WO2011022174A1

WO2011022174A1 - Method of processing blood samples containing 5-fu

Info

Publication number: WO2011022174A1
Application number: PCT/US2010/043378
Authority: WO
Inventors: Carrie Colvin; Michael Overfield; Benjamin Roa; Michael Rhode; Don Allred; Toby Barrus
Original assignee: Myriad Genetics, Inc.
Priority date: 2009-08-21
Filing date: 2010-07-27
Publication date: 2011-02-24

Abstract

A method is disclosed for processing blood samples obtained from a patient administered 5-fluorouracil (5-FU) that is particularly suited for large scale clinical testing for plasma level of 5-FU in a patient.

Description

METHOD OF PROCESSING BLOOD SAMPLES CONTAINING 5-FU

RELATED US APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 61/235,967 filed on August 21 , 2009, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention generally relates to a diagnostic assay, and particularly to a method for preparing human blood samples containing 5-fluorouracil and assaying 5- fluorouracil.

BACKGROUND OF THE INVENTION

[0003] 5-fluorouracil (5-FU) is one of the most important chemotherapeutics in cancer treatment. It is commonly used in various chemotherapy regimens for colorectal, stomach, breast and pancreatic cancers. For example, 5-FU alone or in combination regimens such as FOLFOX and FOFIRI is the standard first line therapy in metastatic colorectal cancer. However, 5-FU is frequently associated with severe adverse effects including myelosuppression, mucositis, dermatitis, diarrhea, cardiac toxicity and even death. These adverse effects are caused at least in part by the current approach to dose determination for individual patients. The current standard of care for dosing-Body Surface Area (BSA)-only takes into account a patient's height and weight. BSA does not account for many of the factors that impact 5-FU metabolism and efficacy such as genotype, age, gender, disease state, drug-drug interactions, organ function, and comorbidities. It is therefore not so surprising that blood concentrations of 5-FU in different patients can vary by more than ten fold despite BSA-based equal dose administration.

[0004] Recent academic studies have shown that individual 5-FU dose adjustment based on pharmacokinetic monitoring of plasma 5-FU concentrations can result in significantly improved objective response rate to 5-FU. See e.g., Gemelin et al. J. Clin. Oncol., 26(13):2099-2105 (2008); Climente-Marti et al , J. Pharmaceut. Sci., 92(6): 1155-1165 (2003); Milano et al. Jnt. J. Cancer, 41 :537-541 (1988). These studies suggest that clinical testing of 5-FU blood concentrations in individual patients undergoing 5-FU therapy is clearly desirable for the purpose of 5-FU dose adjustment, optimization of therapeutic effect, and reduction of severe adverse reactions.

BRIEF SUMMARY OF THE INVENTION

[0005] Academic studies typically involve a small group of well trained research technicians and carefully controlled conditions which may not be easily attained in commercial settings. In contrast, in a commercial diagnostic setting, a large number of clinical nurses of diverse background in a large number clinics and hospitals all over the country and even the world process different patient blood samples under different laboratories conditions. This can present significant challenges to quality control, and maintaining testing consistency. The problems are exacerbated by two factors in 5-FU assays: first, 5-FU is especially unstable in patient blood samples; and second, most samples have to be transported to a distant location or stored for a considerable length of time before the assay is performed.

[0006] The inventors are among the very first ones involved in commercializing a clinical assay of blood 5-FU levels in patients to aid physicians in optimizing 5-FU dosing in clinics. The above problems were unexpectedly encountered during the development of a large scale commercial 5-FU test. In their effort of solving the problems, the inventors surprisingly discovered several steps and parameters that are unexpectedly critical to satisfactory consistency and reliability in large scale

commercial testing of plasma 5-FU levels.

[0007] The present invention therefore provides a method of processing a blood sample obtained from a patient administered 5 -fluoro uracil (5-FU) that incorporates such steps and parameters. The method is particularly suited for large scale clinical testing for plasma level of 5-FU, especially in a clinical reference lab setting.

[0008] Generally, the method comprises the steps of (1) obtaining a blood sample from a patient administered 5-FU or a prodrug thereof; (2) immediately chilling the blood sample at a temperature of about O⁰C for at least about 5 minutes; (3) within about one hour thereafter, separating plasma in the blood sample to form a plasma layer; and (4) collecting no greater than half of the plasma layer for use in an assay for 5-FU level. Preferably, the collected plasma sample is placed under a freezing condition for at least about one hour, preferably at least about 3 hours before being shipped for testing. For shipping, in a preferred embodiment, the frozen plasma sample is placed in a shipping container having a refrigerant sufficient to maintain a

temperature of below about 1O⁰C.

[0009] In another aspect, a method of assaying plasma 5-FU in a patient administered 5-FU or a prodrug thereof is provided. In the assay method, the plasma sample processed in accordance with the method above is provided, and the amount of 5-FU is determined.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0011] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 includes graphs showing the impact on 5-FU stability by different coagulants and chilling blood samples on ice before centrifugation. The y- axis is the measured 5-FU level (ng/ml). On the "Incubation" half of the graph, the labels on the x-axis are as follows: "0"- immediate centrifugation, no incubation at room temperature or on ice; "1"- 20 minutes at room temperature before centrifugation; "20"- 20 minutes on ice before centrifugation; "30"- 30 minutes on ice before centrifugation; "60"- 60 minutes on ice before centrifugation. [0012] Figure 2A is a graph showing the significant impact on 5-FU degradation by spinning immediately or putting on ice for at least 5 minutes before the spinning. The measured 5-FU level (ng/ml) is plotted against the chill time on ice.

[0013] Figure 2B is a graph showing the significantly different effect of chilling on ice and refrigeration in 5-FU stability. The measured 5-FU level (ng/ml) is plotted against the chill type (on ice or in 4⁰C refrigerator for 5 to 20 minutes).

[0014] Figure 3 shows the significant impact of both freezing time and freezing temperature on the stability of the 5-FU in the collected plasma samples.

[0015] Figure 4A shows the significance of freezing time on the stability of the 5-FU in the collected plasma samples as measured at different time points post- shipment.

[0016] Figure 4B shows the significance of freezing temperature on the stability of the 5-FU in the collected plasma samples as measured at different time points post- shipment.

[0017] Figure 5A demonstrates the effect of plasma collection (separation from buffy coat) on the stability of 5-FU in the sample.

[0018] Figure 5B is a graph showing the effect of pre-shipping sample storage condition on the stability of 5-FU in the sample.

[0019] Figure 5C is a graph showing the effect of shipping condition on the stability of 5-FU in the sample.

[0020] Figure 6A is a graph showing the combined effect of plasma collection and pre-shipping sample storage condition on 5-FU stability in the sample. "Good" samples are clean plasma samples with clear separation from buffy coat, whereas "WBC" samples are clean plasma samples mixed with buffy coat simulating the effect of poor separation during plasma collection.

[0021] Figure 6B is a graph showing the combined effect of plasma collection and sample shipping temperature on 5-FU stability in the sample. "Good" samples are clean plasma samples with clear separation from buffy coat, whereas "WBC" samples are clean plasma samples mixed with buffy coat simulating the effect of poor separation during plasma collection. [0022] Figure 6C is a graph showing the combined effect of pre-shipping sample storage condition (4⁰C, -2O⁰C, or room temperature) and sample shipping temperature (4⁰C or room temperature) on 5-FU stability in the sample.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a method for processing a blood sample obtained from a patient administered 5-fluorouracil (5-FU) or a prodrug thereof (e.g., capecitabine, tegafur, S l) for a 5-fluorouracil (5-FU) assay. The method is particularly suited for clinical testing for plasma level of 5-FU, especially in a clinical reference lab setting. Generally, the method comprises the steps of (1) obtaining a blood sample from a patient administered 5-FU or a prodrug thereof; (2) immediately chilling the blood sample at a temperature of about O⁰C for at least about 5 minutes; (3) within about one hour thereafter, separating plasma in the blood sample to form a plasma layer; and (4) collecting no greater than half of the plasma layer for use in an assay for 5-FU level. Preferably, before being shipped for testing, the collected plasma sample is placed under a freezing condition sufficient for the sample to become frozen within about one hour.

[0022] The method of the present invention can be used to process blood samples from any patients administered 5-FU or a prodrug thereof in whom 5-FU level is to be determined. For example, 5-FU and its prodrugs are commonly used in chemotherapy for cancer patients including patients with breast cancer, colorectal cancer, pancreatic cancer or head and neck cancer. 5-FU is also used in ophthalmic surgery in glaucoma patients as an anti-scarring agent at the trabeculectomy site. In addition, 5-FU is also used topically as a cream for treating Bowen's disease, solar keratoses, and basal cell carcinomas of the skin.

[0023] Patients from whom a blood sample is drawn for use in the method of the present invention can be administered with 5-FU or a prodrug alone, or in combination therapies. For example, 5-FU is typically administered along with folic acid or leucovorin. In metastatic colon cancer, 5-FU is often used in combination regimens such as FOLFOX (5-FU plus Oxaliplatin and leucovorin) and FOLFIRI (5-FU plus irinotecan and leucovorin). Newer drugs such as bevacizumab, panitumumab and cetuximab may also be added to the regimens.

[0024] In cancer therapies, 5 -fluoro uracil is typically administered by intravenous bolus injection or intravenous infusion. The administration can be repeated daily, weekly, every four weeks, depending on the choice of treatment regimen. Bolus injection can be given over 1 to 10 minutes or over 20 to 60 minutes, and for example, can be repeated for 5 consecutive days, every four week for 6 cycles. In continuous IV infusion, 5-FU can be administered over several hours, e.g., in 8 hours on a weekly basis. Blood samples can be drawn from a patient at any time before, after or during the administration of 5-FU. Often times, measuring blood 5-FU level at a steady state is desirable, in which case blood samples are drawn after a steady state is reached. For example in an eight-hour continuous infusion, a steady state can be reached two hours after the initiation of the infusion.

[0025] Typically, blood samples can be collected in any conventional ways of drawing peripheral venous blood. For example, a blood sample can be collected into a tube or vial typically used in clinics. The amount of blood collected depends on the amount required for a particular 5-FU assay. For example, 1 milliliter to 2 milliliters to 5 milliliters to 10 milliliters can be sufficient for most types of 5-FU assays.

Optionally, EDTA or heparin may be mixed with the blood sample to prevent clotting. The mixing can be achieved by e.g., using a heparinized tube or an EDTA tube.

[0026] In accordance with the method of the present invention, the collected blood sample should be immediately chilled at a temperature of about O⁰C for at least about 5 minutes. That is, after the blood sample is separated from the patient's body, the collected blood sample should be as soon as practical placed in an environment having a temperature of about O⁰C for at least about 5 minutes, 10 minutes or 15 minutes, and preferably from about 5 minutes to about one to two hours. This can be done e.g., by placing the blood sample on ice or immersed in ice water, or in a O⁰C refrigerator. It has been surprisingly discovered that such a chill for 5 minutes or longer results in a significant increase in the stability of 5-FU in blood.

[0027] In accordance with the method of the present invention, after the chilling step, plasma is separated in the chilled blood sample. This can be conveniently achieved by centrifuging the chilled blood sample at a sufficient speed and for a sufficient amount of time so as to form a clear plasma layer. Separating plasma in blood samples is well known in the art and would be apparent to skilled artisans.

Generally, centrifugation at from about 1000 g to about 5000 g for about ten minutes may be sufficient. While spinning at room temperature is acceptable, it is preferably done at a temperature below room temperature, e.g., at about 4⁰C.

[0028] After centrifugation, the blood sample is typically divided into three layers: the plasma layer at the top, the buffy coat or cellular layer (with lymphocytes) in the middle, and the red blood cells at the bottom. In accordance with the present invention, the plasma layer is collected and placed in a new container. Importantly, only a portion, but not all, of the plasma layer is collected and care must be taken so as not to disturb the buffy coat/cellular layer. In preferred embodiments, no more than two thirds by volume, more preferably no more than one half by volume of the plasma layer is collected. More preferably, no more than the top two thirds or one half layer of the plasma layer is collected for further processing. It has been discovered that limiting the collection of the plasma player as such significantly increases 5-FU stability in the sample, and substantially improves test consistency and accuracy, particularly in large scale commercial testing. In preferred embodiments, at least 1 ml of plasma sample is collected.

[0029] The collected plasma sample can be used in a biochemical assay for 5- FU. In a typical commercial setting, plasma samples are collected in physicians' clinics, and the 5-FU assays are performed separately in a laboratory distant from the clinics. As such, the plasma samples have to be sent from the clinics to the laboratory, often by a courier. In those situations, before being sent or shipped to a distant location, such as a laboratory, preferably the collected plasma sample is placed under a freezing condition and for a time sufficient for the collected plasma sample to become completely frozen. For this purpose, for example, the sample can be stored in a freezer with a temperature of about -12⁰C or lower for at least about 3 hours, or preferably in a freezer with a temperature of about -2O⁰C or lower for at least about 1 hour, and preferably for 3 hours or longer. Alternatively, the sample can be quickly frozen in liquid nitrogen or dry ice, and either immediately shipped or stored thereafter in a freezer (-12⁰C or lower, -2O⁰C or lower) for storage thereafter for 1 hour or 2 hours or 3 hours or longer.

[0030] During shipping, preferably the frozen plasma sample is placed in a shipping container having a refrigerant sufficient to maintain a temperature of below about 1O⁰C, preferably below about 8⁰C for at least about 24 hours in the container . Any conventional shipping containers can be used, but preferably those containers with good heat insulating properties are used, e.g., Styrofoam boxes. For cooling, any suitable refrigerants may be used, e.g., PolarPack™ foam brick refrigerant gel packs (VWR), and dry ice. Typically, the gel packs are stored with the collected plasma samples at a freezing temperature and before shipping placed into the container together with the sample. Upon receipt after overnight shipping, the temperature inside the container can be below about 1O⁰C or below 8⁰C.

[0031] The samples processed by the method of the present invention are particularly suitable for use in assays for 5 -FU level, as 5 -FU degradation in the samples after they are drawn but before assaying is minimized. Any 5 -FU assay methods known in the art may be used in measuring 5 -FU levels in the samples obtained by the method of present invention. For example, methods of quantitatively assaying 5 -FU by HPLC are generally known in the art. Alternatively, U.S. Patent No. 7,205,1 16, which is incorporated herein by reference, discloses 5-FU immunoassays for quantification of 5-FU in plasma samples using antibodies specifically immunoreactive with 5-FU.

[0032] In the following illustrative examples, 5-FU was measured using an antibody-based agglutination assay on the Olympus AU680 Analyzer using an antibody disclosed in U.S. Patent No. 7,205, 116. Specifically, the concentration of 5-FU in a sample is determined based on the absorbance of a sample, which is determined by the amount of aggregation by the nanoparticles. The system requires two reagents, which when mixed in the absence of free 5-FU will form large aggregates of nanoparticles and scatter light to result in a higher absorption value. In the presence of free 5-FU, the agglutination reaction becomes somewhat inhibited and scattering of light is less, causing a lower absorption value. The instrument is calibrated twice monthly, and a curve is generated (Absorbance vs. 5 -FU concentration). Absorbance values from individual samples are compared against this curve to determine concentration.

[0033] Generally in the agglutination assay, 95 μl of a buffer containing 5-FU- labeled amino-dextran is pipetted to a cuvette, followed by 7μL of a filtered plasma sample. The sample and reagent are mixed and then incubated for about 3.5 minutes. 95 μl of 5-FU antibody-conjugated nanoparticles is pipetted into the cuvette and mixed. The complete reaction incubates for about 6.2 minutes. A series of 27 measurements at 600 nm are taken at regular intervals by the AU680 analyzer throughout this process, and 5-FU concentration is calculated using the measurements from two specific time points.

EXAMPLE 1

This experiment tested 3 different sample collection tubes (spray-on K₂-EDTA, K3-EDTA, and NaHeparin) under the following 5 different incubation conditions after the blood is drawn:

1) immediate centrifugation

2) 20 minutes at RT prior to centrifugation

3) 20 minutes on ice prior to centrifugation

4) 30 minutes on ice prior to centrifugation

5) 60 minutes on ice prior to centrifugation

Two donors were required to complete the experiment. Each donor provided 8 tubes of blood for the K₂-EDTA tubes, and 4 tubes each for the K3-EDTA and

NaHeparin tubes. Tube types were staggered during collection in the event the donor was unable to donate the full spectrum of tubes, and the tubes were inverted gently 5 times after collection to mix the anti-coagulant into the blood. The tubes for each tube type were consolidated into its respective 50 ml conical tube. From each pooled sample, 21 ml was transferred into a clean 50 ml conical tube using a graduated serological pipet. Each sample (21 ml) was spiked with lOOμl of 63 ng/μl 5-FU and inverted gently 5 times to mix. The spiked pools were aliquotted into five 3 ml serum tubes, one each to follow the incubation parameters outlined above. After incubation, samples were spun at 2500 rpm for 10 minutes at room temperature. Each plasma sample was transferred into a separate 15 ml conical tube using a transfer pipet. A 125 μl aliquot was taken from each sample for a time-zero (TO) measurement on Olympus AU680 analyzer. Plasma was centrifuged for 15 minutes at 12,500rpm prior to being loaded onto the analyzer. Plasma samples (15 ml corneals) were stored at room temperature covered in foil, and 5-FU level was assayed in each sample 24 hours later.

[0034] As shown in Figure 1 , significant degradation of 5-FU was associated with samples that were immediately centrifuged (no ice incubation) or centrifuged after a 20-minute incubation at room temperature. In Figure 1 , on the "Incubation" half of the graph, the labels on the x-axis are as follows:

"0": immediate centrifugation, no incubation at room temperature or on ice;

"1": 20 minutes at room temperature before centrifugation;

"20": 20 minutes on ice before centrifugation;

"30": 30 minutes on ice before centrifugation;

"60": 60 minutes on ice before centrifugation.

EXAMPLE 2: The Effect of Ice Chill on 5-FU Stability

[0035] Blood was collected from donors into K2-EDTA tubes, and blood draws were spaced every 20 minutes. Blood collection tubes were inverted gently 5 times to mix the anti-coagulant into the blood and kept at room temperature throughout the duration of the preparation time. Blood was pooled by individual into 50 ml conical tubes, and as much blood as possible was measured and transferred into a new 50 ml conical tube using a graduated serological pipet. Calculations were made to adjust the volume of 5-FU to be spiked into the blood with the goal of getting a whole blood concentration of 300 ng/ml of 5-FU for each pool. The blood was inverted gently 5 times and aliquotted into multiple serum tubes per donor. The conditions being tested are as follows:

1) spin immediately (2 tubes per donor)

2) 5 minutes on ice

3) 10 minutes on ice

4) 15 minutes on ice

5) 20 minutes on ice 6) 5 minutes in refrigerator

7) 10 minutes in refrigerator

8) 15 minutes in refrigerator

9) 20 minutes in refrigerator

[0036] The blood was then either spun immediately or put in a refrigerator at 4⁰C or on ice for the designated amount of time prior to centrifugation to separate plasma. The blood samples were spun at 2500 rpm for 10 minutes, and the plasma was aspirated off of each sample into a 15 ml conical tube using a transfer pipet. A 125 μl aliquot was taken for a baseline measurement, spun in a Nanosep IOOK filter tube for 15 minutes at 12,500 rpm, transferred into a 12x75mm tube containing a nesting cup, and loaded onto Olympus AU680 analyzer. The remaining plasma was stored at room temperature covered in foil, and 5 -FU level was measured in the samples 24 hours after centrifugation.

[0037] Figure 2A is a graph showing the significant impact on 5-FU degradation by spinning immediately or putting on ice for at least 5 minutes before the spinning. The measured 5-FU level (ng/ml) is plotted against the chill time on ice.

[0038] Figure 2B is a graph showing the significantly different effect of chilling on ice and refrigeration in 5-FU stability. The measured 5-FU level (ng/ml) is plotted against the chill type (on ice or in refrigerator for 5 to 20 minutes). In Figure 2B, each data point is an average of multiple data at different lengths of chill time between 5 and 20 minutes.

[0039] As shown in Figure 2, there is a significant difference in 5-FU

degradation between spinning immediately and putting on ice for at least 5 minutes before the spinning. There is also a significant difference between chilling on ice and refrigeration.

EXAMPLE 3 : Effect of Pre-shipment Freezing Temperature and Length of Time

[0040] Five tubes (spray-on K₂EDTA tubes, 6 ml) of blood was drawn from each blood donor. The tubes were gently inverted 5 times to mix anti-coagulant with the blood. The blood tubes were placed on ice for at least 20 minutes before centrifuged at 2000 g, for 10 minutes at room temperature. The plasma was aspirated and pooled by donor into 50 ml conical tubes. All of each donor's plasma was transferred into a new 50 ml conical tube, and the volume transferred was recorded. Based on the volume of the plasma from each donor, it was calculated the volume of 200 ng/μl 5-FU stock to be added to the plasma to achieve a final concentration of about 500 ng/ml 5-FU. The calculated volume of 5-FU stock was added to the plasma and mixed by inversion. An aliquot of each sample was run on the Olympus AU680 analyzer for 5-FU level as a t_zero timepoint. This occurred about 15 minutes after the addition of the buffy coat.

[0041] The sample tubes were placed in designated freezers of different temperatures (-6⁰C, -12 ⁰C, and -20 ⁰C) for either 1 or 3 hours. After 1 hour of freezing, samples at -6⁰C were all liquid, two out of six samples at -12⁰C were liquid, and all samples at -2O⁰C were frozen. After 3 hours of freezing, one sample was completely frozen at -6⁰C; the other five were partially frozen, and all samples at -12⁰C and -2O⁰C were frozen. After the specified length of freezing, the tubes were transferred out of the freezers, and put in a Styrofoam shipping container with four gel ice packs for overnight cold shipping with UPS. Two different boxes were prepared: one for the samples frozen for 1 hour, and the other for the samples frozen for 3 hours.

[0042] Upon receipt, the samples were stored at room temperature until the end of the experiment. For 5-FU assays, the received samples were run under normal conditions for the Olympus AU680 analyzer, as described above. The assays were performed at about 24 hours after initial centrifugation of the blood samples.

[0043] As shown in Figures 3, 4 A, and 4B both freezing time and freezing temperature the collected plasma samples were subjected to prior to shipping had significant effect on the stability of the 5-FU in the samples. In Figure 4A, each data point is an average of multiple data at freezing temperatures of -6 ⁰C, -12⁰C, and -2O⁰C. In Figure 4B, the freezing times were 1 hour and 3 hours for each temperature, and the data points were averaged so that each set of data points per temperature is a combined value of the 1 hour and 3 hours of incubation. As shown in Figures 4A and 4B, the effect of freezing temperature is more pronounced by delays in assaying the samples after receipt from shipment.

[0044] EXAMPLE 4: COMBINATION EFFECT [0045] This experiment is a follow-up to the experiment in Example 3 that tested multiple shipping and storage conditions. The difference in this experiment is that "sloppy" plasma aspiration conditions will also be tested in addition to the standard "good" conditions. "Sloppy" samples will have WBC/buffy coat added to the plasma sample to induce degradation. After plasma is aspirated from all samples, they will be placed at one of three incubation conditions (room temperature, 4⁰C, and frozen) and then shipped either cold (gel ice) or at room temperature.

[0046] Five tubes (spray-on K₂EDTA tubes, 6 ml) of blood was drawn from each blood donor. The tubes were gently inverted 5 times to mix anti-coagulant with the blood. The blood tubes were placed on ice for about 30 minutes before centrifuged at 2000 g, for 10 minutes at room temperature. The plasma was aspirated and pooled by donor into 50 ml conical tubes. The buffy coat from all donors was also pooled into new a serum tube, and spun at 2000 g for 10 minutes at room temperature. The enriched buffy coat was collected into a small cryovial or Eppendorf tube. All of each donor's plasma was transferred into a new 50 ml conical tube, and the volume transferred was recorded. Based on the volume of the plasma from each donor, it was calculated the volume of 200 ng/μl 5-FU stock to be added to the plasma to achieve a final concentration of about 500 ng/ml 5-FU. The calculated volume of 5-FU stock was added to the plasma and mixed by inversion. Half of each spiked plasma sample was transferred into a new separate conical tube (by donor).

[0047] For "sloppy" samples, the enriched WBC/buffy coat was spiked into the plasma sample in an amount of 50 μl buffy coat per 3 ml plasma, and mixed thoroughly. An aliquot of each sample was run on the analyzer for 5-FU level as a t_zero timepoint. This occurred about 30 minutes after the addition of the buffy coat.

[0048] The sample tubes were placed under designated temperature conditions (benchtop for room temperature, refrigerator, and freezer (-2O⁰C)) for a predetermined length of time. After the specified length of time, the tubes were placed in plastic bags, and put in separate Styrofoam shipping containers with four frozen gel ice packs for overnight cold shipping with UPS. The temperature inside the containers upon receipt is typically below about 8⁰C. [0049] Upon receipt, the samples were stored at room temperature until the end of the experiment. For 5-FU assays, the received samples were run under normal conditions for the Olympus AU680 analyzer, as described above. The assays were performed at about 24 hours after initial centrifugation of the blood samples.

[0050] As shown in Figures 5 and 6, plasma separation quality, storage conditions (prior to shipping), and shipping conditions are each clinically and statistically significant in their effect on 5-FU stability and 5-FU assay accuracy. The interaction between separation and storage, separation and shipping, and storage and shipping are all statistically and clinically significant. In Figures 5 C, 6B and 6C, the "cold" shipping indicates that a plasma sample was placed in a container having four frozen gel ice packs for overnight shipping with UPS. The temperature inside the containers upon receipt was below about 8⁰C. "RT" means that no gel ice packs were used in the overnight UPS shipping.

[0051] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.

[0052] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of processing a blood sample from a patient administered 5- fluorouracil (5-FU) or a prodrug thereof for a 5 -fluoro uracil (5-FU) assay, said method comprising:

obtaining a blood sample from the patient administered 5 -FU or a prodrug thereof;

immediately chilling the blood sample at a temperature of about O⁰C for at least about 5 minutes;

centrifuging the blood sample within about one hour to separate plasma from the blood sample and form a plasma layer;

collecting no greater than the top half of the plasma layer;

placing the collected plasma sample under a freezing condition and for a time sufficient for the collected plasma sample to become completely frozen; and

placing the frozen collected plasma sample in a shipping container having a refrigerant sufficient to maintain a temperature of below about 1O⁰C within the container for at least about 24 hours.

2. The method of Claim 1 , wherein the patient is undergoing continuous intravenous infusion of 5-FU and the blood sample is drawn at about two hours or longer after the beginning of the infusion.

3. The method of Claim 1 , wherein said collected plasma sample is at least 1 ml.

4. A method of processing a blood sample from a patient administered 5- fluorouracil (5-FU) or a prodrug thereof for a 5 -fluoro uracil (5-FU) assay, said method comprising: obtaining a blood sample from the patient administered 5 -FU or a prodrug thereof;

collecting no greater than the top half of the plasma layer; and

placing the collected plasma sample in a temperature of -2O⁰C or lower for at least about one hour or a temperature of -12⁰C or lower for at least about 3 hours.

5. The method of Claim 4, further comprising placing the frozen collected plasma sample in a shipping container having a refrigerant sufficient to maintain a temperature of below about 1O⁰C inside the container for at least about 24 hours.