Method and apparatus for assessing bilirubin toxicity levels in vivo and diagnosing bilirubin neurotoxicity risk

文档序号：1661621 发布日期：2019-12-27 浏览：20次中文

阅读说明：本技术 评估胆红素体内毒性水平以及诊断胆红素神经毒性风险增加的方法和设备 (Method and apparatus for assessing bilirubin toxicity levels in vivo and diagnosing bilirubin neurotoxicity risk ) 是由 C·阿尔福斯于 2019-04-02 设计创作，主要内容包括：在替代实施方案中,提供了使用临床数据确定患有高胆红素血症的新生儿体内胆红素结合是否正常来检测体内胆红素神经毒性水平并确定是否需要治疗以预防胆红素引起的神经损伤(例如脑病)的方法、设备和系统。在替代实施方案中,还提供了包括自动化微流体处理技术(例如区域流体系统)的装置和系统,以获得胆红素结合组。在替代实施方案中,还提供了使用胆红素结合小组来确定是否需要治疗来改善、逆转或预防有需要的个体(例如患有高胆红素血症(黄疸)的新生儿)的由胆红素引起的神经系统损伤(例如脑病)的方法,以及在需要时开始治疗的方法。(In alternative embodiments, methods, devices and systems are provided for detecting levels of bilirubin neurotoxicity and determining whether treatment is needed to prevent bilirubin-induced nerve damage (e.g., encephalopathy) using clinical data to determine whether bilirubin binding is normal in a neonate with hyperbilirubinemia. In alternative embodiments, devices and systems are also provided that include automated microfluidic processing techniques (e.g., regional fluidic systems) to obtain bilirubin-binding moieties. In alternative embodiments, methods of using a bilirubin-binding panel to determine whether a treatment is needed to ameliorate, reverse or prevent bilirubin-induced neurological damage (e.g., encephalopathy) in an individual in need thereof (e.g., a newborn with hyperbilirubinemia (jaundice)) and to initiate treatment when needed are also provided.)

1. A method for quantifying:

the extent (or clinically effective extent) of bilirubin binding of the plasma, serum or blood of the individual, including the determination of the bilirubin binding constant, or the maximum total bilirubin concentration (B)_Tmax) And equilibrium association constant (K)_A) To quantify binding, an

-a subject's risk of bilirubin toxicity, optionally a risk of bilirubin neurotoxicity, optionally a risk of bilirubin-induced neurological dysfunction (BIND), toAnd the extent or necessity of treatment, including determining B_Free，

Wherein the content of the first and second substances,

B_Freeand B_TotalRespectively, the measured concentration of non-albumin bound or free bilirubin and the total bilirubin concentration,

and measuring B before and after the sample is enriched in bilirubin_FreeAnd B_TotalTo obtain B_Total、B_FreeAnd B_{Total_2}、B_{Free_2}To provide a signal having two unknowns (B)_TmaxAnd K_A) The two alternative equations of (a) are,solving to obtain B_TmaxUsing measured B_TotalAnd B_FreeAnd B is obtained by calculation_TmaxTo obtainOr as an alternative, becauseIs a linear equationK_AIs a negative intercept, B_TmaxIs the negative slope divided byAndthe intercept of the relational expression is,

wherein optionally enriching comprises increasing the bilirubin content of the sample from a value between about 5 to 25mg/dL to about the relevant current clinical treatment B of the relevant cohort_TotalOr will beSample B_TotalRelevant current clinical treatment B to increase to approximately relevant cohort_TotalWherein optionally the relevant group comprises gestational age less than (A)<) Crossover transfusion threshold B for 28 week neonates_Total: 14mg/dL (Table 1), or as Pediatrics 2004; 114:297-_Total：25mg/dL，

Optionally, the method includes:

(a) providing or drawing, or having provided a plasma, blood or serum sample from an individual;

(b) measurement B_TotalAnd B_Free，

wherein optionally enriching comprises increasing the bilirubin content of the sample from a value between about 5 and 25mg/dL to a value near the relevant current clinical threshold B for the relevant cohort_TotalOr let sample B_TotalRelevant current clinical threshold B enriched to near relevant cohort_TotalWherein optionally the relevant group comprises gestational age less than (A)<) Crossover transfusion threshold B for 28 week neonates_Total: 14mg/dL (Table 1), either according to the term neonate as prescribed by the American academy of Pediatrics or as Pediatrics 2004; 114:297-_Total: 25 mg/dL; and

(d) measurement of B in bilirubin-enriched plasma_TotalAnd B_Free，

(e) Determination of the maximum Total bilirubin concentration (B)_Tmax) And equilibrium association constant (K)_A)，

Wherein, if B is individual_TmaxAnd K_AAlternative B lower than comparable group_TmaxAnd K_AAverage, arithmetic average or median of (e.g., according to table 2, for gestational age less than: (a)<) Newborn baby of 28 weeks, B_TmaxAnd K_AMedian 22.0mg/dL and 1.16dL/μ g, respectively), the individual had poor bilirubin binding (clinical grade)Invalid), and

wherein, if B is individual_FreeEqual to or greater than the comparable group is achieving current treatment B_Total(optionally treatment B as shown in Table 1_Total(optionally for gestational age less than: (<)28 week neonate 14mg/dL exchange transfusion B_Total) And optionally B of the group_TmaxAnd K_AAverage, arithmetic average or median of (A)_FreeStandard(for gestational age according to Table 2 <<) Alternative 22.0mg/dL of B in 28 week neonates_TmaxMedian and K of 1.16 dL/. mu.g_AMedian, Current exchange transfusion B at 14mg/dL_TotalWhen B is present_FreeStandardIs composed of This indicates that there is a risk of BIND sufficient to justify the need for treatment,

and optionally, the method further comprises if at any B_TotalIndividual B_FreeEqual to or greater than the appropriate comparable neonatal cohort at the current treatment B_Total(optionally a threshold value as shown in Table 1, and optionally for gestational age less than: (<)28 week neonate 14mg/dL exchange transfusion B_Total) And optionally B of the group_TmaxAnd K_AAverage, arithmetic average or median of (A)_FreeStandard(for gestational age according to Table 2 <<) Alternative 22.0mg/dL of B in 28 week neonates_TmaxMedian and K of 1.16 dL/. mu.g_AMedian, Current exchange transfusion B at 14mg/dL_TotalWhen B is present_FreeStandardIs composed ofEvaluation at any B_TotalThe need for treatment of hyperbilirubinemia (optionally jaundice) in a patient,

wherein B is_FreeIs equal to or greater than B_FreeStandardIs shown to be sufficiently largeThe brain of (a) is exposed to bilirubin and has a risk of bilirubin toxicity, optionally a risk of bilirubin neurotoxicity, optionally a risk of bilirubin-induced neurological dysfunction (BIND), indicating a need for treatment of hyperbilirubinemia, optionally jaundice,

and optionally, the method further comprises when patient B is_TotalBelow the current treatment threshold the patient's need for treatment of hyperbilirubinemia (and optionally jaundice) is assessed,

wherein optionally for gestational age according to table 1 less than: (a)<)28 week neonates, forced exchange transfusion B_TotalIs 14mg/dL, wherein, the B of the individual_FreeEqual to or greater than the appropriate comparable neonatal cohort at the current treatment B_Total(optionally treatment B as shown in Table 1_TotalAnd optionally for gestational age less than: (a)<)28 week neonate 14mg/dL exchange transfusion B_Total) When and in alternative B of the group_TmaxAnd K_AIs determined under the mean, arithmetic mean or median of_FreeStandard(for gestational age according to Table 2 <<) Alternative 22.0mg/dL of B in 28 week neonates_TmaxMedian and K of 1.16 dL/. mu.g_AMedian, Current exchange transfusion B at 14mg/dL_TotalWhen B is present_FreeStandardIs composed of Wherein if B is_FreeIs less than B_FreeStandardThen using patient B_TmaxAnd K_ADetermining attainment of B_FreeStandardAnd demonstrates the specificity B in need of treatment_TotalWhen is coming into contact withLess than treatment B_TotalOptionally B_TotalLess gestational age according to table 1 <<) At 14mg/dL in a 28 week newborn, adequate brain exposure to bilirubin and a risk of bilirubin toxicity is indicatedOptionally a bilirubin neurotoxic risk, optionally a bilirubin-induced neurological dysfunction (BIND) risk, is indicative of a need for treatment of hyperbilirubinemia, optionally jaundice.

2. Method for quantifying the degree of plasma-bound bilirubin, comprising determining the maximum total bilirubin concentration (B)_Tmax) And equilibrium association constant (K)_A)，

Wherein the content of the first and second substances,

B_Freeand B_TotalMeasurement of non-albumin bound or free bilirubin and Total bilirubin concentration, respectively, B is measured before and after the sample is enriched for bilirubin_TotalAnd B_FreeTo obtain B_Total、B_FreeAnd B_Total_₂、B_Free_₂To provide a signal having two unknowns (B)_TmaxAnd K_A) Two equations of (a) are solved to obtain B_TmaxAnd K_A：Using measured B_TotalAnd B_FreeAnd B is obtained by calculation_TmaxTo obtain Or as an alternative, becauseIs a linear equationK_AIs a negative intercept, B_TmaxIs the negative slope divided byAndthe intercept of the relational expression is,

and optionally the method by administering to patient B_TmaxAnd K_AB of comparable group_TmaxAnd K_AMaking a comparison to determine whether bilirubin binding is at a normal level, or below a normal level, wherein optionally the patient is a neonate, wherein B_TmaxAnd K_AA patient who is at a lower than normal level is reaching B of the patient_TotalWhen, it indicates that there is more brain exposure to bilirubin and a greater risk of jaundice and bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)), or jaundice and bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)),

and optionally the method further comprises treating the patient B with a pharmaceutical composition comprising_FreeB of group of newborns comparable to the appropriate_FreeStandardMaking a comparative assessment of whether any significant current threshold B is reached or fallen below_TotalIn a patient in need of a treatment for hyperbilirubinemia (optionally jaundice), wherein B is equal to or higher than B_FreeStandardB of (A)_FreeIndicating that there is sufficient exposure of the brain to bilirubin and a greater risk of bilirubin toxicity, optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND), indicating the need for treatment of hyperbilirubinemia, optionally jaundice,

and optionally the method further comprises detecting a change in the current treatment threshold B_TotalB of (A)_TotalAssessing the patient's need for treatment of hyperbilirubinemia (optionally jaundice),

optionally for gestational age less than (A) according to Table 1<)28 week neonates, exchange transfusion B_TotalIs 14mg/dL, wherein, the B of the individual_FreeEqual to or greater than the appropriate comparable neonatal cohort at the current treatment B_Total(optionally treatment B as shown in Table 1_TotalAnd optionally for gestational age less than: (a)<)28 week neonate 14mg/dL exchange transfusion B_Total) And optionally B of the group_TmaxAnd K_AAverage, arithmetic average or median of (A)_FreeStandard(for gestational age according to Table 2 <<) Alternative 22.0mg/dL of B in 28 week neonates_TmaxMedian and K of 1.16 dL/. mu.g_AThe median number is the number of the median,wherein when is equal to or greater than B_FreeStandardB of (A)_FreeBIND Risk indicates a sufficient brain exposure to bilirubin and a risk of bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)), thereby indicating a need for treatment of hyperbilirubinemia (optionally jaundice) and a need for treatment of less than a sufficient brain exposure to bilirubin and a risk of bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)))_FreeStandardB of (A)_FreeWhen it is, it is expressed in specificityThe need to treat hyperbilirubinemia (optionally jaundice) is addressed, where B_TmaxAnd K_AIs an individual B_TmaxAnd K_A。

3. A computer-implemented method comprising the method of claim 1 or claim 2, or for performing the method of claim 1 to determine B_TmaxAnd K_AAnd reach B_FreeStandardWhen B is present_TotalOptionally further comprising: receiving a data element; and storing the data elements.

4. A computer program product for processing data and determining B_TmaxAnd K_AAnd reach B_FreeStandardWhen B is present_TotalSaid computer program product comprising rightsThe computer-implemented method of claim 3.

5. A Graphical User Interface (GUI) computer program product for determining a BBC/KD ratio, comprising the computer-implemented method of claim 3.

6. A computer system comprising a processor and a data storage device, wherein said data storage device has stored thereon: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

7. A non-transitory storage medium comprising program instructions for execution, processing, and/or implementation: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

8. A non-transitory computer readable medium storing a computer program product for inputting data and executing for determining B_Tmax·K_ACalculation of a product comprising the computer-implemented method of claim 3.

9. A non-transitory computer readable storage medium comprising computer readable instructions that, when executed by a processor of a computing device, cause the computing device to perform, process and/or implement: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

10. A computer program product, comprising: a non-transitory computer-readable storage medium; and program instructions residing in the storage medium, which when executed by a computer, operate, process, and/or implement: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

11. A computer program storage device, embodied on a tangible computer readable medium, comprising: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

12. A computer or equivalent electronic system comprising: a memory; and a processor operatively coupled to the memory, the processor adapted to execute program code stored in the memory to run, process and/or implement: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

13. A system, comprising: a memory configured to store values associated with a plurality of data points and/or a plurality of data elements; and a processor adapted to execute the program code stored in the memory to run, process and/or implement: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

14. A computer-implemented system for providing an application for accessing an external data source or an external server process through a connection server, for providing the ability to store values associated with a plurality of data points and/or a plurality of data elements, and for running, processing and/or implementing an application program for: (a) the computer-implemented method of claim 3; (b) the computer program product of claim 4; (c) a Graphical User Interface (GUI) computer program product of claim 5; or (d) combinations of the foregoing.

15. A device capable of quantifying the degree of plasma binding to bilirubin, wherein said device is capable of measuring B before and after a plasma or blood sample is enriched for bilirubin_Free(non-albumin bound or free bilirubin concentration) and B_Total(total bilirubin concentration) and communicating this data to the computer-implemented system of claim 14, the system of claim 13, or the computer or equivalent electronic system of claim 12, which is capable of performing the computer-implemented method of claim 3 to determine or calculate and output B to, for example, a user_TmaxAnd K_AAnd reach B_FreeStandardWhen B is present_Total，

Wherein optionally the computer-implemented system of claim 14, the system of claim 13, or the computer or equivalent electronic system of claim 12 is part of or within the apparatus, or remote (e.g., only directly connected or wirelessly connected) from the apparatus,

wherein the device comprises a component, optionally a robotic chemical component, capable of measuring: total serum bilirubin concentration (B) of a sample (optionally, a plasma or blood sample)_Total) And unbound bilirubin or free bilirubin concentration (B)_Free)，

Wherein optionally said computer-implemented system, said system or said computer or equivalent electronic system is an integral part of the apparatus, or is operatively remotely connected to the apparatus,

wherein optionally the apparatus comprises automated microfluidic processing technology, optionally a regional fluidic system or a robotic regional fluidic analysis system.

16. A method for diagnosing or prognosing (or predicting the likelihood of acquiring) the following:

severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-neurodevelopmental disorders caused by bilirubin, or alternatively neurodevelopmental disorders caused in newborns by toxic levels of bilirubin as a causative agent, optionally including encephalopathy or kernicterus, or sudden neurotoxicity (acute bilirubin encephalopathy), or choreoathetosis-like cerebral palsy,

-defects optionally caused in the neonate by toxic levels of bilirubin as a causative agent, optionally including encephalopathy or kernal jaundice, or sudden neurotoxicity (acute bilirubin encephalopathy), or choreoathetosis-like cerebral palsy,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-the yellowing of teeth caused by bilirubin,

the method comprises the following steps:

quantifying the extent to which the plasma of the subject binds bilirubin comprises:

(a) using the method of claim 1 or claim 2; or

(b) Determination of the maximum Total bilirubin concentration (B)_Tmax) And equilibrium association constant (K)_A)，

Wherein

Wherein the method is performed by comparing the calculated B of the patient_TmaxAnd K_AAnd comparable newborn group B_TmaxAnd K_AComparing to determine whether bilirubin binding is at or below normal levels, and determining the patient's condition by comparing B_FreeIs comparable withNewborn group B_FreeStandardMaking a comparison and at B_FreeStandardOf the patient_TotalIt is determined whether the risk of BIND is increased,

wherein optionally the patient is a neonate,

wherein, is lower than normal B_TmaxAnd K_AB of (A)_TmaxAnd K_AAnd at the arrival of B_FreeStandardSpecific patient B_TotalIndicating that there is more exposure of the brain to bilirubin and greater risk of, or the following:

severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

17. A method for treating, ameliorating, reversing or preventing in a subject in need thereof (optionally a icteric neonate or young child) the following:

severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-the yellowing of teeth caused by bilirubin,

the method comprises the following steps:

(a) use of the method of claim 1, claim 2 or claim 16 or the apparatus of claim 15 by comparing the patient's calculated B_TmaxAnd K_AAnd comparable newborn group B_TmaxAnd K_AMaking a comparison to determine whether bilirubin binding is at or below normal levels, and by calculating B for the patient in an individual in need thereof_FreeAnd comparable newborn group B_FreeStandardMaking a comparison to determine if BIND Risk is increased, wherein B_TmaxAnd K_AB of an individual in need thereof with a product lower than normal_FreeHigher than group B_FreeStandardIndicating that there is more exposure of the brain to bilirubin and greater or the following risks:

severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-bilirubin-induced tooth yellowing; and

(b) if necessary, the individual has a calculated B below the normal value_TmaxAnd K_AOr equal to or greater than B determined in step A_FreeStandardB of (A)_FreeThen the treatment, amelioration, reversal or prevention of:

jaundice (hyperbilirubinemia) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-a neurodevelopmental disorder or neurodevelopmental disorder caused by bilirubin,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

18. An apparatus for:

-treating, ameliorating, reversing or preventing an individual in need thereof for:

severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-the yellowing of teeth caused by bilirubin,

wherein the apparatus comprises the apparatus of claim 15, the computer-implemented system of claim 14, the system of claim 13 or the computer or equivalent electronic system of claim 12,

and the device can pass the patient's calculation B_TmaxAnd K_AAnd comparable newborn group B_TmaxAnd K_AA comparison is made to determine whether bilirubin binding is at or below a normal level,and by B of the patient in an individual in need thereof_FreeAnd comparable newborn group B_FreeStandardA comparison is made to determine if BIND risk is increased, wherein individuals in need thereof have a calculated B below normal_TmaxAnd K_AAnd has a radical of formula B_FreeStandardB in comparison with the normal value_FreeIndicating that there is more exposure of the brain to bilirubin and greater or the following risks:

severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

19. Use of a device for:

-treating, ameliorating, reversing or preventing an individual in need thereof for:

severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-the yellowing of teeth caused by bilirubin,

wherein the apparatus comprises the apparatus of claim 15, the computer-implemented system of claim 14, the system of claim 13 or the computer or equivalent electronic system of claim 12,

and the device can pass the patient's calculation B_TmaxAnd K_AProduct and B of comparable neonatal cohorts_TmaxAnd K_AMaking a comparison to determine whether bilirubin binding is at or below normal levels, and by comparing B to a subject in need thereof_FreeAnd comparable newborn group B_FreeStandardA comparison is made to determine if BIND risk is increased, wherein individuals in need thereof have a calculated B below normal_TmaxAnd K_AProduct and having a value of B higher than normal_FreeIndicating that there is more exposure of the brain to bilirubin and greater or the following risks:

jaundice (hyperbilirubinemia) or bilirubin toxicity, optionally bilirubin neurotoxicity,

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

Technical Field

The present invention relates generally to medical, medical diagnostic and medical devices. In alternative embodiments, methods, devices and systems are provided for assessing and treating bilirubin toxicity levels in vivo and diagnosing the relative risk of developing or having bilirubin encephalopathy by processing clinical data to accurately determine whether bilirubin binding is normal in a patient. In alternative embodiments, methods, devices and systems are provided for detecting levels of bilirubin neurotoxicity and determining whether treatment is needed to prevent bilirubin-induced nerve damage (e.g., encephalopathy) using clinical data to determine whether bilirubin binding is normal in a neonate with hyperbilirubinemia. Also provided is a computer-implemented method for converting clinical laboratory data into a bilirubin-binding group (including conventional serum or plasma total bilirubin concentrations (B)_Total) And in two B_TotalThe obtained serum or plasma has unbound bilirubin or free bilirubin concentration (B)_Free) Measured value) to calculate a new clinically relevant maximum B_TotalAnd capacity constant (B)_Tmax) And the equilibrium association constant (K) corresponding thereto_A) So as to be at any B_Total<B_TmaxWhile usingAccurate acquisition of B_Free. In alternative embodiments, apparatus and systems are also provided, including automated microfluidic processing (such as zone fluidics) to perform operations on two B' s_TotalMeasuring serum or plasmaB in the sample_TotalAnd B_FreeTo obtain a bilirubin-binding group, while the device and system further comprise the computer-implemented method provided herein for analyzing such data and outputting B for the bilirubin-binding group_TmaxAnd K_ATo determine B in the relevant group of neonates_FreeWhether or not criterion B is met or exceeded_Free(B_FreeStandard) If not, useCalculate B that will happen_Total。B_Free≥B_FreeStandardOrIndicating that the relative risk of bilirubin-induced neurological damage is increased in newborns with hyperbilirubinemia. In alternative embodiments, methods for treating, ameliorating, reversing, or preventing a bilirubin-associated disorder are also provided, as are methods of using the methods provided herein, including using a bilirubin-binding group to determine whether treatment is needed to ameliorate, reverse, or prevent bilirubin-induced neurological damage (e.g., encephalopathy) in a subject in need thereof, such as a neonate with hyperbilirubinemia (jaundice).

Background

In the first two weeks of birth, approximately 60% of newborns develop visible jaundice. Jaundice is the result of the normal transient accumulation of yellow unbound bilirubin IX-alpha (hereinafter referred to as bilirubin), a product of hemoglobin catabolism. Bilirubin accumulation is due to increased bilirubin production due to the shorter life span of fetal red blood cells compared to adult red blood cells and to delayed bilirubin excretion due to the immature metabolic pathways for bilirubin clearance during the first few days of neonatal birth. Thus, in the first few days after birth of a newborn, a brief rise in blood bilirubin levels (known as hyperbilirubinemia), often accompanied by visible jaundice, is generally normal and harmless. However, bilirubin is neurotoxic and in some cases can cause severe nerve damage and thus death or serious sequelae, and clinicians will closely monitor newborns with hyperbilirubinemia.

Neurotoxic levels of bilirubin can cause a range of severe neurological damage, such as acute bilirubin encephalopathy, which can lead to death, from kernal jaundice (specific brain nuclei have yellow staining) found at necropsy; or a series of chronic neurological sequelae (also known as kernicterus) including choreoathetosis-like cerebral palsy, treble hearing loss, upper gaze paralysis, and yellowing of teeth. In addition, a recent focus has been on the neurotoxicity of bilirubin to cause other neurological disorders, including disorders of the auditory neuropathy spectrum, apnea in premature infants and possible autism. Nerve damage in this range is collectively referred to as bilirubin-induced neurological dysfunction (BIND).

BIND can be prevented or improved by increasing the excretion of bilirubin in the body using phototherapy or more risky invasive therapy known as blood exchange transfusion, during which treatment the blood of a newborn with a high bilirubin level is slowly removed and replaced with a compatible blood supply with a low bilirubin level. For newborns less than 35weeks, the clinician currently uses the serum or plasma total bilirubin concentrations (B) shown in Table 1 below_Total) (see, e.g., Maisels MJ, et al, an approach to the management of hyperbilirubinemia in the predetermined influence loss of a 35weeks of gelatin of gelatin.J. Peramino 2012; 32:660).

TABLE 1

Treatment B used in Table 1_TotalIn the treatment of (relative to the use of monotherapy B)_TotalIs based on clinical experience and expert opinion, not on evidence, and introduces considerable uncertainty as to when treatment is required, as shown in figure 1, e.g., less than (at gestational age)<) In 28 week neonates, in B_TotalExchange of blood was considered necessary at 11mg/dL, but not mandatory until B_TotalIt was forced to 14 mg/dL. ThenHow the clinician determines that gestational age is less than 28 weeks and B_TotalThe uncertainty of whether a 12mg/dL neonate needs a revascularization? neonate with gestational age greater than or equal to (≧)35 weeks is even greater, with no mandatory B for phototherapy or revascularization_TotalOnly when B is present_TotalUp to 25mg/dL, the exchange of blood is "considered" (see American Academy of Pediatrics, Management of hyperbilirubinemia in the new born factor 35or more roads of age of Pediatrics 2004; 114: 297. sup. 316). These uncertainties lead to over-treatment, thereby incurring significant social and economic costs, and this approach also does not eliminate BIND.

Using treatment B as for example in Table 1_TotalIs because of B_TotalThe correlation with BIND is poor (see, for example, Watchko JF et al, the origin of low bilirubin kernicters in preliminary fates: low dos it sticu and is it predicted? Semin period 2014; 38: 397) 406 and Ip et al, an evaluation-based review of transfusion isolated synergistic depletion neurological depletion 2004. Pediatrics 2004; 114: e 130). since there is no risk (including death) of phototherapy or exchange transfusion, newborns may develop BIND or complications due to unnecessary treatment.

As shown in FIG. 2, measurement of plasma bilirubin binding is important because there is only non-albumin bound or free plasma bilirubin (B)_Free) Can cross the capillaries and the blood-brain barrier into the tissues of the brain. As shown in fig. 3, at any B_TotalB below_FreeThe higher the corresponding tissue level of bilirubin, the more exposed the brain is to bilirubin and therefore the risk of BIND. Bilirubin binding varies greatly in the plasma of a newborn, and a newborn with poor bilirubin binding is at any B as compared to a newborn with normal binding levels_TotalWith a relatively higher B_FreeAnd tissue bilirubin levels because a given B is achieved when there is poor bilirubin as compared to a similar neonate with normal bilirubin binding_TotalMore bilirubin is accumulated and is in the B_TotalBilirubin of tissueThe levels will be higher, increasing exposure of the brain to bilirubin and the risk of BIND (see figures 2 and 3).

Recent studies have shown that it is possible to obtain a peptide with a similar B_TotalB of hyperbilirubinemia neonates_FreeTo predict BIND (see FIG. 3, and for example, Amin SB, et al, pharmaceutical audio therapy in membrane prediction mechanisms with signaling hyperbilirubinemia. Pediatrics 2017; 140: e20164009), demonstrates that bilirubin binding assessments can be increased in routine assessments of these neonates. In addition, bilirubin binding is routinely detected in Japan and is reported to be clinically very helpful (see, e.g., Morioka I et al. serum unbound bilirubin as a predictor for clinical key invasion low height antigens a late in the new endogenous carbon unit. BBrain Dev 2015; 37: 753).

B_TotalAnd B_FreeIs often mistakenly regarded as an independent alternative to guide clinical care and misunderstands B_FreeThe therapeutic criteria will replace the current B to some extent_TotalTreatment criteria, such as table 1. But B_TotalAnd B_FreeAre not independent of each other but rather interdependent measurements that are chemically closely related to the plasma bilirubin binding site (e.g. albumin) by the law of mass action. The risk of BIND depends on the cumulative amount of bilirubin and the way bilirubin is distributed between the blood and tissue, which depends on B_Free(FIG. 2), in turn, as described in detail below, B_FreeIs B_TotalAnd the concentration of plasma bilirubin binding sites (e.g., albumin) and the inherent binding capacity. A possible way to incorporate bilirubin binding into clinical care is to quantify bilirubin binding in a manner that enables identification of newborns with bilirubin binding below average or with poor bilirubin binding and adjust the current B accordingly_TotalTreatment guidelines. This reduces the use of B alone for personalized care_TotalTo determine the uncertainty inherent in BIND risk (e.g., fig. 1).

Quantification of plasma bilirubin binding requires determination of (1) a likely bindingMaximum amount of bilirubin combined (B)_Tmax) And (2) the tightness of binding, which is usually quantified using equilibrium association or dissociation constants. B is_TmaxDepending on the concentration of functional bilirubin binding sites, it is generally referred to as the bilirubin binding capacity or B when the binding sites are "saturated" with bilirubin_Total(for example, if the concentration of binding sites is 453. mu. mol/L, B_Tmax26.5mg/dL 453 μmol/L). The closeness of bilirubin binding at the binding site is determined by the binding constant (e.g., equilibrium association constant K)_n) Where n is the number of sites with different intrinsic bilirubin binding capacity, and represents the constant K for each site₁，K₂，……，K_n. Chemical equilibrium is

Wherein, B_Total–B_FreeIs the bilirubin concentration bound by the binding site, B_Tmax–(B_Total–B_Free) Is the concentration of unoccupied (available) bilirubin binding sites. Albumin is known to have at least two bilirubin binding sites, and bilirubin binding is quantified using standard methods to obtain B_TmaxAnd corresponding equilibrium constants need to be at multiple B_TotalMeasure B_Free(see Jacobsen J. binding of bilirubin to human serum albumin-Determination of the association constants FEBS Lett 1969; 5: 112-. Clinical laboratories are unable to routinely quantify bilirubin binding using standard methods due to the large amount of testing time, large numbers of samples, and complexity of data analysis.

Disclosure of Invention

In alternative embodiments, methods, devices and systems are provided for assessing bilirubin toxicity levels in vivo and diagnosing the relative risk of developing bilirubin-associated disorders, particularly newborns with hyperbilirubinemia (jaundice), such as neuropathy, e.g., encephalopathy or bilirubin-induced neurological dysfunction (BIND), including encephalopathy, deafness, or choreoathetosis-like cerebral palsy.

In an alternative embodiment, the methods, devices, and systems provided herein include processing and analyzing clinical data to pass the bilirubin binding group (BBP) of the combination test: measurement of B before and after enrichment of bilirubin in the sample_TotalAnd B_FreeAnd determining the current B using an instrumental computer algorithm_TotalTreatment under the guidelines B_TmaxAnd K_ATo accurately determine whether plasma bilirubin binding is normal in the patient and to assess the relative risk of BIND. These data are provided at present B_TotalTwo important new assessment values for evaluating BIND Risk, and the Standard Risk B of the related group_Free(B_FreeStandard) B for comparison_FreeAnd occurrence of B_FreeStandardB is reached_Total，

If B is present_Free＝B_FreeStandardOr

In icteric newborns with severe hyperbilirubinemia, the relative risk of BIND is increased (in other embodiments, the term "severe hyperbilirubinemia" is hyperbilirubinemia that requires treatment to maintain the health of an individual (e.g., a patient such as a newborn), or treatment to reduce hyperbilirubinemia to improve the health of an individual and/or to prevent further negative effects on the health of an individual due to hyperbilirubinemia, or to alleviate symptoms of hyperbilirubinemia).

In alternative embodiments, methods (steps), devices and systems are provided for quantifying the extent of plasma-bound bilirubin, including determining a maximum total bilirubin concentration (B)_Tmax) And the equilibrium association constant (K) associated therewith_A)，

Wherein the content of the first and second substances,

and B_FreeAnd B_TotalMeasured unbound bilirubin plasma concentration or free bilirubin plasma concentration and total bilirubin plasma concentration respectively,

and optionally, the method by first obtaining B of the patient using a novel method_TmaxAnd K_ATo determine if there is an increased risk of bilirubin neurotoxicity (optionally BIND), to obtain patient B_TmaxAnd K_AThe method comprises the following steps: by measuring plasma samples before bilirubin enrichment (B)_Total，B_Free) And thereafter (B)_{Total_2}，B_{Free_2}) B of (A)_TotalAnd B_FreeTwo unknowns (B) are obtained_TmaxAnd K_A) Two sets of equations of, B_TmaxThe solution can be found as follows:

then calculating B_Tmax、B_TotalAnd B_FreeSubstitution intoTo obtain Or as an alternative, becauseB_FreeIs a linear equationK_AIs a negative intercept, B_TmaxIs the negative slope divided byAndintercept of the relation, then_FreeTreatment with comparable group B_TotalAnd optionally B_TmaxMedian sum K_AB reached at the median_FreeStandardMaking comparisons, e.g.As shown in fig. 4, and determines that B is reached_FreeStandardPatient B at the time of day_TotalI.e. byIf B is present_Free＝B_FreeStandardOrIndicates the presence of BIND risk, whether B_TotalTo do so (see, e.g., Table 2, showing gestational age less than: (A)<) Bilirubin binding in 31 neonates at 28 weeks (see Ahlfors CE, et al, Bilirubin binding capacity and Bilirubin binding in neonatal plasma E-PAS 20172017: 2718.2715),

TABLE 2

Wherein B is_TmaxMedian 22.0mg/dL, K_AA median of 16dL/μ g, wherein optionally the patient is a neonate, wherein as shown in figure 4, for gestational age less than (dL/μ g)<) Neonate in 28 weeks achieving mandatory phototherapy B_Total6mg/dL (Table 1) and optionally B_TmaxMedian sum K_AB in the median_FreeStandardWill be

As shown in fig. 4, for gestational age less than: (a)<) Neonate of 28 weeks achieving forced intercourse transfusion B_TotalB at 14mg/dL (Table 1)_FreeStandardWill beHaving the 25 th percentile B_Tmax(14.3mg/dL) and K_ANeonates in the group of (0.75 dL/ug) (i.e., poorly bound bilirubin) will be in each caseAndachieve corresponding phototherapy B_FreeStandard0.32. mu.g/dL and exchange transfusion B_FreeStandard 1.51μg/dL，Far below theCurrent phototherapy B in Table 1_TotalThreshold 5mg/dL and crossover transfusion threshold 11 mg/dL. On the other hand, has a 75 th percentile B_Tmax(24.8mg/dL) and K_ANeonates in the group of (2.20 dL/ug) (i.e., superior bilirubin binding) will be separately in phototherapyAnd exchange transfusionAchieve corresponding phototherapy B_FreeStandard0.32. mu.g/dL and exchange transfusion B_FreeStandard 1.51μg/dL，Is far higher thanCurrent phototherapy B in Table 1_TotalThreshold 5mg/dL and exchange transfusion B_TotalThe threshold is 11 mg/dL. B is_TotalThe BIND risk will be significantly different for two newborns who are identical but have significantly different bilirubin binding capacities and this difference can only be detected by measuring bilirubin binding. Having the 25 th percentile B_Tmax(14.3mg/dL) and K_A(0.75 dL/. mu.g) (i.e.bilirubin binding failure) with a gestational age of less than (<)28 week newborn infant at B_TotalB at 7.6mg/dL had reached for exchange transfusion (1.51. mu.g/dL)_FreeStandardBut without measuring bilirubin formationIf appropriate, follow current B_TotalAn unsuspecting clinician in the treatment guidelines (table 1) will only perform phototherapy and will not consider exchanging transfusions.

In alternative embodiments, the methods provided herein further comprise administering to the subject a_FreeAnd B in a comparable group (e.g., wherein optionally the comparable group is the same gestational age group as shown in Table 1)_FreeStandardA comparison was made to assess the attainment of any B_TotalIn patients in need of treatment for hyperbilirubinemia (whether or not current clinical practice is deemed to be a consideration for treatment), wherein, when patient B is in need of treatment_FreeEqual to or greater than that shown in FIG. 4 Or B_TotalIs equal to or greater than(e.g., for gestational age less than: (A)<) A 28 week newborn achieves forced intercourse transfusion B_Total14mg/dL (Table 1) and optionally B_TmaxMedian (22.0mg/dL) and K_AB at median (1.16 dL/. mu.g) (Table 2)_FreeStandardIs composed of ) Indicating that mandatory treatment B was achieved_Total(e.g., for gestational age less than: (A)<) Newborn at 28 weeks, reached the 25 th percentile B in Table 2_Tmax(14.3mg/dL) and K_A(0.75 dL/. mu.g) and forced crossover transfusion B in Table 1_Total14mg/dL of B_FreeIs composed of Over B_FreeStandard40 times 1.51 ug/dL) and up to any B_TotalWith greater risk of BIND, as shown in fig. 3 and 4, B_FreeAnd BIND Risk with B_TmaxAnd K_AIs decreased and increased, and B_TotalIndependently, i.e. in any B_TotalWith B_TmaxAnd K_AReduced, there is more risk of brain exposure to bilirubin and increased hyperbilirubinemia and bilirubin toxicity (optionally bilirubin neurotoxicity, optionally BIND) or hyperbilirubinemia and bilirubin toxicity (optionally bilirubin neurotoxicity, optionally BIND).

In an alternative embodiment, there is provided a method of quantifying the extent (or clinically effective extent) of bilirubin binding in the plasma, serum or blood of an individual comprising comparing to a comparable group of optionally B_TmaxAnd K_AMedian comparison to determine maximum total bilirubin concentration (B)_Tmax) And the equilibrium association constant (K) corresponding thereto_A). If R is in the process of achieving mandatory treatment B_TotalAnd optionally B of said group_TmaxAnd K_AB obtained at the median_FreeStandardOf patients in whom R occurs Wherein B is_TmaxAnd K_AIs B of an individual_TmaxAnd K_A，

Wherein the content of the first and second substances,

and B_TotalRespectively a non-albumin bound or free bilirubin concentration and a total bilirubin concentration,

and B is measured before and after the sample is enriched in bilirubin_TotalAnd B_FreeProviding B_{Total_1}、B_{Free_1}And B_{Total_2}、B_{Free_2}To obtain two unknowns (B)_TmaxAnd K_A) Equation (A) of_TmaxThe solution can be as follows:

then calculating B_Tmax、B_TotalAnd B_FreeSubstitution intoTo obtainOr as an alternative, becauseIs a linear equationK_AIs a negative intercept, B_TmaxIs the negative slope divided byRatio ofThe intercept of the relational expression is,

wherein the optional enrichment comprises increasing the amount of bilirubin in the sample to near that required for mandatory treatment B_TotalFor gestational age less than (A)<) Up to about 20mg/dL for newborns at 35weeks (table 1) and about 30mg/dL for newborns older than 35weeks or more, see, e.g., wickremaininghe AC, et al, skin of sensorineural intake and bilirubin exchange transfusion threshold. 136:505-512,

optionally, the method includes:

(a) providing or withdrawing a plasma, blood or serum sample from the individual;

(b) measuring B of a sample_TotalAnd B_FreeAnd an

wherein the optional enrichment comprises increasing the amount of bilirubin in the sample to a concentration near that which necessitates forced crossover transfusion, e.g., to about 20mg/dL for neonates with gestational age less than (<)35 weeks (Table 1), to about 30mg/dL for neonates with gestational age greater than or equal to 35weeks,

(d) measurement of B in samples after bilirubin enrichment_TotalAnd B_FreeAnd an

(e) Determination of the maximum Total bilirubin concentration (B)_Tmax) And corresponding equilibrium association constant (K)_A)，

Wherein, regardless of B of the individual_TotalTo what is, if the individual's B_FreeGreater than to current treatment B_Total(optional mandatory treatment B as shown in Table 1_TotalAnd for gestational age of less than: (<) Newborn at 28 weeks, forced B optional exchange transfusion_Total14mg/dL) and optionally B for comparable groups_TmaxAnd K_AMedian (optionally B)_TmaxMedian 22.0mg/dL, K_AA median of 1.16. mu.g/dL, and optionally

See fig. 4) B_FreeStandardThis indicates that plasma or serum is not clinically effective in binding or retaining bilirubin, and that hyperbilirubinemia (jaundice) is in need of treatment,

and optionally, the method further comprises quantifying bilirubin binding and assigning individual B_TmaxAnd K_AB of an optionally suitable group of comparable neonates_TmaxAnd K_AMedian (for gestational age less than: (as shown in table 2)<)28 week neonate, optionally B_TmaxMedian 22.0mg/dL, K_AMedian 1.16 μ g/dL) was compared to assess reaching any B_TotalIn a subject in need of treatment for hyperbilirubinemia, wherein B of the patient_TmaxAnd K_ALower than B_TmaxAnd K_AMedian is expressed in any B_TotalThere may be more brain exposure to bilirubin and have a lower than current treatment B_Total(e.g., Table 1) B_TotalThere may be a higher risk of bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)), while indicating the potential need for treatment of hyperbilirubinemia, including jaundice,

and optionally, the method further comprises assessing specificityIn a patient in need of treatment of hyperbilirubinemia (including jaundice), which is a drug for treating hyperbilirubinemia_TotalFor patients with B_TmaxAnd K_AIs specific, wherein B_TotalPossibly with current treatment B_Total(e.g., Table 1) are different, and B_FreeStandardIs to achieve current treatment B_TotalTo the same age of the patient (optionally the cohort) to reach optionally B_TmaxAnd K_AAt a median value (optionally, if the patient is of gestational age less than: (a)<) For 28 weeks, forced intercourse transfusion B, as shown in Table 1_Total14mg/dL and optionally B with 22.0mg/dL_TmaxMedian and K of 1.16 dL/. mu.g_AA median of then) (ii) a Reach an individualBIND-related risks ofThe group achieves treatment B_Total(Table 1) and optionally B_TmaxAnd K_AThe same occurs in the median and there is a sufficient risk of brain exposure to bilirubin and bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)) to justify the need for treatment of hyperbilirubinemia or jaundice.

In an alternative embodiment, a method for quantifying the extent to which plasma, serum or blood binds bilirubin is provided, comprising determining the maximum total bilirubin concentration (B)_Tmax) And equilibrium association constant (K)_A)，

Wherein the content of the first and second substances,

B_Freeand B_TotalRespectively a non-albumin bound or free bilirubin concentration and a total bilirubin concentration,

and optionally, the method is performed by administering to patient B_TmaxAnd K_ABilirubin binding (i.e., B) is determined by comparison to an average or median value of optionally comparable individuals_TmaxAnd K_A) Whether or not it is a normal level (e.g., B)_TmaxAnd K_AEqual to or higher than the median or average of comparable individuals), or lower than normal, wherein optionally the patient is a neonate, wherein patient B_TmaxAnd K_ALower than normal B_TmaxAnd K_AIndicated at a lower than current treatment B_Total(e.g., Table 1) with greater brain exposure to bilirubin and greater risk of hyperbilirubinemia and bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)), or with hyperbilirubinemia and bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)),

and optionally the method further comprises the step of removing B_FreeB of comparable individuals_FreeStandardMaking a comparison to assess the need for hyperbilirubinemia (or jaundice) treatment, optionally forIndividuals may be compared for determination at treatment B_TotalAnd B_TmaxAnd K_AB in the median_FreeStandardWherein the patient is a neonate, wherein B_FreeGreater than or equal to B_FreeStandardIndicating a sufficient exposure of the brain to bilirubin and a risk of bilirubin toxicity, optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND), evidences a need for treatment of hyperbilirubinemia or jaundice,

and optionally, the method further comprises determining a specificity B_TotalTo evaluate B having the specificity_TotalThe patient in need of treatment of hyperbilirubinemia (including jaundice), the specificity B_TotalLower than B achieved when treatment is considered to be considered according to current clinical practice_TotalTo achieve the specificity B_TotalAt the same time, the patientBy using B of the patient_TmaxAnd K_AAnd patients of the same age (optionally a cohort) B_FreeStandardTo determine (e.g., if the patient is less than gestational age: (a))<)28 weeks, B for forced intercourse transfusion_Total14mg/dL (Table 1) and optionally 22.0mg/dL of B_TmaxMedian and K of 1.16 dL/. mu.g_AB in median (Table 2)_FreeStandardIs composed of And gestational age of less than<)28 weeks B with e.g. 25 th percentile_Tmax14.3mg/dL and K_A0.75 dL/. mu.g of newborn infantWill reach 1.51. mu.g/dL of B_FreeStandardApproximately current for gestational age less than (C) according to Table 1<) B for forced intercourse transfusion of 28-week newborn_TotalHalf of 14 mg/dL); and reachSpecificity B to the patient_TotalAt this time, exposure of the brain to bilirubin and the risk of bilirubin toxicity (optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND)) are reaching current mandatory treatments B_TotalThe same happens at all times, proving despite B of the patient_TotalLess than mandatory treatment B_TotalThere is still a need for treatment.

In alternative embodiments, a computer-implemented method is provided, comprising a method as provided herein (e.g., a method as provided herein), or for performing a method as provided herein to determine B_TmaxAnd K_AAnd optionally further comprising: receiving the data element; and storing the data elements.

In an alternative embodiment, a computer program product is provided for processing data and determining B_TmaxAnd K_ASaid B is_TmaxAnd K_AB measured before and after enrichment of bilirubin by use of plasma samples_TotalAnd B_FreeThe computer program product comprising the computer-implemented method provided herein.

In an alternative embodiment, methods for determining B are provided_TmaxAnd K_AThe Graphical User Interface (GUI) computer program product of, B_TmaxAnd K_AB measured before and after enrichment of bilirubin by use of plasma samples_TotalAnd B_FreeThe Graphical User Interface (GUI) computer program product comprising the computer-implemented method provided herein.

In an alternative embodiment, a computer system is provided that includes a processor and a data storage device, wherein the data storage device has stored thereon: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, a non-transitory storage medium is provided that includes program instructions for executing, processing, and/or implementing: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, a non-transitory computer readable medium storing a computer program product for inputting data and performing calculations for determining B measured before and after bilirubin enrichment by use of a plasma sample is provided_TotalAnd B_FreeB obtained by the novel process of_TmaxAnd K_AThe non-transitory computer-readable medium includes the computer-implemented method provided herein.

In an alternative embodiment, a non-transitory computer readable storage medium is provided that includes computer readable instructions that, when executed by a processor of a computing device, cause the computing device to perform, process and/or implement: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, a computer program product is provided, comprising: a non-transitory computer-readable storage medium; and program instructions residing in the storage medium, which when executed by a computer, operate, process, and/or implement: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, a computer program storage device, embodied on a tangible computer readable medium, is provided comprising: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, there is provided a computer or equivalent electronic system comprising: a memory; and a processor operatively coupled to the memory, the processor adapted to execute program code stored in the memory to run, process and/or implement: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, a system is provided, comprising: a memory configured to store values associated with a plurality of data points and/or a plurality of data elements; and a processor adapted to execute the program code stored in the memory to run, process and/or implement: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, a computer-implemented system is provided for providing an application for accessing an external data source or an external server process through a connection server, for providing the ability to store values associated with a plurality of data points and/or a plurality of data elements, and for running, processing and/or implementing an application program for: (a) a computer-implemented method provided herein; (b) a computer program product provided herein; (c) a Graphical User Interface (GUI) computer program product provided herein; or (d) combinations of the foregoing.

In an alternative embodiment, a device is provided, such as a medical or analytical device, capable of quantifying the degree of plasma binding to bilirubin, wherein the device is capable of measuring B_Free(non-albumin bound or free bilirubin concentration) and B_Total(total bilirubin concentration) and communicate such data to a computer-implemented system provided herein, a system provided herein, or a computer or equivalent electronic system provided herein capable of performing the computer-implemented methods provided herein to determine or calculate and output B to, for example, a user_TmaxAnd K_AThe B is_TmaxAnd K_AIs B measured before and after enriching bilirubin using a plasma sample_TotalAnd B_FreeThe method is characterized in that the method is obtained by the novel method,

wherein optionally the computer-implemented system provided herein, the system provided herein, or the computer or equivalent electronic system provided herein is part of or within the apparatus, or remote (e.g., only directly connected or wirelessly connected) to the apparatus,

wherein the apparatus comprises components, optionally robotic chemical components (robotic chemical components), capable of measuring: total serum bilirubin levels (B)_Total) (ii) a And unbound bilirubin concentration or free bilirubin concentration before and after bilirubin enrichment (B)_Free) To obtain B of a sample (optionally a plasma or blood sample)_TmaxAnd K_A，

wherein optionally the apparatus comprises automated microfluidic processing technology, optionally a regional fluidic system or a robotic regional fluidic analysis system.

In alternative embodiments, methods or procedures, or systems or devices, are provided for diagnosing or prognosing (or predicting the likelihood of developing) an individual in need thereof:

-a bilirubin toxicity risk, optionally a bilirubin neurotoxicity risk, optionally a BIND risk,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-the yellowing of teeth caused by bilirubin,

the method comprises the following steps:

quantifying the extent to which the plasma of the subject binds bilirubin comprises:

(a) using the methods provided herein; or

(b) Determination of the maximum Total bilirubin concentration (B)_Tmax) And corresponding equilibrium association constant (K)_A)，

WhereinB_FreeAnd B_TotalRespectively a non-albumin bound or free bilirubin concentration and a total bilirubin concentration,

wherein the method determines whether bilirubin binding is at or below normal levels and determines the relative risk of BIND by combining B_FreeGroup B comparable to peer_FreeStandard(e.g., for gestational age according to Table 1. less than: (A)<) A 28 week newborn achieves forced intercourse transfusion B_Total14mg/dL and optionally 22.0mg/dL of B according to Table 2_TmaxMedian and K of 1.16 dL/. mu.g_AA median of then ) Make a comparison and will reach B_FreeStandardSpecificity of time B_TotalCurrent treatment with age-comparable cohort B_TotalComparison is made (e.g., for gestational age less than: (a)<)28 weeks at 14mg/dL B according to Table 1_TotalNeonates with forced intercourse transfusions, 25 th percentile B_Tmax(14.3mg/dL) and K_A(0.75 dL/. mu.g) will beWhen the concentration reaches 1.51 mu g/dL B_FreeStandard) Therein can beOptionally the patient is a neonate, wherein if B_FreeIs equal to or greater than B_FreeStandardOr B_TotalLess than treatment B_TotalThen it means that there is more brain exposure to bilirubin and there is an increased risk of, or the presence of:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

In alternative embodiments, methods are provided for treating, ameliorating, reversing, or preventing in an individual in need thereof (optionally a icteric neonate) the following:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

The method comprises the following steps:

(a) whether bilirubin binding is at or below normal levels using the methods provided herein or the devices provided herein is determined by administering to patient B_TmaxAnd K_AAnd optionally B of a comparable neonatal group_TmaxAnd K_AComparing the mean or median of the patients B_FreeB of comparable group_FreeStandardMaking a comparison and comparing B_Free＝B_FreeStandardPatient B at the time of day_TotalWherein, if necessary, B of the individual_FreeIs equal to or greater than B_FreeStandardOr B_TotalLess than current treatment B_TotalThen it means that there is more brain exposure to bilirubin and there is a higher risk of, or the presence of:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-bilirubin-induced tooth yellowing;

and

(b) b of an individual if required_FreeIs equal to or greater than B_FreeStandardOr B_TotalLess than current treatment B_TotalTreating (or initiating treatment), ameliorating, reversing or preventing an individual in need thereof for:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

In an alternative embodiment, an apparatus is provided for:

-treating, ameliorating, reversing or preventing an individual in need thereof for:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

Wherein the apparatus comprises an apparatus as provided herein, a computer-implemented system as provided herein, a system as provided herein or a computer or equivalent electronic system as provided herein,

and the device can determine whether bilirubin binding is normal or below normal by using a novel method of measuring B before and after bilirubin enrichment of a sample_TotalAnd B_FreeTo obtain B_TmaxAnd K_AAnd B_FreeThen patient B_TmaxAnd K_AAnd comparable newborn group B_TmaxAnd K_AMaking a comparison and comparing B of individuals in need thereof_FreeAnd comparable newborn group B_FreeStandardMaking a comparison in which individuals in need thereof are under current treatment B_TotalB of (A)_TotalHas a higher than normal B_FreeOr B_FreeStandardB of (A)_FreeIndicating that there is more brain exposure to bilirubin and a higher risk of, or the presence of:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

In an alternative embodiment, use of the apparatus in:

-treating, ameliorating, reversing or preventing an individual in need thereof for:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

-the yellowing of teeth caused by bilirubin,

wherein the apparatus comprises an apparatus as provided herein, a computer-implemented system as provided herein, a system as provided herein or a computer or equivalent electronic system as provided herein,

and the device can determine whether bilirubin binding is normal or below normal by using a novel method of measuring B before and after bilirubin enrichment of a sample_TotalAnd B_FreeTo obtain B_TmaxAnd K_AAnd B_FreeThen patient B_TmaxAnd K_AAnd optionally comparable neonate cohort mean or median B_TmaxAnd K_AMaking a comparison and comparing B of individuals in need thereof_FreeAnd comparable newborn group B_FreeStandardMaking a comparison in which there is B in the individual in need_TmaxAnd K_ALower than normal B_TmaxAnd K_AAnd B_FreeIs equal to or greater than B_FreeStandardTime indicates that there is more brain exposure to bilirubin and a higher risk of, or the presence of:

severe hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND,

bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

Bilirubin-binding group (BBP) described herein uses a novel plasma laboratory test group (Total bilirubin concentration B)_TotalAnd unbound or free bilirubin concentrations B measured before and after enrichment of the plasma sample with bilirubin_Free) To calculate B_TmaxAnd K_ATo provide B to the clinician_TmaxAnd K_AAnd B_FreeIn which B is_TmaxAnd K_AFor quantifying bilirubin binding in a patient, and B_FreeFor reaching said B_TotalThe relative risk of bilirubin-induced neurological dysfunction or BIND was quantified. These data were obtained by modifying the current methods to measure the sample-enriched bilirubinBilirubin binding before and after (e.g. B)_TotalAnd B_Free) And (4) obtaining the product. Significantly more sample (typically 25. mu.L of plasma) will be required using standard methods (see, e.g., Ahlfors CE, et al.measurement of unbounded bilirubin by the peroxidisesting using Zone fluids. clin Chim Acta 2006; 365:78-85), but the Zone fluid/SIA analysis requires only a few samples and the sample volume needs to be increased only minimally to allow other measurements to be made (less than 25. mu.L of plasma). At present, clinicians use only B_TotalTo assess BIND risk, while increasing B_Free、B_TmaxAnd K_ATo quantify bilirubin binding and assess BIND risk, thereby allowing for individualized patient care and improved determination of when and how to treat hyperbilirubinemia newborns.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences and ATCC deposits cited herein are expressly incorporated herein by reference for all purposes.

Brief description of the drawings

The following drawings are illustrative of various aspects of the invention and are not intended to limit the scope of the embodiments covered by the claims.

FIG. 1 shows the results for gestational age less than: (A), (B), (C<) Current B of 28 week neonates_TotalTreatment guidelines. In any B_TotalThe risk of bilirubin-induced neurological dysfunction (BIND) is unknown, but with B_TotalAnd increased by an increase. When B is present_TotalPhototherapy was considered but not mandatory at 5mg/dL up to B_TotalMandatory phototherapy up to 6 mg/dL; when B is_TotalExchange transfusion is contemplated but not mandatory when 11mg/dL is reached until B_TotalExchange transfusion was mandatory to reach 14 mg/dL. The gray areas indicate that there is a large uncertainty, and it is not clear that B is present in the gray areas_TotalHow the clinician isIt is determined whether the risk of BIND is great enough to justify treatment. For example, how the clinician determines B_TotalWhether or not a neonate at 12mg/dL requires crossover transfusion

FIG. 2 schematically illustrates the concentration of non-albumin bound or free bilirubin (B)_Free) Dominates the movement of bilirubin between the tissue (brain) and the blood. Poor binding of plasma bilirubin (B at any total bilirubin concentration)_FreeHigher) infants need to accumulate more bilirubin to reach a given B_TotalThus, said B is achieved in individuals with normal bilirubin binding_TotalIn contrast, patients achieved the B_TotalWith higher tissue levels of bilirubin and more exposure of the brain to bilirubin. Therefore, poorly bilirubin-bound neonates are in any of B_TotalThe greater the risk of BIND.

FIG. 3 shows a graph with B_TotalAnd B_FreeBoth increases the risk of BIND and, with B alone, also increases_TotalComparing fig. 1, which evaluates risk, both are known to improve risk assessment.

FIG. 4 shows B across the gray region of FIG. 1 (blue to orange) that would occur when the median, 25 th percentile, and 75 th percentile of the cohort are reached_FreeIncrease, while showing B at 6mg/dL and 14mg/dL respectively, to achieve forced phototherapy (0.32. mu.g/dL) and exchange transfusion (1.51. mu.g/dL) according to Table 1_TotalAnd a median B according to Table 2_Tmax(22.0mg/dL) and K_A(1.16. mu.g/dL) using B obtained by calculation_FreeStandard。

FIG. 5 plots measured B from Table 3_FREE(*)vs B_TotalAnd (4) relationship. Also shown is B in 1mg/dL increments_TotalB obtained by time calculation_FreeOr as used in B_Total8.3 and31.3mg/dL B from paired data_Tmax36.9 and K_A0.57 dL/. mu.g, at this timeOr suppose B_Tmax＝A_Total26.4mg/dL andand use paired data B_TmaxAnd K_AB obtained by calculation_FreeIn contrast, B calculated using the albumin model for a single site_FreeSignificant deviation from measured B_Free。

Fig. 6 shows the current treatment guidelines for neonates of gestational age less than 28 weeks shown in fig. 1 using bilirubin binding modification, in particular using mandatory phototherapy B for a group of 31 neonates of gestational age less than 28 weeks in table 2_Total(6mg/dL) and forced intercourse transfusion B_Total(14mg/dL) and median B_Tmax(22.0mg/dL) and K_A(1.16 dL/. mu.g) obtained This eliminates B in fig. 1 where the treatment is considered as appropriate (uncertain)_TotalThe gray area.

FIG. 7 shows the measured B from Table 2_Free(*)vs B_TotalA relationship; also shown is B in 1mg/dL increments_TotalB obtained by time calculation_FreeOr using the equationWherein MR is B_Total/A_TotalMolar ratio (Table 3), K₁(0.93 dL/. mu.g) and K₂(0.04 dL/. mu.g) is the stoichiometric mass equation of actionIs the most important ofGood fitting nonlinear regression equilibrium constants, or using a method of fitting B_TotalBinding data pairs at 8.3 and 31.3mg/dL determined B_Tmax36.9 and K_A0.57 dL/. mu.g(Table 3). The novel method of quantifying bilirubin binding described herein compares very well with standard chemometric methods.

FIG. 8 shows the measurement of Kp for horseradish peroxidase-catalyzed peroxidized bilirubin in a bilirubin solution without albumin (i.e., total bilirubin concentration equal to unbound or free bilirubin concentration). Since the total bilirubin concentration is the absorbance at 440nm divided by the extinction coefficient, Kp is determined by integrating the velocity equation

And (4) determining.

FIG. 9 shows the absorbance of bilirubin/albumin solution at 460nm as a function of time before and after addition of horseradish peroxidase (HRP) and peroxide. B was obtained using the initial absorbance at 460nm_TotalThe change in absorbance after addition of HRP and peroxide was used to obtain B_Free。

Like reference symbols in the various drawings indicate like elements.

Detailed Description

In alternative embodiments, methods, devices and multiplexing systems are provided for assessing whether bilirubin binding is normal in a patient (e.g., a newborn infant), whether there is a risk of bilirubin-induced neurological dysfunction (BIND), and whether bilirubin levels in the patient's plasma require treatment, and for diagnosing severe hyperbilirubinemia with an increased risk of neurotoxicity of bilirubin, including acute bilirubin encephalopathy and BIND. In alternative embodiments, methods are provided for treating or ameliorating or preventing the effects of bilirubin toxicity levels in a subject identified by the methods provided herein, or for treating or ameliorating or preventing bilirubin-induced neurological dysfunction (BIND) in a subject identified by the methods provided herein.

In an alternative embodiment, methods are provided, which may be computer-implemented methods, for scaling a plasma bilirubin-binding group (including: total serum bilirubin concentration (B))_Total) Unbound bilirubin or free bilirubin concentrations measured before and after bilirubin enrichment (B)_Free) To calculate a clinically relevant maximum total bilirubin concentration B)_TmaxAnd equilibrium association constant (K) corresponding thereto_A) Output B_TmaxAnd K_ATo the extent that bilirubin is incorporated into the patient, B_FreeAnd the group reaches B_FreeStandardB of (A)_TotalQuantification was performed to determine if the risk of BIND was high enough to justify treatment.

In an alternative embodiment, there is also provided an analytical device comprising an automated microfluidic processing process, such as a zone fluidics system, for measuring: total serum bilirubin levels (B)_Total) And unbound bilirubin or free bilirubin concentration of the plasma, serum or blood sample before and after bilirubin enrichment (B)_Free) And the analysis device also incorporates the computer-implemented method provided herein for analyzing the data and outputting: including B measured before and after bilirubin enrichment_TotalAnd B_FreeThe bilirubin-binding group of (a); clinically relevant maximum total bilirubin (B)_Tmax) And equilibrium association constant (K) corresponding thereto_A) To compare B of individuals_TmaxAnd K_AMaking a comparison to accurately determine whether bilirubin binding in the patient is normal; and at which B_FreeStandardAchieve clinically relevant diagnosis B_FreeAnd B_TmaxWhen the B is substituted_FreeStandardB of individuals comparable to each other_FreeStandardAnd current treatment B_TotalIn contrast, the risk of bilirubin-induced neurological dysfunction (BIND) is accurately determined. In an alternative embodiment, the device has built into it a computer or processor capacity to perform the computer-implemented methods for analyzing measured clinical data provided herein. In other placesIn an embodiment, a system is provided that houses a computer or processor capacity to perform the computer-implemented methods provided herein, the system being remote from the device (e.g., a regional fluid analysis device).

In an alternative embodiment, methods, devices and multiplexing systems are provided for assessing whether bilirubin binding in a patient (e.g., a neonate) is normal to accurately assess the presence or risk of the patient for bilirubin-induced neurological dysfunction (BIND). the clinical application of bilirubin binding depends on the measured bilirubin binding and bilirubin binding parameters of a known comparable neonatal cohort (e.g., a full-term neonate, a neonate of the same gestational age shown in Table 1, etc.)_TmaxAnd K_AIs optionally B_TmaxAnd K_AMedian, then B in the 25 th percentile_TmaxAnd K_AHas poor bilirubin binding relative to the group (75% of the group has a higher B than the patient)_TmaxAnd K_A). In mandatory therapy B_TotalFor example according to Table 1, wherein for gestational age less than: (<)28 week neonates in B_TotalExchange transfusion was mandatory at 14mg/dL, reaching B of Table 2_TmaxMedian (22.0mg/dL) and K_AB at the median (1.16 dL/. mu.g)_FreeIs composed of B up to the 25 th percentile_Tmax(14.3mg/dL) and K_A(0.75 dL/. mu.g) B_FreeIs composed ofWith a significantly higher risk of BIND. Thus, determining B for an individual in need thereof (optionally, a neonate)_Total、B_Free、B_TmaxAnd K_AThe degree of neonatal bilirubin binding is quantified and by comparing these indices with those of a cohort of similar ages it can be determined whether there is an increased risk of BIND in an individual in need thereof. To obtain B of the comparison group_TmaxAnd K_ANorm (norm) (e.g., mean, SD, range, median, quartile, etc.) requires measurement of an appropriate number of comparable neonates, typically about 400 patients, see, e.g., Lott JA, et al_Free、B_TmaxAnd K_ACan be administered to an individual in need thereof in B_TotalThe risk of bilirubin-induced neurological dysfunction (BIND) was quantified and the B when it had was determined_TotalB below the current standard recommendation for treatment for this group_TotalWhether it is needed for the individual in need.

In an alternative embodiment, each component of the bilirubin-binding group (BBP) includes B measured before and after the sample is enriched for bilirubin_TotalAnd B_FreeAnd calculated clinically relevant B_TmaxAnd equilibrium association constant (K) corresponding thereto_A) For passing B through_TmaxAnd K_AB of optionally comparable population_TmaxAnd K_AComparing the median to determine whether bilirubin binding is normal, and determining the median by comparing B to the median_FreeWith current treatment B to reach said group_TotalAnd optionally B_TmaxAnd K_AB of the group determined at median_FreeStandardA comparison is made to determine if BIND risk is increased. In addition, B may be used_FreeStandardAnd B_TmaxAnd K_ATo determine the actual B that needs treatment_Total. The BBP provided herein more reasonably quantifies bilirubin binding and can be used to determine whether bilirubin binding is normal when assessing whether treatment for hyperbilirubinemia (including jaundice) is required. The BBP can also be used as a screening assay to determine attainment of the B_FreeStandardAnd demonstrate the actual need for treatment B_Total(e.g., if less than (according to tables 1 and 2)<) B for 28-week neonates on exchange transfusion_FreeStandard1.51. mu.g/dL, with a B of 3.0mg/dL in this group_Total0.18B_Free20mg/dL of B_TmaxAnd K at 1.00 dL/. mu.g_AWill be inTo said B_FreeStandardBelow B requiring forced intercourse for transfusion_Total(14 mg/dL)). The bilirubin-binding group identified by the methods provided herein comprises B_TotalAnd is adjunct B in determining need of treatment_TotalInstead of with B_TotalCompeting against each other.

In an alternative embodiment, methods and systems are provided that overcome difficulties in quantifying bilirubin binding using simple techniques that can be used in clinically relevant patients with B_TotalUnder range (e.g. for gestational age less than: (a)<)35 weeks of newborn, B_TotalLess than 20mg/dL (see table 1)) reasonably quantitated bilirubin binding. In this process, B_TmaxIs not at allAll ofB when plasma binding sites are all occupied by bilirubin_TotalBut is clinically relevant B_TotalUpper limit of functional bilirubin binding sites in the range B_Total，K_AIs K corresponding thereto₁…K_nBalancing the combination of association constants. The chemical balance is:

and due to clinically relevant B_TotalWhen, B_FreeRatio B_TotalIs several orders of magnitude smaller than the total weight of the polymer,the resulting mass action equation is shown below,

can be determined by measuring B before and after the sample is enriched in bilirubin_TotalAnd B_FreeTo provide B_Total、B_FreeAnd B_{Total_2}、B_{Free_2}Can easily alignSolving to obtain B_TmaxAnd K_A. They provide a signal with two unknowns (B)_TmaxAnd K_A) Can be solved to obtain B_TmaxAs follows:

Table 3 shows the concentration of human defatted albumin (A) from bilirubin and having a concentration of 3.0g/dL_Total) Bilirubin binding isotherms were obtained from the artificial sera. FIG. 5 shows following B_TotalIncrease of (B)_Free(black dots). 1/B_FreeRatio 1/B_TotalNegative intercept (i.e. K)_A) 0.53. mu.g/dL, and a negative slope/intercept (i.e., B)_Tmax) It was 37.5 mg/dL. Using the lowest B_Total(8.3mg/dL) and B_Free(0.51. mu.g/dL) with five further groups B_TotalAnd B_FreeThe measured value pairs were calculated as described above to obtain B in Table 3_TmaxAnd K_A. Average B of all 15 possible pairings in Table 4_TmaxAnd K_A39.1mg/dL and 0.56 dL/mug, respectively. Using slave pair B_TotalNot higher than 8.3mg/dL and B_TotalB was obtained at 31.3mg/dL_Tmax(36.9mg/dL) and K_A(0.57 dL/. mu.g) in B in 1mg/dL increments_TotalB calculated in the range_FreeOverlap with the measured binding site as indicated by the hollow orange circle in fig. 5. But if B is assumed_TmaxIs A_Total＝26.4mg/dL， Calculated B_FreeSignificant deviation from the measured binding points is shown by the open blue circles in fig. 5. This indicates that even though bilirubin is known to bind primarily to plasma albumin, B_TmaxB in 31 neonates with gestational age less than 28 weeks, not closely related to albumin concentration but to plasma bilirubin binding site_TmaxAnd A_TotalIs not significantly correlated (r)²＝0.02)。

TABLE 3

By determining B of a group of neonates_TmaxAnd K_AQuantifying bilirubin binding may be used to reduce the dependence on current B_TotalGuidelines for uncertainty in treatment (e.g., table 1, fig. 1). Table 2 summarizes gestational age less than: (A)<) Binding data of 31 neonates at 28 weeks, from which, for example, median was knownNumber, optionally group B_TmaxAnd K_AIs then on the current treatment B_Total(see Table 1) can be found to be a standard B_FreeI.e. B_FreeStandardE.g. in group B_TmaxAnd K_AB in the median_FreeStandardComprises the following steps:

where all other aspects are peer-to-peer with at B_FreeStandardHalf of the population has a lower risk of BIND than (usually unknown) and the general population has a higher risk of BIND. For the half with higher BIND risk, it is lower than treatment B_TotalB of (A)_TotalWhen it reaches B_FreeStandardI.e. in individual B_Total、B_TmaxAnd K_AWhereinWhen measured B of an individual_FreeIs equal to or greater than B_FreeStandardOr when B is present in an individual_TotalIs equal to patient B_TmaxAnd K_ACalculatedThe BIND risk of the individual and reaching B_FreeStandardThe BIND risk for this group was the same as the tissue level of bilirubin, brain bilirubin exposure and BIND risk would be similar, as for B_TotalThe size is irrelevant.

Provided herein are serum or plasma B using measurements_TotalAnd B_FreeObtaining B_TmaxAnd K_AAnd methods of using same in the treatment of neonatal hyperbilirubinemia_TotalUse of methods for quantifying bilirubin binding and assessing BIND risk in the context of treatment guidelines and use of these methods for reducing current B in neonatal hyperbilirubinemia_TotalUse in the uncertainty of treatment guidelines, as illustrated in FIG. 6 compared to FIG. 1. These data, including the bilirubin Binding group (BBP) (see Ahlfors CE. the bilirubin Binding Panel: A Henderson-Hasselbalch aproach to neonatal hyperbilirubinemia. Pediatrics 2016; 138: e20154378) (B)_Total、B_Free、B_TmaxAnd K_A) Will greatly reduce the use of only B_TotalInherent in current treatment guidelines.

Quantification of plasma bilirubin binding:

defining normal bilirubin binding requires determining (1) how much bilirubin can be bound, and (2) the degree of "tightness" of bilirubin binding. Since bilirubin is mainly bound to plasma albumin, albumin concentration (A) has long been used_Total) To estimate how much bilirubin can be bound, it is generally assumed that one albumin molecule binds one bilirubin molecule. However, an albumin molecule may bind to more than one bilirubin molecule, and A_TotalIt is not "how much bilirubin can be bound" (i.e. B)_Tmax) A clinically useful estimate of.

Due to the clinical relevance of B in the hyperbilirubinemia neonate_TotalIn range of at least two bilirubin molecules per albumin molecule (see FIG. 5), bilirubin binding is quantified using graphical analysis (e.g., Jacobsen J. binding of bilirubin to human serum albumin-Determination of the association constancy. FEBS Lett 1969; 5: 112-; 114), or non-linear regression analysis using polynomial mass-action equations associated with multi-site binding (e.g., see Honor. B, Brodersen R. Albubmin binding anti-infinitional drugs. Utility of a site-oriented university a biological chemistry. mol Pharmacol 1984; Klotz 150and 5: 80. molecular chemistry. Hunston DL. protein molecules for molecules: binding interactions and 76; Biophys 328: Biophys 314). Stoichiometric two-point binding model measurements at multiple B_TotalPlasma albumin concentration (A) at time_Total) Total bilirubin concentration (B)_Total) And non-albumin bound or free bilirubin concentration (B)_Free) And used to determine two equilibrium association constants: for binding a bilirubinMolecular Albumin molecule (K)₁) And for albumin molecules (K) that bind two bilirubin molecules₂). In this model, B_TotalIs the concentration of albumin molecules that bind to one bilirubin molecule (A: B)₁) And 2 times the concentration of albumin molecules that bind to both bilirubin molecules (2 xA: b is₂) Sum of (A) plus B_Free，A_TotalIs A: b is₁+A：B₂+ concentration of unoccupied or free albumin binding sites not bound to bilirubin (A)_Free) The sum of (a) and (b). The chemical equilibrium formula is:

the mass action equation is:

the following equation may be used to solve for B_FreeWherein MR is the molar ratio:

the molar ratios from Table 3 with B were used_FreeBy using the stoichiometric equationTo determine the best fit K₁(0.93 dL/. mu.g) and K₂(0.04 dL/. mu.g). FIG. 7 will use8.3mg/dL of B from Table 3 was used_TotalAnd 31.3mg/dL of B_TotalB obtained by pairing_Tmax(36.9mg/dL) and K_A(0.57) at 1mg/dL of B_TotalB calculated in increments_FreeAndcomparing and displayingThe new method of quantifying bilirubin binding is very comparable to the standard chemometric method of quantifying binding. The clear advantage of this new method is that it provides a reasonable binding assay, but only requires two data points and does not require the measurement of A_TotalThus reducing the time and materials required for many of the measurements needed to quantify bilirubin binding.

A more clinically useful method of quantifying bilirubin binding is to quantify how much bilirubin will be bound (B)_Tmax) Degree of "tightness" of binding to bilirubin (K)_A) Are all considered as unknowns and pass through B_TotalAnd B_FreeThe measurements derive these unknowns. This needs to be for B_TotalAnd B_FreeBy measuring B of plasma samples before and after the sample is enriched with bilirubin_TotalAnd B_Free。

In any given plasma B_TotalAnd (unknown) B_TmaxThe plasma equilibrium concentration of (a) is:

wherein, B_TmaxHow much bilirubin, B, can be bound_Tmax–B_TotalIs the concentration of available (unoccupied) bilirubin binding sites, B_Total–B_FreeIs the bilirubin concentration associated with the plasma binding site (because of B_FreeIs orders of magnitude smaller than B_TotalSo as to be combined)。

The mass action equation isWherein B is_TotalAnd B_FreeIs measured, B_TmaxAnd K_AIs unknown. Such asB is measured before and after the sample is enriched in bilirubin_TotalAnd B_FreeGiving a measured value B_Total、B_FreeAnd B_{Total_2}、B_{Free_2}Then a signal with two unknowns (B) is provided_TmaxAnd K_A) Two equations of (A), B_TmaxAnd K_ACan be solved as follows.

Then calculating B_Tmax、B_TotalAnd B_FreeSubstitution intoTo obtainOr as an alternative, becauseIs a linear equationK_AIs a negative intercept, B_TmaxIs the negative slope divided byAndthe intercept of the relation of (a) to (b),

clinically relevant quantification of bilirubin binding is the above quality effect variable (B)_Total、B_Free、B_{Total_2}、B_{Free_2}、B_TmaxAnd K_A) They constitute the bilirubin-binding group (BBP). Alternatively, B_TmaxAnd K_ACan be used to determine whether neonatal bilirubin binding is normal. Table 4 below shows the results determined before and after the addition of sulfisoxazole (sulfanilamide) to bilirubin/human albumin samples containing 3.0g/dL albuminB_TmaxAnd K_AIn which B is_FreeApproximately double and B_TmaxAnd K_ASignificantly changing. When B is present_TotalTo achieveWhen the gestational age according to tables 1 and 2 is less than (<)28 weeks with 8.3B_Total0.51. mu.g/dL of B_Free24.3mg/dL of B_TmaxAnd K of 1.01_AThe newborn will reach the exchange transfusion B_FreeStandard1.51. mu.g/dL (FIG. 6), but if B is present_Tmax42.1mg/dL, K_AIs 0.20dL/ug,indicating that despite having the same B of 8.3mg/dL_TotalBut there is still a significant risk of BIND.

TABLE 4

Total bilirubin concentration and unbound bilirubin concentration were measured (B)_TotalAnd B_Free)：

Peroxidase assays (see, e.g., Jacobsen J, Wennberg RP. determination of unbound bilirubin in the serum of newborns. Clin Chem 1974; 20:783-_TotalAnd B_FreeAnd both. This test is used clinically in japan. In an alternative embodiment, the new and improved method provided herein measures B at two levels of horseradish peroxidase_FreeTo accurately determine B_FreeAnd B is measured for plasma or other blood samples before and after bilirubin enrichment_TotalAnd B_FreeTo provide B_TmaxAnd K_ATo complete the bilirubin-binding group (BBP) described herein. BBP Using B of a determined comparable group_FreeStandardFor bilirubin binding (B)_TmaxAnd K_A) And BIND risk were quantified. Peroxidase test peroxidase-based peroxidase-catalyzed peroxidase-oxidation-bilirubin reaction by horseradish peroxidase (HRP). Bilirubin absorbs light maximally at 440nm when albumin is not present; when bound to albumin, bilirubin absorbs maximum light at 460 nm. Albumin-bound bilirubin is protected from oxidation, only B_FreeIs oxidized. As bilirubin is oxidized, the absorbance at 440nm (no albumin) or 460nm (albumin present) decreases, and the reaction rate constant Kp can be determined using the known bilirubin concentration and HRP concentration in a solution in the absence of albumin (i.e. all bilirubin is unbound or "free") as shown by the equivalent velocity equation:

determination of K_P: FIG. 8 graphically shows the change in bilirubin absorbance per second at 440nm and 460nm as recorded using an HP8452 computer-directed spectrophotometer during HRP catalyzed oxidation of bilirubin in the absence of albumin (reaction: 3.0mL containing 128. mu. mol/L H₂O₂0.1M phosphate buffer (pH 7.4), 25. mu.L of HRP (reaction [ HRP ]]0.061. mu.g/mL), 5. mu.L of 1mg/mL bilirubin solution (reaction [ B)_Total]163. mu.g/dL, cuvette with 1cm optical path, 30 ℃).

By integrating the equation of speed between time t-0 and t-t, the K of the reaction can be easily calculated_P·HRP：-

K_pHRP ofThe negative slope of the time dependence, divided by the HRP concentration of the reaction, gives K_p。

Determination of B_TOTALAnd B_FREE: FIG. 9 is a diagrammatic representation ofThe absorbance at 460nm as a function of time (seconds) is shown and the use of HP8452 is shown^TMChange in bilirubin absorbance at 460nm in bilirubin-albumin solution before and after addition of HRP and peroxide as recorded by a computer directed spectrophotometer. B was obtained using the initial absorbance at 460nm as described below_TotalAnd B was obtained using the change in absorbance at 460nm after HRP/peroxide addition_Free。

The standard reaction is carried out in a cuvette with a 1cm light path, containing 1.0mL of 0.1M phosphate buffer, pH 7.4, to which 25. mu.L of sample (e.g., plasma or serum) is added, followed by 25. mu.L of HRP (typically 1.5mg/mL) and 5. mu.L of 26mmol/L H₂O₂Thus obtaining 120. mu. mol/L H₂O₂Reaction H of₂O₂. After adding HRP and H₂O₂Previously calculated from the absorbance B_TotalAfter adding HRP/H₂O₂Then B is calculated from the change in absorbance_FreeAs described further below. A new variation of the method involves repeating the test at another HRP concentration (typically 0.75mg/mL is used) and then enriching the sample with bilirubin (typically B_TotalIncrease 5 to 20mg/dL) and repeat the test at two HRP concentrations.

B_TotalDividing the initial absorbance by the known extinction coefficient (B)_Total(mg/mL) optical path length of 0.827/cm), B_FreeBy adding HRP/H₂O₂This is calculated from the change in absorbance at 460 nm. Due to only B_FreeIs oxidized (bound bilirubin is protected from oxidation) and the velocity equation is thusHowever, since B_FreeThe oxidation of (A) breaks the equilibrium from

Become into

Balance B_FreeReduced to an unknown lower steady state free bilirubin (B)_Fss) Thus, therefore, it is

Wherein the content of the first and second substances,the integration speed is:

And

whereinIt is from

The relation with t is obtained.

By measuring B at additional HRP concentration_FssAnd useTo obtain B_FreeWherein from the above B_FssThe reciprocal of the equation gives 1/B_FssIntercept of relation with HRPThe inverse of the intercept is

As seen in tables 2 and 3, the samples were enriched for bilirubin, after which B was measured_{Total_2}And B_{Free_2}And B before enrichment is used_TotalAnd B_FreeTo obtain B_TmaxAnd K_A. Then, a signal with two unknowns (B) is used_TmaxAnd K_A) Two equations of (A) yield B_TmaxAnd K_AThereby can be easily alignedSolution, B_TmaxThe solution is performed as follows:

Bilirubin binding group：

The bilirubin binding group is formed by mass equation of actionMathematically related, it makes no assumptions about the stoichiometry or chemistry of the actual plasma bilirubin binding site, but the constant B_TmaxAnd K_AFor being lower than B_TmaxB of (A)_TotalB of (A)_FreeProviding an accurate estimate as shown in fig. 5 and 7. In one embodiment, the peroxidase assay measures serum or plasma B_TotalAnd B_FreeFor example, as in Jacobsen J, Wennberg RP.determination of unbounded bilirubin in the serum of newborns. Clin Chem 1974; 20: 783-789. In an alternative embodiment, B is measured at the second peroxidase concentration_FreeTo ensure B_FreeThen enriching the sample with bilirubin and at a higher B_TotalAnd B_FreeRepeating the test to use two equations and two unknowns or to useLinear analysis of to obtain B_TmaxAnd K_AWherein the negative intercept is K_AAnd is and

subjecting an individual B_TmaxAnd K_AAnd optionally comparable B of the group_TmaxAnd K_AThe median is compared to determine whether the individual has normal bilirubin binding. If B of the individual_FreeGreater than or equal to B of the group_FreeStandardThen the risk of BIND increases, determining B of said group_FreeStandardIn order to determine whether an individual has normal bilirubin binding. If B of the individual_FreeGreater than or equal to B of the group_FreeStandardThen, thenIncreased risk of BIND, use of Current B_TotalTreatment guidelines and optionally B of said group_TmaxAnd K_AMedian determines B of the group_FreeStandardFor example for gestational age according to Table 1 less than: (A)<) Neonates of 28 weeks, B of forced intercourse transfusions in 31 neonates according to Table 2_Total(14mg/dL)、B_TmaxMedian (22.0mg/dL) and K_AThe median (1.16 dL/. mu.g),if personal B_FreeB is equal to or greater than (not less than)_FreeStandardThen no matter B_TotalTo what extent, the need for treatment is evidenced; if B is present_FreeIs less than B_FreeStandardDue to the fact that By using B of an individual_Tmax、K_AAnd B_FreeStandardCan be obtained up to B_FreeStandardAnd individual specificity B in need of treatment_Total。

B_TmaxAnd K_AThe extent of plasma-bound bilirubin was reasonably quantified, as both were responsible for the amount of plasma-bound bilirubin (B)_Tmax) And degree of compaction (K)_A) Quantification was performed. The newborn B_TmaxAnd K_AB with age group_TmaxAnd K_AMake a comparison (e.g., compare them to B_TmaxAnd K_AMedian comparison) determined the degree of bilirubin binding of the neonate compared to a peer, as compared to the normal values for the cohort in any blood test, for the detection of potential disease. If B of the newborn_FreeEqual to or exceed B of the group_FreeStandardThen no matter B_TotalTo what extent, the need for treatment is demonstrated. If B of the newborn_FreeIs less than B_FreeStandardAs indicated above, can be based onB_FreeStandardAnd B of newborn_TmaxAnd K_AObtaining the specificity B that the newborn should be treated_Total. This method reduces the use of B alone_TotalAnd (2) uncertainty in current treatment guidelines (see fig. 1) and individualize care.

Various neonatal cohorts (gestation, preterm birth shown in table 2)<28 weeks, disease, etc.) of B_TmaxAnd K_AThe group parameters (mean, standard deviation, median, etc.) can be readily obtained to provide for improvement of the current B-only basis_TotalGroup-specific bilirubin binding data as required for therapeutic decision making.

Equipment: regional fluid analysis instrument

Initially described for measurement B_TotalAnd B_FreeThe manual peroxidase test of (1) required 25. mu.L of sample, whereas the four tests described herein (measurement of B at two peroxidase concentrations before and after bilirubin enrichment)_TotalAnd B_Free) 100 μ L of sample is required. The new technology herein is a technology that automates the testing and reduces the sample size.

In an alternative embodiment, systems and devices for processing and manipulating samples (serum, plasma, or whole blood samples from patients) are provided that include automated microfluidic processing techniques, such as regional fluidic systems, and suitable chemicals (e.g., robotic chemistry), that measure: total serum bilirubin concentration of plasma or blood samples (B)_Total) And unbound bilirubin or free bilirubin concentration (B)_Free) (Jacobsen J, Wennberg RP.determination of unbounded bilirubin in the serum of newborns. Clin chem 1974; 783, Ahlfors CE, et al.measurement of unbounded bilirubin by the peroxidase test using Zone fluids. Clin Chim Acta 2006; 365: 78) and further includes a computer-implemented method provided herein, either directly incorporated into the device or indirectly incorporated into the device as a multiplexing system operatively connected to the device, that analyzes the data and outputs a maximum bilirubin concentration (B)_Tmax) Hedan bladderErythrosine binding constant (K)_A) Both of which are capable of accurately determining the degree of bilirubin binding of the patient when compared to the product of a cohort of similar ages and B_FreeTo determine whether bilirubin-induced neurological dysfunction (BIND) is at higher risk than in cohorts at B_FreeStandardThe risk of the time.

In an alternative embodiment, an apparatus is provided comprising Sequential Injection Analysis (SIA) and/or regional fluid techniques and equivalent automated microfluidic processing techniques for processing and analyzing blood, serum or plasma of a patient and extending these techniques to include titration with bilirubin to enable calculation of B_TmaxAnd K_A。

In an alternative embodiment, an apparatus is provided that includes a component (e.g., a robotic chemical component) for measuring: total serum bilirubin concentration (B) of a sample (e.g., plasma, serum, or blood sample)_Total) (ii) a Unbound bilirubin or free bilirubin concentration (B)_Free). Any chemical, device, or robotic chemical assembly known in the art may be used or incorporated into the devices used and/or provided herein, for example, as described in the following U.S. patents: 7,939,333 (describing a metal-enhanced fluorescence based sensing method); 7,767,467 (describing, for example, methods and devices for separating small particles or cells from a fluid suspension); 7,416,896 (describing, for example, methods and devices for determining total plasma bilirubin and bound plasma bilirubin); 7,625,762 (describing, for example, methods and devices for separating small particles or cells from a fluid suspension); 6,887,429 (describing, for example, methods and apparatus for automating existing medical diagnostic tests); 6,692,702 (describing, for example, methods and apparatus for removing interferents from a sample comprising cells in an automated device using a filtration device); and 6,613,579; alternatively, as described in the following U.S. patent publications: for example, U.S. patent application No. 2018/0045723 a1 (describing, for example, lateral flow devices and methods for analyzing fluid samples); U.S. patent application No. 2018/0052093 a1 (describing, for example, apparatus and methods for analyzing particles in a sample); U.S. patent application No. 2016/0245799; or, as described in the following documents: amin, s.b., Clinical diagnostics 43(2016) 241-257 (describing, for example, the peroxidase method for measuring plasma bilirubin binding); ahlfors, et al, clinical biochemistry 40(2007) 261-267 (describing the effect of, for example, sample dilution, peroxidase concentration, and chloride ion on unbound bilirubin measurements in preterm infants); ahlfors, c.e., Analytical Biochemistry 279, 130-135 (2000) (describing the determination of unbound, unbound bilirubin, e.g., plasma); ahlfors, et al, ClinicaChimica Acta 365(2006) 78-85 (describing unbound bilirubin assays such as by peroxidase assays using zone fluids); (by peroxidase assay using Zone Fluidics); wennberg et al, Pediatrics 117(2006) 474-; or WO 2013032953A 2, Huber et al, Clinical Chemistry 58(2012)869-876 (describing fluorescent probes for quantifying unbound bilirubin, e.g., by fluorescence quenching upon binding bilirubin).

In an alternative embodiment, a device is provided with the capability to output or send relevant data to a device integrated or stand-alone device or system to perform the computer-implemented methods provided herein, which then calculates and outputs: bilirubin binding constant, maximum total bilirubin concentration, and clinically relevant diagnostic product B obtained from the measured components of the bilirubin-binding group_Tmax·K_A。

In an alternative embodiment, a zone fluidic system with a flow manifold is provided that is simple and robust, e.g., including pumps, selection valves, and detectors connected by microporous tubing. The same manifold can be used with a variety of different chemicals with only changes to the flow program and without changes to the piping structure and hardware. In an alternative embodiment, a zone fluidic technique is provided that acts as a fluidic analysis robotic system. In alternative embodiments, particular advantages of exemplary embodiments of microfluidic technology may include one, several or all of the following features or benefits:

can handle sample volumes in the smaller microliter range;

＝>bilirubin may be added to the sample to enable measurement before and after the sample is enriched for bilirubinB_TotalAnd B_Free；

The performance of high-end clinical chemistry systems or robotically implemented systems can be reached at significantly lower prices;

the expansibility of expanding to the aspect of health care instruments can be realized, and the sale cost is low;

may be computer controlled and automated;

improved methods can be easily developed-workflow is flexible; and

the dynamics allow the method to be optimized to produce the highest quality data without being limited by the hardware design;

complex methods can be fully automated;

can provide higher reliability and ease of maintenance;

the use of reagents can be greatly reduced (many other methods typically use 1 to 100mL of reagent per measurement) -SIA typically uses 1 to 100 μ L per measurement.

In alternative embodiments, a zonal fluid system as described in U.S. patent No. 7,416,896, or an apparatus or assembly as described in U.S. patent application No. 2016/0245799, may be used to practice alternative apparatus embodiments provided herein.

A computer system for performing the computer-implemented method:

in an alternative embodiment, a computer-implemented method is provided to analyze laboratory data and output a bilirubin-binding group (including B before and after enrichment of bilirubin in a plasma or blood sample)_TotalAnd B_Free) Max B_Total(B_Tmax) And bilirubin binding constant (K)_A) Providing clinically relevant B_TmaxAnd K_ABy matching it with B from a cohort of the same age_TmaxAnd K_AA comparison is made to determine whether bilirubin binding is normal and whether the patient's BIND risk is increased (B)_FreeIs equal to or greater than B_FreeStandard) And if not, determining the specificity B of the patient at the time the patient is proven to require treatment_Total. Using e.g. notA transitory computer readable medium performs a computer-implemented method, including for example using a computer or processor, which may be integrated into the apparatus provided herein or separately but operatively connected to the apparatus, for example, as a system.

Alternative embodiments, including computer-implemented methods, are presented in terms of algorithms and symbolic representations of operations on data bits within a computer memory. These algorithmic descriptions and representations are the means used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art. An algorithm is here, and generally, considered to be a self-consistent sequence of steps leading to a result. The steps are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities may take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, elements, symbols, characters, terms, numbers, or the like.

It should be borne in mind, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. Unless specifically stated otherwise as apparent from the following discussions, it is appreciated that throughout the specification discussions utilizing terms such as "processing," "computing," "calculating," "determining," "displaying," or the like, refer to the action and processes of a computer system, or similar electronic computing device, that manipulate and transform data represented as physical (electronic) quantities within the computer system's registers and memories into other data similarly represented as physical quantities within the computer system memories or registers or other such information storage, transmission or display devices.

In alternative embodiments, an apparatus for performing the operations or computer-implemented methods provided herein is provided. This apparatus may be specially constructed for the required purposes, or it may comprise a general-purpose computer selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored in a computer readable storage medium, such as, but is not limited to, any type of disk including floppy disks, optical disks, CD-ROMs, and magnetic-optical disks, read-only memories (ROMs), Random Access Memories (RAMs), EPROMs, EEPROMs, magnetic or optical cards, or any type of media suitable for storing electronic instructions.

The algorithms and displays presented herein are not inherently related to any particular computer or other apparatus. Various general-purpose systems may be used with programs taught herein, or it may prove convenient to construct more specialized apparatus to perform the method steps. The following description will present the structure of various of these systems. In addition, embodiments herein are not described with reference to any particular programming language. It will be appreciated that a variety of programming languages may be used to implement the teachings of the embodiments as described herein.

In alternative embodiments, a machine-readable medium includes any mechanism for storing or transmitting information in a form readable by a machine (e.g., a computer). For example, a machine-readable medium includes a machine-readable storage medium (e.g., read only memory ("ROM"), random access memory ("RAM"), magnetic disk storage media, optical storage media, flash memory devices, etc.), a machine-readable transmission medium (e.g., electrical, optical, acoustical or other form of propagated signals (e.g., carrier waves, infrared signals, digital signals, etc.)), and others.

In an alternative embodiment, the methods provided herein are implemented in a computer system within which a set of instructions for causing a machine to perform any one or more of the protocols or methods provided herein may be executed. In alternative embodiments, the machine may be connected (e.g., networked) to other machines in a LAN, an intranet, an extranet, or the internet, or any equivalent thereof. The machine may operate in the capacity of a server or a client machine in a client-server network environment, or as a peer machine in a peer-to-peer (or distributed) network environment. The machine may be a Personal Computer (PC), a tablet PC, a set-top box (STB), a Personal Digital Assistant (PDA), a cellular telephone, a Web appliance, a server, a network router, switch or bridge, or any machine capable of executing a set of instructions (sequential or otherwise) that specify operations to be performed by that machine. The term "machine" shall also be taken to include any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one or more of the methodologies discussed herein.

In alternative embodiments, the exemplary computer system provided herein includes a processing device (processor), a main memory (e.g., Read Only Memory (ROM), flash memory, Dynamic Random Access Memory (DRAM), such as synchronous DRAM (sdram) or Rambus DRAM (RDRAM), etc.), a static memory (e.g., flash memory, Static Random Access Memory (SRAM), etc.), and a data storage device, which communicate with each other over a bus.

In alternative embodiments, the processor represents one or more general-purpose processing devices, such as a microprocessor, central processing unit, or the like. More specifically, the processor may be a Complex Instruction Set Computing (CISC) microprocessor, Reduced Instruction Set Computing (RISC) microprocessor, Very Long Instruction Word (VLIW) microprocessor, or a processor implementing other instruction sets or processors implementing a combination of instruction sets. The processor may also be one or more special-purpose processing devices such as an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA), a Digital Signal Processor (DSP), network processor, or the like. In an alternative embodiment, the processor is configured to execute instructions (e.g., processing logic) for performing the operations and steps discussed herein.

In an alternative embodiment, the computer system further comprises a network interface device. The computer system may further include a video display unit (e.g., a Liquid Crystal Display (LCD) or a Cathode Ray Tube (CRT)), an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse)), and a signal generation device (e.g., a speaker).

In an alternative embodiment, a data storage device (e.g., a drive unit) comprises a computer-readable storage medium having stored thereon one or more sets of instructions (e.g., software) embodying any one or more of the protocols, methods, or functions provided herein. The instructions may also reside, completely or at least partially, within a main memory and/or within a processor during execution thereof by the computer system, the main memory and the processor also constituting machine-accessible storage media. The instructions may further be transmitted or received over a network via a network interface device.

In an alternative embodiment, a computer-readable storage medium is used to store a set of data structures defining user identification states and user preferences, which define a user profile. The data structure set and user profile may also be stored in other parts of the computer system, such as static memory.

In alternative embodiments, the term "machine-accessible storage medium" may be taken to include a single medium or multiple media (e.g., a centralized or distributed database, and/or associated caches and servers) that store the one or more sets of instructions, although the computer-readable storage medium in the exemplary embodiments is a single medium. In alternative embodiments, the term "machine-accessible storage medium" may also be considered to include any medium that is capable of storing, encoding or carrying a set of instructions for execution by the machine and that cause the machine to perform any one or more of the methodologies provided herein. In alternative embodiments, the term "machine accessible storage medium" shall accordingly be taken to include, but not be limited to, solid-state memories, and optical and magnetic media.

Treating BIND and hyperbilirubinemia

In an alternative embodiment, a method for treating, ameliorating, reversing or preventing the following in an individual in need thereof (optionally a icteric neonate or young child) is provided:

-severe hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity or bilirubin-induced neurological dysfunction (BIND),

-bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent,

-bilirubin-induced autism, or with toxic levels of bilirubin as a causative agent,

-bilirubin-induced high-pitch hearing loss,

-bilirubin-induced upward gaze paralysis, or

Bilirubin-induced tooth yellowing.

The methods provided herein indicate when treatment of an individual in need thereof should be initiated and provide guidance to the physician regarding appropriate treatment of the individual in need thereof, e.g., requiring the use of phototherapy and/or the exchange of transfusions, when a concurrent clinical condition does not indicate a high risk of BIND.

In addition, the methods provided herein can be used to monitor treatment to determine that bilirubin levels have been sufficiently reduced to significantly reduce the risk of BIND, thereby signaling to the physician that treatment can be altered, interrupted, or discontinued. If BIND does not have obvious symptoms, the methods provided herein can alert the clinician that early treatment can be performed, thereby reversing or mitigating the lesion (see Johnson L, et al, Clinical report from the pilot USA key house registry (1992) 2004. J Perinatol 2009; 29: S25-45), where the patient can be a neonate, child, or adult (see, for example, Blanchke TF, et al, Crigler-Najjar syndrome: an unused core with depth of neural damage at facility. Peditar. Res. 1974; 8: 573-.

Thus, the diagnostic and therapeutic methods provided herein help address the problem that symptoms of BIND are often confused with other conditions (e.g., infection) (see Ahlfors et al, unbounded bilirubin in a term newborn with kernicters. Pediatrics 2003; 111: Pediatrics 2003; 111: 1110-.

Any method known in the art may be used to treat or ameliorate or prevent significant hyperbilirubinemia, such as jaundice, bilirubin toxicity (including bilirubin neurotoxicity), bilirubin-induced neurological dysfunction (BIND), bilirubin-induced neurodevelopmental disorder, or alternatively neurodevelopmental disorder in a newborn caused by toxic levels of bilirubin as a causative agent, optionally including encephalopathy or kernal jaundice, or sudden neurotoxicity (acute bilirubin encephalopathy), or athetosis-like cerebral palsy; optionally a defect in the neonate caused by bilirubin as a causative agent at a toxic level, optionally including encephalopathy or kernal jaundice, or sudden neurotoxicity (acute bilirubin encephalopathy), or choreoathetosis-like cerebral palsy; bilirubin-induced hearing impairment, or bilirubin at toxic levels as a causative agent; autism caused by bilirubin, or with toxic levels of bilirubin as a causative agent; bilirubin-induced treble hearing loss; bilirubin-induced upward gaze paralysis; and/or bilirubin-induced tooth yellowing.

For example, severe hyperbilirubinemia (such as jaundice, e.g., neonatal jaundice) can be treated by phototherapy or colored light, which can act by converting trans-bilirubin to a water-soluble cis-bilirubin isomer, or by exchange transfusion, which involves repeatedly drawing small amounts of blood and replacing it with donor blood, thereby diluting bilirubin and maternal antibodies. In an alternative embodiment, intravenous immunoglobulin (IVIg) is used when severe hyperbilirubinemia, such as jaundice, may be associated with blood group differences between mothers and infants. This condition results in the infant carrying antibodies from the mother, which results in rapid breakdown of the infant's red blood cells. Intravenous infusion of anti-maternal Ig immunoglobulins can alleviate hyperbilirubinemia or jaundice and reduce the need or extent of crossover transfusion.

Those of skill in the art would understand that information and signals may be represented using any of a variety of different technologies and techniques. For example, data, instructions, commands, information, signals, bits, symbols, and chips that may be referenced throughout the above description may be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields or particles, or any combination thereof.

Those of skill would further appreciate that the various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.

In view of these teachings, those skilled in the art will readily recognize the improved methods of the present invention. The above description is illustrative and not restrictive. The invention is limited only by the accompanying claims, which include all such embodiments and modifications when viewed in conjunction with the above specification and accompanying drawings. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

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Method and apparatus for assessing bilirubin toxicity levels in vivo and diagnosing bilirubin neurotoxicity risk

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