METHODS AND DEVICES FOR ASSESSING IN VIVO TOXIC LEVELS OF BILIRUBIN AND DIAGNOSING INCREASED RISK OF

BILIRUBIN NEUROTOXICITY RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. (USSN) 62/652,266 April 3, 2018. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

TECHNICAL FIELD

This invention generally relates to medicine, medical diagnostics and medical devices. In alternative embodiments, provided are methods, devices and systems for assessing and treating in vivo toxic levels of bilirubin, and diagnosing the relative risk for developing bilirubin encephalopathy, or, having a bilirubin encephalopathy, by processing clinical data to accurately determine whether bilirubin binding is normal in a patient. In alternative embodiments, provided are 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 such as encephalopathy. Also provided are computer- implemented methods for converting clinical laboratory data into a bilirubin binding panel that comprises conventional serum or plasma total bilirubin concentration (B Total) and serum or plasma unbound bilirubin or free bilirubin concentration (BFree) measurements at two B Total, to calculate a novel, clinically relevant maximum BTotai and capacity constant (BTmax), and its corresponding equilibrium association constant (KA), in order to accurately obtain BFree at any BTo _tai < BTmax using

. 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 by measuring B _Totai and B _Free in a serum or a plasma sample at two BTotai and incorporating computer-implemented methods as provided herein to analyze these data and output for the bilirubin binding panel a B _Tmax and K _{A ΪO} determine whether B _Free has reached or exceeded a standard BFree (BFreeS _tandard ) in the relevant newborn population, and, if not to calculate the Bio _tai at which that will occur using

A BFree /i BFreeStandard OG a

B FreeStandard K _L · B Tmax

^Total ³

1 (^A ^FreeStandard·^

indicate the relative risk of a bilirubin-induced neurological injury in a newborn with hyperbilirubinemia is increased. In alternative embodiments, also provided are methods for treating, ameliorating, reversing or preventing a bilirubin-related pathology and for using methods as provided herein, including use of a 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).

BACKGROUND

About 60% of all newborns become visibly jaundiced during the first two weeks of life. The jaundice is due to a normal, transient accumulation of the yellow pigment unconjugated bilirubin IX-a (referred to as bilirubin henceforth), a product of hemoglobin catabolism. The bilirubin accumulation is due increased bilirubin production as fetal red blood cells have shorter life spans versus adult red blood cells and delayed bilirubin excretion as the metabolic pathways for eliminating bilirubin mature over the first few days of life. Therefore, a transiently elevated blood bilirubin level, referred to as hyperbilirubinemia, that is often accompanied by visible jaundice, is a normal, usually harmless event in newborns during the first few days of life. However, bilirubin is neurotoxic, and in some circumstances causes severe neurological injury resulting in death or serious sequelae, and clinicians therefore closely monitor newborns with hyperbilirubinemia.

Neurol ogically toxic levels of bilirubin cause a spectrum of serious neurological injuries such as acute bilirubin encephalopathy resulting in death with kernicterus at autopsy (yellow staining of specific brain nuclei) or chronic

neurological sequelae (also referred to as kernicterus) including choreoathetotic cerebral palsy, high tone hearing loss, paralysis of upward gaze, and yellow staining of the teeth. In addition, there is recent concern that bilirubin neurotoxicity contributes to other neurological disorders including auditory neuropathy spectrum disorder, apnea in premature newborns, and possibly autism. This spectrum of neurological damage is collectively referred to as a bilirubin- induced neurologic dysfunction (BIND).

BIND can be prevented or ameliorated by increasing bilirubin excretion from the body using phototherapy or the more risky and invasive procedure known as blood exchange transfusion in which the newborn’s blood with high bilirubin levels is slowly removed and replaced by compatible donor blood with low bilirubin levels. Clinicians currently use the serum or plasma total bilirubin concentration (Biotai) as shown in Table 1 below for newborns less than 35 weeks (see, e.g., Maisels MJ, et al. An approach to the management of hyperbilirubinemia in the preterm infant less than 35 weeks of gestation. J Perinatol 20l2;32:660).

TABLE 1

The ranges of treatment Bi _otai used in TABLE 1 (versus using a single treatment

B _Total ) are based on clinical experience and expert opinion rather than evidence-based, and introduce considerable uncertainty as to when treatment is needed as illustrated in FIG. 1 where, for example, in newborns less than (<) 28 weeks gestation, exchange transfusion is considered at Bi _otai = 11 mg/dL but not mandatory until B _totai reaches 14 mg/dL. How does a clinician decide whether a newborn less than 28 weeks gestation and a Bi _otai = 12 mg/dL needs an exchange transfusion? The uncertainties are even greater in newborns greater than or equal to (>) 35 weeks gestation wherein there are no mandatory Bi _otai for phototherapy or exchange transfusion, the latter being only being“considered” when the Bi _otai reaches 25 mg/dL (see American Academy of Pediatrics, Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 2004; 114:297-316). These uncertainties lead to excessive treatment resulting in significant social and financial costs, yet this approach has not eliminated BIND.

Ranges of treatment Bi _otai are used, e.g. in TABLE 1 because Bi _otai correlates poorly with BIND (e.g. see Watchko JF et al. The enigma of low bilirubin kernicterus in premature infants: why does it still occur, and is it preventable? Semin Perinatol 2014; 38: 397-406 and Ip S et al. An evidence-based review of important issues concerning neonatal hyperbilirubinemia. Pediatrics 2004; 114: el30). Since neither phototherapy or exchange transfusion are without risk (including death), newborns may suffer from BIND or complications from unnecessary treatments.

As illustrated in FIG. 2, measuring plasma bilirubin binding is important since only the non-albumin bound or free plasma bilirubin (B _Free ) crosses capillaries and the blood-brain barrier to enter the tissues where the brain resides. The higher the B _Free at any B _totai , the higher the corresponding tissue levels of bilirubin with greater brain exposure to bilirubin and, therefore, the risk of BIND, as illustrated in FIG. 3.

Bilirubin binding is highly variable in newborn plasma, and newborns with poor bilirubin binding will have relatively higher B _Free and tissue bilirubin levels at any Bi _otai compared to newborns with normal binding, since, when poor bilirubin is present, the accumulated bilirubin needed to reach a given Bi _otai is greater, and the higher tissue bilirubin levels at that Bi _otai , increase the brain exposure to bilirubin and the risk of BIND relative to comparable newborns with normal bilirubin binding (see FIG. 2 and FIG. 3).

Recent studies document that BIND is predicted by B _Free in newborns with hyperbilirubinemia that have similar Bi _otai (see FIG. 3, and e.g. Amin SB, et al.

Chronic auditory toxicity in late preterm and term infants with significant

hyperbilirubinemia. Pediatrics 2017; 140: e20164009), validating adding bilirubin binding to the routine evaluation of these newborns. Furthermore, bilirubin binding is routinely measured in Japan and has been reported be very helpful clinically (e.g. see Mori oka I et al. Serum unbound bilirubin as a predictor for clinical kernicterus in extremely low birth weight infants at a late age in the neonatal intensive care unit. Brain Dev 2015; 37:753).

Bi _otai and B _Free are commonly but mistakenly viewed as independent alternatives for guiding clinical care, with the misconception that B _Free treatment criteria would somehow replace current Bi _otai treatment criteria, e.g. TABLE 1. Bi _otai and B _Free are not independent but rather interdependent measurements, inextricably linked chemically with plasma bilirubin binding sites (e.g. albumin) through the law of mass action. The risk of BIND depends on how much bilirubin has accumulated and how it distributed between blood and tissue, which is determined by B _Free (FIG. 2) which in turn is a mathematical function of the Bi _otai and the concentration and inherent binding ability of plasma bilirubin binding sites (e.g. albumin) as described in detail below. A workable approach for incorporating bilirubin binding into clinical care is to quantify bilirubin binding in a manner that allows identification of those newborns with below average or poor bilirubin binding and adjusting the current Bi _otai treatment guidelines accordingly. This reduces the uncertainty inherent in using Bi _otai alone to determine the risk of BIND (e.g. FIG. 1) by individualizing care.

Quantifying plasma bilirubin binding requires determining (1) the maximum amount of bilirubin that can be bound (Bimax) and (2) how tightly it can be bound, which is typically quantified using equilibrium association or dissociation constants. Bi _max depends on the concentration of functioning bilirubin binding sites and is often referred to as the bilirubin binding capacity or the Bi _otai at which the binding sites are “saturated” with bilirubin (e.g. if the concentration of binding sites is 453 pmol/L, Bi _max = 26.5 mg/dL = 453 pmol/L). How tightly bilirubin is bound at a binding site is quantified by a binding constant, e.g. an equilibrium association constant K _n , where n is the number of sites with different inherent abilities to bind bilirubin, and the constants representing each site are Ki, K ₂ ...K _n . The chemical equilibrium is

(Bimax ^— (Biotai ^— BFree )) + B _F ree B Total - B Free wherein Bi _{otai -} B _Free is the concentration of bilirubin bound to binding sites and Bi _max - (Bio _{tai -} BFree) is the concentration of the unoccupied (available) bilirubin binding sites. Albumin is known to have at least two bilirubin binding sites, and quantifying bilirubin binding using standard methods to obtain Bi _max and the corresponding equilibrium constants requires measurement of B _Free at several Bi _otai (see Jacobsen J. Binding of bilirubin to human serum albumin - Determination of the Dissociation Constants. FEBS Lett 1969; 5: 112-114). The significant testing time, large sample volumes, and complexity of data analysis preclude routine quantification of bilirubin binding in clinical laboratories using standard methods. SUMMARY

In alternative embodiments, provided are methods, devices and systems for assessing in vivo toxic levels of bilirubin, and diagnosing the relative risk for developing a bilirubin-related pathology such as a neuropathy, e.g., an

encephalopathy or bilirubin-induced neurological dysfunction (BIND), which can include encephalopathy, deafness, or choreoathetotic cerebral palsy, particularly in a newborn with hyperbilirubinemia (jaundice).

In alternative embodiments, methods, devices and systems as provided herein comprise processing and analyzing clinical data to accurately determine whether plasma bilirubin binding is normal and to assess the relative risk of BIND in a patient by coupling the Bilirubin Binding Panel (BBP) of tests: Bi _otai and B _Free measured before and after sample enrichment with bilirubin, Bimax, and KA with current Biotai treatment guidelines as determined with instrument implementation with computer algorithms. These data provide two important new assessments of the risk of BIND at the current Bi _otai , the B _Free for comparison with the standard risk B _Free for the relevant population (B _F reeStandaKi) and the Biotai at which B _F reeStandard occurs

B Total

If BFree BFreeStandard OG

B

Total 1 + K _A · B FreeStandard

the relative risk of BIND is increased in jaundiced newborns with significant hyperbilirubinemia (in alternative embodiments, the term“significant

hyperbilirubinemia” is a hyperbilirubinemia that requires a treatment to maintain the health of the individual, e.g., a patient such as a newborn infant, or requires a treatment to lower the hyperbilirubinemia to improve the health of the individual and/or to prevent further negative effects of the individual’s health because of the hyperbilirubinemia, or to ameliorate symptoms of hyperbilirubinemia).

In alternative embodiments, provided are methods (processes), devices and systems for quantifying how well plasma binds bilirubin comprising determining the maximum total bilirubin concentration (Bi _max ) and its associated equilibrium association constant (KA),

wherein B Total

B Free

, and BFree and Bio _tai are the measured plasma concentrations of unbound or free bilirubin and total bilirubin, respectively,

and optionally the method determines whether the risk of bilirubin

neurotoxicity, optionally BIND, is increased, by first using a novel method to obtain Bimax and KA in a patient by measuring Bio _tai and BFree before (Bio _tai , BFree) and after (B _Totai-2 , B _Free-2 ) bilirubin enrichment of a plasma sample to obtain two equations with two unknowns (Bi _max and KA), that can be solved for Bi _max as shown below:

n

Tmax

The calculated Bimax, Bio _tai , and BFree are then entered into to obtain KA or alternatively, KA IS the negative intercept and Bi _max is the negative slope divided

1 1

by the intercept of versus as the reciprocal of B free

Bpret ^Total

and then comparing BFree with the BFreeS _tandard occurring at the treatment Bio _tai and, optionally, the median Bi _max and median KA for the comparable population, e.g.

Treatment B _Xotal

B FreeStandard median K _A (median B _Tmax — Treatment B _Totai ) as illustrated in FIG. 4 and determining the patient’s Bio _tai at which the BFreeS _tandard occurs, i.e. the B ^FreeStandard ^{' '} Bimax

Total . If B _F ree = BFreeStandard OG ^Total —

1 ⁺ (KA BpreeStandard)

BFreeStandard 'FA '^Tmax , . ,

- the risk ot BIND warrants treatment irrespective ot Bi _otai + FA ^‘ ^ FreeStandard

(e.g., see Table 2, showing bilirubin binding in 31 newborns less than (<) 28 weeks gestation (see Ahlfors CE, et ak, Bilirubin binding capacity and bilirubin binding in neonatal plasma E-PAS2017 2017:2718.2715) wherein the

TABLE 2

median Bi _max is 22.0 mg/dL and median K _A is 16 dL/pg, wherein optionally the patient is a newborn, wherein the BFreeS _tandard at the mandatory phototherapy Bio _tai of 6 mg/dL (Table 1) and optionally, the median Bi _max and K _A for newborns less than (<) 28 weeks gestation (Table 1) would be 0.32 pg/dL as illustrated in FIG. 4, and the BFreeS _tandard at

the mandatory exchange transfusion Bi _otai of 14 mg/dL for newborns less than (<) 28

,mg

14

dL

weeks gestation (Table 1) would be FreeStandard 1.51

_1.16 ^ \ig ₍ 22.0 ^ dL 14 ¾ dL ' pg/dL as illustrated in FIG. 4. A newborn in this population with a 25 ^th percentile Bi _max (14.3 mg/dL) and K _A (0.75 dL/pg), i.e. poor bilirubin binding, would reach the phototherapy and exchange transfusion B _FreeStandard of 0.32 pg/dL and 1.51 pg/dL,

0 32^ 0 75^ 14 3^

_ pree Standard ^‘ K A ^' ^Tmax ^' dL respectively, g dL

2.8

1 + (KA ^’ BFreeStandard) l+(0.75—-0.32¾

\ g ^, d ¹ L ^{t J} '

mg

-14.3 ..

dL

mg/dL and B _Total - = 7.6

1 G ^I ^·G FreeStandard ) l+(0.75— \ig-1.51¾ dL'

mg/dL, respectively, well below the current phototherapy Bi _otai threshold of 5 mg/dL and exchange transfusion threshold of 11 mg/dL in Table 1. On the other hand, a newborn in this population with a 75 ^th percentile Bi _max (24.8 mg/dL) and K _A (2.20 dL/pg), i.e. excellent bilirubin binding, would reach the phototherapy and exchange transfusion B _FreeStandard of 0.32 pg/dL and 1.51 pg/dL, respectively, at phototherapy

n 3 'Ί M. .'y ?n ^dL T> L IUM.

b _ BFreeStandard-^A-BTmax _ cLL "yg ° dL

’Total = 10.2 mg/dL and

1 + (K _A -B _FreeStandard ) l+(2.20—-0.32¾

\ig dL ^J exchange transfusion

= 19.1 mg/dL, well above the current phototherapy Bi _otai threshold of 5 mg/dL and Bi _otai exchange transfusion threshold of 11 mg/dL in Table 1.) Two newborns with the same Bi _otai but significantly different abilities to bind bilirubin will be at significantly different risks of BIND, and that difference can only be detected by measuring bilirubin binding. A newborn less than (<) 28 weeks gestation with 25 ^th percentile Bi _max (14.3 mg/dL) and KA (0.75 dL/pg), i.e. poor bilirubin binding, and a Bio _tai of 7.6 mg/dL has reached the BFreeS _tandard for exchange transfusion (1.51 pg/dL) but without measuring bilirubin binding an unsuspecting clinician following current B _Totai treatment guidelines (Table 1), would only administer phototherapy and not consider exchange transfusion.

In alternative embodiments, methods as provided herein further comprise assessing the need for hyperbilirubinemia treatment in a patient at any Bi _otai (irrespective of whether current clinical practice deems treatment should be considered) by comparing a the BFree in a patient versus the BFreeS _tandard in a comparable population (e.g. wherein optionally a comparable population is a population of the same gestational ages as shown in Table 1), wherein a B _Free in the patient equal to or greater than the illustrated in

FIG. 4 or a

B _F reeStandard ^{‘ ‘} BTmax

B _To tai equal to or greater than

(e.g. for newborns less than (<) 28 weeks gestation, the B _FreeStandard at the mandatory exchange transfusion Bi _otai of 14 mg/dL (Table 1), and optionally the median Bi _max

BTotai

(22.0 mg/dL) and KA (1.16 dL/pg) (Table 2) is BFreeS _tandard

^A^Tmax ^Total)

14 mg/dL

1.51 pg/dL), indicates a greater risk of

1.16 dL/p,g(22.0 mg / dL-14mg / dL) BIND at the mandatory treatment Bi _otai (e.g. for newborns less than (<) 28 weeks gestation, the B _Free at the 25 ^th percentile Bi _max (14.3 mg/dL) and KA (0.75 dL/pg) per

Table 2 and the mandatory exchange transfusion Bi _otai of 14 mg/dL per Table 1 is

_ ^Total _ 14 mg/dL

BFree 62.2

K·A(Bthiac ^~ Btoΐaΐ) 0.75 dL/\ig(143 mg / dL-14mg / dL

pg/dL, over 40 times than BFreeS _tandard = 1.51 pg/dL) and that at any Bio _tai , the BFree and risk of BIND increase as Bimax and KA decrease irrespective of the Bio _tai as illustrated in FIG. 3 and FIG. 4, i.e. at any Bi _otai there is more brain exposure to bilirubin and increased risk of, or presence of, hyperbilirubinemia and bilirubin toxicity, optionally bilirubin neurotoxicity, optionally BIND as Bi _max and KA decrease.

In alternative embodiments, provided are methods for quantifying how well (or how clinically efficiently) plasma, serum or blood binds bilirubin in an individual, comprising determining the maximum total bilirubin concentration (Bi _max ) and its corresponding equilibrium association constant (KA) for comparison with the, optionally median Bimax and KA for a comparable population. If R is the BFreeS _tandard obtained at a mandatory treatment Bi _otai and optionally the median Bi _max and KA for

?-KA~BTmax

the population, the patient’s Bi _otai at which R occurs wherein Bimax

I +(KA-R

and KA are the individual’s Bimax and KA),

wherein

_ BTotal _

BFree and Bio _tai are the concentrations of the

^A^Tmsx ^~ ^Total)

non-albumin bound or free bilirubin and total bilirubin, respectively,

and measuring Bi _otai and B _Free before and after enrichment of the sample with bilirubin to provide Bi _0tai _i, BFree_i and Bi _0tai _2, BFree_2 to obtain two equations with two unknowns (Bi _max and KA), that can be solved for Bi _max as shown below:

„

Tmax

The calculated Bimax, Bio _tai , and BFree are then entered into B _ree —

BTotal . is _ BTotal . . .

, _{i D} - - - r to obtain K _L — — - - - - r, or alternatively,

^A ^Tmsx ^B Total) ^FreeK^Tmsx ^Total)

KA IS the negative intercept and Bi _max is the negative slope divided by the intercept of versus as the reciprocal of B _Free - « the

BFree ^Total linear equation

wherein optionally enrichment comprises increasing the amount of bilirubin in the sample to approximately the is mandatory treatment Bi _otai , e.g., up to about 20 mg/dL in newborns less than (<) 35 weeks gestation (TABLE 1) and up to about 30 mg/dL in newborns greater than 35 or more weeks of gestation, see e.g., see

Wickremasinghe AC, et al. Risk of sensorineural hearing loss and bilirubin exchange transfusion thresholds. Pediatrics 2015; 136: 505-512,

wherein optionally the method comprises:

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

(b) measuring Bio _tai and BFree in the sample, and

(c) enriching the plasma, blood or serum sample with bilirubin (or, adding

exogenous bilirubin to the sample),

wherein optionally enrichment comprises increasing the amount of bilirubin in the sample to approximately the concentration at which exchange transfusion is mandatory, e.g. up to about 20 mg/dL in newborns less (<) 35 weeks gestation (TABLE 1) and up to about 30 mg/dL in newborns greater than or equal to 35 weeks gestation,

(d) measuring Bi _otai and B _Free in the sample after bilirubin enrichment, and (e) determining the maximum total bilirubin concentration (Bimax) and the corresponding equilibrium association constant (KA),

wherein if the individual’s BFree is above the BFrees _tandard determined at a current treatment Bi _otai (optionally the mandatory treatment Bi _otai as set forth in Table 1, and optionally the mandatory Bi _otai for exchange transfusion is 14 mg/dL for newborns less than (<) 28 weeks gestation) and optionally the median Bi _max and K _A for the comparable population (and optionally the median Bi _max is 22.0 mg/dL and median

KA is 1 . 1 6 pg/dL, and optionally

14 mg/dL

= 1.51 pg/dL, see FIG. 4 ) irrespective of the

1.16 dL/p^(22.0 mg / dL-l mg / dL)

individual’s Bi _otai , this indicates that the plasma or serum is not clinically efficient in binding or retaining bilirubin and that treatment for hyperbilirubinemia (jaundice) is indicated,

and optionally the method further comprises assessing the need for treatment of hyperbilirubinemia in an individual at any Bi _otai by quantifying bilirubin binding and comparing the individual’s Bi _max and KA with, optionally, the median Bi _max and KA in the appropriate comparable newborn population (optionally the median Bi _max = 22.0 mg/dL and KA = 1.16 dL/pg for newborns less than (<) 28 weeks gestation as set forth in TABLE 2), wherein a Bi _max and KA in the patient that are lower than the median Bi _max and KA indicate more brain exposure to bilirubin at any Bi _otai and more risk of bilirubin toxicity, optionally bilirubin neurotoxicity, optionally bilirubin- induced neurological dysfunction (BIND), and treatment of hyperbilirubinemia, including jaundice, at Bio _tai below current treatment Bio _tai , e.g. Table 1, may be warranted,

and optionally the method further comprises assessing the need for treatment of hyperbilirubinemia, including jaundice, in a patient at

B _To tal ⁼ ^ — -— unique for that individual at the patient’s Bi _max

1 T (^A ^' Bp _r eeStandard^

and KA and wherein Biotai may be different than a current treatment Biotai, e.g. Table 1 , and BFreestandard is the BFree at optionally the median Bimax and KA for the patient’s peers, optionally the population, at a current treatment Bi _otai

Treatment Bi _ot ai

(B FreeStandard -) (optionally, if a median K _A (median B _Tmax -Treatment B _Totai ) patient is less than (<) 28 weeks gestation, the mandatory exchange transfusion is Bi _otai = 14 mg/dL as shown in TABLE 1, and at the optionally median Bi _max of 22.0 mg/dL and median KA of 1.16 dL/pg, BFrees _tandard =

14 mg/dL

= 1.51 pg/dL); and the relative risk of BIND

1.16 dL/p^(22.0 mg / dL-lAmg / dL)

p _ ^B FreeStandard ^’K A ^’B Tmax , , , , at the individual s L> _0tai — - - - — would be the same as that

1 T (^A ^'B p _re eStandard/

occurring at the treatment Bi _otai (Table 1) and optionally the median Bi _max and KA for the population and there is sufficient brain exposure to bilirubin and a risk of bilirubin toxicity, optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND), to warrant treatment for the hyperbilirubinemia, or jaundice. In alternative embodiments, provided are methods for quantifying how well plasma, serum, or blood binds bilirubin, comprising determining the maximum total bilirubin concentration (Bi _max ) and the equilibrium association constant (KA),

wherein

concentrations of the non-albumin bound or free bilirubin and total bilirubin, respectively,

and optionally the method determines whether bilirubin binding, i.e.

Bimax and KA, is normal (e.g. Bimax and KA at or above the median or average for comparable individuals), or below normal, by comparing the Bi _max and KA in a patient with optionally the average or median values in comparable individuals, wherein optionally the patient is a newborn infant, wherein lower than normal Bi _max and KA in the patient indicates that at Bi _otai below current treatment Bi _otai (e.g. Table 1) there is more brain exposure to bilirubin and more risk of, or the presence of,

hyperbilirubinemia and bilirubin toxicity, optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND),

and optionally the method further comprises assessing the need for hyperbilirubinemia (or jaundice) treatment by comparing the BFree with BFreeS _tandard for comparable individuals, optionally BFreeS _tandard determined at the treatment Bio _tai and median Bi _max and KA for comparable individuals, wherein the patient is a newborn infant, wherein a B _Free greater than or equal to B _FreeStandard indicates sufficient brain exposure to bilirubin and risk of bilirubin toxicity, optionally bilirubin neurotoxicity, optionally bilirubin-induced neurological dysfunction (BIND), to warrant treatment for hyperbilirubinemia (e.g., jaundice),

and optionally the method further comprises assessing the need for

hyperbilirubinemia (including jaundice) treatment in a patient at a unique Bi _otai below that at which current clinical practice deems treatment should be considered by determining the unique Bi _otai at which the patient’s

_ BFreeStandard '^A'^Tmax . . . , , _ .

Total ^— - - - ^~ as determined using the patient s Bimax and

1 T ^A ^' Bp _j -eeStandard)

K _A and the patient’s peers, optionally population, B _FreeStandard (e.g., if the patient is less than (<) 28 weeks gestation, B _FreeStandard at the mandatory exchange transfusion Bi _otai of 14 mg/dL (TABLE 1) and optionally the median Bi _max of 22.0 mg/dL and KA of 1.16 dL/pg (TABLE 2) is BFreeStandard ^— median newborn less than 28

1.16

weeks gestation with e.g. a 25 ^th percentile Bi _max of 14.3 mg/dL and K _A of 0.75 dL/pg

BFreeStandard ' ' ^Tm ax would reach the BFreeStandard of 1.51 pg/dL at B _ota l—

1 T (^A ^' Bp _r eeStandard^

- - T -— - = 7.6 mg/dL, about half of the current mandatory exchange l+(0.75— \ig -1.51¾ d¾L ^J

transfusion Bi _otai of 14 mg/dL for newborns less than (<) 28 weeks gestation per Table 1); and at the patient’s unique Bi _otai , the brain exposure to bilirubin and of risk bilirubin toxicity, optionally bilirubin neurotoxicity, and optionally bilirubin-induced neurological dysfunction (BIND), is the same as that occurs at the current mandatory treatment Bi _otai , and treatment is warranted despite a patient’s Bi _otai below the mandatory treatment Bi _otai.

In alternative embodiments, provided are computer-implemented methods comprising a method as provided herein (e.g., a method as provided herein), or for executing a method as provided herein to determine a Bima _x and KA, and optionally further comprising: receiving the data elements; and storing the data elements.

In alternative embodiments, provided are computer program products for processing data and determining a Bi _max and K _A obtained by a novel method using Bi _otai and B _Free measured before and after bilirubin enrichment of a plasma sample, the computer program product comprising a computer-implemented method as provided herein.

In alternative embodiments, provided are Graphical User Interface (GUI) computer program products for determining Bima _x and KA obtained by a novel method using Bi _otai and B _Free measured before and after bilirubin enrichment of a plasma sample, comprising the computer-implemented method as provided herein.

In alternative embodiments, provided are computer systems comprising a processor and a data storage device wherein said data storage device has stored thereon: (a) a computer-implemented method as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or, (d) a combination thereof.

In alternative embodiments, provided are non-transitory memory medium comprising program instructions for running, processing and/or implementing: (a) a computer-implemented method as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or, (d) a combination thereof.

In alternative embodiments, provided are non-transitory computer readable medium storing a computer program product for inputting data and performing calculations for determining a Bima _x and KA obtained by a novel method using

B _Total and B _Free measured before and after bilirubin enrichment of a plasma sample, comprising the computer-implemented method as provided herein.

In alternative embodiments, provided are 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 run, process and/or implement: (a) a computer-implemented method as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or, (d) a combination thereof.

In alternative embodiments, provided are computer program products comprising: a non-transitory computer-readable storage medium; and program instructions residing in said storage medium which, when executed by a computer, run, process and/ or implement: (a) a computer-implemented method as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or, (d) a combination thereof.

In alternative embodiments, provided are computer program storage devices, embodied on a tangible computer readable medium, comprising: (a) a computer- implemented method as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or, (d) a combination thereof.

In alternative embodiments, provided are computers or equivalent electronic systems, 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 as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or, (d) a combination thereof.

In alternative embodiments, provided are systems, 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 program code stored in the memory to: run, process and/ or implement: (a) a computer-implemented method as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or,

(d) a combination thereof.

In alternative embodiments, provided are computer-implemented systems for providing an application access to an external data source or an external server process via a connection server, and providing the ability to store values associated with the plurality of data points and/or the plurality of data elements, and an application for running, processing and/or implementing: (a) a computer-implemented method as provided herein; (b) a computer program product as provided herein; (c) a Graphical User Interface (GUI) computer program product as provided herein; or, (d) a combination thereof.

In alternative embodiments, provided are devices, e.g., medical or analytical devices, capable of quantifying how well plasma binds bilirubin, wherein the device is capable of measuring B _Free (non-albumin bound or free bilirubin concentration), and B _Total (total bilirubin concentration), and communicating this data to a computer- implemented system as provided herein, a system as provided herein, or a computer or equivalent electronic system as provided herein, which can execute the computer- implemented method as provided herein, to determine or calculate, and output, e.g., to a user, a Bx _max and KA obtained by a novel method using Bt-o _tai and BFree measured before and after bilirubin enrichment of a plasma sample,

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

wherein the device comprises components, optionally robotic chemistry components, capable of measuring: total serum bilirubin concentration (Biotai);

unbound bilirubin or free bilirubin concentration (B _Free ) before and after bilirubin enrichment to obtain Bi _max , and K _A from a sample, optionally a plasma or a blood sample,

wherein optionally the computer-implemented system, the system, or the computer or equivalent electronic system is an integral part of the device, or is operatively linked remotely to the device,

wherein optionally the device comprises an automated micro-fluid handling technology, optionally a zone fluidics system or a robotic zone fluidics analytical system.

In alternative embodiments, provided are methods or processes, or systems or devices, for the diagnosis or prognosis of (or predicting the likelihood of acquiring):

- the risk of bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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,

in an individual in need thereof, comprising:

quantifying how well that individual’s plasma binds bilirubin

comprising:

(a) using a method as provided herein; or

(b) determining the maximum total bilirubin concentration (Bi _max ) and the corresponding equilibrium association constant (K _A ), _ BTotal _

wherein Bp _ree and BF ree and Biotai are the

^A^Tmsx ^~ BTotal)’

concentrations of the non-albumin bound or free bilirubin and total bilirubin, respectively,

wherein the method determines whether bilirubin binding is normal, or below normal, and the relative risk of BIND by comparing a BFree with BFreeS _tandard for the comparable population of peers (e.g. for newborns less than (<) 28 gestation per Table 1, at the mandatory exchange transfusion Bi _otai of 14 mg/dL and optionally the median Bimax of 22.0 mg/dL and KA of 1.16 dL/pg per Table 2, BFreeS _tandard = median

- unique Bi _otai at which

1.16

BFreeStandard occurs (B^tal = with a current treatment

B _Total for the comparable population of peers (e.g. for newborns less than (<) 28 gestation with a mandatory exchange transfusion at Bi _otai of 14 mg/dL per Table 1, a patient with a 25 ^th percentile Bi _max (14.3 mg/dL) and KA (0.75 dL/pg) will reach the

BFreeStandard 'KA ^' BTPΊ ax

BFreeStandard of 1 .5 1 pg/dL at B'J’ _Q ^ —

f T (^A Bp _{reeStancjarcj} ) - 7.6 mg/dL), wherein optionally the patient is a newborn,

wherein if a BFree is equal to or greater than BFreeS _tandard or a Bio _tai is less than a treatment Biotai , this indicates more brain exposure to bilirubin and increased risk of, or presence of,

- significant hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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.

In alternative embodiments, provided are methods for treating, ameliorating, reversing or preventing in an individual in need thereof (optionally a jaundiced newborn):

-significant hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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) using methods as provided herein or a device as provided herein to determine whether bilirubin binding is normal, or below normal, by comparing a Bi _max and KA in a patient versus optionally an average or median Bi _max and KA ΪP a comparable newborn population and the BFree in a patient with a BFrees _tandard in the comparable population and the Bio _tai in a patient at which BFree = BFrees _tandard , wherein if the individual in need thereof has a BFree equal to or greater BFrees _tandard or Bio _tai below a current treatment Bi _otai , this indicates more brain bilirubin exposure and more risk of, or the presence of:

-significant hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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;

and

(b) if the individual in need thereof has a B _Free equal to or greater than BFreeS _tandard or a Bio _tai below a current treatment B To _tal , then treating (or commencing treatment for), ameliorating, reversing or preventing the individual in need thereof for:

-significant hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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.

In alternative embodiments, provided are devices for use in: - treating, ameliorating, reversing or preventing the individual in need thereof for:

-significant hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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.

wherein the device comprises a device 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 for measuring Bi _otai and B _Free before and after bilirubin enrichment of a sample to obtain and then compare a Bi _max and KA in a patient versus a Bi _max and Kxun a comparable newborn population and B _Free in the individual in need thereof versus a B _Free s _tandard in a comparable newborn population, wherein a higher than normal B _Free or a B _Free s _tandard occurring in the individual in need thereof at a Bi _otai below a current treatment Bi _otai indicates more brain bilirubin exposure and more risk of, or the presence of:

-significant hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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.

In alternative embodiments, provided are uses of device for:

- treating, ameliorating, reversing or preventing the individual in need thereof for: -significant hyperbilirubinemia (including j aundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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,

wherein the device comprises a device 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 for measuring Bi _otai and B _Free before and after bilirubin enrichment of a sample to obtain and then compare a Bi _max and KA in a patient versus optionally the average or median Bi _max and K _{A i} n a comparable newborn population and BFree in the individual in need thereof versus BFreeS _tandard in a comparable newborn population, wherein a lower than normal Bi _max and KA and B _Free equal to or greater than B _FreeStandani in the individual in need thereof indicates more brain bilirubin exposure and more risk of, or the presence of:

-significant hyperbilirubinemia (including jaundice) or bilirubin toxicity, optionally bilirubin neurotoxicity, optionally 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,

- 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 Bilirubin Binding Panel (BBP) described herein uses a novel panel of plasma laboratory tests (total bilirubin concentration, Bt _o o _ΐ , and unbound or free bilirubin concentration, B _Free measured before and after enrichment of a plasma sample with bilirubin) to calculate Bi _max and K _A to provide clinicians with the Bi _max K _A that quantify a patient’s bilirubin binding and the B _Free that quantifies the relative risk of bilirubin-induced neurological dysfunction or BIND at that Biotai. These data are obtained by modifying the current method for measuring bilirubin binding such that B _Total and B _Free are measured before and after sample enrichment with bilirubin. This would require significantly more sample using standard methods (typically 25 pL of plasma) (see, e.g., see Ahlfors CE, et al. Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics. Clin Chim Acta 2006; 365: 78-85), but Zone Fluidics/SIA analysis requires very small samples and can be adapted to performing the additional measurements using minimal increases in sample volume (less than 25 pL of plasma). Currently, clinicians have only Bi _otai to assess the risk of BIND, and adding B _Free , Bi _max and K _A to quantify binding assess the risk of BIND individualizes patient care and improves the determination of when and how to treat newborns with hyperbilirubinemia. 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 hereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIG. 1 illustrates current Bi _otai treatment guidelines for newborns less than (<)

28 weeks gestation per TABLE 1. The risk of bilirubin-induced neurological dysfunction (BIND) at any B _T0tai is unknown but increases as Bi _otai increases.

Phototherapy is considered at Bi _otai = 5 mg/dL but not mandatory until it reaches 6 mg/dL and exchange transfusion is considered at Bi _otai = 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 Bi _otai in the gray zones. For example, how does a clinician decide whether a newborn with a Bi _otai = 12 mg/dL needs an exchange transfusion?

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

FIG. 3 illustrates that the risk of BIND increases as both Bi _otai and B _Free increase, and knowing both improves the assessment of risk as compared Figure 1 wherein only Bi _otai is used to assess risk.

FIG 4 illustrates the increase in B _Free across the gray zones of FIG. 1 (blue dots to orange dots) that would occur at the median, 25 ^th , and 75 ^th percentiles for a population and shows the B _FreeStandaid that occur at the mandatory phototherapy (0.32 pg/dL) and exchange transfusion (1.51 pg/dL) Bi _otai of 6 mg/dL and 14 mg/dL, respectively, per Table 1 and median Bi _max (22.0 mg/dL) and K _A (1.16 pg/dL) per Table 2 calculated using BFreeStandard = median

FIG. 5 plots the measured B _Free from TABLE 3 (·) versus Bi _otai . Also shown are BFree calculated at 1 mg/dL increments in Bio _tai using either Bimax = 36.9 and KA =

0.57 dL/pg from the pairing data at Bi _otai = 8.3 and 31.3 mg/dL where Bp _ree = assuming B _Tm ax Aio _tai = 26.4 mg/dL and K _A —

8.3 mg/dL „„„ , r> _

0.51 \ig/dL (26.4 mg/dL -8.3 mg/dL ) °· ^{90 dL/} ^§ ^and Free ^~

_ BTotai _

(®). The B _F ree calculated using the single site albumin model

^Ai^Tmsx ^~ ^Total)

deviate significantly from the measured B _Free compared with B _Free calculated using the paired data Bimax and KA.

FIG. 6 illustrates the current treatment guidelines for newborns < 28 weeks gestation shown in FIG. 1 modified using bilirubin binding, specifically the

BFreeS _tandard obtained using the mandatory Bio _tai phototherapy (6 mg/dL) and exchange transfusion (14 mg/dL) and median Bi _max (22.0 mg/dL) and KA (1.16 dL/pg) for the population of 31 newborns < 28 weeks gestation in TABLE 2 (B _FreeStandard =

Treatment Bc _0ΐa1

, , - -). This eliminates the Bio _tai median K ^(median Bp _max -Treatment Bp _ota i)

gray zones where treatment is considered discretionary (uncertain) in FIG. 1.

FIG. 7 illustrates measured B _Free from TABLE 2 (#) versus Bi _otai ; also shown are B _Free calculated at 1 mg/dL increments in Bi _otai using either the stoichiometric model using the equation Bp _ree — the

Bi _otai / Atotal molar ratio (TABLE 3) and Ki (0.93 dL/pg)and K ₂ (0.04 dL/pg) are the best-fit non-linear regression equilibrium constants to the stoichiometric mass action equation

using Bi _max = 36.9 and K _A = 0.57 dL/pg determined by pairing the binding data at Bi _otai = 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.

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

dAbsorbance 440 nm

integrating the velocity equation— = k _n · HRP

dt

Absorbance 440 nm

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 Bi _otai and the change in absorbance after adding HRP and peroxide is used to obtain the B _Free.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

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.

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 (Bi _otai ) and unbound bilirubin or free bilirubin concentration (BFree) measured before and after bilirubin enrichment to calculate the clinically relevant maximum total bilirubin concentration Bi _max and its corresponding equilibrium association constant (K _A ) outputting the Bi _max and K _A to quantify how well a patient binds bilirubin and B _Free and B _Total at which the B _FreeStandaid for the population occurs to determine whether the risk of BIND is high enough to warrant treatment.

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 _Total ) and unbound bilirubin or free bilirubin concentration (B _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 Bi _otai and B _Free measured before and after bilirubin enrichment, the clinically relevant maximum total bilirubin (Bi _max ) and its corresponding equilibrium association constant (KA) to compare with Bimax and

KA in comparable individuals to accurately determine whether bilirubin binding is normal in a patient, and the clinically relevant diagnostics B _Free and B _Totai at which BFreeS _tandaid occurs, which when compared to the BFreeS _tandard in comparable

individuals and the current treatment B _Totai , 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.

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 Bimax and KA for the population is optionally the median Bimax and KA, a newborn with Bimax and KA at the 25 ^th percentile has poor bilirubin binding relative to the population (75% of the population have higher Bima _x and KA than the patient). At a mandatory treatment B _T o _t a _l , e.g. per Table 1, wherein exchange transfusion is mandatory at Bi _otai = 14 mg/dL for newborns less than (<) 28 weeks gestational age, the B _Free at the Table 2 median Bi _max

(22 0 mg/dL) and K ₄ (1.16 dL/gg) is B _Pree = _{K Bt} - _BToia ύ

14 mg/dL

= 1.51 pg/dL and at the 25th percentile

1.16 dL/\ig(22.Q mg / dL-14mg / dL)

^Total

- = 62.2 pg/dL, with much higher risk of BIND. Therefore,

determining Bio _tai , BFree, Bimax and KA 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 Bi _max and KA 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 JA, et al. Estimation of reference ranges: how many subjects are needed? Clin Chem 1992; 38:648-650), and the B _Free , Bi _max and KA in a sample from an individual in need thereof quantify the risk of bilirubin-induced neurological dysfunction (BIND) at the Bi _otai of the individual in need thereof and determine whether treatment is needed in the individual in need thereof at a Bi _otai below that at which treatment is currently recommended for the population.

In alternative embodiments, the components of the bilirubin binding panel (BBP) including the measured Bi _otai and B _Free before and after bilirubin enrichment of the sample and the calculated clinically relevant Bi _max and its corresponding equilibrium association constant (KA) are used to determine whether bilirubin binding is normal by comparing Bima _x and KA with

optionally the median Bi _max and KA for the comparable population, and

whether the risk of BIND is increased by comparing BFree with BFreeS _tandard as determined for the population at a current treatment Bi _otai and optionally the median Bi _max and KA for the population. Additionally, the actual Bi _otai at which treatment is needed can be determined using BFreeS _t an _d ar _d and the Bimax and KA. 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 Bio _tai at which the BFrees _tandard and at which treatment may be warranted (e.g. if B _Free s _tandard for exchange

transfusion is 1.51 pg/dL for newborns (<) 28 weeks per TABLES 1 and 2, a newborn in this group with a Bi _otai of 3.0 mg/dL, a B _Free of 0.18, a Bi _max of 20 mg/dL, and a KA of 1.00 dL/pg would reach the BFrees _tandard at 12.0

mg/dL, below the mandatory Biotai exchange transfusion of 14 mg/dL). The

Bilirubin Binding Panel as determined by methods provided herein, includes and assists rather than competes with Biotai in determining the need for

treatment.

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 Biotai, e.g. Biotai less than 20 mg/dL for newborns less than (<) 35 weeks gestation (see TABLE 1). In this approach, Bimax is not Biotai at which the all the plasma binding sites are occupied with bilirubin but instead the upper limit B _to tai of the functioning bilirubin binding sites within the clinically relevant range of Biotai, and KA is the corresponding composite of the Ki . . K _n equilibrium association constants. The chemical equilibrium is:

K _{A ^}

(Bimax ^— (Biotai ^— BFree)) BFree ^ ^ Biotai ^— BFree

and since BFree is orders of magnitude less than Biotai at clinically relevant Biotai, Biotai - BFree = Biotai, the resulting mass action equations are shown below,

B BTotal

Free can be readily solved by for Bimax and KA by

K·A(Bthiac ^~ Btoΐaύ

measuring Biotai and B _Free before and after enrichment of the sample with bilirubin to provide Biotai, BFree and Biotai 2, BFree_2. These provide two equations with two unknowns (Bimax and KA), that can be solved for Bimax as shown below: B Total ^Total 2

B Tmax

^ Total ^Free_2

The calculated Bimax, Bio _tai , and BFree are then entered into Bp _ree =

alternatively, K _{A IS} the negative intercept and Bi _max is the negative slope divided by

1 1

the intercept of— - versus— - as the reciprocal of tfp _ree —

^B Free ^B Total

^B Total

„ - - - T is the linear equation

^K Av ^B Tmsx ^B Total)

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

_ ^B Total _ 8.3 mg/dL

0.90

^B Freei ^B Tmsx ^{~ B} Total ) ^_ 0.51 \ig/dL (26.4 mg/dL -8.3 mg/dL )

dL/pg, the calculated B _Free deviate deviated significantly from the measured binding points as illustrated by the open blue circles in FIG. 5. This suggests Bi _max 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 Bi _max and Ai _otai in the 31 newborns less than 28 weeks gestation did not correlate significantly (r ² = 0.02).

TABLE 3

Quantifying bilirubin binding by determining Bi _max and KA in a population of newborns can be used to reduce the uncertainties in the current Bi _otai 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 Bi _max and KA of a population, then a standard B _Free , i.e.

BFrees _tandard can be designated at a current treatment Bio _tai (e.g. TABLE 1) and, e.g. the BFrees _tandard at the median Bimax and KA of the population is: 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 _Free s _tandard . For the half at greater risk of BIND BFrees _tandard occurs at a Bio _tai below the treatment Bio _tai , i.e. at the individual’s B _Totai , Bimax, and KA where BFreestandard = _D ^BTotal _D - - . The risk

^A PTmax “Total)

of BIND in an individual is the same as that for the population at B _Free s _tandard when the individual’ s measured BFree is equal to or greater BFrees _tandard or when the individual’ s

Bio _tai is equal to calculated using the patient’s Bimax and KA as the

tissue levels of bilirubin, brain bilirubin exposure, and risk of BIND will be similar irrespective of the Bio _tai .

Novel methods for using measured serum or plasma Bi _otai and B _Free to obtain Bi _max and KA 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 Bi _otai guidelines for treatment of newborn hyperbilirubinemia as illustrated in FIG. 6 compared with FIG. 1. These data (Bio _tai , BFree, Bimax, and KA) comprise a Bilirubin Binding Panel (BBP) (see Ahlfors CE. The Bilirubin Binding Panel: A Henderson- Hasselbalch approach to neonatal hyperbilirubinemia. Pediatrics 2016; 138 : e20l54378) that will significantly reduce the uncertainties inherent in current treatment guidelines that use Bi _otai only.

Quantifying Plasma Bilirubin Binding:

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 (Aiotai) 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 Ai _otai per se is not a clinically useful estimate of how much bilirubin can be bound (i.e. Bi _max ).

Since each albumin molecule binds at least two bilirubin molecules over the clinically relevant range Bi _otai 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 albumin - Determination 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 Honore 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 IM, Hunston DL. Protein affinities for small molecules: Conceptions and misconceptions. Arch Biochem Biophys 1979; 193: 314-

328). The stoichiometric two-site bindin ' — easures the concentrations the

Ki, K _{2 ^}

plasma albumin (Aiotai), the total biliru ii. ...is the non-albumin bound or free bilirubin (BFree) measured at multiple Bio _tai and uses them to determine the two equilibrium association constants for the albumin molecules binding one (Ki) and those binding two bilirubin molecules (K ₂ ). In this model the Bi _otai is the sum of the concentrations of albumin binding one (A:Bi) and twice that binding two bilirubin molecules (2xA:B ₂ ) plus B _Free and the Ai _otai is the sum of A:Bi + A:B ₂ + the concentration of unoccupied or free albumin binding sites (A _Free ) binding no bilirubin. The chemical equilibrium is:

AFree + BFree A:Bl + 2A:B ₂ ,

and the mass action equations are: which can be solved for B _Free using the equation below wherein MR is the molar ratio:

Non-linear regression analysis of the molar ratios versus the B _Free from TABLE 3 were used to determine the best fit Ki (0.93 dL/pg) and K ₂ (0.04 dL/pg) using the n r 1 F^ B p ^p -FZK^ Ki Bjirpp

stoichiometric equation Molar Rat o = - 5— . FIG. 7 compares

1 +K ₁ B _Free +K ₁ K ₂ B* _ree

_ Brotal _

[lie calculated B _Free at 1 mg/dL Bi _otai increments using B Free ¾(®Tmsx ^_ B Total) using the Bi _max (36.9 mg/dL) and KA (0.57) obtained from the pairing the Bi _otai of 8.3 mg/dL with Bi _otai of 31.3 mg/dL in Table 3 versus 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 Ai _otai and therefore much less time and materials for the measurements needed to quantify bilirubin binding.

A more clinically applicable approach to quantifying bilirubin binding is to consider both how much (Bi _max ) and how“tightly” (KA) bilirubin can be bound as unknowns and derive these unknowns from Bio _tai and BFree measurements. This requires a novel approach to the routine measurement of Bi _otai and B _Free , which is to measure Bi _otai and B _Free in a plasma sample before and after enrichment of the sample with bilirubin.

The plasma equilibrium concentrations at any given plasma Bi _otai and

(unknown) B _{T max} are:

K _A

(Bimax ^— Biotai) BFree ^ ^ Biotai ^— BFree

K _A

(Bimax ^— Biotai) BFree ^ ^ Biotai wherein Bimax is how much bilirubin can be bound, Bimax - Bio _tai is the concentration of available (unoccupied) bilirubin binding sites, and Bi _{otai -} B _Free is the concentration of bilirubin bound to plasma binding sites (since B _Free is orders of magnitude less than Biotai, bound bilirubin = Biotai - BFree ³ Biotai). The mass action equation is wherein Bio _t ai and B _F ree

are measured and Bimax and KA are unknown. If Bio _t ai and BFree are measured before and after sample enrichment with bilirubin to give measured values Bio _t ai, B _F ree and Biotai 2, BFree_2, two equations with two unknowns (Bimax and KA) are provided that can be solved for Bima _x and KA as shown below.

P B Total

Tmax _T 3 i _ o . o

Total F5p _ree _2 ^ Total *FFree_ l

The calculated Bimax, Bio _t ai, and BFree are then entered into B _ree =

B oίaI , i y BTotal , . ,

, _{t i D} - - - 7 to obtain K A — — - rr - - - r, or alternatively,

^K A BTmsx Bf _Q tal) Bp _ree tl _’ p _rnsx Bp ₀ ^ _a i)

KA IS the negative intercept and Bima _x is the negative slope divided by the intercept of versus as the reciprocal of B _Free - « the

^Free ^Total linear equation

The clinically relevant quantification of bilirubin binding are the mass action variables above (Biotai, BFree, Biotai_2, BFree_2, Bimax, and KA) constitute a Bilirubin Binding Panel (BBP). Optionally, Bima _x and KA can be used to determine whether a newborn binds bilirubin normally. TABLE 4 below shows Bima _x and KA determined before and after adding sulfisoxazole (sulfa) to a bilirubin/human albumin sample containing 3.0 g/dL albumin, which about doubles the B _F ree and significantly changes Bima _x and KA. A newborn less than (<) 28 weeks gestation with a Bio _t ai of 8.3, B _F ree of 0.51 pg/dL, Bima _x of 24.3 mg/dL and KA of 1.01 per Tables 1 and 2 would reach the exchange transfusion BFrees _t an _d a _d of 1.51 pg/dL (FIG. 6) at when the Bio _t ai reaches 24.2 mg/dL, but if B _{T max IS}

1.51* ·0.2L2.1»

dL \ g dL

42.1 mg/dL and KA is 0.20 dL/ug, = 9.8 mg/dL showing the

l+(0.20— \ g-1.51¾ d¾L

much risk of BIND despite identical Bio _t ai of 8.3 mg/dL. TABLE 4

Measuring Total and Unbound Bilirubin

The peroxidase test (see, e.g., Jacobsen J, Wennberg RP. Determination of unbound bilirubin in the serum of newborns. Clin Chem 1974; 20:783-789) measures both B _Total and B _Free . This test is used clinically in Japan. In alternative embodiments, novel modifications of methods as provided herein measure B _Free at two horseradish peroxidase levels to accurately determine BFree and measure B _to tai and BFree before and after bilirubin enrichment of a plasma or other blood sample to provide Bx _max and K _{. A} to complete the Bilirubin Binding Panel (BBP) described herein. The BBP quantifies bilirubin binding (B imax and KA) and the risk of BIND using BFreeS _tandard 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 _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:

dB Total

— K _p HRP B _Totai .

dt

dAbsorbance 440 nm

= K _p HRP · Absorbance 440 nm.

dt

DETERMINATION OF K _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 pmol/L H2O2, 25 pL HRP with

reaction [HRP] = 0.061 pg/mL, 5 pL of 1 mg/mL bilirubin solution with

reaction [B _T0tai ] = 163 pg/dL, 1 cm path length cuvette, 30° C). The Kp HRP for the reaction is easily calculated by integrating the

velocity equation above between times t = 0 and t = t: - _{p Totai}

in tit J _n

Brotal Absorbance 440 nm

ln ) = -Kp HRP t

B ) = ln(

Total at t= 0 Absorbance 440 nm at t = 0 Absorbance 440 nm

Kp HRP is the negative slope of the ln( - )

Absorbance 440 nm at t= 0 versus time, which divided by the reaction HRP concentration provides the K _p.

DETERMINATION OF B _TO TAL AND BFREE: 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 _Totai and the change in absorbance at 460 nm after adding HRP/peroxide is used to obtain the

BFree as described below.

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 pL of sample (e.g. plasma or serum), followed by 25 pL of HRP (typically 1.5 mg/mL) and 5 pL of 26 mmol/L H ₂ 0 ₂ ΐo provide a reaction H ₂ 0 ₂ of 120 pmol/L H ₂ 0 _2. Bi _otai is calculated from the absorbance prior to adding HRP and H ₂ 0 ₂ and B _Free from the change in absorbance following addition of HRP/H ₂ 0 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 Bi _otai by 5 to 20 mg/dL) and repeating the test again at two HRP concentrations.

Bi _otai is calculated by dividing the initial absorbance by the known extinction coefficient (0.827/cm light path length for BTotai in mg/mL) and BFree is calculated from the change in absorbance at 460 nm after adding HRP/ H ₂ 0 _2. Since only B _Free is oxidized (bound bilirubin is protected from oxidation), the velocity equation is p HRP · B _Free . However, the equilibrium BFree falls to an

unknown lower steady state free bilirubin (BFSS) as the oxidation of B _Free disrupts the equilibrium from K _A (Blmax ^— BTotal) t BFI Btooΐ, to

HRP

(BTmax - B Total) + BF _SS + H ₂ O2 ^ ^► Biotai + oxidized bilirubin.

K _A

and, therefore — ^dBT d ^o t ^tal = K , HRP B _FSS

BTotal

wherein: B Fss , and the integrated

K-A( ^B Tmax ^B Total) ^R p ^BRP

velocity is:

or

B _Free is obtained by measuring B _FSS at the additional HRP concentration and using , which is the reciprocal of the

B _FSS equation above to obtain the intercept of l/B _Fss versus HRP ( the reciprocal of which is BFree ( Bfree

^B Total

A ( ^B TVISX ^B Total )

The sample is enriched with bilirubin and B _Totai-2 and B _Free-2 are measured and used with the pre-enrichment Bio _tai and BFree to obtain B imax and KA as seen in

BTotal

TABLES 2 and 3. B _Free can then be readily solved by for

^B Total) Bimax and KA using two equations with two unknowns (Bimax and KA) and solving for Bi _max as shown below:

B Tmax

^Total ^Frcc 2 - B Total 2 ^ Free

The calculated Bimax, Bio _tai , and BFree are then entered into B _ree - alternatively,

K _{A IS} the negative intercept and Bi _max is the negative slope divided by the intercept of

1 1

versus as the reciprocal of is the

Bpret ^B Total

K A

linear equation - K A.

The Bilirubin Binding Panel:

The components of bilirubin binding are linked mathematically by the mass

_ B oίaI _

action equation B _ree , which makes no assumptions

^K A( ^B Tmsx ^{~ B} Total )

about the stoichiometric or chemical nature of the actual plasma bilirubin binding sites, yet the constants Bimax and KA provide accurate estimates of BFree at Biotai below Bi _max as illustrated in FIG. 5 and 7. In one embodiment, the peroxidase test measures serum or plasma Bi _otai and B _Free , e.g., as described by Jacobsen J, Wennberg RP. Determination of unbound bilirubin in the serum of newborns. Clin Chem 1974; 20:783-789. In alternative embodiments, B _Free is measured at a second peroxidase concentration to insure accurate measurement of B _Free and the sample is then enriched with bilirubin and the test repeated at the higher Bio _tai and BFree to provide Bimax and

1 _

KA using two equations and two unknowns or using linear analysis of -—

BFree

A ^B Tmax

KA wherein the negative of the intercept = - intercept = K _A , and

^B Total

- slope A B Tmax

the negative of the slope/intercept = - Bimax- intercept - K _A

An individual’s Bi _max and K _A are compared with, optionally the median B Tmax and K _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 _{F ree} is greater than or equal to a B _Free s _tandard for the population that is determined determine whether the individual has normal bilirubin binding. The risk of BIND is increased if the individual’s BFree is greater than or equal to a BFrees _tandard for the population that is determined using current Bi _otai treatment guidelines and optionally the population’s median Bi _max and KA, e.g. for newborns less than (<) 28 weeks gestation per Table 1, at the mandatory exchange transfusion Bi _otai of 14 mg/dL and the median Bi _max (22.0 mg/dL) and KA (1.16 dL/pg) for the 31 newborns in Table 1.51 pg/dL, and if an

individual’s BFree is equal to or greater than (>) BFrees _tandard , treatment is warranted irrespective of the Bio _tai , and if BFree is less than BFrees _tandard , the individual’s unique

Bi _otai at which B _Free s _tandard occurs and treatment is needed can be obtained using the

. - , _^ _ ^B Tmax K A ^B FreeStandard individual S Bimax, KA and BFreestandard as Biotai— .

⁴ + (.K A ^B FreeStandard)

Bi _max and KA robustly quantify how well the plasma binds bilirubin as they quantify how much (Bi _max ) and how tightly (KA) plasma binds bilirubin. Comparing Bi _max and KA in a newborn with Bi _max and KA in a population of peers (e.g.

comparing them with the median Bi _max and KA) 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 BFree is equal to or exceeds BFreestandard in the population as described above, treatment is warranted irrespective of the Bio _tai . If the newborn’s BFree is less than BFrees _tandard the unique Bio _tai at which that newborn should be treated is obtained from the BFreestandard and that newborn’s Bimax and KA as shown above. This approach reduces the uncertainties in the current treatment guidelines that use Bi _otai alone (see FIG. 1) and individualizes care.

Bi _max and KA 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

Biotai- Devices: Zone Fluidics Analytical Instalments

The manual peroxidase test as originally described to measure Bio _tai and BFree requires 25 pL sample, and for the four tests described herein (Bi _otai and B _Free measured at two peroxidase concentrations before and after bilirubin enrichment) would require 100 pL of sample. Novel herein are technologies that automate the tests and reduce sample volumes.

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 (Bi _otai ) and unbound bilirubin or free bilirubin concentration (B _Free ) from a plasma or blood sample (Jacobsen J, Wennberg RP. Determination of unbound bilirubin in the serum of newborns. Clin Chem 1974; 20:783, Ahlfors CE, et. al. Measurement of unbound bilirubin by the peroxidase test using Zone Fluidics. Clin Chim Acta 2006;365:78), and also incorporating - directly in the device or indirectly as a multiplexed system operatively connected to the device - computer-implemented methods as provided herein to analyze this data and output a maximum bilirubin concentration (Bi _max ) and a bilirubin binding constant (K _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 _Free is greater than the risk at B _FreeStandard for the population of peers.

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 BT ma _x and KA.

In alternative embodiments, provided are devices comprising components, e.g., robotic chemistry components, for measuring: total serum bilirubin concentration

(Biotai); unbound bilirubin or free bilirubin concentration (BFree) 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 USPNs: 7,939,333 (describing metal enhanced fluorescence-based sensing methods); 7,767,467 (describing e.g., methods and device for the separation of small particles or cells from a fluid suspension); 7,416,896 (describing e.g., methods and devices for determining total and bound plasma bilirubin); 7,625,762 (describing e.g., methods and device for the separation of small particles or cells from a fluid suspension); 6,887,429 (describing e.g., methods and apparatus for the automation of existing medical diagnostic tests); 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,

6,613,579; or, as described in U.S. patent publications: e.g., U.S. Pat App no.

2018/0045723 Al (describing e.g., lateral flow devices and methods for analyzing a fluid sample); U.S. Pat App no. 2018/0052093 Al (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).

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 Bi _max K _A obtained from the measured components of the Bilirubin Binding Panel described above.

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 _T0tai and B _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 instrument - low cost of goods sold;

=> can be computer controlled and automated;

=> can be easily developed with modified methods— the 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 m L per measurement.

In alternative embodiments, a Zone Fluidics system as described in USPN 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.

Computer systems for executing computer-implemented methods:

In alternative embodiments, provided are computer-implemented methods to analyze laboratory data and output a Bilirubin Binding Panel including Biotai and BFree before and after bilirubin enrichment of plasma or blood sample, the maximum Bi _otai (Bimax) and the bilirubin binding constant (KA) that provide the clinically relevant Bimax and KA to compare with Bimax and KA from the population of peers to determine whether bilirubin binding is normal, whether the risk of BIND is increased in a patient (BFree is equal to or greater than BFreeStandard), and if not, the unique Biotai 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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

Treating BIND and hyperbilirubinemia

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 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 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.

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.

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 kernicterus 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 TF, et al, Crigler-Najjar syndrome: an unusual course with development of neurologic damage at age eighteen. Pediatr. Res. 1974; 8:573- 890).

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 kernicterus. Pediatrics 2003; 111 : 1110-1112), and that symptoms of BIND are often absent in premature newborns (see Watchko JF et al. The enigma of low bilirubin kernicterus in premature infants: why does it still occur, and is it preventable? Semin Perinatol 2014; 38: 397-406).

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.

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 maternal 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. 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.

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.