization of analyte molecules.
  • SELDI Surface Enhanced Laser Desorption and Ionization
  • probe refers to a device adapted to engage a probe interface and to present an analyte to ionizing energy for ionization and introduction into a gas phase ion spectrometer, such as a mass spectrometer.
  • a probe typically includes a solid substrate, either flexible or rigid, that has a sample-presenting surface, on which an analyte is presented to the source of ionizing energy.
  • SELDI Surface-Enhanced Affinity Capture
  • SEEC Surface-Enhanced Affinity Capture
  • a “chemically selective surface” is one to which is bound either the adsorbent, also called a “binding moiety,”' or “capture reagent,” or a reactive moiety that is capable of binding a capture reagent, e.g., through a reaction forming a covalent or coordinate covalent bond.
  • reactive moiety here denotes a chemical moiety that is capable of binding a capture reagent.
  • Epoxide and carbodiimidizole are useful reactive moieties to covalently bind polypeptide capture reagents such as antibodies or cellular receptors.
  • Nitriloacetic acid and iminodiacetic acid are useful reactive moieties that function as chelating agents to bind metal ions that interact noncovalently with histidine containing peptides.
  • a “reactive surface” is a surface to which a reactive moiety is bound.
  • An “adsorbent” or “capture reagent” can be any material capable of binding a biomarker of the invention. Suitable adsorbents for use in SELDI, according to the invention, are described in U.S. Pat. No. 6,225,047.
  • a method of diagnosing a newborn as having an increased risk of developing cerebral palsy includes comparing the levels of a plurality of biomarkers in a biological sample obtained from a subject to corresponding control values to determine if there is an increased risk for the newborn of developing cerebral palsy.
  • the biomarkers include Hp and/or HpRP, IL-6, and in some embodiments, C-reactive protein (CRP).
  • Allelic subtypes of Hp i.e., Hpl-1, Hpl-2, and Hp2-2
  • the corresponding control values are therefore an Hp and/or HpRP control values, control values for the Hp allelic subtypes, an IL-6 control value, and in some embodiments, a CRP control value.
  • Control values are based upon the level of the risk predictor (e.g., Hp and/or HpRP or IL-6) in comparable samples obtained from a reference cohort.
  • the reference cohort is the general population.
  • the reference cohort can be a select population of human subjects.
  • Control values for particular biomarkers may in some cases already be known to those skilled in the art.
  • the reference cohort is comprised of newborns who went on to become healthy children without cerebral palsy or retinopathy of preumaturity.
  • the IL-6 control value is useful for identifying newborns that were exposed to infection in utero. Accordingly, in some embodiments, the control value for IL-6 is a value of about 100 pg/mL.
  • the control value can take a variety of forms.
  • the control value can be a single cutoff value, such as a median or mean.
  • Control values of risk predictors in biological samples obtained are established by assaying a large sample of individuals in the general population or the select population and using a statistical model such as the predictive value method for selecting a positivity criterion or receiver operator characteristic curve that defines optimum specificity (highest true negative rate) and sensitivity (highest true positive rate) as described in Knapp, R. G., and Miller, M. C. (1992). Clinical Epidemiology and Biostatistics. William and Wilkins, Harual Publishing Co. Malvern, Pa., which is specifically incorporated herein by reference.
  • a "cutoff" value can be determined for each biomarker that is assayed.
  • the method of diagnosis can be used to indicate or guide treatment of cerebral palsy.
  • the method can further comprise the step of providing treatment for the newborn having an increased risk of having or developing cerebral palsy.
  • Suitable treatment can include use of a variety of different methods of treatment. Examples of methods of treatment include administration of therapeutic agents, surgery, and various forms of therapy intended to treat symptoms of cerebral palsy, such as physiotherapy, speech therapy, and massage therapy. Examples of therapeutic agents include Botulinum toxin, to reduce muscle tone, and magnesium sulfate.
  • a variety of methods of treatment are known for retinopathy of prematurity (ROP).
  • ROP retinopathy of prematurity
  • One type of treatment is peripheral retinal ablation, which involves the destruction of the avascular retina using a solid state laser photocoagulation device.
  • Cryotherapy in which regional retinal destruction was done using a probe to freeze the desired areas, can also be used, but is less preferred.
  • Scleral buckling and/or vitrectomy surgery may be considered for severe ROP where the eyes have progressed to retinal detachment.
  • Intravitreal injection of bevacizumab (avastin) has been reported as a supportive measure in aggressive posterior retinopathy of prematurity. Shah et al., Indian journal of ophthalmology 55 (1): 75-76 (2007).
  • kits for diagnosing a newborn as having an increased risk of developing cerebral palsy includes a first antibody or binding fragment thereof specific for Hp and/or HpRP, a second antibody or binding fragment thereof specific for IL-6, reagents for conducting the diagnosis, and a package for holding the antibodies and the reagents.
  • a further aspect of the invention provides a kit for diagnosing a newborn as having an increased risk of developing retinopathy of prematurity.
  • This kit includes a first antibody or binding fragment thereof specific for Hpl-2, a second antibody or binding fragment thereof specific for Hp2-2, reagents for conducting the diagnosis, and a package for holding the antibodies and the reagents.
  • the kit lacks either the first or second antibody.
  • a kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow.
  • the kit can also include other material(s), which may be desirable from a user standpoint, such as a buffers), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay.
  • the kit includes a lateral flow strip which can be used to conduct an immunoassay using the antibodies included in the kit.
  • the reagents include antibodies capable of specifically binding to the compound they are capable of detecting.
  • the kit can include an antibodies specific for Hp, HpRP, and IL-6.
  • multiple concentrations of each antibody can be included in the kit to facilitate the generation of a standard curve to which the signal detected in the test sample can be compared.
  • a standard curve can be generated by preparing dilutions of a single antibody solution provided in the kit.
  • the antibodies comprise surface-enhanced Raman scattering probes.
  • the terms “specific binding” or “specifically binding”, refer to the interaction of an antibody, a protein, or a peptide with a second chemical species, wherein the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.
  • a particular structure e.g., an antigenic determinant or epitope
  • the kit can also include instructions for using the kit to determine the levels of Hp and/or HpRP, Hp allelic subtypes, and the level of IL-6, and Hp and/or HpRP and IL-6 controls.
  • Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
  • the term "instructions" can include the address of an internet site that provides the instructions.
  • Hp is a known inflammation-inducible plasma protein so the finding that EONS associates with elevated Hp and HpRP immunoreactivity would not have appeared exciting unless corroborated with the knowledge that: (a) normal newborns at term should not have detectable levels of circulating Hp; (b) the switch from the physiological neonatal ahaptoglobinemia to the adult serum level of Hp (100-150 mg/dL) occurs within the first year of life.
  • the mechanism responsible for Hp gene silencing in the fetal period and its activation switch after delivery remains unknown and is the subject of our ongoing research.
  • Hp switched-off pattern HpO-0 was characterized by significantly lower ELISA immunoreactivity compared to all other phenotypes (P ⁇ 0.001, Fig. 1C).
  • Hp switch pattern had the highest discriminative power while gestational age at birth, sex, PPROM and Apgar scores were excluded.
  • Hp switch pattern alone was able to drive the cluster assignment (as "likely” or "unlikely exposed to inflammation") for 97% of newborns.
  • Other significant participants to clustering the newborns were cord blood IL-6 and presumed EONS.
  • cord blood IL-6 to Hp switch pattern improved the model by driving the cluster assignment as "likely exposed” for a minority of 5 (3%) newborns whose Hp expression remained switched-off despite high levels of cord blood IL-6 (range -100-1,500 pg/mL).
  • the cluster algorithm was significantly better than the clinical diagnosis of EONS at predicting several neonatal morbidities including IVH. Equally important was that in the context of antenatal exposure to intra-amniotic inflammation (chorioamnionitis), the Hp phenotype (but not gestational age at birth) was an independent predictor of short-term neonatal morbidities and especially of IVH and ROP. Despite earlier gestational age at birth, newborns carrying the Hp2-2 exhibited significantly less incidence of major morbidities compared to those with Hpl-1 phenotype (Fig. 2B). This observation was consistent with the known function of circulating Hp and HpRP as antimicrobial, antioxidant and immunomodulatory proteins. Moreover, our analysis underlined the ability of the Hp-switch pattern to reflect the interaction between the individual genetic makeup and exposure to an environmental antenatal insult consistent with the definition of "exposome”.
  • Example 2 Cord Blood Haptoglobin as Biomarker of Neonatal Exposure to Intrauterine Inflammation
  • Inclusion criteria for the original clinical trial were: singletons or twins, GA 24 - 31 weeks, high risk for spontaneous PTB due to PPROM or advanced cervical dilatation of 4 to 8 cm in the setting of intact membranes, and delivery anticipated within 2 - 24 hours. Exclusion criteria included: birth anticipated within less than 2 hours, cervical dilatation > 8 cm, PPROM ⁇ 22 weeks, congenital fetal anomalies or death, hypertension or preeclampsia, contraindications to magnesium sulfate and receipt of intravenous magnesium sulfate within the previous 12 hours.
  • Surviving infants underwent neurologic evaluation by an annually certified pediatrician or pediatric neurologist at 6, 12, and 24 months of age (corrected for GA). Infants with a normal neurologic examination at 1 year and who could walk 10 steps independently and had a bilateral pincher grasp were considered normal and did not undergo further physical examinations, although the scheduled neurodevelopmental examination was performed.
  • CP The diagnosis of CP was made by certified pediatrician or pediatric neurologist using well established criteria. Bax et al., Dev Med Child Neurol 47 (8) 571-576 (2005). For severity assessment, children diagnosed with CP were further classified by the Gross Motor Function Classification System (GMFCS). Palisano et al., Dev Med Child Neurol 39 (4) 214- 223 (1997). Scores of > 1 were considered mild CP; moderate CP > 2-3; severe CP > 4-5. Neurodevelopmental stages were assessed by a trained psychologist or psychometrist using the Bayley Scales of Infant Development II (BSID- ⁇ ) test. Varner et al., Am J Perinatol. 30(2): 115-22 (2015).
  • BSID- ⁇ Bayley Scales of Infant Development II
  • Components included a Mental Development Index (MDI) and a psychomotor development index (PDI).
  • MDI Mental Development Index
  • PDI psychomotor development index
  • the diagnoses of IVH, ROP, NEC, BPD, patent ductus arteriosus, and neonatal proven sepsis were established based on well recognized clinical criteria. Rouse et al, N Engl J Med. 359(9):895-905 (2008). This secondary analysis was considered exempt by the Ohio State University Office of responsible Research Practices.
  • the primary outcome was defined as death and/or IVH category 3 and 4.
  • CB serum Umbilical cord blood (CB) serum. CB was retrieved by sterile puncture of the umbilical vein after delivery. Samples were centrifuged for 10 min (1,000 g, 4°C), the serum aliquoted and stored at -80°C.
  • Blocking buffer (5% non-fat dry milk) was used for sample dilution: 100, 1,000 or 10,000-fold.
  • Hpl-2 a mixed phenotype Hp standard
  • IL-6 (Pierce-Endogen, Rockford, IL) was quantified to assess the fetal inflammatory status. All samples were tested by laboratory investigators unaware of the clinical outcome.
  • Bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL) was employed to quantify the CB serum total protein.
  • Haptoglobin is a tetrameric protein with two a and two ⁇ -chains linked by disulfide bonds.
  • Hp occurs in two co-dominant allelic forms, Hpl and Hp2, which differ in the length of the a-chain.
  • the human population has 3 major Hp phenotypes (Hpl-1, Hp2-2 and the heterozygous Hpl-2), derived from variations in the a-chain with identical ⁇ -chains. Maeda N, Smithies O., Annu Rev Genet 20: 81-108 (1986). Absence of Hp at protein level denotes HpO-0 phenotype (ahaptoglobinemia).
  • SDS-PAGE gels (10-20%, InVitrogen, Carlsbad, CA) were loaded with equal amounts of CB protein (2 ⁇ g/lane) mixed 1:2 with reducing sample buffer (Bio-Rad, La Jolla, CA) and boiled for 5 min.. After electrophoretic transfer, nitrocellulose membranes (Bio- Rad) were blocked with 5% milk and then incubated overnight at 4°C rabbit anti-Hp polyclonal antibody (1:3,000, Sigma, St Louis, MO).
  • Detection was performed using biotinylated goat anti-rabbit secondary antibody (1:5,000, Jackson Immunoresearch, West Grove, PA) followed by streptavidin-linked horseradish peroxidase, (1:8,000, Amersham Biosystems, Piscataway, NJ), chemiluminescence (ECL-Plus, Amersham) and a timed 3 min. exposure to film (Kodak Biomax).
  • Optical density of the bands of interest was analyzed with Image J software, available online from the research services branch of the National Institutes of Health. The results were expressed as the optical density of each band and as total optical density calculated from the sum of a- and ⁇ -chains.
  • Purified Hp from blood of adults with known phenotypes Hpl-1, Hpl-2 and Hp2-2, Sigma, St. Louis, MO was used as positive control.
  • Hp phenotypes were defined by additional presence of a-chain bands at either ⁇ 9 kDa (a 1 : Hpl-1), -20 kDa (a 2 : Hp2-2) or both (Hpl- 2).
  • LCA Latent-class analysis
  • Laboratory analyses and cluster assignments were done by investigators blinded to clinical data.
  • Odds ratios OR
  • positive likelihood ratio (+LR number of times more likely that a newborn with a positive diagnosis will develop the adverse outcome compared to newborn with a negative diagnosis
  • NNH number of subjects needed to be exposed to a risk factor to cause harm in one subject that otherwise would not have been harmed
  • Table 1 Baseline characteristics by treatment group
  • Hp phenotype did not modify the Hp and IL-6 levels among groups, there was a significant relationship among Hp status and CB IL-6 levels with higher levels of the pro-inflammatory cytokine when Hp was switch-on (P ⁇ 0.001) (Table 2).
  • magnesium CB levels was higher in the treatment group compared to placebo (placebo: 1.56 [1.52 - 1.60] vs. magnesium 2.55 [2.45 - 2.64] mg/dL, P ⁇ 0.001). Data was corrected for twins.
  • Figure 3 shows the relationship between CP and cluster 3 was only identified as part of the new study, as the prior study investigating primarily EONS did not have CP as outcome.
  • Figure 4 shows the relationships among the timing of the infectious trigger, cord blood IL-6 and Hp levels which generate the 3 newborns clusters described in this study.
  • Newborns in Cluster 1 are identified at birth as having switch-off Hp and HpRP (identified phenotypically as haptoglobin 0-0) and low IL-6. These newborns did not have an antenatal encounter with an infectious agent.
  • Newborns in Cluster 2 on the other hand, have had an antenatal encounter with infection yet they were able to mount an adequate inflammatory response which includes IL-6 as well as Hp and HpRP. Based amounted evidence from us and others we expect IL-6 immunoreacitity in cord blood peaks earlier than Hp and HpRP and is transitory.

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Abstract

L'invention concerne un procédé de diagnostic d'un nouveau-né pour détecter un risque accru de développer une infirmité motrice cérébrale. Le procédé consiste à obtenir un échantillon biologique provenant du nouveau-né ; déterminer le niveau de Hp et/ou de HpRP dans l'échantillon biologique et le comparer à une valeur de contrôle ; déterminer le niveau de IL-6 dans l'échantillon biologique et le comparer à une valeur de contrôle ; et diagnostiquer le nouveau-né pour détecter un risque accru de développer une infirmité motrice cérébrale si le niveau de Hp et/ou de HpRP est inférieur à la valeur de contrôle, et si le niveau de IL-6 est supérieur à la valeur de contrôle. L'invention concerne également un procédé de diagnostic d'un nouveau-né pour détecter un risque accru de développer une rétinopathie de prématurité.
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WO2021141698A1 (fr) * 2020-01-07 2021-07-15 Movsas Tammy Utilisation d'indicateurs de régulation du glucose pour l'évaluation du risque et le traitement de troubles neurodéveloppementaux
RU2809909C1 (ru) * 2023-06-15 2023-12-19 Федеральное государственное бюджетное образовательное учреждение высшего образования "Ярославский государственный медицинский университет" Министерства здравоохранения Российской Федерации Способ прогнозирования риска развития детского церебрального паралича у детей, рожденных с экстремально низкой массой тела

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