A process of preparing endogenous liquid photostable bilirubin control material from pooled human serum for its utility in a clinical laboratory

Abstract

A process for preparation of in-house liquid photostable bilirubin control material from left over human serum samples of all genders and all age groups included whose total bilirubin concentration is within the range between ≥3mg / dl (≥0.2mmol / l) to ≤20mg / dl (≤1.1mmol / l) in each sample. Each sample is pretested by rapid kits for HIV I / II, HbsAg, Anti-HCV prior pooling, while excluding haemolysed, lipemic(turbid) and insufficient samples. Pooling is done in a falcon tube and baseline mean is established after twenty consecutive pre-runs for total bilirubin concentration estimated by diazo method in the pooled serum. The pool is transferred in a generic amber vial(s) which has a polypropylene stopper and a polypropylene screw cap. This amber vial(s) is shielded within aluminium packet with zip lock which has a silica gel packet (not exceeding 2gms) enclosed inside the aluminium packet with zip lock. This complete unit is stored in fridge door at a fixed temperature range 2-8°c. The endogenous bilirubin control material prepared with this method has very less amount of variation and is said to be stable for its utility in a clinical laboratory as a internal quality control material.

Description

[0001] TITLE - A PROCESS OF PREPARING ENDOGENOUS LIQUID PHOTOSTABLE BILIRUBIN CONTROL MATERIAL FROM POOLED HUMAN SERUM FOR ITS UTILITY IN A CLINICAL LABORATORY FIELD OF INVENTION - Biochemistry & its applications OBJECTIVE OF THE INVENTION - A procedure to prepare in-house liquid photostable bilirubin control material from left over human serum post analysis and its usage as control material for autoanalyzer in a clinical laboratory. STATEMENT OF THE INVENTION-The invention described herein is a process of pooling left over human serum samples from all genders and all age groups of which each sample pertains to have total bilirubin concentration within the value range of >3mg / dl (>0.2mmol / l) to <20mg / dl (<l.lmmol / l). Prior pooling each sample is screened for HIV I / II, HbsAg, Anti-HCV by any validated rapid kit methods. All Sero-positive, haemolysed, lipemic & insufficient samples are excluded before unifying the serum. Pooling is carefully done in a falcon tube with screw cap and mixed well with gentle inversions two to three times & then transferred into amber vial(s) which has polypropylene material stopper along with additional screw cap which is also a polypropylene material. This amber vial is shielded in aluminium cover with zip lock. This aluminium packet encloses silica gel (not more than 2gms) in a sealed packet (commercially available). This complete packet is stored in 2-8°c in a fridge door compartment. All vial(s), aluminium packets are properly labelled and allocated a proper LOT number as it would be easier to trace back the source of the samples. Twenty consecutive pre run estimation is done for total bilirubin from this pooled serum in the method diazo reaction in autoanalyzer, to establish a mean value in mg / dl (or mmol / 1) which is within maximum ±1.5 standard deviation from the mean. The total bilirubin concentration measured from the pooled serum will have very low coefficient of variation %. Any coefficient of variation if is <1.0 is usually considered a good stability for this abovementioned process. This pool is stable for 30 days as per the experimental example provided. If there is any microbial contamination, it is strictly recommended to generate a fresh batch of pool from the above-mentioned pooling process. If pooled serum is encroached with microbial contamination or unfit for its usage, it can be identified as change of pool colour from yellowish to green or pungent odour from the pool sample. SUMMARY OF THE INVENTION-The invention described here is a process of unifying left-over human serum from all genders and all age groups in which the total bilirubin concentration post analysis was within range of >3mg / dl (>0.2mmol / l) to <20mg / dl (<l. lmmol / l) in each sample. Before pooling it is ensured that all samples are tested negative for HIV I / II, HbsAg, Anti-HCV by any rapid kit methods. All haemolysed, lipemic and insufficient samples are excluded from this process. The pooling can be done in Falcon® Centrifuge Tube, Disposable, Conical, 50mL, Sterile, Corning® Approximate dimensions are 30mm O.D.; 115mm length and capped off. After 2-3 gentle inversions the pooled serum is tested for 20 times pre run consecutive observations for total bilirubin concentration in it for establishing the baseline mean value. Once Baseline mean is achieved pooled serum is transferred in to a generic amber vial of volume 10ml, 20ml or 30ml (maximum) as per requirements. This amber vial is labelled with proper lot number, date of pooling, and mean at pooling date. The amber vial is kept in the aluminium packet with zip lock which has silica gel packet of (2gms) encased inside of aluminium packet. This whole content is kept in door fridge in fixed temperature range 2-8°c. The containers are not frozen. The endogenous bilirubin control material made from pooled human serum is stable in these conditions for 30days and its utility is justifiable. Fresh batch of control can be prepared easily if the older batch deemed unfit for further analytical procedures. DETAILED DESCRIPTION

[0002] 6.1 PREAMBLE- Bilirubin is a degraded product formed from heme enclosed in haemoglobin as the erythrocyte breakdown occurs. It is also formed via myoglobin metabolism [2]. Heme is converted to bilirubin and the former is metabolized in the presence of heme oxygenase to form biliverdin, later is converted to bilirubin with help of biliverdin reductase [2]. Most of the clinical laboratories currently measure total as well as direct bilirubin under spectrophotometric method based on the endpoint diazo reaction [2] . Many photophysical parameters of bilirubin indicates deactivation of electronically excited molecules. Specific optical activity of bilirubin with its confirmational relation and binding properties are provided by circular dichroism spectroscopy [7]. Bilirubin is a highly photosensitive and photooxidative material [7,2]. Aluminium reflects UV radiation and light material efficiently than any other metal [5]. Silica gel adsorbs water effectively from its surroundings [9]. All commercially prepared bilirubin controls usually are either spiked with artificial bilirubin in protein matrix or it is dissolved in artificial synthetic substances to achieve the desired concentrations of standard, where the absorbance peak and its absorptivity might yield different results [8]. Hence preparing endogenous bilirubin control material from pooled human serum and its proper handling with storage is given in this said invention where bilirubin is stable for its analytical utility in a clinical laboratory. SUMMARY- The invention described here is a process of unifying left-over human serum from all genders and all age groups in which the total bilirubin concentration post analysis was >3mg / dl (>0.2mmol / l) to <20mg / dl (<l. lmmol / l) in each sample. Before pooling it is ensured that all samples are tested negative for HIV I / II, HbsAg, Anti-HCV by any rapid kit methods. All Haemolysed, lipemic and insufficient samples are excluded from this process. The pooling can be done in Falcon® Centrifuge Tube, Disposable, Conical, 50mL, Sterile, Corning® Approximate dimensions are 30mm O.D.; 115mm length and capped off. After 2-3 gentle inversions the pooled serum is tested 20 times pre run consecutive observations for total bilirubin concentration in it for establishing the baseline mean value. Once Baseline mean is achieved pooled serum is transferred in to a generic amber vial of volume 10ml, 20ml or 30ml (maximum) as per requirements. This amber vial is labelled with proper lot number, date of pooling, and mean at pooling date. The amber vial is kept in the aluminium packet with zip lock which has silica gel packet of (2gms) encased inside of aluminium packet. This whole content is kept in door fridge in fixed temperature range 2-8°c. The containers are not frozen. The endogenous bilirubin control material made from pooled human serum is stable in these conditions for 30days and its utility is justifiable. Fresh batch of control can be prepared easily if the older batch deemed unfit for further analytical procedures. PRIOR ART-Bilirubin by nature is a yellow-coloured compound which has an absorbance of approximately 450nm and highly unstable photosensitive compound [8,2]. Bilirubin endures both photoisomerization as well as photooxidation, and usually stable until 24hrs [2]. With excitation it has greater value for configurational photoisomerization quantum yield observed in bilirubin human serum albumin complex at room temperature symbolising to radiationless deactivation mechanisms. The exact quantum yields of both fluorescence and configurational isomerization of bilirubin varies in opposite directions as the temperature is changed and the radiative deactivation are deemed to be as competitive processes. The bilirubin photoisomerization quantum observations vary significantly along with the excitation wavelength [7]. Bilirubin bound with the human serum albumin is a stable complex which has approximately a diastereomeric excess of the P-helicity enantiomer of bilirubin structure about 20%. Configurational photoisomerization confirmation of bilirubin can be destabilized by light. At excitation exocyclic double bonds at C4=C5 & C15=C16 behaves as single bond, which in turn allows free rotation at C=C bonds breaking the Intramolecular hydrogen bonds after which the intermediates are formed and these either go to the initial confirmations or get isomer configurations. The native confirmations as described are 4Z,15Z and the twisted confirmations are 4E,15Z and 4Z,15E [7]. Protection against photooxidation of bilirubin is by albumin when the bilirubin is bound with human serum albumin [7]. If there is substantial photoisomerization of bilirubin then the photolysis might be detectable, where photolysis is a slower process than the photoisomerization [3]. Usually, human based serum or protein-based material is preferred for building of standards and its usage for spectrophotometric methods. Different preparations of same proteins may yield different results. Synthetic bilirubin may have any impurities while its preparation. The solubility of bilirubin is markedly increased in aqueous solution which has a protein matrix, here the absorbance peak along with absorptivity completely depends on source and nature of proteins [8]. Human serum-based materials are recommended for effective internal quality control of spectrophotometric methods [8]. Proper CLSI C37 guideline was followed for the preparation of pooled off the clot serum [6]. Bilirubin was unaffected by precentrifugation serum-cells contact time for about 48 hours (at room temperature). In post centrifugation phase serum should be separated from contact of cells within maximum 2 hours from the time of collection. Repeated freeze-thaw cycle of serum would lead to analyte deterioration [1]. Any Radiating energy from either UV or visible light critically affects the stability of photosensitive products, because it accelerates the photochemical degradation reactions and induces a deterioration effect [4]. A photosensitive drug like riboflavin is more degraded in transparent glass and plastic bottle as compared to amber, semi opaque, and opaque LDPE and HDPE bottles as transparent glass and bottle absorb UV and visible light which might affect the drug stability as well as shelf life [4] . Leaching of any coloured materials from glass bottles or any pigments from plastic bottles into the solution during storage appeared to increase the transmission of light which can be destructive to photosensitive drugs [4]. Aluminium when its properly prepared it reflects UV radiation more efficiently than any other metal. Aluminium surfaces can be efficient in reflecting light and UV radiation [5]. Silica gel can be utilised as desiccant as it has the adsorbent properties towards water molecules [9]. METHODOLOGY - Left over human serum post analysis from all age groups, all genders which pertain to be negative for HIV I / II, HbsAg, Anti-HCV and have range of total bilirubin between >3mg / dl (>0.2mmol / l) to <20mg / dl (<l. lmmol / l) which are free from haemolysis & turbid (lipemic) are pooled in equal volumes from each sample. However insufficient samples are not included in this pooled serum. The pooling process is done in Falcon® Centrifuge Tube, Disposable, Conical, 50mL, Sterile, Corning® Approximate dimensions are 30mm O.D.; 115mm length. For preparation of large batch large tubes are recommended. Once the pooling process is complete the falcon tube is gently inversed 2-3 times. The pooled serum is tested for 20 times pre run consecutive observations for the total bilirubin concentration in it, and a baseline mean value is established for this pooled serum. The pooled serum is transferred to generic amber vial with polypropylene material stopper along with additional screw cap which is also a polypropylene material. Amber vial(s) for smaller quantity preparation should be within 10ml and for larger batch it should not exceed 30ml. As fresh lot can be easily prepared by this method. This pooled serum should not be frozen or kept below 0°c, as freezing or thawing may lead to analyte deterioration and change in bilirubin present in this serum, it may also alter the chemistry for its further utility. Recommended storage temperature for this endogenous prepared pooled serum would be 2-8°c. No preservatives or additives should be added to this pooled serum as it might alter the matrix and not yield the desired results. After the amber vial(s) are sealed properly with stoppers and caps, proper labelling should be mentioned over the amber vial(s) like, Date of pooling for each batch, mean concentration at the time of capping for each batch, LOT number assigned for each batch and biohazard symbol on every vial(s) these parameters will enable the pool to trace back the source and will help in further proper batch segregation. All vial(s) must be considered biohazard material and should be strictly handled by professionals only. Complete physical / Digital record should be taken into consideration while data compilation / data storage. Once the process of this labelling is complete then these amber vial(s) can be transferred carefully in upright position in to the aluminium packets which have zip locks. The aluminium packets having zip locks can be easily obtained from commercial stores. The packet dimensions should be fair enough to accommodate the amber vial(s) and not be overcrowded. The zip lock must be a type of resealable open / close mechanism without affecting the accommodating area for the given amber vial(s) and justifiable. The aluminium packet with zip lock must have a silica gel packet which is commercially available and this mentioned packet of silica gel must not exceed 2gm in weight per packet of aluminium with zip lock. This silica gel has properties to adsorb water / moisture around it. This is a simple process for preparation of endogenous bilirubin control material which is a cost-effective method.

[0003] Example Experimentation:

[0004] About eighty left over human serum samples from all genders and all age groups were carefully selected which had total bilirubin concentrations between >3.0mg / dl (>0.2mmol / l) to <10mg / dl (>0.6mmol / l). The sample assay was done in diazo method, concentrations were estimated with the help of spectrophotometer in an autoanalyzer (EM 360 ERBA). From each left-over human serum sample 200pl serum was drawn which were negative for HIV I / II, HbsAg, Anti-HCV and not haemo lysed or turbid (lipemic). Serum was pooled in Falcon® Centrifuge Tube, Disposable, Conical, 50mL, Sterile, Corning® Approximate dimensions are 30mm O.D.; 115mm length. Total pooled serum volume collected was 16000pl, from which 2000pl (lOOpl per run 20times) was utilised for pre run establishment of baseline mean value for this pooled serum. Once the baseline mean was established, remaining 14,000pl of this pooled serum was equally divided into 7,000pl each to two different storage containers 1) Falcon® Centrifuge Tube, Disposable, Conical, 50mL, Sterile, Corning® Approximate dimensions are 30mm O.D.; 115mm length. 2) Generic amber vial 10ml in volume with polypropylene material stopper along with additional screw cap which is also a polypropylene material. This amber vial was shielded specifically within aluminium packet (opaque & reflective on overall surface) with zip lock having silica gel (2gms) enclosures which was obtained commercially. Both the containers were stored in 2-8°c fixed temperature range. There was no addition of preservatives nor the samples were spiked with artificial bilirubin materials to achieve desired concentration. Samples were not frozen and were kept in the above-mentioned fixed temperature range in fridge door. The samples from both the containers were measured starting from 24hr up till daily for about 30 days. The means and standard deviation were calculated for all the observations from 24hr to 30thday in both the containers. The observations are given in Table 1. Observations for Total bilirubin (Diazo method) in Falcon tube vs Amber / in Aluminium packet.

[0005] Table 1. Observations for Total bilirubin (Diazo method) in Falcon tube vs Amber vial / in Aluminium packet. There were no changes observed with respect to total bilirubin concentration within 24hr in both the storage containers. All the observations recorded was evaluated for mean, standard deviation and coefficient of variation in SPSS version 20.0 for stability assessment. Here coefficient of variation for stability study was considered for a value of <1.0 specifically for this above-mentioned experiment. The results at end of 30thday in both the containers at fixed temperature range is given in Table 2. 30thday Observations for stability study in Falcon vs Amber vial / in Aluminium packet.

[0006] Table 2. 30thday Observations for stability study in Falcon vs Amber vial / in Aluminium packet.

[0007] Results for this experimental study give out the conclusion regarding the endogenous pooled serum which was taken for this study and stored specifically by the given above methodology is stable for its usage as endogenous control material for autoanalyzer in a clinical laboratory. The pooled serum kept in falcon tube experienced a detrimental colour changed from yellowish tinge to greenish colour at the end of 30thday of this experiment. Whereas there was no change in the colour from yellowish tinge in the serum present in the amber vial within aluminium packet.

[0008] 7. References:

[0009] 1. NCCLS. Procedures for the Handling and Processing of Blood Specimens; Approved Guideline— Third Edition. NCCLS document H18-A3 (ISBN 1-56238-555-0). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.

[0010] 2. A.G. Sofronescu, T. Loebs, Y. Zhu, Effects of temperature and light on the stability of bilirubin in plasma samples, Clin. Chim. Acta 413 (3-4) (2012) 463-466.

[0011] 3. McDonagh AF. Photolysis and photoisomerization of bilirubin in serum specimens exposed to room lighting. Clin Chim Acta. 2008 Jul 17;393(2):130; author reply 131. doi: 10.1016 / j.cca.2008.03.005. Epub 2008 Mar 20. PMID: 18397760.

[0012] 4. Sabah A, Shah A, Tanwir S, Arsalan A, Malik A, Ahmad I. Light transmission properties of pharmaceutical liquid bottles and evaluation of their photoprotective efficacy. Pak J Pharm Sci. 2020 Mar;33(2(Supplementary)):877-885. PMID: 32863265.

[0013] 5. A. H. Taylor and Junius D. Edwards, "Ultraviolet and Light Reflecting Properties of Aluminum," J. Opt. Soc. Am. 21, 677-684 (1931) Danilenko U, Vesper HW, Myers GL, Clapshaw PA, Camara J E, Miller WG. An updated protocol based on CLSI document C37 for preparation of off-the-clot serum from individual units for use alone or to prepare commutable pooled serum reference materials. Clin Chem Lab Med. 2020 Feb 25;58(3):368-374. doi: 10.1515 / cclm-2019-0732. PMID: 31665109; PMCID: PMC7153737. G. Agati, F. Fusi, New trends in photobiology recent advances in bilirubin photophysics, J. Photochem. Photobiol. B Biol. 7 (1990) 1-14, https: / / doi.org / 10.1016 / 1011-1344(90)85138- M. Westwood A. The analysis of bilirubin in serum. Ann Clin Biochem. 1991 Mar;28 ( Pt 2):119- 30. doi: 10.1177 / 000456329102800202. PMID: 1859150 Chirsty A, Sivarukshy P. Comparison of adsorption properties of commercial silica and rice husk ash (RHA) silica: A study by NIR spectroscopy. Open Chemistry. 2021;19(l): 426-431. https: / / doi.org / 10.1515 / chem-2021-0044

Claims

CLAIMS-I, CLAIM:

1. A process of preparing endogenous bilirubin control material from left-over pooled human serum for its utility in a clinical laboratory comprising, a) The Claim 1, where each left-over human serum sample from all genders and all age groups prior unifying would be having total bilirubin concentration within the range >3mg / dl (>0.2mmol / l) to <20mg / dl (<l. lmmol / l). b) The Claim 1, where each screened selected sample should be in same quantity prior unifying the human serum, (example a+b = c, where a=l,b=l,c=2). c) The Claim 1, where this left-over human pooled serum would test negative for HIV I / II, HbsAg, Anti-HCV by any rapid kit method prior unification of the serum. d) The Claim 1, where each selected left-over human serum sourced would not be a haemolysed serum, lipemic(turbid) serum in quality and seropositive. e) The Claim 1, where this left-over pooled human serum material would be utilised for the purpose of internal quality control for autoanalyzer in a clinical laboratory. f) The Claim 1, where this unified left over human serum can be utilised for the purpose of accuracy and precession check of any autoanalyzer.

2. A generic amber vial(s) is / are considered for storage of this prepared endogenous bilirubin control material from left-over pooled human serum. a) The Claim 2, where the generic amber vial(s) volume can be 10ml ,20ml or 30ml maximum. b) The Claim 2, where the generic amber vial(s) should have a stopper which is of polypropylene material along with a polypropylene material screw cap, where cap seals off the amber vial(s). c) The Claim 2, where it will contain the contents from Claim 1.

3. An aluminium packet with zip lock for the purpose of shielding the amber vial, a) The Claim 3, where the amber vial(s) from the claim 2 will be placed inside the aluminium packet covering amber vial(s) completely. b) The Claim 3, where the aluminium packet with zip lock intended for the storage of Claim 2 should be opaque on all surface, reflective on all surface. c) The Claim 3, where the aluminium packet with zip lock encloses a silica gel packet not exceeding 2grams. d) The Claim 3, where zip lock covers only 20% area of the intended aluminium packet for storage. e) The Claim 3, where zip lock on the aluminium packet is a type of resealable open / close mechanism without affecting the accommodating area for the given amber vial(s) and justifiable. f) The Claim 3, where the whole content that are, aluminium packet, amber vial(s), silica gel packet is kept in the fridge door in a fixed temperature range 2- 8 °c and the contents here mentioned are not frozen or intended to be kept in deep freezer.

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