WO2018220601A1

WO2018220601A1 - Sample storage system and methods of use thereof

Info

Publication number: WO2018220601A1
Application number: PCT/IB2018/053958
Authority: WO
Inventors: Anthony TSAI; Tatsuya Matsunaga
Original assignee: Hitachi Chemical Co., Ltd.
Priority date: 2017-06-02
Filing date: 2018-06-01
Publication date: 2018-12-06

Abstract

The disclosure relates to storage systems using citrate, theorphylline, adenosine and dipyridamole (CTAD) for circulating tumor cells (CTCs). The disclosure also relates to kits using citrate, theorphylline, adenosine and dipyridamole (CTAD) for circulating tumor cells (CTCs). The disclosure further relates to methods of using citrate, theorphylline, adenosine and dipyridamole (CTAD) for storing circulating tumor cells (CTCs).

Description

Sample Storage System and Methods of Use Thereof

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention relates to storage systems for circulating tumor cells (CTCs) and methods of use thereof.

Description of the Related Art

[0002] The CTCs have the potential to be an indicator to monitor therapy response and predict drug efficiency. The CTCs can be collected by a noninvasive blood collection method, enabling multi-sampling for the continuous tracking of therapeutic response. However, the number of CTCs in blood is small compared to the common blood cells, such as platelets, red blood cells, and white blood cells (WBCs).

[0003] Current US Food and Drug Administration (FDA)-approved CellSearch system (Veridex, Raritan, NJ, USA) analysis is using magnetic beads coated with a monoclonal antibody-targeting epithelial cell marker, such as the epithelial cell-adhesion molecule (EpCAM) to enrich CTCs. This method, however, does not capture all types of CTCs, and the recovery of the captured cells for the downstream molecular or cellular analysis is limited. Thus, it is important to develop an improved methodology for CTC isolation that enables subsequent molecular analysis even at the single cell level in order to understand the origin and role of CTCs in cancer progression and treatment response.

[0004] Recently, devices using microfabrication method for size-based separation of tumor cells have been widely developed to enable precise and efficient enrichment of CTCs from whole blood. These devices include a miniaturized micro filter array system that can be used to trap rare cells by filtration based on differences in the sizes of cells.

[0005] An automated filter-based system is able to deplete the majority of those common cells and retain the CTCs for further analysis including enumeration with immunostaining and molecular analysis, such as gene expression from fresh sample. The filter pore size of our system can be precisely controlled by photolithography and the metal plating technology. Due to the fact that cancer cells are isolated solely based on their size and deformability, not their surface markers, our filter system can be applied to a broad range of cancers unlike an antigen-antibody based system (e.g., EpCAM-based).

[0006] In order to efficiently deplete the common cells from human whole blood, samples are processed through the filter-based system within 6 hours. The number of remaining WBCs will drastically increase after 8 hours' storage. The longer the sample stores after drawing from a body, the more WBCs can be found on the filter after processing. The background noise from WBCs may affect the results of molecular analysis. In this disclose, we described a sample storage system, for example, to extend the sample storage time for CTCs, and methods of use thereof.

SUMMARY OF THE INVENTION

[0007] The disclosure is related to a method of preparing a circulating tumor cell (CTC) storage sample, comprising adding citrate, theorphylline, adenosine and dipyridamole (CTAD) to a blood sample containing CTCs to prepare the CTC storage sample. In some embodiments, the concentration of CTAD in the storage sample is from 0.01% to 1%; and/or the CTC storage sample comprises microbeads including, for example, magnetic beads.

[0008] The disclosure is also related to a method of detecting circulating tumor cells (CTCs), comprising preparing the CTC storage sample as described herein; and detecting CTCs in the CTC storage sample. The disclosure is also related to a method of retaining circulating tumor cells (CTCs), comprising preparing the CTC storage sample as described herein; and storing the CTC storage sample. The disclosure is further related to a method of detecting circulating tumor cells (CTCs), comprising retaining the CTCs as described herein; and detecting CTCs in the CTC storage sample.

[0009] The disclosure is also related to a method of reducing proliferation of white blood cells (WBCs) in a blood sample containing CTCs, comprising preparing the CTC storage sample as described herein; and storing the CTC storage sample. The disclosure is further related to a method of detecting circulating tumor cells (CTCs), comprising reducing proliferation of WBCs as described herein; and detecting CTCs in the CTC storage sample. [00010] In some embodiments, the methods described herein may comprise storing the CTC storage sample for at least 1 hour. In additional embodiments, after 2 days of storing, a number of CTCs in the CTC storage sample is decreased by less than 30% compared to an original number of CTCs in the blood sample. In yet additional embodiments, after 2 days of storing, the ratio of the number of CTCs to the number of WBCs in the CTC storage sample is decreased by less than 10 times compared to an original ratio of the number of CTCs to the number of WBCs in the blood sample. In further embodiments, wherein after 2 days of storing, a number of WBCs in the CTC storage sample increases less than 20 times compared to an original number of WBCs in the blood sample. In yet further embodiments, the CTC storage sample is stored at room temperature.

[00011] The disclosure is also related to a storage system comprising CTCs and CTAD. In some embodiments, the concentration of CTAD in the storage sample is from 0.01% to 1%; and/or the CTC storage sample comprises microbeads including, for example, magnetic beads.

[00012] The disclosure is also related to a kit comprising CTAD and at least one probe for CTCs. In some embodiments, the at least one probe is an immunostaining material for a marker selected from the group consisting of DAPI, cytokeratin (CK) and CD45.

BRIEF DESCRIPTION OF THE DRAWINGS

[00013] These and other aspects of the invention will be readily apparent from the following description and from the appended drawings (not to scale), which are meant to illustrate and not to limit the invention, and wherein:

[00014] Figure 1 demonstrates the recovery of 100 spiked cancer cells and the remaining WBCs on the filter from 3 mL whole blood with different storage time and additives including EDTA, CTAD, Cell-Free DNA, Heparin, citric acid, ACD-A and glucose (concentration 200 mM-1000 mM).

[00015] Figure 2 presents the recovery of 100 spiked cancer cells and the remaining WBCs on the filter from the same donor's 3 mL whole blood aliquots with different storage time and selected additives including EDTA, CTAD, Cell-Free DNA and Heparin. [00016] Figure 3 A is the recovery of 100 spiked cancer cells and the remaining WBCs on the filter from the same donor's 3 mL whole blood aliquots with different storage time and selected additives including EDTA and CTAD.

[00017] Figure 3B shows the recovery of 100 spiked cancer cells and the remaining WBCs on the filter from the same donor's 3 mL whole blood aliquots as Figure 3 A with magnetic beads pretreatment, different storage time and selected additives including EDTA and CTAD.

[00018] Figure 4A performs the comparison of spiked cell recovery rate from the same donor's 3 mL whole blood aliquots, stored in EDTA tubes, with and without magnetic beads pretreatment (left: EDTA without magnetic beads; right: EDTA with magnetic beads (EDTA-MB)).

[00019] While Figure 4B performs the comparison of spiked cell recovery rate from the same donor's 3 mL whole blood aliquots, stored in CTAD tubes, with and without magnetic beads pretreatment (left: EDTA without magnetic beads; right: EDTA with magnetic beads (EDTA- MB)).

[00020] Figure 5A displays the comparison of remaining WBCs on the filter from the same donor's 3 mL whole blood aliquots, stored in EDTA tubes, with and without magnetic beads pretreatment (left: EDTA without magnetic beads; right: EDTA with magnetic beads (EDTA- MB)).

[00021] Figure 5B presents the comparison of remaining WBCs on the filter from the same donor's 3 mL whole blood aliquots as Figure 5 A, stored in CTAD tubes, with and without magnetic beads pretreatment (left: EDTA without magnetic beads; right: EDTA with magnetic beads (EDTA-MB)).

[00022] Figure 5C demonstrates displays the comparison of remaining WBCs on the filter from the same donor's 3 mL whole blood aliquots as Figure 5 A, stored in EDTA and CTAD tubes with magnetic beads pretreatment (left: EDTA without magnetic beads; right: EDTA with magnetic beads (EDTA-MB)).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[00023] The following detailed description is directed to certain specific embodiments of the invention. However, the invention can be embodied in a multitude of different ways. In this description, reference is made to the drawing wherein like parts are designated with like numerals throughout.

[00024] In one aspect, the disclosure is related to a method of preparing a circulating tumor cell (CTC) storage sample, comprising adding at least one, two, three or four of Citrate,

Theorphylline, Adenosine and Dipyridamole (CTAD) to a sample containing CTCs to prepare the CTC storage sample. The "circulating tumor cell" (CTC) is a cancer cell that detach from a primary tumor and travel though blood stream or lymphatic system to other parts of the body. The CTC may be detected by using methods known in the art, for example, by detecting cells that are DAPI positive, cytokeratin (CK) positive and CD45 negative. The methods described herein may comprise staining the CTCs immunochemically with DAPI, CK and/or CD45.

[00025] The CTC storage sample is a sample containing at least one CTC drawn outside of a body. In some embodiments, the CTC storage sample has at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500 or 600 CTCs per mL before or after any filtering process. In additional embodiments, the CTC storage sample may from about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or 5.0 mL to about 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 10.0, 20.0, 30.0, 40.0 or 50.0 mL. For example, the CTC storage sample may be a blood sample having 100/3mL CTCs.

[00026] In some embodiments, the concentration of the at least one, two, three or four of citrate, theorphylline, adenosine and dipyridamole (CTAD) added in the CTC storage sample is from about 0.0001, 0.001, 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0% to about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0 or 5.0%. In additional embodiments, the concentration of the at least one, two, three or four of citrate, theorphylline, adenosine and dipyridamole (CTAD) added in the CTC storage sample is from about 0.0001 to about 5.0%, 0.01 to about 2.0%, or about 0.01 to about 1.0%. In additional embodiments, the citrate in CTAD may be sodium citrate. For example, using 50-200 mmol/L buffered citrate containing theophylline, adenosine, and dipyridamole (CTAD) may minimize platelet activation. In order to minimize platelet activation and cancer cell survivability, the concentration of sodium citrate for CTAD solution may be from about 0.1% to about 3%, from about 0.1 % to about 2.5 %, from about 1 % to about 2.5 %; the concentration of theophylline for CTAD solution may be from about 0.01% to about 10%, from about 0.1 % to about 1 %, from about 0.15 % to 0.5 %; the concentration of adenosine for CTAD solution may be from about 0.01% to about 10%, from about 0.02 % to about 1 %, from about 0.05 % to about 0.5 %; and the concentration of dipyridamole for CTAD solution may be from about 0.001% to about 20%, from about 0.002 % to 10%, from about 0.005 % to about 5 %. In some embodiments, the concentration of sodium citrate for CTAD solution may be from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 or 1.1 % to about 1, 1.5, 2, 2.5, or 3%; the concentration of theophylline for CTAD solution may be from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 % to about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 %; the concentration of adenosine for CTAD solution may be from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 % to about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 %; and the concentration of dipyridamole for CTAD solution may be from about 0.0001, 0.001, 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0% to about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 %.

[00027] In further embodiments, citrate, theorphylline, adenosine and dipyridamole (CTAD) are added in the CTC storage sample, and the concentration of the CTAD is from about 0.01% to about 2%, from about 1 % to 3 %, from about 0.001 % to 5%, from about 0.01% to 2% or from about 0.01% to 1 %.

[00028] As used herein, the term "about" modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, diameters, lengths, and like values, and ranges thereof, refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term "about" also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term "about" the claims appended hereto include equivalents to these quantities. The term "about" further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term "about" refers to a range of values that fall within 10, 9, 8,7, 6, 5,4, 3, 2, 1 percent or less of the stated reference value.

[0001] In some embodiments, the sample described herein may be blood sample. In some embodiments, the sample is selected from the group consisting of blood, serum, plasma, urine, sweat, and saliva. In further embodiments, the method described herein may comprise collecting a sample from a subject. The subject may be human or animal.

[00029] In some embodiments, the CTC storage sample may comprise microbeads. The microbeads described herein may have an average dimension from about 1, 5, 10, 50, 80, 100, 150, 200, 300 or 400 microns to about 0.3, 0.5, 0.8, 1, 5, 8, or 10 mm; from about 10 microns to about 10 mm; about 100 microns to about 10 mm, from about 1 micron to about 0.1 mm, from about 1 micron to about 1 mm, or from about 1 micron to about 5 mm. In additional

embodiments, the microbeads may be magnetic beads. In additional embodiments, the microbeads may include one or more materials selected from the group consisting of metal, glass, plastic, agar, carrageenan and agarose. In further embodiments, the concentration of microbeads in the CTC storage sample may be from about 0.5, 1, 5, 10, 20 or 30 to about 40, 50, 80, 100 or 200

sample. In yet further embodiments, the microbeads may be labeled with a marker, for example, including antibody, such as CD45.

[00030] In another aspect, the disclosure includes a method of detecting circulating tumor cells (CTCs), comprising preparing the CTC storage sample described herein; and detecting CTCs in the CTC storage sample. In additional embodiments, the CTCs may be filtered and/or detected by methods described in U.S. Patent Application Publication Nos. 2014-0178890; 2014- 0238863; 2015-0004687; 2014-0299539; 2015-0111293; 2016-0195458; and 2016-0169781, all of which are herein incorporated by reference in their entirety. For example, based on the methods described herein, the number of CTCs recovered and/or detected after storing is about 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 98, 99 or 100 % or more of the number of CTCs collected from the initial sample drawn from a subject. In some embodiments, based on the methods described herein, the number of CTCs recovered and/or detected after storing and filtering is about 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 98, 99 or 100 % or more of the number of CTCs collected from the initial sample drawn from a subject. Moreover, the background noise may be less than 3000, 2000, 1000, or 500 WBC background noise. In further embodiments, the detecting described herein may include molecular analysis of the CTC storage sample described herein, for example, for nucleic acids (e.g. DNA, RNA or mRNA) or proteins before or after filtering.

[00031] In another aspect, the disclosure includes a method of retaining circulating tumor cells (CTCs), comprising preparing the CTC storage sample as described herein; and storing the CTC storage sample. In another aspect, the disclosure includes a method of detecting circulating tumor cells (CTCs), comprising retaining the CTCs as described herein; and detecting CTCs in the CTC storage sample. In another aspect, the disclosure includes a method of reducing proliferation of white blood cells (WBCs) in a blood sample containing CTCs, comprising preparing the CTC storage sample described herein; and storing the CTC storage sample. In another aspect, the disclosure includes a method of detecting circulating tumor cells (CTCs), comprising reducing proliferation of WBCs as described herein; and detecting CTCs in the CTC storage sample.

[00032] For example, the retention described herein may extend the sample storage time (e.g. half-life) for CTCs before processing, for example, before filtering or detecting CTCs.

[00033]In some embodiments, the CTC storage sample is stored for at least about 10, 20 or 30 minutes, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, or 20 hours, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 26, 28 or 30 days. The CTC storage sample may be stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, or 20 hours, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 26, 28 or 30 days, or about 1, 2 or 3 months or less.

[00034]In some embodiments, after 2, 4, 6 or 12 hours, or 1, 2, 3, 4, 5, 10, 15, 20 or 30 days of storing, the number of CTCs in the CTC storage sample is decreased by less than 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % compared to an original number of CTCs in the blood sample.

[00035]In some embodiments, after 2, 4, 6 or 12 hours, or 1, 2, 3, 4, 5, 10, 15, 20 or 30 days of storing, the ratio of the number of CTCs to the number of WBCs in the CTC storage sample is decreased by less than 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.2 or 1.1 times compared to an original ratio of the number of CTCs to the number of WBCs in the blood sample. [00036]In some embodiments, after 2, 4, 6 or 12 hours, or 1, 2, 3, 4, 5, 10, 15, 20 or 30 days of storing, the number of WBCs in the CTC storage sample is increased less than 1.2, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 or 60 times compared to an original number of WBCs in the blood sample.

[00037] In some embodiments, the CTC storage sample is stored at or under room temperature (i.e. about 23 °C). In additional embodiments, the CTC storage sample is stored in liquid nitrogen. In further embodiments, the CTC storage sample is stored at about -196, 0, 20, 21, 22, 23, 24, 25 or 36 °C; at a temperature of about -190, -100, -50, -10, -5, -3, -1, 0, 1, 5, 10, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 °C or below; or at a temperature of about -200, -190, -100, -50, -10, -5, -3, -1, 0, 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,

22, 23, 24, 25, 26, 30, 35 or 37 °C or above. In yet further embodiments, the CTC storage sample is stored at from about 20 to about 25 °C, from about 19 to 26 °C, from about -5 to 5 °C, from about -200 to 190 °C, from about 0 to 10 °C, from about -20 to 19 °C, from about -200 to 19 °C, from about 26 to 36 °C, or from about 20 to 36 °C.

[00038] In another aspect, the disclosure also relates to a storage system comprising the CTCs and the at least one, two, three or four of CTAD described herein.

[00039] In another aspect, the disclosure also relates to a kit for storing comprising (i) the at least one, two, three or four of CTAD described herein; and (ii) one or more probes for the CTCs. In some embodiments, the at least one probe comprises an immunostaining material for a marker selected from the group consisting of DAPI, cytokeratin (CK) and CD45. In some embodiments, the at least one probe comprises an antibody. In additional embodiments, the at least one probe comprises an antibody for CK or CD45.

[00040] In some embodiments, the disclosure is related to additives in the sample storage system described herein and sample pretreatment procedure.

[00041] For example, first, sample with additives may be split into 3 ML aliquots. Second, cultured cancer cells may be diluted to 1000 cells/mL for spiking at 100 cells/100

Third, sample with spiked cancer cells may be processed through the filter-based CTC enrichment system. The machine reagent rack may contain the following: wash buffer in tube 1, CD45 primary antibody in tube 2, CD45 secondary antibody in tube 3, lysing solution (R2) in tube 4, permeabilizing solution (R3) in tube 5, and CK-DAPI staining solution in tube 6. After the sample injection, the filter cartridge may be washed with wash buffer, followed by incubation with CD45 stain, injection of lysing and permeabilizing solution, and incubation with CK-DAPI stain. A wash step with wash buffer may follow each incubation step. The filter aperture area may be scanned by fluorescence microscope and processed for cell nucleus stain in blue (DAPI), cytokeratin stain in green (FITC), and CD45 stain in red (Alexa594).

[00042] The additives described herein may include, but not limited to,

Ethylenediaminetetraacetic Acid (EDTA), poly-anethol- sulfonate, Sodium Citrate, Heparine, Potassium oxalate, glucose, Citrate dextrose acid, Citrate theorphylline adenosine dipyridamole (CTAD). The preservative additive for filter-based CTC enrichment system may be CTAD.

[00043] In some cases, whole blood sample with spiked cancer cells may require pretreatment which is incubating the mixture of whole blood and magnetic beads labeled with CD45 antibody.

Example 1

[00044] Cultured NCI-H358 lung cancer cells are diluted to 1000 cells/mL for spiking at 100 cells/100 μL·. Blood samples are collected in the following blood collection tubes: EDTA, CTAD, Cell-Free DNA, Heparin, citric acid, ACD-A and glucose with concentration 200 mM- 1000 mM.

[00045] Fresh blood samples were dispensed into 3 mL aliquots and spiked with 100 H358 cancer cells. The blood-cell samples were stored at room temperature. Samples were processed through Hitachi Chemical filter-based system after 2, 24, 48 and 72 hours incubation at room

temperature.

[00046] The filtration procedure included sample injection, washing, lysing, permeabilization, and staining. Processed sample was scanned through fluorescence microscope. Counting of cancer cells is based on DAPI positive, cytokeratin positive, and CD45 negative. While counting of WBCs is based on DAPI positive, cytokeratin negative and CD45 positive.

[00047] Figure 1 shows sample in tube with CTAD as additives results the consistent CTC recovery rate and relatively low remaining WBCs on the filter after up to 72 hours storage period, followed by EDTA, Cell-Free DNA, heparin, citric acid, ACD-A and glucose.

Example 2 [00048] This experiment was based on the selected tubes with different additive from example 1. Cultured SKBR3 breast cancer cells are diluted to 1000 cells/mL, for spiking at 100 cells/100 μΚ Blood samples were collected in the following blood collection tubes: EDTA, CTAD, Cell- Free DNA and Heparin. Same donors' fresh blood samples were dispensed into 3 rriL aliquots and spiked with 100 SKBR3 cancer cells. The blood-cell samples are stored at room temperature. Samples were processed through Hitachi Chemical filter-based system after 2, 24 and 48 hours incubation at room temperature.

[00049] The filtration procedure included sample injection, washing, lysing, permeabilization, and staining. Processed sample was scanned through fluorescence microscope. Counting of cancer cells is based on DAPI positive, cytokeratin positive, and CD45 negative. While counting of WBCs is based on DAPI positive, cytokeratin negative and CD45 positive.

[00050] Figure 2 presents sample in tube with CTAD as additives results the best CTC recovery rate and relatively low remaining WBCs on the filter after up to 48 hours storage period, followed by EDTA, Cell-Free DNA and heparin.

Example 3

[00051] This experiment was based on the selected tubes with different additive from example 2.

[00052] Cultured SKBR3 breast cancer cells were diluted to 1000 cells/mL, for spiking at 100 cells/100 μL·. Blood samples were collected in the following blood collection tubes: EDTA and CTAD. Same donors' fresh blood samples were dispensed into 3 rriL aliquots and spiked with 100 SKBR3 cancer cells. The blood-cell samples are stored at room temperature. Magnetic beads (50 μΕ/ηιΕ of sample) were added to the blood-cell mix and incubated on a rotator at room temperature for 20 minutes as sample pretreatment. The samples were then passed through a whole blood filter column to remove the magnetic beads. Pretreated and no pretreatment samples were processed through Hitachi Chemical filter-based system after 2, 24, 48 and 72 hours incubation at room temperature (Figure 3 A and 3B).

[00053] The filtration procedure included sample injection, washing, lysing, permeabilization, and staining. Processed sample was scanned through fluorescence microscope. Counting of CTC is based on DAPI positive, cytokeratin positive, and CD45 negative. While counting of WBCs is based on DAPI positive, cytokeratin negative and CD45 positive. The experiment was repeated 2 more times for statistical purpose.

[00054] Figure 4A shows no significant effect of magnetic beads for EDTA samples in terms of cancer cell recovery rate at 72 hours. While figure 4B shows CTAD samples cancer cell recovery rate increased by 93% after 72 hours at room temperature. Both Figures 5A and 5B demonstrate magnetic beads show positive effect in WBCs depletion. While Figure 5C clearly shows the effect of magnetic beads is more significant in samples with CTAD as additives than sample with EDTA as additive.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a circulating tumor cell (CTC) storage sample, comprising adding citrate, theorphylline, adenosine and dipyridamole (CTAD) to a blood sample containing CTCs to prepare the CTC storage sample.

2. The method according to claim 1, wherein the concentration of CTAD in the storage sample is from 0.01% to 1%.

3. The method according to any one of the preceding claims, wherein the CTC storage sample comprises microbeads.

4. The method according to any one of the preceding claims, wherein the CTC storage sample comprises magnetic beads.

5. A method of detecting circulating tumor cells (CTCs), comprising

preparing the CTC storage sample in accordance with the method of any one of claims 1-

4; and

detecting CTCs in the CTC storage sample.

6. A method of retaining circulating tumor cells (CTCs), comprising

4; and

storing the CTC storage sample.

7. A method of detecting circulating tumor cells (CTCs), comprising

retaining the CTCs in accordance with the method of claim 6; and

detecting CTCs in the CTC storage sample.

8. A method of reducing proliferation of white blood cells (WBCs) in a blood sample containing CTCs, comprising preparing the CTC storage sample in accordance with the method of any one of claims 1-

4; and

storing the CTC storage sample.

9. A method of detecting circulating tumor cells (CTCs), comprising

reducing proliferation of WBCs in accordance with the method of claim 8; and detecting CTCs in the CTC storage sample.

10. The method according to any one of the preceding claims, comprising storing the CTC storage sample for at least 1 hour.

11. The method according to any one of the preceding claims, wherein after 2 days of storing, a number of CTCs in the CTC storage sample is decreased by less than 30% compared to an original number of CTCs in the blood sample.

12. The method according to any one of the preceding claims, wherein after 2 days of storing, the ratio of the number of CTCs to the number of WBCs in the CTC storage sample is decreased by less than 10 times compared to an original ratio of the number of CTCs to the number of WBCs in the blood sample.

13. The method according to any one of the preceding claims, wherein after 2 days of storing, a number of WBCs in the CTC storage sample increases less than 20 times compared to an original number of WBCs in the blood sample.

14. The method according to any one of the preceding claims, wherein the CTC storage sample is stored at room temperature.

15. A storage system comprising CTCs and CTAD.

16. The storage system according to claim 15, wherein the concentration of CTAD in the storage sample is from 0.01% to 1%.

17. The storage system according to claim 15 or 16, wherein the CTC storage sample comprises microbeads.

18. The storage system according to any one of claims 15-17, wherein the CTC storage sample comprises magnetic beads.

19. A kit comprising CTAD and at least one probe for CTCs.

20. The kit according to claim 19, wherein the at least one probe comprises an immunostaining material for a marker selected from the group consisting of DAPI, cytokeratin (CK) and CD45.