Methods and devices for assessing in vivo toxic levels of bilirubin and diagnosing increased risk of bilirubin neurotoxicity

Abstract

In alternative embodiments are provided methods, devices and systems that use clinical data to determine whether bilirubin binding is normal in a newborn infant with hyperbilirubinemia in order to detect in vivo neurologically toxic levels of bilirubin and to determine whether treatment is needed to prevent a bilirubin-induced neurological injury (e.g. encephalopathy). In alternative embodiments, also provided are devices and systems comprising automated micro-fluid handling technologies such as zone fluidics systems to obtain a bilirubin binding panel. In alternative embodiments, also provided are methods for using the bilirubin binding panel to determine if treatments are needed to ameliorate, reverse, or prevent a bilirubin-induced neurological injury (e.g. encephalopathy) in an individual in need thereof such as a newborn with hyperbilirubinemia (jaundice), and for commencing the treatment, if needed.

Claims

1. A method for treating, ameliorating, reversing or preventing in an individual in need thereof: a hyperbilirubinemia or bilirubin toxicity, a bilirubin neurotoxicity, a bilirubin-induced neurodevelopmental impairment, a neurodevelopmental impairment having toxic levels of bilirubin as a causative agent, a sudden bilirubin-induced neurotoxicity, an acute bilirubin encephalopathy, a choreoathetotic cerebral palsy, a bilirubin-induced hearing impairment, or a hearing impairment having toxic levels of bilirubin as a causative agent, a bilirubin-induced autism, or an autism having toxic levels of bilirubin as a causative agent, a bilirubin-induced high tone hearing loss, a bilirubin-induced paralysis of upward gaze, or a bilirubin-induced yellow staining of the teeth, the method comprising: (a) determining whether bilirubin binding is normal, or below normal in the individual in need thereof by a method comprising: (i) providing or taking, or having provided, a plasma, blood or serum sample from the individual; (ii) measuring B.sub.Total and B.sub.Free, (iii) enriching the plasma, blood or serum sample with bilirubin, (iv) measuring B.sub.Total and B.sub.Free in the bilirubin enriched plasma; and, (v) determining the maximum total bilirubin concentration (B.sub.Tmax) and the equilibrium association constant (K.sub.A), wherein if the individual's B.sub.Tmax and K.sub.A are below the mean, average, or median B.sub.Tmax and K.sub.A for a comparable population, the individual has poor or clinically inefficient bilirubin binding, and if the individual's B.sub.Free is equal to or greater than the B.sub.FreeStandard for the comparable population that occurs at a current treatment B.sub.Total this indicates the presence of a risk of BIND that is sufficient to warrant treatment, wherein $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$ and B.sub.Free and B.sub.Total are the measured concentrations of non-albumin bound or free bilirubin and total bilirubin concentration, respectively, and measuring B.sub.Total and B.sub.Free before and after enrichment of the sample with bilirubin to obtain B.sub.Total, B.sub.Free and B.sub.Total 2, B.sub.Free 2 to provide two equations with two unknowns (B.sub.Tmax and K.sub.A), that are solved for B.sub.Tmax $B_{\mathrm{Tmax}}=\frac{B_{\mathrm{Total}}{B}_{\mathrm{Total}\_2}\left(B_{\mathrm{Free\_}2}-B_{\mathrm{Free}}\right)}{B_{\mathrm{Total}}{B}_{\mathrm{Free\_}2}-B_{\mathrm{Total\_}2}{B}_{\mathrm{Free}}}$ and the measured B.sub.Total and B.sub.Free are used with the calculated B.sub.Tmax to obtain $K_A=\frac{B_{\mathrm{Total}}}{B_{\mathrm{Free}}\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)},$ or K.sub.A is the negative intercept and B.sub.Tmax is the negative slope divided by the intercept of $\frac{1}{B_{\mathrm{Free}}}\mathrm{versus}\frac{1}{B_{\mathrm{Free}}}$ as the reciprocal of $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$ is the linear equation $\frac{1}{B_{\mathrm{Free}}}=\frac{B_{\mathrm{Tmax}}.Math.K_A}{B_{\mathrm{Total}}}-K_A,$ and (b) commencing treating, ameliorating, reversing or preventing the individual in need thereof for: the jaundice or bilirubin toxicity, the bilirubin neurotoxicity, the bilirubin-induced neurodevelopmental impairment, the impairment having toxic levels of bilirubin as a causative agent, the acute bilirubin encephalopathy, the choreoathetotic cerebral palsy, the bilirubin-induced hearing impairment, or hearing impairment having toxic levels of bilirubin as a causative agent, the bilirubin-induced autism, or the autism having toxic levels of bilirubin as a causative agent, the bilirubin-induced high tone hearing loss, the bilirubin-induced paralysis of upward gaze, or the bilirubin-induced yellow staining of the teeth, if the individual in need thereof has a lower than normal calculated B.sub.Tmax and K.sub.A or a B.sub.Free equal to or greater than B.sub.FreeStandard as determined in step (a).

2. The method of claim 1, wherein in step (c) enriching the plasma, blood or serum sample with bilirubin comprises increasing the amount of bilirubin in the sample from between about 5 to 25 mg/dL, or to enrich B.sub.Total near the relevant current clinical threshold B.sub.Total for a relevant population.

3. The method of claim 1, wherein the individual in need thereof is a jaundiced newborn or infant.

4. The method of claim 1, wherein the significant hyperbilirubinemia comprises jaundice.

5. The method of claim 1, wherein the neurodevelopmental impairment having toxic levels of bilirubin as a causative agent comprises an encephalopathy or kernicterus.

6. The method of claim 1, wherein if the comparable population is a newborn less than 28 weeks gestation, the mean, average, or median B.sub.Tmax and K.sub.A for the comparable population is: the median B.sub.Tmax and K.sub.A of 22.0 mg/dL and 1.16 dL/g.

7. The method of claim 2, wherein the relevant population comprises: the exchange transfusion threshold B.sub.Total of 14 mg/dL in newborns less than 28 weeks gestation, or the exchange transfusion threshold of 25 mg/dL in well term newborns.

8. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for jaundice or bilirubin toxicity.

9. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for bilirubin neurotoxicity.

10. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for bilirubin-induced neurodevelopmental impairment.

11. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for impairment having toxic levels of bilirubin as a causative agent.

12. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for acute bilirubin encephalopathy.

13. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for choreoathetotic cerebral palsy.

14. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for bilirubin-induced hearing impairment, or hearing impairment having toxic levels of bilirubin as a causative agent.

15. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for bilirubin-induced autism or for autism having toxic levels of bilirubin as a causative agent.

16. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for bilirubin-induced high tone hearing loss.

17. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for bilirubin-induced paralysis of upward gaze.

18. The method of claim 1, wherein the treating, ameliorating, reversing or preventing is for bilirubin-induced yellow staining of the teeth.

19. The method of claim 1, wherein the treating, ameliorating, reversing or preventing comprises phototherapy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following drawings are illustrative of aspects of the invention and are not meant to limit the scope of the embodiments as encompassed by the claims.

(2) FIG. 1 illustrates current B.sub.Total treatment guidelines for newborns less than (<) 28 weeks gestation per TABLE 1. The risk of bilirubin-induced neurological dysfunction (BIND) at any B.sub.Total is unknown but increases as B.sub.Total increases. Phototherapy is considered at B.sub.Total=5 mg/dL but not mandatory until it reaches 6 mg/dL and exchange transfusion is considered at B.sub.Total=11 mg/dL but not mandatory until it reaches 14 mg/dL. The gray zones indicate considerable uncertainty and it is unclear how clinicians determine whether the risk of BIND is sufficient to warrant treatment at B.sub.Total in the gray zones. For example, how does a clinician decide whether a newborn with a B.sub.Total=12 mg/dL needs an exchange transfusion?

(3) FIG. 2 schematically illustrates that the non-albumin bound or free bilirubin concentration (B.sub.Free) governs the movement of bilirubin between tissues (brain) and blood. A baby with poor plasma bilirubin binding (higher B.sub.Free at any total bilirubin concentration) requires more accumulated bilirubin to reach a given B.sub.Total and will have, therefore, higher tissue levels of and brain exposure to bilirubin at that B.sub.Total relative to a patient with normal bilirubin binding that reaches that B.sub.Total. Therefore the risk of BIND at any B.sub.Total is greater in a newborn with poor bilirubin binding.

(4) FIG. 3 illustrates that the risk of BIND increases as both B.sub.Total and B.sub.Free increase, and knowing both improves the assessment of risk as compared FIG. 1 wherein only B.sub.Total is used to assess risk.

(5) FIG. 4 illustrates the increase in B.sub.Free across the gray zones of FIG. 1 (blue dots to orange dots) that would occur at the median, 25.sup.th, and 75.sup.h percentiles for a population and shows the B.sub.FreeStandard that occur at the mandatory phototherapy (0.32 g/dL) and exchange transfusion (1.51 g/dL) B.sub.Total of 6 mg/dL and 14 mg/dL, respectively, per Table 1 and median B.sub.Tmax (22.0 mg/dL) and K.sub.A (1.16 g/dL) per Table 2 calculated using

(6) $B_{\mathrm{FreeStandard}}=\frac{\mathrm{Treatment}{B}_{\mathrm{Total}}}{\mathrm{median}{K}_A\left(\mathrm{median}{B}_{\mathrm{Tmax}}-\mathrm{Treatment}{B}_{\mathrm{Total}}\right)}.$

(7) FIG. 5 plots the measured B.sub.Free from TABLE 3 (.circle-solid.) versus B.sub.Total. Also shown are B.sub.Free calculated at 1 mg/dL increments in B.sub.Total using either B.sub.Tmax=36.9 and K.sub.A=0.57 dL/g from the pairing data at B.sub.Total=8.3 and 31.3 mg/dL where

(8) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}\left(\right)$
or assuming B.sub.Tmax=A.sub.Total=26.4 mg/dL and

(9) $K_A=\frac{8.3\mathrm{mg}\text{/}dL}{0.51\mathrm{.Math.}g\text{/}dL\left(26.4\mathrm{mg}\text{/}dL-8.3\mathrm{mg}\text{/}dL\right)}=0.90dL\text{/}\mathrm{.Math.}g\mathrm{and}$$B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}\left(\right).$
The B.sub.Free calculated using the single site albumin model deviate significantly from the measured B.sub.Free compared with B.sub.Free calculated using the paired data B.sub.Tmax and K.sub.A.

(10) FIG. 6 illustrates the current treatment guidelines for newborns <28 weeks gestation shown in FIG. 1 modified using bilirubin binding, specifically the B.sub.FreeStandard obtained using the mandatory B.sub.Total phototherapy (6 mg/dL) and exchange transfusion (14 mg/dL) and median B.sub.Tmax (22.0 mg/dL) and K.sub.A (1.16 dL/g) for the population of 31 newborns <28 weeks gestation in TABLE 2

(11) $B_{\mathrm{FreeStandard}}=\frac{\mathrm{Treatment}{B}_{\mathrm{Total}}}{\mathrm{median}{K}_A\left(\mathrm{median}{B}_{\mathrm{Tmax}}-\mathrm{Treatment}{B}_{\mathrm{Total}}\right)}.$
This eliminates the B.sub.Total gray zones where treatment is considered discretionary (uncertain) in FIG. 1.

(12) FIG. 7 illustrates measured B.sub.Free from TABLE 2 (.circle-solid.) versus B.sub.Total; also shown are B.sub.Free calculated at 1 mg/dL increments in B.sub.Total using either the stoichiometric model using the equation

(13) 0$B_{\mathrm{Free}}=\frac{\left(-K_1\left(\mathrm{MR}-1\right)\sqrt{{K_1\left(\mathrm{MR}-1\right)}^2-4(K_1{K}_2\mathrm{MR}\left(\mathrm{MR}-2\right)}\right)}{2K_1{K}_2\left(\mathrm{MR}-2\right)}\left(\right)$
where MR is the B.sub.Total/Atotal molar ratio (TABLE 3) and K.sub.1 (0.93 dL/g) and K.sub.2 (0.04 dL/g) are the best-fit on-linear regression equilibrium constants to the stoichiometric mass action equation

(14) $\mathrm{MR}=\frac{K_1{B}_{\mathrm{Free}}+2K_1{K}_2{B}_{\mathrm{Free}}^2}{1+K_1{B}_{\mathrm{Free}}+K_1{K}_2{B}_{\mathrm{Free}}^2}\mathrm{or}{B}_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}\left(\right)$
using B.sub.Tmax=36.9 and K.sub.A=0.57 dL/g determined by pairing the binding data at B.sub.Total=8.3 and 31.3 mg/dL (TABLE 3). The novel method for quantifying bilirubin binding described herein compares extremely well with the standard stoichiometric method.

(15) FIG. 8 illustrates that the Kp for the horseradish peroxidase catalyzed oxidation of bilirubin by peroxide is determine in bilirubin solutions containing no albumin (i.e. the total bilirubin concentration is equal to the unbound or free bilirubin concentration. Since the total bilirubin concentration is the absorbance at 440 nm divided by the extinction coefficient, the Kp is determined by integrating the velocity equation

(16) $-\frac{\mathrm{dAbsorbance}440\mathrm{nm}}{\mathrm{dt}}=K_p.Math.\mathrm{HRP}.$
Absorbance 440 nm.

(17) FIG. 9 illustrates the light absorbance at 460 nm of a bilirubin/albumin solution as a function of time before and after adding horseradish peroxidase (HRP) and peroxide. The initial absorbance at 460 nm is used to obtain B.sub.Total and the change in absorbance after adding HRP and peroxide is used to obtain the B.sub.Free.

(18) Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

(19) In alternative embodiments, provided are methods, devices and multiplexed systems for assessing whether bilirubin binding is normal in a patient, e.g., a newborn infant, at and risk of bilirubin-induced neurological dysfunction (BIND), and whether the patient has plasma levels of bilirubin requiring treatment, and for diagnosing significant hyperbilirubinemia with increased risk of bilirubin neurotoxicity, including acute bilirubin encephalopathy and BIND. In alternative embodiments, provided are methods for treating or ameliorating, or preventing, the effects of in vivo toxic levels of bilirubin, or treating or ameliorating, or preventing bilirubin-induced neurological dysfunction (BIND), in individuals identified by methods as provided herein.

(20) In alternative embodiments, provided are methods, which can be computer-implemented methods, for converting clinical laboratory data contained in a plasma bilirubin binding panel including: total serum bilirubin concentration (B.sub.Total) and unbound bilirubin or free bilirubin concentration (B.sub.Free) measured before and after bilirubin enrichment to calculate the clinically relevant maximum total bilirubin concentration B.sub.Tmax and its corresponding equilibrium association constant (K.sub.A) outputting the B.sub.Tmax and K.sub.A to quantify how well a patient binds bilirubin and B.sub.Free and B.sub.Total at which the B.sub.FreeStandard for the population occurs to determine whether the risk of BIND is high enough to warrant treatment.

(21) In alternative embodiments, also provided are analytical devices comprising automated micro-fluid handling technologies such as zone fluidics systems, for measuring: total serum bilirubin concentration (B.sub.Total) and unbound bilirubin or free bilirubin concentration (B.sub.Free) from a plasma, serum or blood sample before and after bilirubin enrichment, and also incorporating computer-implemented methods as provided herein to analyze this data and output a bilirubin binding panel including B.sub.Total and B.sub.Free measured before and after bilirubin enrichment, the clinically relevant maximum total bilirubin (B.sub.Tmax) and its corresponding equilibrium association constant (K.sub.A) to compare with B.sub.Tmax and K.sub.A in comparable individuals to accurately determine whether bilirubin binding is normal in a patient, and the clinically relevant diagnostics B.sub.Free and B.sub.Total at which B.sub.FreeStandard occurs, which when compared to the B.sub.FreeStandard in comparable individuals and the current treatment B.sub.Total, respectively, accurately determine the risk of bilirubin-induced neurological dysfunction (BIND). In alternative embodiments, the computer or processor capacity to execute computer-implemented methods as provided herein for analyzing the measured clinical data is built within the device. In other embodiments, provided are systems where the computer or processor capacity to execute computer-implemented methods as provided herein is remote to the device, e.g., a zone fluidics analytical device.

(22) In alternative embodiments, provided are methods, devices and multiplexed systems for assessing whether bilirubin binding is normal in a patient, for example, a newborn infant for the purpose of accurately assessing the presence or risk of acquiring bilirubin-induced neurological dysfunction (BIND) in that patient. The clinical use of bilirubin binding depends on measuring bilirubin binding and knowing the bilirubin binding parameters of the comparable population of newborns (e.g., well term newborns, newborns of the same gestational age as shown in Table 1, etc.). These data answer the questions: (1) Is bilirubin binding normal in a newborn with hyperbilirubinemia)?, and (2) What is the risk of bilirubin-induced neurological dysfunction (BIND)?. For example, if the normal B.sub.Tmax and K.sub.A for the population is optionally the median B.sub.Tmax and K.sub.A, a newborn with B.sub.Tmax and K.sub.A at the 25.sup.th percentile has poor bilirubin binding relative to the population (75% of the population have higher B.sub.Tmax and K.sub.A than the patient). At a mandatory treatment B.sub.Total, e.g. per Table 1, wherein exchange transfusion is mandatory at B.sub.Total=14 mg/dL for newborns less than (<) 28 weeks gestational age, the B.sub.Free at the Table 2 median B.sub.Tmax (22.0 mg/dL) and K.sub.A (1.16 dL/g) is

(23) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}=\frac{14\mathrm{mg}\text{/}dL}{1.16dL\text{/}\mathrm{.Math.}g\left(22.0\mathrm{mg}\text{/}dL-14\mathrm{mg}\text{/}dL\right)}=1.51\mathrm{.Math.}g\text{/}dL$
and at the 25th percentile B.sub.Tmax (14.3 mg/dL) and K.sub.A (0.75 dL/g)

(24) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}=62.2$
g/dL, with much higher risk of BIND. Therefore, determining B.sub.Total, B.sub.Free, B.sub.Tmax and K.sub.A in an individual in need thereof, optionally a newborn infant, quantify how well a newborn binds bilirubin and by comparing these metrics with those in a population of peers, it is possible to determine whether the risk of BIND is increased in the individual in need thereof. Obtaining the comparative population B.sub.Tmax and K.sub.A norms (e.g. mean, SD, range, median, quartiles, etc.) requires measuring them in an appropriate number of comparable newborns, typically about 400 patients, see e.g., Lott J A, et al. Estimation of reference ranges: how many subjects are needed? Clin Chem 1992; 38:648-650), and the B.sub.Free, B.sub.Tmax and K.sub.A in a sample from an individual in need thereof quantify the risk of bilirubin-induced neurological dysfunction (BIND) at the B.sub.Total of the individual in need thereof and determine whether treatment is needed in the individual in need thereof at a B.sub.Total below that at which treatment is currently recommended for the population.

(25) In alternative embodiments, the components of the bilirubin binding panel (BBP) including the measured B.sub.Total and B.sub.Free before and after bilirubin enrichment of the sample and the calculated clinically relevant B.sub.Tmax and its corresponding equilibrium association constant (K.sub.A) are used to determine whether bilirubin binding is normal by comparing B.sub.Tmax and K.sub.A with optionally the median B.sub.Tmax and K.sub.A for the comparable population, and whether the risk of BIND is increased by comparing B.sub.Free with B.sub.FreeStandard as determined for the population at a current treatment B.sub.Total and optionally the median B.sub.Tmax and K.sub.A for the population. Additionally, the actual B.sub.Total at which treatment is needed can be determined using B.sub.FreeStandard and the B.sub.Tmax and K.sub.A. The BBP as provided herein robustly quantifies bilirubin binding and can be used to determine whether bilirubin binding is normal when assessing the need for treatment of hyperbilirubinemia, including jaundice. The BBP can also be used as a screening test to determine the actual B.sub.Total at which the B.sub.FreeStandard and at which treatment may be warranted (e.g. if B.sub.FreeStandard for exchange transfusion is 1.51 g/dL for newborns (<) 28 weeks per TABLES 1 and 2, a newborn in this group with a B.sub.Total of 3.0 mg/dL, a B.sub.Free of 0.18, a B.sub.Tmax of 20 mg/dL, and a K.sub.A of 1.00 dL/g would reach the B.sub.FreeStandard at

(26) $B_{\mathrm{Total}}=\frac{B_{\mathrm{FreeStandard}}.Math.K_A.Math.B_{\mathrm{Tmax}}}{1+\left(K_A.Math.B_{\mathrm{FreeStandard}}\right)}=\frac{1.51\frac{\mathrm{.Math.}g}{dL}.Math.1.00\frac{dL}{\mathrm{.Math.}g}.Math.20.0\frac{\mathrm{mg}}{dL}}{1+\left(1.00\frac{dL}{\mathrm{.Math.}g}.Math.1.51\frac{\mathrm{.Math.}g}{dL}\right)}=12.0\mathrm{mg}\text{/}dL,$
below the mandatory B.sub.Total exchange transfusion of 14 mg/dL). The Bilirubin Binding Panel as determined by methods provided herein, includes and assists rather than competes with B.sub.Total in determining the need for treatment.

(27) In alternative embodiments, provided are methods and systems overcome difficulties in quantifying bilirubin binding using a simple technique that robustly quantifies bilirubin binding over the clinically relevant range of B.sub.Total, e.g. B.sub.Total less than 20 mg/dL for newborns less than (<) 35 weeks gestation (see TABLE 1). In this approach, B.sub.Tmax is not B.sub.Total at which the all the plasma binding sites are occupied with bilirubin but instead the upper limit B.sub.Total of the functioning bilirubin binding sites within the clinically relevant range of B.sub.Total, and K.sub.A is the corresponding composite of the K.sub.1 . . . K.sub.n equilibrium association constants. The chemical equilibrium is:

(28) ##STR00002##
and since B.sub.Free is orders of magnitude less than B.sub.Total at clinically relevant B.sub.Total, B.sub.TotalB.sub.FreeB.sub.Total, the resulting mass action equations are shown below,

(29) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}-B_{\mathrm{Free}}}{K_A\left(B_{\mathrm{Tmax}}-\left(B_{\mathrm{Total}}-B_{\mathrm{Free}}\right)\right)}\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)}.Math.B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)}$
can be readily solved by for B.sub.Tmax and K.sub.A by measuring B.sub.Total and B.sub.Free before and after enrichment of the sample with bilirubin to provide B.sub.Total, B.sub.Free, and B.sub.Total_2, B.sub.Free_2. These provide two equations with two unknowns (B.sub.Tmax and K.sub.A), that can be solved for B.sub.Tmax as shown below:

(30) $B_{\mathrm{Tmax}}=\frac{B_{\mathrm{Total}}{B}_{\mathrm{Total\_}2}\left(B_{\mathrm{Free\_}2}-B_{\mathrm{Free}}\right)}{B_{\mathrm{Total}}{B}_{\mathrm{Free\_}2}-B_{\mathrm{Total\_}2}{B}_{\mathrm{Free}}}$
The calculated B.sub.Tmax, B.sub.Total, and B.sub.Free are then entered into

(31) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$
to obtain K.sub.A

(32) $K_A\left(K_A=\frac{B_{\mathrm{Total}}}{B_{\mathrm{Free}}\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}\right),$
or alternatively, K.sub.A is the negative intercept and B.sub.Tmax is the negative slope divided by the intercept of

(33) 0$\frac{1}{B_{\mathrm{Free}}}\mathrm{versus}\frac{1}{B_{\mathrm{Total}}}$
as the reciprocal of

(34) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$
is the linear equation

(35) $\frac{1}{B_{\mathrm{Free}}}=\frac{B_{\mathrm{Tmax}}.Math.K_A}{B_{\mathrm{Total}}}-K_A.$

(36) TABLE 3 shows a bilirubin binding isotherm obtained in artificial serum containing bilirubin and with a human defatted albumin concentration (A.sub.Total) of 3.0 g/dL. FIG. 5 illustrates the change in B.sub.Free (black dots) as B.sub.Total increases. The negative intercept of 1/B.sub.Free versus 1/B.sub.Total, i.e. K.sub.A, is 0.53 g/dL and the negative slope/intercept, i.e. B.sub.Tmax, is 37.5 mg/dL. The B.sub.Tmax and K.sub.A in Table 3 are calculated as described above using the lowest B.sub.Total (8.3 mg/dL) and B.sub.Free (0.51 g/dL) paired with each of the other five measures of B.sub.Total and B.sub.Free. The mean B.sub.Tmax and K.sub.A of all 15 possible pairings in TABLE 4 are 39.1 mg/dL and 0.56 dL/g, respectively. The B.sub.Free calculated over 1 mg/dL increases in B.sub.Total using the B.sub.Tmax (36.9 mg/dL) and K.sub.A (0.57 dL/g) obtained from pairing B.sub.Total=8.3 mg/dL and B.sub.Total=31.3 mg/dL overlap the measured binding points illustrated by the open orange circles in FIG. 5, but if B.sub.Tmax=is assumed to be A.sub.Total=26.4 mg/dL, and

(37) $K_A=\frac{B_{\mathrm{Total}}}{B_{\mathrm{Free}}\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}=\frac{8.3\mathrm{mg}\text{/}dL}{0.51\mathrm{.Math.}g\text{/}dL\left(26.4\mathrm{mg}\text{/}dL-8.3\mathrm{mg}\text{/}dL\right)}=0.90dL\text{/}\mathrm{.Math.}g,$
the calculated B.sub.Free deviate deviated significantly from the measured binding points as illustrated by the open blue circles in FIG. 5. This suggests B.sub.Tmax and the albumin concentration are not closely related, and plasma bilirubin binding sites are closely related to even though bilirubin is known to bind primarily to plasma albumin, and B.sub.Tmax and A.sub.Total in the 31 newborns less than 28 weeks gestation did not correlate significantly (r.sup.2=0.02).

(38) TABLE-US-00003 TABLE 3 B.sub.Total/A.sub.Total B.sub.Total B.sub.Free B.sub.Tmax K.sub.A Molar Ratio mg/dL g/dL mg/dL dL/g 0.31 8.3 0.51 0.44 11.7 0.92 24.3 1.01 0.69 18.5 1.70 39.1 0.53 0.83 22.2 2.39 40.8 0.50 1.06 28.3 6.05 36.4 0.58 1.18 31.3 9.88 36.9 0.57

(39) Quantifying bilirubin binding by determining B.sub.Tmax and K.sub.A in a population of newborns can be used to reduce the uncertainties in the current B.sub.Total guidelines for treatment (e.g. TABLE 1, FIG. 1). TABLE 2 summarizes binding data from 31 newborns less than (<) 28 weeks gestation, and knowing, e.g. the median, optionally the mean or average B.sub.Tmax and K.sub.A of a population, then a standard B.sub.Free, i.e. B.sub.FreeStandard can be designated at a current treatment B.sub.Total (e.g. TABLE 1) and, e.g. the B.sub.FreeStandard at the median B.sub.Tmax and K.sub.A of the population is:

(40) $B_{\mathrm{FreeStandard}}=\frac{\mathrm{Treatment}{B}_{\mathrm{Total}}}{\mathrm{median}{K}_A\left(\mathrm{median}{B}_{\mathrm{Tmax}}-\mathrm{Treatment}{B}_{\mathrm{Total}}\right)}$
wherein, all else being comparable, half the population has a lower and half a higher risk of BIND versus the (usually unknown) risk of BIND at B.sub.FreeStandard. For the half at greater risk of BIND B.sub.FreeStandard occurs at a B.sub.Total below the treatment B.sub.Total, i.e. at the individual's B.sub.Total, B.sub.Tmax, and K.sub.A where

(41) $B_{\mathrm{FreeStandard}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)}.$
The risk of BIND in an individual is the same as that for the population at B.sub.FreeStandard when the individual's measured B.sub.Free is equal to or greater B.sub.FreeStandard or when the individual's B.sub.Total is equal to

(42) $\frac{B_{\mathrm{FreeStandard}}.Math.K_A.Math.B_{\mathrm{Tmax}}}{1+\left(K_A.Math.B_{\mathrm{FreeStandard}}\right)}$
calculated using the patient's B.sub.Tmax, and K.sub.A as the tissue levels of bilirubin, brain bilirubin exposure, and risk of BIND will be similar irrespective of the B.sub.Total.

(43) Novel methods for using measured serum or plasma B.sub.Total and B.sub.Free to obtain B.sub.Tmax and K.sub.A are provided herein as are their use to quantify bilirubin binding and assess the risk of BIND within the context of and reducing the uncertainties of current B.sub.Total guidelines for treatment of newborn hyperbilirubinemia as illustrated in FIG. 6 compared with FIG. 1. These data (B.sub.Total, B.sub.Free, B.sub.Tmax, and K.sub.A) comprise a Bilirubin Binding Panel (BBP) (see Ahlfors C E. The Bilirubin Binding Panel: A Henderson-Hasselbalch approach to neonatal hyperbilirubinemia. Pediatrics 2016; 138: e20154378) that will significantly reduce the uncertainties inherent in current treatment guidelines that use B.sub.Total only.

(44) Quantifying Plasma Bilirubin Binding:

(45) Defining normal bilirubin binding requires determining (1) how much bilirubin can be bound, and (2) how tightly bilirubin is bound. Since bilirubin binds mostly to plasma albumin, the concentration of albumin (A.sub.Total) has long been used to estimate how much bilirubin can be bound, usually assuming that one albumin molecule binds one bilirubin molecule. However, albumin molecules can bind more than one bilirubin molecule and A.sub.Total per se is not a clinically useful estimate of how much bilirubin can be bound (i.e. B.sub.Tmax).

(46) Since each albumin molecule binds at least two bilirubin molecules over the clinically relevant range B.sub.Total encountered in newborns with hyperbilirubinemia (see FIG. 5), graphic analysis has often been used to quantify bilirubin binding (e.g. Jacobsen J. Binding of bilirubin to human serum albuminDetermination of the Dissociation Constants. FEBS Lett 1969; 5: 112-114), or alternatively non-linear regression analysis of the polynomial mass action equations associated with multiple site binding are used (e.g. see Honor B, Brodersen R. Albumin binding of anti-inflammatory drugs. Utility of a site-oriented versus a stoichiometric analysis. Mol Pharmacol 1984; 25: 137-150 and Klotz I M, Hunston D L. Protein affinities for small molecules: Conceptions and misconceptions. Arch Biochem Biophys 1979; 193: 314-328). The stoichiometric two-site binding model measures the concentrations the plasma albumin (A.sub.Total), the total bilirubin (B.sub.Total) and the non-albumin bound or free bilirubin (B.sub.Free) measured at multiple B.sub.Total and uses them to determine the two equilibrium association constants for the albumin molecules binding one (K.sub.1) and those binding two bilirubin molecules (K.sub.2). In this model the B.sub.Total is the sum of the concentrations of albumin binding one (A:B.sub.1) and twice that binding two bilirubin molecules (2A:B.sub.2) plus B.sub.Free and the A.sub.Total is the sum of A:B.sub.1+A:B.sub.2+the concentration of unoccupied or free albumin binding sites (A.sub.Free) binding no bilirubin. The chemical equilibrium is:
A.sub.Free+B.sub.Free

(47) $\underset{}{\underset{.fwdarw.}{K_1,K_2}}$
A:B.sub.1+2A:B.sub.2,

(48) and the mass action equations are:

(49) $\frac{A\text{:}{B}_1+2A\text{:}{B}_2}{A_{\mathrm{Total}}}\frac{B_{\mathrm{Total}}}{A_{\mathrm{Total}}}=\mathrm{Molar}\mathrm{Ratio}\frac{K_1{B}_{\mathrm{Free}}+2K_1{K}_2{B}_{\mathrm{Free}}^2}{1+K_1{B}_{\mathrm{Free}}+K_1{K}_2{B}_{\mathrm{Free}}^2}$
which can be solved for B.sub.Free using the equation below wherein MR is the molar ratio:

(50) $B_{\mathrm{Free}}=\frac{\left(-K_1\left(\mathrm{MR}-1\right)\sqrt{{K_1\left(\mathrm{MR}-1\right)}^2-4(K_1{K}_2\mathrm{MR}\left(\mathrm{MR}-2\right)}\right)}{2K_1{K}_2\left(\mathrm{MR}-2\right)}$

(51) Non-linear regression analysis of the molar ratios versus the B.sub.Free from TABLE 3 were used to determine the best fit K.sub.1 (0.93 dL/g) and K.sub.2 (0.04 dL/g) using the stoichiometric equation

(52) 0$\mathrm{Molar}\mathrm{Ratio}\frac{K_1{B}_{\mathrm{Free}}+2K_1{K}_2{B}_{\mathrm{Free}}^2}{1+K_1{B}_{\mathrm{Free}}+K_1{K}_2{B}_{\mathrm{Free}}^2}.$
FIG. 7 compares the calculated B.sub.Free at 1 mg/dL B.sub.Total increments using

(53) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$
using the B.sub.Tmax (36.9 mg/dL) and K.sub.A (0.57) obtained from the pairing the B.sub.Total of 8.3 mg/dL with B.sub.Total of 31.3 mg/dL in Table 3 versus

(54) $B_{\mathrm{Free}}=\frac{\left(-K_1\left(\mathrm{MR}-1\right)\sqrt{{K_1\left(\mathrm{MR}-1\right)}^2-4(K_1{K}_2\mathrm{MR}\left(\mathrm{MR}-2\right)}\right)}{2K_1{K}_2\left(\mathrm{MR}-2\right)}$
and shows that the novel method for quantifying bilirubin binding compares quite favorably with the standard stochiometric method for quantifying binding. The clear advantage of the novel method is that it provides robust binding analysis yet requires only two data points and no measurement of A.sub.Total and therefore much less time and materials for the measurements needed to quantify bilirubin binding.

(55) A more clinically applicable approach to quantifying bilirubin binding is to consider both how much (B.sub.Tmax) and how tightly (K.sub.A) bilirubin can be bound as unknowns and derive these unknowns from B.sub.Total and B.sub.Free measurements. This requires a novel approach to the routine measurement of B.sub.Total and B.sub.Free, which is to measure B.sub.Total and B.sub.Free in a plasma sample before and after enrichment of the sample with bilirubin.

(56) The plasma equilibrium concentrations at any given plasma B.sub.Total and (unknown) B.sub.Tmax are:

(57) ##STR00003##

(58) wherein B.sub.Tmax is how much bilirubin can be bound, B.sub.TmaxB.sub.Total is the concentration of available (unoccupied) bilirubin binding sites, and B.sub.TotalB.sub.Free is the concentration of bilirubin bound to plasma binding sites (since B.sub.Free is orders of magnitude less than B.sub.Total, bound bilirubin=B.sub.TotalB.sub.FreeB.sub.Total).

(59) The mass action equation is

(60) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)}$
wherein B.sub.total and B.sub.Free are measured and B.sub.Tmax and K.sub.A are unknown. If B.sub.Total and B.sub.Free are measured before and after sample enrichment with bilirubin to give measured values B.sub.Total, B.sub.Free and B.sub.Total_2, B.sub.Free_2, two equations with two unknowns (B.sub.Tmax and K.sub.A) are provided that can be solved for B.sub.Tmax and K.sub.A as shown below.

(61) $B_{\mathrm{Tmax}}=\frac{B_{\mathrm{Total}}.Math.B_{\mathrm{Total\_}2}\left(B_{\mathrm{Free\_}2}-B_{\mathrm{Free}}\right)}{B_{\mathrm{Total}}.Math.B_{\mathrm{Free\_}2}-B_{\mathrm{Total}}.Math.B_{\mathrm{Free\_}1}}$
The calculated B.sub.Tmax, B.sub.Total, and B.sub.Free are then entered into

(62) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$
to obtain

(63) $K_A=\frac{B_{\mathrm{Total}}}{B_{\mathrm{Free}}\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)},$
or alternatively, K.sub.A is the negative intercept and B.sub.Tmax is the negative slope divided by the intercept of

(64) $\frac{1}{B_{\mathrm{Free}}}\mathrm{versus}\frac{1}{B_{\mathrm{Total}}}$
as the
reciprocal of

(65) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$
is the linear equation

(66) $\frac{1}{B_{\mathrm{Free}}}=\frac{B_{\mathrm{Tmax}}.Math.K_A}{B_{\mathrm{Total}}}-K_A.$

(67) The clinically relevant quantification of bilirubin binding are the mass action variables above (B.sub.Total, B.sub.Free, B.sub.Total_2, B.sub.Free_2, B.sub.Tmax, and K.sub.A) constitute a Bilirubin Binding Panel (BBP). Optionally, B.sub.Tmax and K.sub.A can be used to determine whether a newborn binds bilirubin normally. TABLE 4 below shows B.sub.Tmax and K.sub.A determined before and after adding sulfisoxazole (sulfa) to a bilirubin/human albumin sample containing 3.0 g/dL albumin, which about doubles the B.sub.Free and significantly changes B.sub.Tmax and K.sub.A. A newborn less than (<) 28 weeks gestation with a B.sub.Total of 8.3, B.sub.Free of 0.51 g/dL, B.sub.Tmax of 24.3 mg/dL and K.sub.A of 1.01 per Tables 1 and 2 would reach the exchange transfusion B.sub.FreeStandard of 1.51 g/dL (FIG. 6) at when the B.sub.Total reaches

(68) 0$\frac{B_{\mathrm{FreeStandard}}.Math.K_A.Math.B_{\mathrm{Tmax}}}{1+\left(K_A.Math.B_{\mathrm{FreeStandard}}\right)}=\frac{1.51\frac{\mathrm{.Math.}g}{dL}.Math.1.01\frac{dL}{\mathrm{.Math.}g}.Math.24.3\frac{\mathrm{mg}}{dL}}{1+\left(1.01\frac{dL}{\mathrm{.Math.}g}.Math.1.51\frac{\mathrm{.Math.}g}{dL}\right)}=24.2\mathrm{mg}\text{/}dL,$
but if B.sub.Tmax is 42.1 mg/dL and K.sub.A is 0.20 dL/ug,

(69) $\frac{1.51\frac{\mathrm{.Math.}g}{dL}.Math.0.20\frac{dL}{\mathrm{.Math.}g}42.1\frac{\mathrm{mg}}{dL}}{1+\left(0.20\frac{dL}{\mathrm{.Math.}g}1.51\frac{\mathrm{.Math.}g}{dL}\right)}=9.8\mathrm{mg}\text{/}dL$
showing the much risk of BIND despite identical B.sub.Total of 8.3 mg/dL.

(70) TABLE-US-00004 TABLE 4 B.sub.Total B.sub.Total.sub..sub.2 B.sub.Free B.sub.Freel.sub..sub.2 B.sub.Tmax K.sub.A mg/dL mg/dL g/dL g/dL mg/dL dL/g No sulfa 8.3 11.7 0.51 0.92 24.3 1.01 + sulfa 8.3 12.3 1.22 2.05 42.1 0.20
Measuring Total and Unbound Bilirubin (B.sub.Total and B.sub.Free): The peroxidase test (see, e.g., Jacobsen J, Wennberg R P. Determination of unbound bilirubin in the serum of newborns. Clin Chem 1974; 20:783-789) measures both B.sub.Total and B.sub.Free. This test is used clinically in Japan. In alternative embodiments, novel modifications of methods as provided herein measure B.sub.Free at two horseradish peroxidase levels to accurately determine B.sub.Free and measure B.sub.Total and B.sub.Free before and after bilirubin enrichment of a plasma or other blood sample to provide B.sub.Tmax and K.sub.A to complete the Bilirubin Binding Panel (BBP) described herein. The BBP quantifies bilirubin binding (B.sub.Tmax and K.sub.A) and the risk of BIND using B.sub.FreeStandard determined for a comparable population. The peroxidase test is based on the horse radish peroxidase (HRP) catalyzed oxidation of bilirubin by peroxide. Bilirubin absorbs light maximally at 440 nm when no albumin is present and at 460 nm when bound to albumin. Bilirubin bound to albumin is protected from oxidation and only B.sub.Free is oxidized. The light absorbance at 440 nm (no albumin) or 460 nm (albumin present) decreases as bilirubin is oxidized, and the reaction rate constant, Kp, can be determined using known bilirubin and HRP concentrations in solutions without albumin present (i.e. all the bilirubin is unbound or free) as shown in the equivalent velocity equations below:

(71) $-\frac{d{B}_{\mathrm{Total}}}{\mathrm{dt}}=K_p.Math.\mathrm{HRP}.Math.B_{\mathrm{Total}}.-\frac{\mathrm{dAbsorbance}440\mathrm{nm}}{\mathrm{dt}}=K_p.Math.\mathrm{HRP}.Math.\mathrm{Absorbance}440\mathrm{nm}.$

(72) DETERMINATION OF K.sub.P: FIG. 8 graphically illustrates the change in bilirubin absorbance per second (s) at 440 nm and 460 nm during HRP catalyzed oxidation of bilirubin by peroxide without albumin present as recorded using an HP8452 computer directed spectrophotometer (reaction: 3.0 mL of 0.1 M phosphate buffer, pH 7.4 containing 128 mol/L H.sub.2O.sub.2, 25 L HRP with reaction [HRP]=0.061 g/mL, 5 L of 1 mg/mL bilirubin solution with reaction [B.sub.Total]=163 g/dL, 1 cm path length cuvette, 30 C.).

(73) The K.sub.P.Math.HRP for the reaction is easily calculated by integrating the velocity equation above between times t=0 and t=t:

(74) $-{}_0^t\frac{d{B}_{\mathrm{Total}}}{\mathrm{dt}}={}_0^t{K}_p.Math.\mathrm{HRP}.Math.B_{\mathrm{Total}}$$\ln \left(\frac{B_{\mathrm{Total}}}{B_{\mathrm{Total}\mathrm{at}t=0}}\right)=\ln \left(\frac{\mathrm{Absorbance}440\mathrm{nm}}{\mathrm{Absorbance}440\mathrm{nm}\mathrm{at}t=0}\right)=-K_p.Math.\mathrm{HRP}.Math.t$

(75) K.sub.p.Math.HRP is the negative slope of the

(76) $\ln \left(\frac{\mathrm{Absorbance}440\mathrm{nm}}{\mathrm{Absorbance}440\mathrm{nm}\mathrm{at}t=0}\right)$
versus time, which divided by the reaction HRP concentration provides the K.sub.p.

(77) DETERMINATION OF B.sub.Total AND B.sub.Free: FIG. 9 graphically illustrates light absorbance at 460 nm as a function of time in seconds and shows the change in bilirubin absorbance at 460 nm in a bilirubin-albumin solution before and after adding HRP and peroxide as recorded using an HP8452 computer directed spectrophotometer. The initial absorbance at 460 nm is used to obtain B.sub.Total and the change in absorbance at 460 nm after adding HRP/peroxide is used to obtain the B.sub.Free as described below.

(78) The standard reaction is conducted in a 1 cm path cuvette containing 1.0 mL of 0.1 M phosphate buffer, pH 7.4 to which 25 L of sample (e.g. plasma or serum), followed by 25 L of HRP (typically 1.5 mg/mL) and 5 L of 26 mmol/f H.sub.2O.sub.2 to provide a reaction H.sub.2O.sub.2 of 120 mol/L H.sub.2O.sub.2. B.sub.Total is calculated from the absorbance prior to adding HRP and H.sub.2O.sub.2 and B.sub.Free from the change in absorbance following addition of HRP/H.sub.2O.sub.2 as further described below. The novel changes to the method involve repeating the test at another HRP concentration (typically using 0.75 mg/mL) and then enriching the sample with bilirubin (typically to increase the B.sub.Total by 5 to 20 mg/dL) and repeating the test again at two HRP concentrations.

(79) B.sub.Total is calculated by dividing the initial absorbance by the known extinction coefficient (0.827/cm light path length for B.sub.Total in mg/mL) and B.sub.Free is calculated from the change in absorbance at 460 nm after adding HRP/H.sub.2O.sub.2. Since only B.sub.Free is oxidized (bound bilirubin is protected from oxidation), the velocity equation is

(80) $-\frac{{\mathrm{dB}}_{\mathrm{Total}}}{\mathrm{dt}}=K_p.Math.\mathrm{HRP}.Math.B_{\mathrm{Free}}.$
However, the equilibrium B.sub.Free falls to an unknown lower steady state free bilirubin (B.sub.Fss) as the oxidation of B.sub.Free disrupts the equilibrium from

(81) $\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)+B_{\mathrm{Free}}\stackrel{K_A}{}{B}_{\mathrm{Total},}\mathrm{to}\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)+B_{\mathrm{Fss}}+H_2{O}_2\underset{K_A}{\overset{\mathrm{HRP}}{}}{B}_{\mathrm{Total}}+\mathrm{oxidized}\mathrm{bilirubin}.$

(82) and, therefore

(83) $-\frac{d{B}_{\mathrm{Total}}}{\mathrm{dt}}=K_p.Math.\mathrm{HRP}.Math.B_{\mathrm{Fss}}$

(84) wherein:

(85) $B_{\mathrm{Fss}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)+K_p.Math.\mathrm{HRP}},$
and the integrated velocity is:

(86) ${}_0^t\frac{d{B}_{\mathrm{Total}}}{\mathrm{dt}}=-{}_0^t{K}_p.Math.\mathrm{HRP}.Math.B_{\mathrm{Fss}}=-{}_0^t\frac{K_p.Math.\mathrm{HRP}.Math.B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)+K_p.Math.\mathrm{HRP}},\mathrm{or}$${}_0^t\frac{d{B}_{\mathrm{Total}}}{B_{\mathrm{Total}}}=-{}_0^t\frac{K_p.Math.\mathrm{HRP}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)+K_p.Math.\mathrm{HRP}}\mathrm{dt}$$\ln \left(\frac{B_{\mathrm{Total}}}{B_{\mathrm{Total}}\mathrm{at}t=0}\right)=-\frac{K_p.Math.\mathrm{HRP}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)+K_p.Math.\mathrm{HRP}}t\mathrm{and}$$B_{\mathrm{Fss}}=\frac{-B_{\mathrm{Total}}.Math.\mathrm{Slope}}{\mathrm{Kp}.Math.\mathrm{HRP}}$

(87) wherein

(88) 0$\mathrm{Slope}=-\frac{K_p.Math.\mathrm{HRP}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)+K_p.Math.\mathrm{HRP}}$
obtained from ln

(89) $\left(\frac{B_{\mathrm{Total}}}{B_{\mathrm{Total}}\mathrm{at}t=0}\right)$ versus t.

(90) B.sub.Free is obtained by measuring B.sub.Fss at the additional HRP concentration and using

(91) $\frac{1}{B_{\mathrm{Fss}}}=\frac{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)}{B_{\mathrm{Total}}}+\frac{K_p.Math.\mathrm{HRP}}{B_{\mathrm{Total}}},$
which is the reciprocal of the B.sub.Fss equation above to obtain the intercept of 1/B.sub.Fss versus

(92) $\mathrm{HRP}\left(\frac{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)}{B_{\mathrm{Total}}}\right),$
the reciprocal of which is B.sub.Free

(93) $(B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$

(94) The sample is enriched with bilirubin and B.sub.Total_2 and B.sub.Free_2 are measured and used with the pre-enrichment B.sub.Total and B.sub.Free to obtain B.sub.Tmax and K.sub.A as seen in TABLES 2 and 3.

(95) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmax}}-B_{\mathrm{Total}}\right)}$
can then be readily solved by for B.sub.Tmax and K.sub.A using two equations with two unknowns (B.sub.Tmax and K.sub.A) and solving for B.sub.Tmax as shown below:

(96) $B_{\mathrm{Tmax}}=\frac{B_{\mathrm{Total}}{B}_{\mathrm{Total\_}2}\left(B_{\mathrm{Free\_}2}-B_{\mathrm{Free}}\right)}{B_{\mathrm{Total}}{B}_{\mathrm{Free\_}2}-B_{\mathrm{Total\_}2}{B}_{\mathrm{Free}}}$
The calculated B.sub.Tmax, B.sub.Total, and B.sub.Free are then entered into

(97) $B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$
to obtain

(98) $K_A=\frac{B_{\mathrm{Total}}}{B_{\mathrm{Free}}\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)},$
or alternatively, K.sub.A is the negative intercept and B.sub.Tmax is the negative slope divided by the intercept of

(99) $\frac{1}{B_{\mathrm{Free}}}\mathrm{versus}\frac{1}{B_{\mathrm{Total}}}$
as the reciprocal of

(100) 00$B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)}$
is the linear equation

(101) 01$\frac{1}{B_{\mathrm{Free}}}=\frac{B_{\mathrm{Tmax}}.Math.K_A}{B_{\mathrm{Total}}}-K_A$
The Bilirubin Binding Panel:

(102) The components of bilirubin binding are linked mathematically by the mass action equation

(103) 02$B_{\mathrm{Free}}=\frac{B_{\mathrm{Total}}}{K_A\left(B_{\mathrm{Tmsx}}-B_{\mathrm{Total}}\right)},$
which makes no assumptions about the stoichiometric or chemical nature of the actual plasma bilirubin binding sites, yet the constants B.sub.Tmax and K.sub.A provide accurate estimates of B.sub.Free at B.sub.Total below B.sub.Tmax as illustrated in FIGS. 5 and 7. In one embodiment, the peroxidase test measures serum or plasma B.sub.Total and B.sub.Free, e.g., as described by Jacobsen J. Wennberg R P. Determination of unbound bilirubin in the serum of newborns. Clin Chem 1974; 20:783-789. In alternative embodiments, B.sub.Free is measured at a second peroxidase concentration to insure accurate measurement of B.sub.Free and the sample is then enriched with bilirubin and the test repeated at the higher B.sub.Total and B.sub.Free to provide B.sub.Tmax and K.sub.A using two equations and two unknowns or using linear analysis of

(104) 03$\frac{1}{B_{\mathrm{Free}}}=\frac{K_A.Math.B_{\mathrm{Tmax}}}{B_{\mathrm{Total}}}-K_A$
wherein the negative of the intercept=intercept=K.sub.A, and the negative of the

(105) 04$\mathrm{slope}\text{/}\mathrm{intercept}=\frac{-\mathrm{slope}}{\mathrm{intercept}}=\frac{-K_A.Math.B_{\mathrm{Tmax}}}{-K_A}=B_{\mathrm{Tmax}}.$

(106) An individual's B.sub.Tmax and K.sub.A are compared with, optionally the median B.sub.Tmax and K.sub.A for the comparable population to determine whether the individual has normal bilirubin binding. The risk of BIND is increased if the individual's B.sub.Free is greater than or equal to a B.sub.FreeStandard for the population that is determined determine whether the individual has normal bilirubin binding. The risk of BIND is increased if the individual's B.sub.Free is greater than or equal to a B.sub.FreeStandard for the population that is determined using current B.sub.Total treatment guidelines and optionally the population's median B.sub.Tmax and K.sub.A, e.g. for newborns less than (<) 28 weeks gestation per Table 1, at the mandatory exchange transfusion B.sub.Total of 14 mg/dL and the median B.sub.Tmax (22.0 mg/dL) and K.sub.A (1.16 dL/g) for the 31 newborns in Table 2,

(107) 05$B_{\mathrm{FreeStandard}}=\frac{B_{\mathrm{Total}}\left(14\frac{\mathrm{mg}}{dL}\right)}{K_A\left(1.16\frac{dL}{\mathrm{.Math.}g}\right)\left(B_{\mathrm{Tmsx}}\left(22.0\frac{\mathrm{mg}}{dL}\right)-B_{\mathrm{Total}}\left(14\frac{\mathrm{mg}}{dL}\right)\right)}1.51\mathrm{.Math.}g\text{/}dL,$
and if an individual's B.sub.Free is equal to or greater than () B.sub.FreeStandard treatment is warranted irrespective of the B.sub.Total, and if B.sub.Free is less than B.sub.FreeStandard, the individual's unique B.sub.Total at which B.sub.FreeStandard occurs and treatment is needed can be obtained using the individual's B.sub.Tmax, K.sub.A, and B.sub.FreeStandard as

(108) 06$B_{\mathrm{Total}}=\frac{B_{\mathrm{Tmax}}.Math.K_A.Math.B_{\mathrm{FreeStandard}}}{1+\left(K_A.Math.B_{\mathrm{FreeStandard}}\right)}.$

(109) B.sub.Tmax and K.sub.A robustly quantify how well the plasma binds bilirubin as they quantify how much (B.sub.Tmax) and how tightly (K.sub.A) plasma binds bilirubin. Comparing B.sub.Tmax and K.sub.A in a newborn with B.sub.Tmax and K.sub.A in a population of peers (e.g. comparing them with the median B.sub.Tmax and K.sub.A) determines how well that newborn binds bilirubin compared with its peers, just as any blood test in a patient is compared with normal values in the population to detect underlying conditions. If the newborn's B.sub.Free is equal to or exceeds B.sub.FreeStandard the population as described above, treatment is warranted irrespective of the B.sub.Total. If the newborn's B.sub.Free is less than B.sub.FreeStandard the unique B.sub.Total at which that newborn should be treated is obtained from the B.sub.FreeStandard and that newborn's B.sub.Tmax and K.sub.A as shown above. This approach reduces the uncertainties in the current treatment guidelines that use B.sub.Total alone (see FIG. 1) and individualizes care.

(110) B.sub.Tmax and K.sub.A population parameters (mean, standard deviation, median, etc.) can be readily obtained in the various newborn populations (term, premature <28 weeks as shown in Table 2, ill, etc.) to provide the population specific bilirubin binding data needed to augment treatment decisions that are currently based solely on B.sub.Total.

(111) Devices: Zone Fluidics Analytical Instruments

(112) The manual peroxidase test as originally described to measure B.sub.Total and B.sub.Free requires 25 L sample, and for the four tests described herein (B.sub.Total and B.sub.Free measured at two peroxidase concentrations before and after bilirubin enrichment) would require 100 L of sample. Novel herein are technologies that automate the tests and reduce sample volumes.

(113) In alternative embodiments, provided are devices and systems comprising automated micro-fluid handling technologies such as zone fluidics systems, and the appropriate chemistry, e.g., robotic chemistry, for the handling and manipulation of samples, e.g., serum, plasma or whole blood samples from patients, for measuring: total serum bilirubin concentration (B.sub.Total) and unbound bilirubin or free bilirubin concentration (B.sub.Free) from a plasma or blood sample (Jacobsen J, Wennberg R P. Determination of unbound bilirubin in the serum of newborns. Clin Chem 1974; 20:783, Ahlfors C E, et. al. Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics Clin Chim Acta 2006; 365:78), and also incorporatingdirectly in the device or indirectly as a multiplexed system operatively connected to the devicecomputer-implemented methods as provided herein to analyze this data and output a maximum bilirubin concentration (B.sub.Tmax) and a bilirubin binding constant (K.sub.A), which when compared to the product in a population of peers accurately determines how well a patient binds bilirubin and whether the risk of bilirubin-induced neurological dysfunction (BIND) as measured by the B.sub.Free is greater than the risk at B.sub.FreeStandard for the population of peers.

(114) In alternative embodiments, provided are devices comprising Sequential Injection Analysis (SIA) and/or Zone Fluidics technology, and equivalent automated micro-fluid handling technologies, for handling and analyzing patient blood, serum, or plasma and expanding these technologies to include titration with bilirubin to enable calculation of B.sub.Tmax and K.sub.A.

(115) In alternative embodiments, provided are devices comprising components, e.g., robotic chemistry components, for measuring: total serum bilirubin concentration (B.sub.Total); unbound bilirubin or free bilirubin concentration (B.sub.Free) from a sample, e.g., a plasma, serum, or a blood sample. Any chemistry, device or robotic chemistry component known in the art can be used or incorporated into a device as used and/or provided herein, e.g., as described in U.S. Pat. No. 7,939,333 (describing metal enhanced fluorescence-based sensing methods); U.S. Pat. No. 7,767,467 (describing e.g., methods and device for the separation of small particles or cells from a fluid suspension); U.S. Pat. No. 7,416,896 (describing e.g., methods and devices for determining total and bound plasma bilirubin); U.S. Pat. No. 7,625,762 (describing e.g., methods and device for the separation of small particles or cells from a fluid suspension); U.S. Pat. No. 6,887,429 (describing e.g., methods and apparatus for the automation of existing medical diagnostic tests); U.S. Pat. No. 6,692,702 (describing e.g., methods and apparatus for utilizing a filtration device for removing interferants from a sample containing cells in an automated apparatus); and, U.S. Pat. No. 6,613,579; or, as described in U.S. patent publications: e.g., U.S. Pat App no. 2018/0045723 A1 (describing e.g., lateral flow devices and methods for analyzing a fluid sample); U.S. Pat App no. 2018/0052093 A1 (describing e.g., devices and methods for analyzing particles in a sample); U.S. Pat App no. 2016/0245799; or, as described in: Amin, S. B., Clinical Perinatology 43 (2016) 241-257 (describing e.g., a peroxidase method for measuring plasma bilirubin binding); Ahlfors, et al., Clinical Biochemistry 40 (2007) 261-267 (describing e.g., effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns); Ahlfors, C. E., Analytical Biochemistry 279, 130-135 (2000) (describing e.g., measurement of plasma unbound unconjugated bilirubin; Ahlfors, et al., Clinica Chimica Acta 365 (2006) 78-85 (describing, e.g., measurement of unbound bilirubin by the peroxidase test using Zone Fluidics); Wennberg et al., Pediatrics 117 (2006) 474-485; or, WO 2013032953 A2, Huber et al, Clinical Chemistry 58 (2012) 869-876 (describing e.g., fluorescent probes that undergo fluorescence quenching when binding bilirubin to quantify unbound bilirubin).

(116) In alternative embodiments, provided are devices having the capacity to output or send relevant data to a device-incorporated or separate device or system for executing a computer implemented method as provided herein, which then calculates and outputs: bilirubin binding constant, maximum total bilirubin concentration, and the clinically relevant diagnostic product B.sub.TmaxK.sub.A obtained from the measured components of the Bilirubin Binding Panel described above.

(117) In alternative embodiments, provided are Zone Fluidics systems having flow manifolds that are simple and robust, e.g., comprising a pump, selection valve, and detector connected by micro-bore tubing. The same manifold can be used for widely different chemistries simply by changing the flow program rather than the plumbing architecture and hardware. In alternative embodiments, provided are Zone Fluidics acting as a fluidics analytical robotic system. In alternative embodiments, specific strengths of this exemplary embodiment of a microfluidics technology that can include one, several or all of the following characteristics or advantages: can process sample volumes in the lower microliter range; can add bilirubin to a sample to enable measurement of B.sub.Total and B.sub.Free before and after sample bilirubin enrichment. can achieve the performance of high-end clinical chemistry systems or robotically enabled systems at a significantly lower price point; can achieve scalability to a point of care instrumentlow cost of goods sold; can be computer controlled and automated; can be easily developed with modified methodsthe flexibility in workflow; and kinetics enables the method to be optimized to produce the highest quality data without limitations from the hardware design; can fully automate complex methods; can provide improved reliability and easy maintenance; can drastically reduce reagent use (many other methods typically use 1 to 100 mL of reagents per measurement)SIA typically uses 1 to 100 L per measurement.

(118) In alternative embodiments, a Zone Fluidics system as described in U.S. Pat. No. 7,416,896, or apparatus or components as described in U.S. Pat App no. US 2016/0245799, are used to practice alternative device embodiments as provided herein.

(119) Computer Systems for Executing Computer-Implemented Methods:

(120) In alternative embodiments, provided are computer-implemented methods to analyze laboratory data and output a Bilirubin Binding Panel including B.sub.Total and B.sub.Free before and after bilirubin enrichment of plasma or blood sample, the maximum B.sub.Total (B.sub.Tmax) and the bilirubin binding constant (K.sub.A) that provide the clinically relevant B.sub.Tmax and K.sub.A to compare with B.sub.Tmax and K.sub.A from the population of peers to determine whether bilirubin binding is normal, whether the risk of BIND is increased in a patient (B.sub.Free is equal to or greater than B.sub.FreeStandard), and if not, the unique B.sub.Total for that patient at which treatment is warranted. The computer-implemented methods are executed using e.g., non-transitory computer readable medium, including e.g., use of a computer or processor, which may be incorporated into a device as provided herein, or separately but operatively connected to the device, e.g., as a system.

(121) 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, conceived 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 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.

(122) 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 discussion, it is appreciated that throughout the description, discussions utilizing terms such as processing, computing, calculating, determining, displaying or the like, refer to the actions and processes of a computer system, or similar electronic computing device that manipulates and transforms data represented as physical (e.g., 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.

(123) In alternative embodiments, provided are apparatus for performing the operations or computer implemented methods provided herein. 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 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.

(124) 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 in accordance with the teachings herein, or it may prove convenient to construct a more specialized apparatus to perform the method steps. The structure for a variety of these systems will appear from the description below. In addition, embodiments provided 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 embodiments as described herein.

(125) 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 (electrical, optical, acoustical or other form of propagated signals (e.g., carrier waves, infrared signals, digital signals, etc.)), etc.

(126) In alternative embodiments, methods as provided herein are implemented in a computer system within which a set of instructions, for causing the machine to perform any one or more of the protocols or methodologies as 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 equivalents 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 actions to be taken 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.

(127) In alternative embodiments, an exemplary computer system as provided herein comprises 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 via a bus.

(128) In alternative embodiments, a processor represents one or more general-purpose processing devices such as a microprocessor, central processing unit, or the like. More particularly, 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 alternative embodiments the processor is configured to execute the instructions (e.g., processing logic) for performing the operations and steps discussed herein.

(129) In alternative embodiments the computer system further comprises a network interface device. The computer system also may 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).

(130) In alternative embodiments, the data storage device (e.g., drive unit) comprises a computer-readable storage medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the protocols, methodologies or functions as provided herein. The instructions may also reside, completely or at least partially, within the main memory and/or within the 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 the network interface device.

(131) In alternative embodiments the computer-readable storage medium is used to store data structure sets that define user identifying states and user preferences that define user profiles. Data structure sets and user profiles may also be stored in other sections of computer system, such as static memory.

(132) In alternative embodiments, while the computer-readable storage medium in an exemplary embodiment is a single medium, the term machine-accessible storage medium can 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. In alternative embodiments the term machine-accessible storage medium can also be taken 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 as 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.

(133) Treating BIND and Hyperbilirubinemia

(134) In alternative embodiments, provided are methods for treating, ameliorating, reversing or preventing in an individual in need thereof (optionally a jaundiced newborn or infant): significant hyperbilirubinemia (optionally jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, or a bilirubin-induced neurologic dysfunction (BIND), a bilirubin-induced neurodevelopmental impairment, or a neurodevelopmental impairment having toxic levels of bilirubin as a causative agent, optionally in a newborn, optionally comprising an encephalopathy or kemicterus, or sudden neurotoxicity (acute bilirubin encephalopathy), or choreoathetotic cerebral palsy, impairment having toxic levels of bilirubin as a causative agent, optionally in a newborn, optionally comprising an encephalopathy or kernicterus, or sudden neurotoxicity (acute bilirubin encephalopathy, or choreoathetotic cerebral palsy, a bilirubin-induced hearing impairment, or a hearing impairment having toxic levels of bilirubin as a causative agent, a bilirubin-induced autism, or an autism having toxic levels of bilirubin as a causative agent, a bilirubin-induced high tone hearing loss, a bilirubin-induced paralysis of upward gaze, or a bilirubin-induced yellow staining of the teeth.

(135) Methods as provided herein indicate when therapy should start (commence) on an individual in need thereof, and provide directions to the physician as to the need for an appropriate treatment for an individual in need thereof, for example, with a phototherapy and/or an exchange transfusion, when concurrent clinical circumstances do not indicate a high risk of BIND.

(136) Furthermore, the methods as provided herein can be used to monitor treatment to determine that bilirubin levels have decreased sufficiently to substantially reduce the risk of BIND, and thus signaling to the physician that treatment can be modified, interrupted or stopped. If BIND is occurring without obvious symptoms, the methods as provided herein can alert clinicians, thus allowing for early treatment that may reverse or lessen the damage (see Johnson L, et al, Clinical report from the pilot USA kemicterus registry (1992-2004). J Perinatol 2009; 29: S25-45), wherein the patient may be a newborn infant, a child, or an adult (e.g. see Blaschke T F, et al, Crigler-Najjar syndrome: an unusual course with development of neurologic damage at age eighteen. Pediatr. Res. 1974; 8:573-890).

(137) Thus, diagnostic and treatment methods as provided herein help solve the problem that symptoms of BIND are often confused with other conditions, for example, infection (see Ahlfors et al, Unbound bilirubin in a term newborn with kemicterus. Pediatrics 2003; 111: 1110-1112), and that symptoms of BIND are often absent in premature newborns (see Watchko J F et al. The enigma of low bilirubin kemicterus in premature infants: why does it still occur, and is it preventable? Semin Perinatol 2014; 38: 397-406).

(138) Any method known the art can be used to treat or ameliorate, or prevent, significant hyperbilirubinemia such as jaundice, bilirubin toxicity including bilirubin neurotoxicity, a bilirubin-induced neurologic dysfunction (BIND), a bilirubin-induced neurodevelopmental impairment, or a neurodevelopmental impairment having toxic levels of bilirubin as a causative agent, optionally in a newborn, optionally comprising an encephalopathy or kernicterus, or sudden neurotoxicity (acute bilirubin encephalopathy), or choreoathetotic cerebral palsy; impairment having toxic levels of bilirubin as a causative agent, optionally in a newborn, optionally comprising an encephalopathy or kemicterus, or sudden neurotoxicity (acute bilirubin encephalopathy, or choreoathetotic cerebral palsy; a bilirubin-induced hearing impairment, or a hearing impairment having toxic levels of bilirubin as a causative agent; a bilirubin-induced autism, or an autism having toxic levels of bilirubin as a causative agent; a bilirubin-induced high tone hearing loss; a bilirubin-induced paralysis of upward gaze, and/or a bilirubin-induced yellow staining of the teeth.

(139) For example, significant hyperbilirubinemia such as jaundice, for example, neonatal jaundice, may be treated with phototherapy, or colored light, which works by changing trans-bilirubin into the water-soluble cis-bilirubin isomer, or by exchange transfusions, which can involve repeatedly withdrawing small amounts of blood and replacing it with donor blood, thereby diluting the bilirubin and material antibodies. In alternative embodiments, intravenous immunoglobulin (IVIg) is used in situations where significant hyperbilirubinemia such as jaundice may be related to blood type differences between mother and baby. This condition results in the baby carrying antibodies from the mother that contribute to the rapid breakdown of the baby's red blood cells. Intravenous transfusion of an anti-maternal-Ig immunoglobulin may decrease the hyperbilirubinemia or jaundice and lessen the need for or the extent of exchange transfusion.

(140) 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.

(141) Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary and/or Detailed Description sections.

(142) As used in this specification and the claims, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise.

(143) Unless specifically stated or obvious from context, as used herein, the term or is understood to be inclusive and covers both or and and.

(144) Unless specifically stated or obvious from context, as used herein, the term about is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term about.

(145) The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.

(146) Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms comprising, consisting essentially of, and consisting of may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

(147) 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.

(148) Modifications of this invention will occur readily to those of ordinary skill in the art in view of these teachings. The above description is illustrative and not restrictive. This invention is to be limited only by the following 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.