JP6272476B2 - 赤血球溶解のための試薬の使用ならびに該試薬に関連する方法およびキット - Google Patents
赤血球溶解のための試薬の使用ならびに該試薬に関連する方法およびキット Download PDFInfo
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- JP6272476B2 JP6272476B2 JP2016527179A JP2016527179A JP6272476B2 JP 6272476 B2 JP6272476 B2 JP 6272476B2 JP 2016527179 A JP2016527179 A JP 2016527179A JP 2016527179 A JP2016527179 A JP 2016527179A JP 6272476 B2 JP6272476 B2 JP 6272476B2
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- 229960002897 heparin Drugs 0.000 description 1
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- 229910052742 iron Inorganic materials 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
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- 244000144972 livestock Species 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 101150024228 mdm2 gene Proteins 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
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- 238000012544 monitoring process Methods 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 229910052755 nonmetal Inorganic materials 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 description 1
- 208000015445 pyruvate dehydrogenase deficiency Diseases 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- PPRSVUXPYPBULA-UHFFFAOYSA-N saponin A Natural products CC1(C)CCC2(CCC3(C)C(=CCC4C5(C)CCC(OC6OC(CO)C(O)C(O)C6=O)C(C)(C)C5CCC34C)C2C1)C(=O)O PPRSVUXPYPBULA-UHFFFAOYSA-N 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000005563 spheronization Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
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- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 150000003512 tertiary amines Chemical group 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000003634 thrombocyte concentrate Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
Description
塩化アンモニウムは、全血中の赤血球の溶解を可能にする、浸透性塩として記載された。古典的な塩化アンモニウム溶解緩衝液は、150mM NH4Cl、1mM KHCO3および0.1mM EDTAを含有する。赤血球は、この緩衝液を用いて、定量的に枯渇させることも可能であるが、約30%またはそれより多くの白血球が失われる(例えば、Meryman H. Red Cell Structure and Function 1969; 352−367、Sass M. Am J Physiol. 1979;236(5):C238−43、Claus R.ら Folia Hematol. 1985; 5: 683−688、Terstappenら J Immun Methods. 1989: 103−112を参照されたい)。
− 2.5mmol/l〜12mmol/l HEPES、
− 60mmol/l〜120mmol/l NH4 +/NH3、
− 0.04mmol/l〜0.8mmol/lキレート剤、および
− 存在する場合、0.15mmol/l〜0.8mmol/l CO3 2-/CO3 -
の範囲である、前記試薬によって解決された。
− 2.5mmol/l〜12mmol/l HEPES、
− 60mmol/l〜120mmol/l NH4 +/NH3、
− 0.04mmol/l〜0.8mmol/lキレート剤、および
− 存在する場合、0.15mmol/l〜0.8mmol/l CO3 2-/CO3 -
の範囲である、前記試薬の使用に関する。
− 3mmol/l〜11mmol/l HEPES、好ましくは、3mmol/l〜10mmol/l HEPES、より好ましくは、3.5mmol/l〜4.5mmol/l HEPES、
− 70mmol/l〜100mmol/l NH4 +/NH3、好ましくは75mmol/l〜85mmol/l NH4 +/NH3、
− 0.05mmol/l〜0.5mmol/lキレート剤、好ましくは0.06mmol/l〜0.2mmol/lキレート剤、より好ましくは0.07mmol/l〜0.1mmol/lキレート剤、および/または
− 存在する場合、0.3mmol/l〜0.6mmol/l CO3 2-/CO3 -、好ましくは0.3mmol/l〜0.5mmol/l CO3 2-/CO3 -、より好ましくは0.35mmol/l〜0.45mmol/l CO3 2-/CO3 -
の範囲の最終濃度中で存在する。
特定の態様において、赤血球または赤血球を含有する試料は、哺乳動物から、特に哺乳動物家畜動物、例えばネコ、イヌ、ウサギ、またはモルモット、あるいは農場動物、例えばウシ、ウマ、ヤギ、ヒツジ、ブタまたはラクダから得られる。非常に特定の態様において、赤血球または赤血球を含有する試料は、ヒトから得られる。
a)赤血球を含む試料を提供し;
b)請求項1〜5のいずれかに定義するような試薬と試料をインキュベーションし、それによって赤血球を溶解し;そして
c)赤血球破片を除去してもよい
工程を含む、前記方法を提供する。
d)上に記載するように、赤血球を含む試料から、特に血液試料から、赤血球以外の細胞を検出するかまたは単離する
工程をさらに含む。
好ましくは、赤血球以外の細胞は、白血球または循環腫瘍細胞、特に循環腫瘍細胞である(上記の詳細もまた参照されたい)。
第三の側面において、本開示は
− 本明細書に開示するような使用および方法の文脈において上に定義するような赤血球溶解のための試薬;および
− 赤血球破片を除去するための試薬;および
− 本明細書に開示するような任意の方法を実行するためのあってもよい使用説明書
を含む、赤血球を含む試料から白血球を単離するためのキットに関する。
別に示さない限り、全血の1部分を、溶解緩衝液の部分(以下に定義する通り)と混合し、室温で10分間インキュベーションし、そして300xgで15分間遠心分離する。上清を廃棄し、そして細胞ペレットを4部分の溶解緩衝液に再懸濁し、そして300xgで15分間遠心分離する。上清を廃棄し、そして細胞ペレットを、0.3mM EDTAを含有するPBS 4部分に再懸濁し、そして300xgで15分間遠心分離する。上清を廃棄し、そして細胞ペレットを、0.3mM EDTAを含有するPBSの別個の量に再懸濁する。
最初の試験において、上記プロトコルにしたがって、慣用的製品の適切さを試験した。以下の慣用的製品を用いた。EasySep(登録商標)赤血球溶解緩衝液(StemCell Technologies、カタログ番号20110)、HetaSep(登録商標)(StemCell Technologies、カタログ番号07806)、Stromatolyser NR溶解(Sysmex、カタログ番号SNR−200、SNR−210A)。
表1:白血球(WBC)の回収率に対する赤血球溶解のための慣用的製品の影響
実施例2:NH4Clを含まない試薬中での赤血球溶解
第二の試験において、上記プロトコルにしたがって、NH4Clを含まない試薬の適切さを試験した。結果を以下の表2に示す:
表2:白血球(WBC)の回収に対するNH4Clを含まない試薬の影響
実施例3:NH4Clを含む、異なる試薬中での赤血球溶解
第三の試験において、上記プロトコルにしたがって、NH4Clを含む、異なる試薬の適切さを試験した。結果を以下の表3に示す:
表3:白血球(WBC)の回収に対する、NH4Clを含む異なる試薬の影響
第四の試験において、上記プロトコルにしたがって、NH4ClおよびHEPESを含む、異なる試薬の適切さを試験した。結果を以下の表4に示す:
表4:白血球(WBC)の回収に対する、NH4ClおよびHEPESを含む、異なる試薬の影響
第五の試験において、上記プロトコルにしたがって、80mM NH4Cl+10mM Hepes+0.1mM EDTAを含む試薬の適切さを試験した。WBCおよびRBCの回収に加えて、回収されたWBCの生存性をトリパンブルー排除試験によって測定した。したがって、WBCを、トリパンブルー(Sigma、カタログ番号T8154−20ML)(希釈WBC懸濁物:トリパンブルー=1:2)で染色し、そしてC−Chip計数チャンバー(Biochrom、カタログ番号P DHC−N01)を用いて、染色された細胞(死んだ細胞)を計数した。結果を以下の表5に示す:
表5:白血球(WBC)および赤血球(RBC)の回収および生存度に対する試薬の影響
実施例6:NH4ClおよびHEPESを含む、異なる試薬中での赤血球溶解
第六の試験において、上記プロトコルにしたがって、NH4ClおよびHEPESを含む、異なる試薬の適切さを試験した。結果を以下の表6に示す:
表6:白血球(WBC)の回収および溶解後のpH値に対する試薬組成の影響
第七の試験において、現存する赤血球溶解プロトコルの有効性を、示すように試験した。結果を以下の表7に示す:
表7:白血球の回収に対する、慣用的赤血球溶解プロトコルの影響
Claims (15)
- 赤血球溶解のための試薬の使用であって、試薬がHEPES(4−(2−ヒドロキシエチル)−1−ピペラジンエタンスルホン酸)、NH4 +/NH3、キレート剤およびあってもよいCO3 2−/CO3 −を含むか、またはこれらからなり、赤血球溶解中の最終濃度が
− 2.5mmol/l〜12mmol/l HEPES、
− 60mmol/l〜120mmol/l NH4 +/NH3、
− 0.04mmol/l〜0.8mmol/l キレート剤、および
− 存在する場合、0.15mmol/l〜0.8mmol/l CO3 2−/CO3 −
の範囲であって、
当該試薬はサポニンを含まない、前記試薬の使用。 - 溶解中の最終濃度が
− 3mmol/l〜11mmol/l HEPES、
− 70mmol/l〜100mmol/l NH4 +/NH3、
− 0.05mmol/l〜0.5mmol/l キレート剤、および/または
− 存在する場合、0.3mmol/l〜0.6mmol/l CO3 2−/CO3 −
の範囲である、請求項1の使用。 - キレート剤がエチレンジアミン四酢酸(EDTA)である、請求項1または2の使用。
- 試薬のpHが、6.4〜7.7の範囲である、請求項1〜3のいずれか一項の使用。
- 赤血球溶解中、pHが、6.4〜7.7の範囲に維持される、請求項4の使用。
- 赤血球を含む試料から、赤血球以外の細胞の検出、濃縮、または単離において前記試薬を用いる、請求項1〜5のいずれか一項の使用。
- 赤血球以外の細胞が、白血球、循環内皮細胞、または循環腫瘍細胞である、請求項6の使用。
- 赤血球を溶解する方法であって
a)赤血球を含む試料を提供し;
b)請求項1〜5のいずれか一項に定義される試薬と該試料をインキュベーションし、それによって赤血球を溶解し;そして
c)赤血球破片を除去してもよい
工程を含む、前記方法。 - 前記試料が、血液試料または赤血球および他の細胞を含む試料である、請求項8の方法。
- d)赤血球を含む試料から、赤血球以外の細胞を検出するかまたは単離する
工程をさらに含む、請求項8または9の方法。 - 赤血球以外の細胞が、白血球または循環腫瘍細胞である、請求項10の方法。
- 工程b)のインキュベーションが、最大30分間である、請求項8〜11のいずれか一項の方法。
- − 請求項1〜5のいずれか一項に定義される赤血球溶解のための試薬;および
− 赤血球破片を除去するための試薬
を含む、赤血球を含む試料から白血球を単離するためのキット。 - − 請求項8〜12のいずれか一項の方法を実行するための使用説明書
をさらに含む、請求項13のキット。 - 赤血球破片を除去するための試薬が、キレート剤を含むリン酸緩衝生理食塩水(PBS)である、請求項13または14のキット。
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CN108279145A (zh) * | 2017-12-19 | 2018-07-13 | 中国医学科学院医学生物学研究所 | 树鼩细胞免疫表型分析中外周血红细胞裂解方法 |
CN109632734A (zh) * | 2018-11-30 | 2019-04-16 | 张丽英 | 一种利用atp生物发光反应检测食品中黄曲霉菌的方法 |
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US11623034B2 (en) | 2020-01-03 | 2023-04-11 | Fenwal, Inc. | System and method to lyse and remove red blood cells from a cell product |
CN111781042B (zh) * | 2020-07-08 | 2023-07-07 | 青海省畜牧兽医科学院 | 一种附红细胞体检测试剂盒及样品处理方法 |
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FR2813891B1 (fr) * | 2000-09-14 | 2005-01-14 | Immunotech Sa | Reactif multifonctionnel pour erythrocytes mettant en jeu des carbamates et applications |
JP2002181809A (ja) * | 2000-12-15 | 2002-06-26 | Asahi Medical Co Ltd | 血液処理剤、それによる血液処理方法及び白血球分析方法 |
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- 2014-11-03 ES ES14792815T patent/ES2913337T3/es active Active
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HK1225798B (zh) | 2017-09-15 |
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