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Granulocyte storage and antigen stability
BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time‐consuming, costly, and technically difficult. STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes...
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| Published in: | Transfusion (Philadelphia, Pa.) Pa.), 1999-09, Vol.39 (9), p.983-990 |
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| creator | Chun, H. Cipolone, K. Procter, J. Stroncek, D.F. |
| description | BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time‐consuming, costly, and technically difficult.
STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4°C for 7 days under three conditions: 1‐percent formaldehyde‐fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI‐1640); and cells were fixed and stored with a commercial white cell‐storage solution (Cyto‐Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis.
RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI‐1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell‐storage solution remained. All antigens were detectable by the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)‐conjugated secondary antibody hindered interpretation of test results on Day 4. Nonspecific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC‐conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7‐aminoactinomycin‐D.
CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of nonspecific staining due to enhanced membrane permeability of dying cells. |
| doi_str_mv | 10.1046/j.1537-2995.1999.39090983.x |
| format | article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_69206986</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>69206986</sourcerecordid><originalsourceid>FETCH-LOGICAL-c4453-208e7940eada5dedf10786a150c9c375b626c0a67eb555be0be992e71904b9753</originalsourceid><addsrcrecordid>eNqVkF1LwzAUhoMoOqd_QQRF8KL1JGnSBq9kuikMv1AEb0Kano3Obp1Nh9u_N6WbeishhJM8583hIeSEQkghkheTkAoeB0wpEVKlVMgV-JXwcLlFOj9v26QDENGAUs72yL5zEwBgCugu2aMgOE-Y6JDzQWVmi6K0qxqPXV1WZozHZpb5XedjnPk7k-ZFXq8OyM7IFA4P12eXvPZvXnq3wfBhcNe7GgY2igQPGCQYqwjQZEZkmI0oxIk0VIBVlscilUxaMDLGVAiRIqSoFMOYKohSFQveJWdt7rwqPxfoaj3NncWiMDMsF05LxUCqRHrwsgVtVTpX4UjPq3xqqpWmoBtTeqIbG7qxoRtTemNKL3330fqbRTrF7E9vq8YDp2vAOGuKkfdkc_fLKUEZa6a4brGvvMDVf0bQL8_9TeVjgjYmdzUuf2JM9aFl7LXpt_uBjvi7YMnjk5b8G7QTk3k</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>69206986</pqid></control><display><type>article</type><title>Granulocyte storage and antigen stability</title><source>Wiley-Blackwell Journals</source><creator>Chun, H. ; Cipolone, K. ; Procter, J. ; Stroncek, D.F.</creator><creatorcontrib>Chun, H. ; Cipolone, K. ; Procter, J. ; Stroncek, D.F.</creatorcontrib><description>BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time‐consuming, costly, and technically difficult.
STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4°C for 7 days under three conditions: 1‐percent formaldehyde‐fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI‐1640); and cells were fixed and stored with a commercial white cell‐storage solution (Cyto‐Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis.
RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI‐1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell‐storage solution remained. All antigens were detectable by the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)‐conjugated secondary antibody hindered interpretation of test results on Day 4. Nonspecific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC‐conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7‐aminoactinomycin‐D.
CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of nonspecific staining due to enhanced membrane permeability of dying cells.</description><identifier>ISSN: 0041-1132</identifier><identifier>EISSN: 1537-2995</identifier><identifier>DOI: 10.1046/j.1537-2995.1999.39090983.x</identifier><identifier>PMID: 10533825</identifier><identifier>CODEN: TRANAT</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Science Inc</publisher><subject>7AAD = 7-aminoactinomycin D ; Animals ; Antibodies - blood ; Antibodies, Monoclonal - immunology ; Antigen-Antibody Reactions ; Antigens - blood ; Antigens - immunology ; Biological and medical sciences ; Blood Specimen Collection ; BSA = bovine serum albumin ; Cell Count ; Dactinomycin - analogs & derivatives ; Dactinomycin - blood ; Drug Stability ; FITC = fluorescein isothiocyanate ; Fixatives - pharmacology ; Flow Cytometry - methods ; Fluorescein-5-isothiocyanate ; Fluorescent Antibody Technique, Indirect ; Fluorescent Dyes - analysis ; Formaldehyde - pharmacology ; GAH = goat F(ab′)2 anti-human IgG ; GAM = goat (Fab′)2 anti-mouse IgG ; GAX = goat F(ab′)2 immunoglobulin ; Goats - immunology ; Granulocytes - chemistry ; Granulocytes - cytology ; Granulocytes - immunology ; HBSS = Hanks' balanced salt solution ; Hematology ; Humans ; Investigative techniques, diagnostic techniques (general aspects) ; Isoantibodies - immunology ; Medical sciences ; Mice ; MoAb(s) = monoclonal antibody(ies) ; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques ; PBS = phosphate-buffered saline ; PE = phycoerythrin ; RBC(s) = red cell(s) ; Staining and Labeling ; WBC(s) = white cell(s)</subject><ispartof>Transfusion (Philadelphia, Pa.), 1999-09, Vol.39 (9), p.983-990</ispartof><rights>1999 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4453-208e7940eada5dedf10786a150c9c375b626c0a67eb555be0be992e71904b9753</citedby><cites>FETCH-LOGICAL-c4453-208e7940eada5dedf10786a150c9c375b626c0a67eb555be0be992e71904b9753</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1046%2Fj.1537-2995.1999.39090983.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1046%2Fj.1537-2995.1999.39090983.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>315,786,790,27957,27958,50923,51032</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1951226$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10533825$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chun, H.</creatorcontrib><creatorcontrib>Cipolone, K.</creatorcontrib><creatorcontrib>Procter, J.</creatorcontrib><creatorcontrib>Stroncek, D.F.</creatorcontrib><title>Granulocyte storage and antigen stability</title><title>Transfusion (Philadelphia, Pa.)</title><addtitle>Transfusion</addtitle><description>BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time‐consuming, costly, and technically difficult.
STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4°C for 7 days under three conditions: 1‐percent formaldehyde‐fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI‐1640); and cells were fixed and stored with a commercial white cell‐storage solution (Cyto‐Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis.
RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI‐1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell‐storage solution remained. All antigens were detectable by the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)‐conjugated secondary antibody hindered interpretation of test results on Day 4. Nonspecific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC‐conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7‐aminoactinomycin‐D.
CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of nonspecific staining due to enhanced membrane permeability of dying cells.</description><subject>7AAD = 7-aminoactinomycin D</subject><subject>Animals</subject><subject>Antibodies - blood</subject><subject>Antibodies, Monoclonal - immunology</subject><subject>Antigen-Antibody Reactions</subject><subject>Antigens - blood</subject><subject>Antigens - immunology</subject><subject>Biological and medical sciences</subject><subject>Blood Specimen Collection</subject><subject>BSA = bovine serum albumin</subject><subject>Cell Count</subject><subject>Dactinomycin - analogs & derivatives</subject><subject>Dactinomycin - blood</subject><subject>Drug Stability</subject><subject>FITC = fluorescein isothiocyanate</subject><subject>Fixatives - pharmacology</subject><subject>Flow Cytometry - methods</subject><subject>Fluorescein-5-isothiocyanate</subject><subject>Fluorescent Antibody Technique, Indirect</subject><subject>Fluorescent Dyes - analysis</subject><subject>Formaldehyde - pharmacology</subject><subject>GAH = goat F(ab′)2 anti-human IgG</subject><subject>GAM = goat (Fab′)2 anti-mouse IgG</subject><subject>GAX = goat F(ab′)2 immunoglobulin</subject><subject>Goats - immunology</subject><subject>Granulocytes - chemistry</subject><subject>Granulocytes - cytology</subject><subject>Granulocytes - immunology</subject><subject>HBSS = Hanks' balanced salt solution</subject><subject>Hematology</subject><subject>Humans</subject><subject>Investigative techniques, diagnostic techniques (general aspects)</subject><subject>Isoantibodies - immunology</subject><subject>Medical sciences</subject><subject>Mice</subject><subject>MoAb(s) = monoclonal antibody(ies)</subject><subject>Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques</subject><subject>PBS = phosphate-buffered saline</subject><subject>PE = phycoerythrin</subject><subject>RBC(s) = red cell(s)</subject><subject>Staining and Labeling</subject><subject>WBC(s) = white cell(s)</subject><issn>0041-1132</issn><issn>1537-2995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><recordid>eNqVkF1LwzAUhoMoOqd_QQRF8KL1JGnSBq9kuikMv1AEb0Kano3Obp1Nh9u_N6WbeishhJM8583hIeSEQkghkheTkAoeB0wpEVKlVMgV-JXwcLlFOj9v26QDENGAUs72yL5zEwBgCugu2aMgOE-Y6JDzQWVmi6K0qxqPXV1WZozHZpb5XedjnPk7k-ZFXq8OyM7IFA4P12eXvPZvXnq3wfBhcNe7GgY2igQPGCQYqwjQZEZkmI0oxIk0VIBVlscilUxaMDLGVAiRIqSoFMOYKohSFQveJWdt7rwqPxfoaj3NncWiMDMsF05LxUCqRHrwsgVtVTpX4UjPq3xqqpWmoBtTeqIbG7qxoRtTemNKL3330fqbRTrF7E9vq8YDp2vAOGuKkfdkc_fLKUEZa6a4brGvvMDVf0bQL8_9TeVjgjYmdzUuf2JM9aFl7LXpt_uBjvi7YMnjk5b8G7QTk3k</recordid><startdate>199909</startdate><enddate>199909</enddate><creator>Chun, H.</creator><creator>Cipolone, K.</creator><creator>Procter, J.</creator><creator>Stroncek, D.F.</creator><general>Blackwell Science Inc</general><general>Blackwell Publishing</general><scope>BSCLL</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>199909</creationdate><title>Granulocyte storage and antigen stability</title><author>Chun, H. ; Cipolone, K. ; Procter, J. ; Stroncek, D.F.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4453-208e7940eada5dedf10786a150c9c375b626c0a67eb555be0be992e71904b9753</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>7AAD = 7-aminoactinomycin D</topic><topic>Animals</topic><topic>Antibodies - blood</topic><topic>Antibodies, Monoclonal - immunology</topic><topic>Antigen-Antibody Reactions</topic><topic>Antigens - blood</topic><topic>Antigens - immunology</topic><topic>Biological and medical sciences</topic><topic>Blood Specimen Collection</topic><topic>BSA = bovine serum albumin</topic><topic>Cell Count</topic><topic>Dactinomycin - analogs & derivatives</topic><topic>Dactinomycin - blood</topic><topic>Drug Stability</topic><topic>FITC = fluorescein isothiocyanate</topic><topic>Fixatives - pharmacology</topic><topic>Flow Cytometry - methods</topic><topic>Fluorescein-5-isothiocyanate</topic><topic>Fluorescent Antibody Technique, Indirect</topic><topic>Fluorescent Dyes - analysis</topic><topic>Formaldehyde - pharmacology</topic><topic>GAH = goat F(ab′)2 anti-human IgG</topic><topic>GAM = goat (Fab′)2 anti-mouse IgG</topic><topic>GAX = goat F(ab′)2 immunoglobulin</topic><topic>Goats - immunology</topic><topic>Granulocytes - chemistry</topic><topic>Granulocytes - cytology</topic><topic>Granulocytes - immunology</topic><topic>HBSS = Hanks' balanced salt solution</topic><topic>Hematology</topic><topic>Humans</topic><topic>Investigative techniques, diagnostic techniques (general aspects)</topic><topic>Isoantibodies - immunology</topic><topic>Medical sciences</topic><topic>Mice</topic><topic>MoAb(s) = monoclonal antibody(ies)</topic><topic>Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques</topic><topic>PBS = phosphate-buffered saline</topic><topic>PE = phycoerythrin</topic><topic>RBC(s) = red cell(s)</topic><topic>Staining and Labeling</topic><topic>WBC(s) = white cell(s)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chun, H.</creatorcontrib><creatorcontrib>Cipolone, K.</creatorcontrib><creatorcontrib>Procter, J.</creatorcontrib><creatorcontrib>Stroncek, D.F.</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Transfusion (Philadelphia, Pa.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chun, H.</au><au>Cipolone, K.</au><au>Procter, J.</au><au>Stroncek, D.F.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Granulocyte storage and antigen stability</atitle><jtitle>Transfusion (Philadelphia, Pa.)</jtitle><addtitle>Transfusion</addtitle><date>1999-09</date><risdate>1999</risdate><volume>39</volume><issue>9</issue><spage>983</spage><epage>990</epage><pages>983-990</pages><issn>0041-1132</issn><eissn>1537-2995</eissn><coden>TRANAT</coden><notes>ark:/67375/WNG-43Z528PQ-6</notes><notes>istex:16F71637D6F6E1BA26B645C8E873002944EC8C5F</notes><notes>ArticleID:TRF39090983</notes><notes>Haecin Chun, MT(ASCP), SBB, Department of Transfusion Center, National Institutes of Health, Building 10, Room 1C711, 10 Center Drive MSC1184, Bethesda, MD 20892‐1184;e‐mail</notes><notes>hchun@dtm.cc.nih.gov</notes><notes>Address reprint requests to</notes><notes>.</notes><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time‐consuming, costly, and technically difficult.
STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4°C for 7 days under three conditions: 1‐percent formaldehyde‐fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI‐1640); and cells were fixed and stored with a commercial white cell‐storage solution (Cyto‐Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis.
RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI‐1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell‐storage solution remained. All antigens were detectable by the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)‐conjugated secondary antibody hindered interpretation of test results on Day 4. Nonspecific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC‐conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7‐aminoactinomycin‐D.
CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of nonspecific staining due to enhanced membrane permeability of dying cells.</abstract><cop>Oxford, UK</cop><pub>Blackwell Science Inc</pub><pmid>10533825</pmid><doi>10.1046/j.1537-2995.1999.39090983.x</doi><tpages>8</tpages></addata></record> |
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| ispartof | Transfusion (Philadelphia, Pa.), 1999-09, Vol.39 (9), p.983-990 |
| issn | 0041-1132 1537-2995 |
| language | eng |
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| source | Wiley-Blackwell Journals |
| subjects | 7AAD = 7-aminoactinomycin D Animals Antibodies - blood Antibodies, Monoclonal - immunology Antigen-Antibody Reactions Antigens - blood Antigens - immunology Biological and medical sciences Blood Specimen Collection BSA = bovine serum albumin Cell Count Dactinomycin - analogs & derivatives Dactinomycin - blood Drug Stability FITC = fluorescein isothiocyanate Fixatives - pharmacology Flow Cytometry - methods Fluorescein-5-isothiocyanate Fluorescent Antibody Technique, Indirect Fluorescent Dyes - analysis Formaldehyde - pharmacology GAH = goat F(ab′)2 anti-human IgG GAM = goat (Fab′)2 anti-mouse IgG GAX = goat F(ab′)2 immunoglobulin Goats - immunology Granulocytes - chemistry Granulocytes - cytology Granulocytes - immunology HBSS = Hanks' balanced salt solution Hematology Humans Investigative techniques, diagnostic techniques (general aspects) Isoantibodies - immunology Medical sciences Mice MoAb(s) = monoclonal antibody(ies) Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques PBS = phosphate-buffered saline PE = phycoerythrin RBC(s) = red cell(s) Staining and Labeling WBC(s) = white cell(s) |
| title | Granulocyte storage and antigen stability |
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