우리는이 방법, 원고 및 비디오의 개발에서의 도움을 제시카 존스, 에리카 클락, 콘 스턴스 Ducar, 도나 스미스, 로이 루이스, 릴리 Apedaile, 조앤 Wiesner, 데빈 아담스, 코리 McBain와 스티븐 Voght 감사드립니다. 이 작품은 빌과 멜린다 게이츠 재단 CAVD 보조금 38,645 (MJM)와 건강 교부금의 국립 연구소 UM1 AI068618 및 AI069481 U01 (MJM)에 의해 지원되었다. EA-N. NIH 그랜트 T32 AI007140에서 지원됩니다. 우리는 그들의 관대 한 장비 기증을 위해 제임스 B. 펜들턴 자선 신탁 감사드립니다.

참고 : 빛의 형광 - 복합 항체를 보호하기 위해 라이트와 바이오 안전 캐비넷의 모든 단계를 수행합니다. 1. 항체 염색법 패널 준비 <ol><li> 항체 염색법 패널은 표 1에서 찾을 수 있습니다. 항체 농도는 전체 피와 적정에 의해 정의하고 동일한 유동 세포 계측법 장비와 스테인드 phenotyping 샘플을 취득하는 데 사용됩니다 절차를 사용해야합니다. </li><li> 적절한 착색의 titers가 결정되면 잠금 캡 튜브에서 하나의 혼합으로 모든 항체가 조화를 이루고 있습니다. 100 μl에 총 볼륨을 가져다 흐름 세척 버퍼 (2 % 열 inactivated 태아 소 혈청과 Dulbecco의 PBS)를 추가합니다. 물들되는 샘플 수에 대한 혼합물을 확장. 이 혼합물은 최대 8 주까지에 대해 4 ° C에 저장할 수 있습니다. </li></ol> 2. 더럽히는 것 <ol><li> 수집 된 혈액은 광고에 다른 목적을 위해 사용하는 경우시간에 민감한 절차 남아있는 혈액 수행하는 동안이 분석에 dition은 따로 나누어지는을 설정할 수 있습니다. 나누어지는이 중요한 세포의 손실없이 venipuncture 후 4 시간 동안 실온에서 최대 저장할 수 있습니다. </li><li> 그대로 비드 펠렛은 Trucount 튜브 및 라벨 샘플 묻은되는 식별 할 수있는 튜브의 하단에 있다는 것을 확인합니다. HVTN 임상 실험에서 실험 데이터 관리 시스템 (프론티어 과학 기술 연구 재단, 애 머스트, NY)은 스테인드 샘플을 라벨 및 추적하는 데 사용됩니다. </li><li> 많은 번호와 모든 시약의 유효 날짜를 기록합니다. 튜브의 가방에있는 제조업체에서 제공 Trucount 관 구슬 카운트 번호를 기록하고, 가방에있는 많은 번호가 튜브에 많은 번호를 일치하는지 확인하십시오. </li><li> 정확하게 단지 금속 리테이너 위에 Trucount 튜브에 전체 혈액의 100 μl를 피펫에 pipetting 반대로 사용합니다. 관의 측면 아래로 번짐의 피를 사용하지 마십시오. </li><li&gt; Trucount 튜브에 일반 (앞으로) pipetting 기술, 혼합 항체 염색법 패널의 피펫 100 μl를 (표 1 참조)를 사용. 약 15 초 혼합하기위한 낮은 속도로 관과 소용돌이를 캡. 시각 비드 펠렛이 완전히 용해되어 있는지 확인하기 위해 튜브를 검사한다. </li><li> 어둠 속에서 실내 온도 (15-30 ° C)에서 15 분 동안 Trucount 관을 품다. </li><li> 필요한 경우, diH 2 O.를 사용하여 1X에 10 나누어지는 × FACS Lysing 솔루션을 희석 900 μl 1 × 튜브에 FACS Lysing 솔루션을 추가 할 수 있습니다. </li><li> 혼합하는 약 15 초에 낮은 속도로 철저하게 관과 소용돌이를 캡. 실험실 필름과 관 및 봉인에 위치를 잠금으로 단단히 뚜껑을 아래로 밀어 넣습니다. </li><li> -65에서 튜브를 저장 ° C -95 ° C의 예제는 중앙 분석 실험실로 배송이나 집에 분석을위한 준비가 될 때까지. 샘플은 적어도 4 주 동안이 단계에서 안정적입니다. imme을 배송 또는 시금 경우diately,이 단계는 생략 할 수 있습니다. </li></ol> 3. 해운 참고 : 다음 지침은 구체적으로 국제 항공 및 운송 협회 (IATA) 규정에 따라 운송 카테고리 B 면제 생물학적 물질을위한 SAF-T-박 주식회사에서 절연 배송 시스템을 사용합니다. 착색이 발생한 같은 위치에 샘플을 분석하는 경우, 섹션 4로 이동합니다. <ol><li> 샘플은 즉시 염색 후 배송하거나 일단은 -65에서 얼 ° C -95 ° C 사이 스테인드 표본 상자에 호일과 장소에 완전히 각 관을 넣어. 흡착제로 leakproof 폴리 가방 내부에 스테인드 표본 상자를 놓으십시오. </li><li> 가방에 가능한 한 공기와 같은과 Tyvek 가방과 인감에 leakproof 폴리 가방과 내용을 넣으십시오. </li><li> 내부 갈색 상자 안에 표본 패키지 (보조 포장 내부의 표본) 놓습니다. </li><li> 내부 갈색 B를 배치스티로폼 상자 내부의 소는 변화를 방지하기 위해 들여 쓰기로 확보. </li><li> 드라이 아이스 (약 8kg)와 스티로폼 상자를 작성하고 가슴에 단단히 뚜껑을 놓습니다. </li><li> 안전하게 테이프 배송 상자와 배는 생물 물질로 적절한 드라이 아이스 (UN1845) 표시가있는 카테고리 B은 (UN3373), IATA PI-650 지시 사항을 따르십시오. </li><li> 수령시, 샘플이 -65에 저장됩니다 ° C -95 ° C에 그들이 분석까지. </li></ol> 4. 해동 및 유동 세포 계측법 분석 <ol><li> 흐름 cytometer에 수집 전에 어둠 속에서 실온에서 해동은 냉장고에서 스테인드 샘플을 제거합니다. 하나 이상의 샘플에 대한 데이터를 수집하면 튜브가 1 시간 이상 각각에 대해 실온에서 앉아하는 일이 없도록 해동 망설으로 모든 튜브에 대한 프로세스를 표준화. </li><li> 샘플은 같은 BD LSR 적절한 필터가 장착 된 네 레이저 흐름 cytometer를 사용하여 취득해야합니다II. 데이터 수집 4 표준 cytometer 보정 및 형광 보상 방법을 사용하십시오. </li></ol> 참고 : 컬렉션 5 중 앞으로 분산 또는 측면 분산 형 임계 값을 설정하지 마십시오. Trucount 비즈는 구슬의 일부는 분석을하는 동안 설명 할 수하지 원인이 이러한 매개 변수에 대한 낮은 임계 값 설정 아래 떨어질 수 있습니다. 임계 값을 설정하려면 악기가 필요한 경우, 가장 낮은가요 시안 채널 임계 값을 설정합니다. 패널 물들 CD45 +의 백혈구는 시안이 긍정적인가되며, Trucount 구슬도 앰 시안 채널에 형광 때문에,이 적절히 수집 할 모든 관련 데이터를 허용해야합니다. <ol start="3"><li> 흐름 cytometer에로드하기 전에 5 초에 소용돌이 샘플 튜브를. Trucount 구슬은 여러 채널에서 높은 형광. PE Cy5와 APC (두 colo에 대한 높은 더블 긍정적 인 인구를 찾아서 게이트 수집하는 동안 구슬을가장 쉽게 세포에서 구슬을 구분하는 RS)은 장비에 따라 달라질 수 있습니다. 최소한 2 만 구슬이 획득되기 전까지는 중단 게이트로 구슬 문을 선택하고 기록 데이터입니다. </li><li> 이러한 FlowJo (Treestar, 애쉬 랜드, OR)와 같은 적절한 소프트웨어를 사용하여 데이터를 분석합니다. 그림 1은 대표적인 제어 혈액 추첨 다른 백혈구 인구의 분석에 활용 게이팅 구조를 보여줍니다. </li></ol> 5. Trucount 계산 <ol><li> 각 Trucount 튜브는 형광 구슬의 동결 건조 된 펠렛이 포함되어 있습니다. 관에 액체를 추가하고 vortexing 후, 구슬 동일하게 시료 전체에 분산 될 것입니다. 펠렛의 구슬의 수는 많은 번호로 약간 다르며 튜브의 저장소 가방에서 찾을 수 있습니다. </li><li> 게이트는 Trucount 비즈와 세포 인구 각 인구에 대한 이벤트 카운트를 결정하기 위해 그림 1과 같이. 이벤트의 수를 비교 전N 관 원래 구슬의 총 개수에 구슬 문은 다음 각 인구의 절대 농도 (즉, 세포 / μl)를 결정하는 데 사용할 수있는 샘플 수집의 비율을 결정 할 수 있습니다. 세포 농도 (# 세포 / μl 전체 혈액) = [/ # 인구 이벤트 (# 비드 이벤트 / # 펠렛의 총 구슬)] / 100 μl 다음 방정식이 목적을 위해 사용할 수 있습니다. </li></ol> 6. 대표 결과 <img src="/files/ftp_upload/4302/4302fig1.jpg" alt="그림 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> 그림 1. 게이팅 제도는 건강한 자원 봉사의 대표 데이터를 보여주는 주요 백혈구 인구의 분석에 활용. A) Trucount 비즈는 (i) 출입을 금지하고 세포에서 제외됩니다. 과립 성 백혈구 (II)이 구분되어 있으며 lympohcytes와 단핵 세포는 3 popluations로 구분됩니다 : CD14부정적인 림프구 (3) 모든 CD14 음성 세포 (IV), 비 림프구 (V). B) CD14 부정적인 림프구는 CD4 + T 세포 (바이올렛), CD8 + T 세포 (VII), B 세포 (VIII), CD56 밝은 NK 세포 (IX), CD56 희미 NK 세포 (X) 및 CD56 부정적인 NK 세포를 구별 할 게이트 아르 (사이). C) 모든 CD14 음성 세포는 골수 양 (XII) 및 plasmacytoid (XIII) 수지상 세포를 구별 할 수 출입을 금지합니다. D) 비 lympocytes은 비 고전 (XIV), 중급 (XV), 그리고 클래식 (XVI) 단핵 세포를 구별 할 수 출입을 금지합니다. <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . 그림 1 (예를 들어, NKT 세포 나 호중구)에 표시되지 않습니다보다 구체적인 셀 집합은 우리가 제시 패널을 사용하여 구별 할 수 있으며, 게이팅 스키마는 확장 또는 특정 학습 요구를 충족하도록 수정할 수 있습니다. 표시 특정 게이팅 단계는이 방법의 특징이다. 특히 메모, 포함 게이트 및제외 게이트는 Trucount 구슬 주위에 그려 서로의 상단에 위치, 계산을위한 게이트 구슬을 한, 하나는 세포 분석 (그림 1A)에서 비즈를 제외 할 수 있습니다. 사람들이 CD45 표현과 사이드 분산은 (그림 1A) 종종 필요가 있습니다를 사용하여 앞으로 분산과 측면 산란, 게이팅 셀에게이 말초 혈 mononuclear 세포에로도, 때문에 림프구, 단핵 세포, 그리고 과립 성 백혈구는 쉽게 전체 혈액에 구별되지 않습니다. CD45과 측면 분산을 사용하여 림프구와 단핵 세포 분리 할 수 없습니다 오염 과립 성 백혈구가 (원) 높은 CD16 표현 (그림 1C와 그림 1D)에 의해 일부 플롯에서 구별됩니다. 오염 과립 성 백혈구의 수는 일반적으로 작고, 그들은 단핵 세포와 NK 세포 게이팅을 방해하지 않습니다.

<ol>
	<li>Taylor, M. J., London, N. J., Thirdborough, S. M., Lake, S. P., James, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryobiology+of+rat+and+human+dendritic+cells:+preservation+and+destruction+of+membrane+integrity+by+freezing.">The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing.</a> Cryobiology. 27, 269-278 (1990).</li><li>Reimann, K. A., Chernoff, M., Wilkening, C. L., Nickerson, C. E., Landay, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+lymphocyte+immunophenotype+and+proliferative+responses+in+cryopreserved+peripheral+blood+mononuclear+cells+from+human+immunodeficiency+virus+type+1-infected+donors:+implications+for+multicenter+clinical+trials.+The+ACTG+Immunology+Advanced+Technology+Laboratories.">Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials. The ACTG Immunology Advanced Technology Laboratories.</a> Clin Diagn Lab Immunol. 7, 352-359 (2000).</li><li>Boonlayangoor, P., Telischi, M., Boonlayangoor, S., Sinclair, T. F., Millhouse, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+granulocytes:+study+of+granulocyte+function+and+ultrastructure.">Cryopreservation of human granulocytes: study of granulocyte function and ultrastructure.</a> Blood. 56, 237-245 (1980).</li><li>Perfetto, S. P., Ambrozak, D., Nguyen, R., Chattopadhyay, P., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+assurance+for+polychromatic+flow+cytometry.">Quality assurance for polychromatic flow cytometry.</a> Nat Protoc. 1, 1522-1530 (2006).</li><li>Brando, B., Barnett, D., Janossy, G., Mandy, F., Autran, B., Rothe, G., Scarpati, B., D'Avanzo, G., D'Hautcourt, J. L., Lenkei, R., Schmitz, G., Kunkl, A., Chianese, R., Papa, S., Gratama, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytofluorometric+methods+for+assessing+absolute+numbers+of+cell+subsets+in+blood.+European+Working+Group+on+Clinical+Cell+Analysis.">Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.</a> Cytometry. 42, 327-346 (2000).</li><li>Bull, M., Lee, D., Stucky, J., Chiu, Y. L., Rubin, A., Horton, H., McElrath, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+blood+processing+parameters+for+optimal+detection+of+cryopreserved+antigen-specific+responses+for+HIV+vaccine+trials.">Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials.</a> J. Immunol Methods. 322, 57-69 (2007).</li><li>Kantor, A. B., Roederer, M., Herzenberg, L., Blackwell, C., Weir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The handbook of Experimental Immunology. 43, 1-49 (1996).</li><li>Hulspas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+the+stability+of+phycoerythrin-tandem+dye+conjugates.">Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.</a> Cytometry A. 75, 966-972 (2009).</li><li>Le Roy, C., Varin-Blank, N., Ajchenbaum-Cymbalista, F., Letestu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+APC-tandem+dyes+are+degraded+through+a+cell-dependent+mechanism.">Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.</a> Cytometry A. 75, 882-890 (2009).</li><li>Mandy, F., Brando, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+absolute+cell+counts+using+immunophenotypic+techniques.">Enumeration of absolute cell counts using immunophenotypic techniques.</a> Curr. Protoc. Cytom. Chapter 6, Unit 6 (2001).</li><li>Vuckovic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+dendritic+cells+in+clinical+practice+using+a+new+whole+blood+single-platform+TruCOUNT+assay.">Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay.</a> J. Immunol Methods. 284, 73-87 (2004).</li><li>Lichtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+dendritic+cells+and+interferon-alpha+production+in+patients+with+tuberculosis:+correlation+with+clinical+outcome+and+treatment+response.">Circulating dendritic cells and interferon-alpha production in patients with tuberculosis: correlation with clinical outcome and treatment response.</a> Clin. Exp. Immunol. 143, 329-337 (2006).</li><li>Hosmalin, A., Lichtner, M., Louis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+analysis+of+dendritic+cell+subsets:+the+dendritogram.">Clinical analysis of dendritic cell subsets: the dendritogram.</a> Methods Mol. Biol. 415, 273-290 (2008).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 보고서에서 우리는 유동 세포 계측법에 의해 신선한 전체 혈액의 백혈구 인구를 열거위한 구슬 기반의 방법을 제시하고 중앙 샘플 분석 multicenter 임상 시험에서의 사용에 대한 매개 변수가 필요 다룹니다. 이 방법은 토대로 및 BD Trucount 프로토콜을 최적화 multicenter 임상 실험 환경에서의 신뢰성 사용할 수 있습니다. 착색 분석은 간단하다하고 가능한 혈액 처리 연구실 기술자가 수행 할 및 분석...

<table border="1"><tbody><tr><td> 시약 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 절대 계산 튜브를 Trucount </td><td> BD Biosciences </td><td> 340334 </td></tr><tr><td> 10X FACS는 솔루션을 Lysing </td><td> BD Biosciences </td><td> 349202 </td></tr><tr><td> 절연 카테고리 B &amp; 면제 운송 시스템, </td><td> SAF-T-박 </td><td> STP-320 </td></tr><tr><td> CD45 단클론 항체 AmCyan </td><td> BD Biosciences </td><td> 339192 </td></tr><tr><td> 3 번 CD FITC 단클론 항체 </td><td> BD Biosciences </td><td> 349201 </td></tr><tr><td> CD8 PerCp - 사 5.5 단클론 항체 </td><td> BD Biosciences </td><td> 341051 </td></tr><tr><td> CD4 알렉사 형석 700 단클론 antibo마구 </td><td> BD Biosciences </td><td> 557922 </td></tr><tr><td> HLA-DR ECD 단클론 항체 </td><td> Beckman 보습 바로 앞에 달린 풀 베는 날 </td><td> IM3636 </td></tr><tr><td> CD14 단클론 항체 v450 </td><td> BD Biosciences </td><td> 560349 </td></tr><tr><td> CD19 PE 단클론 항체 </td><td> BD Biosciences </td><td> 555413 </td></tr><tr><td> CD16 APC-H7 단클론 항체 </td><td> BD Biosciences </td><td> 560195 </td></tr><tr><td> CD56 PE-Cy7 단클론 항체 </td><td> BD Biosciences </td><td> 335791 </td></tr><tr><td> CD11c APC 단클론 항체 </td><td> BD Biosciences </td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5 단클론 항체 </td><td> BD Biosciences </td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

말초 혈 백혈구의 Cryopreservation 널리 임상 실험에 면역 반응 평가를위한 세포를 보존하기 위해 사용하고 면역 평가의 용이성 및 표준화를위한 많은 장점을 제공하지만,이 과정의 해로운 효과는 같은 과립 성 백혈구와 같은 일부 세포 집합, B 세포에 관찰 된 수 있습니다 , 1-3 및 수지상 세포. 신선한 백혈구를 시금하면 세포의 생체 상태에서의보다 정확한 사진을 제공하지만, 종종 큰 임상 시험의 맥락에서 수행 할 수 어렵습니다. 신선한 세포 assays은 시간이 오래 걸릴 경우, 자신의 응용 프로그램으로 인해 실험실 직원의 필요한 노동 시간에 비현실적 일 수 자원 봉사 약속과 시간대에 의존하고. 또한, 재판이 여러 센터에서 실시하는 경우, assays을 수행하기 위해 필요한 자원과 훈련 연구소는 임상 사이트에 충분한 가까이에 위치 할 수 없습니다. 이러한 문제를 해결하기 위해, 우리는 devel이예와 같은 assays보다 더 강력한 세포 유형 특정 정보를하였으며, 표현형과 말초 혈 내의 주요 백혈구 인구를 열거, Trucount 튜브 (산호세, CA Becton 디킨슨)와 함께 사용할 수있는 11 색 항체 염색법 패널을 oped 완전한 혈액 카운트 (CBC) 또는 몇 세포 유형에 얼룩 Trucount 관을위한 상용 가능한 패널 assays. 착색 절차는 간단합니다, 신선한 전체 혈액의 약 100 μl가 필요하고 가능한 표준 혈액 처리 연구소가 수행 할 수있게 약 45 분 정도 소요됩니다. 그것은 BD Trucount 튜브 기술 데이터 시트 (에서 적응 <a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">버전 2,010분의 8</a> ). 착색 항체 칵테일은 중앙 검정 실험실에서 대량으로 사전에 준비 사이트 처리 실험실로 배송 될 수 있습니다. 스테인드 튜브가 해결 될 수 있습니다및 다색의 유동 세포 계측법 분석을위한 중앙 분석 실험실에 배송을 위해 냉동. 이 착색 패널에서 생성 된 데이터는 개입과 관련하여 시간이 지남에 따라 백혈구 농도의 변화를 추적 할 쉽게 더 관심의 특정 세포 유형의 활성화 상태를 평가하기 위해 개발 될 수 사용할 수 있습니다. 이 보고서에서 우리는 신선한 전체 혈액 및 multicenter 임상 실험을 지원하는 중앙 검정 실험실에서 이러한 스테인드 샘플을 분석하는 단계에 착색을 수행 할 혈액 처리 연구실 기술자가 사용하는 절차를 보여줍니다. 비디오 정보는 HIV 백신 평가판 네트워크 (HVTN)에 임상 시험 혈액 추첨의 컨텍스트에서 수행되기 때문에 절차.

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

이 보고서에서, 우리는 주요 타고난 및 적응 백혈구 인구를 열거하는 신선한 전체 혈액에서 수행 phenotyping 분석의 착색 및 분석 단계를 보여줍니다. 우리는 multicenter 임상 실험의 맥락에서이 절차를 수행하기위한 고려 사항을 강조.

전체 혈액 Phenotyping 분석을 사용하여 Multicenter 임상 시험을위한 주요 말초 혈 백혈구 인구의 열거

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

enumeration-major-peripheral-blood-leukocyte-populations-for

Research

JoVE Journal

Immunology and Infection

14.9K 조회수.  Fred Hutchinson Cancer Research Center. 이 보고서에서, 우리는 주요 타고난 및 적응 백혈구 인구를 열거하는 신선한 전체 혈액에서 수행 phenotyping 분석의 착색 및 분석 단계를 보여줍니다. 우리는 multicenter 임상 실험의 맥락에서이 절차를 수행하기위한 고려 사항을 강조.

비디오: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

보완 고갈에 의해 특정 세포 집단의 고갈

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

연구

면역학 및 감염병학

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Zoledronate를 사용하는 사람 말초혈의 확장 γδ T 세포

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Mycobacteria의 가능성을 측정하는 기능 전체 혈액 분석은 리포터 - 진 BCG 또는 M. TB (BCG 태그 사용 럭스 / M. TB 럭스)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

백혈구가 풍부한 혈액 샘플의 할로겐 퍼 옥시 다제 활동의 신속하고 구체적인 평가

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Immunophenotype 및 Cytokine 생산 대량 Cytometry 통해 주변 전 혈의 단일 세포 분석

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...