Expression of functional antigens on neutrophils

Macey, Marion G.; Jiang, Xiaohua; Veys, PA; McCarthy, D.; Newland, Adrian C.

doi:10.1016/s0022-1759(12)80046-9

Cited by 80 publications

(37 citation statements)

References 8 publications

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“…They further demonstrated that fixation with formaldehyde under these conditions did not significantly affect the binding of CD3, CD4, CD8, CDllb, or CD18 antibodies or of antibodies to HLA-DR, although it may have affected the binding of CD14. In a comparative investigation into the effects of different conditions of cell preparation and labelling on the surface expression of five functional antigens on human neutrophils, Macey et al (8) confirmed the results obtained by Hamblin et al (4) for C D l l b and showed that the procedure was suitable also for the quantitation of the antigens recognised by CD14, CD16, and CDw32, although there was some doubt as to whether it was suitable for the quantitating CD13 antigen.…”

mentioning

confidence: 63%

“…However the physical separation procedures commonly used to isolate leucocytes prior to labelling can influence granulocyte surface antigen expression (vide supra; 2, 3, 5) and cell surface antigens can be upregulated when labelling anticoagulated blood, even at 4"C, prior to removing erythrocytes (8). It has recently been shown that fixing blood with formaldehyde immediately it has been drawn can prevent subsequent in vitro changes in the leucocyte integrins and other surface antigens whose expression can be rapidly modulated on granulocyte activation (4).…”

Section: Discussionmentioning

confidence: 99%

“…Consequently it is abundantly clear that anticoagulated blood must be processed with the minimum delay if artefactual changes in the ex-pression of granulocyte surface antigens are to be avoided. Indeed, it is possible that some of the changes in expression of granulocyte surface antigens that were previously thought to be due to dextran sedimentation, to Ficoll-Hypaque centrifugation or to antibody binding (2,8), arose from the necessity to have anticoagulants present during the experiments. A further corollary of these observations is that it is probably impossible to isolate live granulocytes from peripheral blood by currently available techniques without artefactually altering the expression of their surface antigens.…”

Section: Discussionmentioning

confidence: 99%

“…In conclusion, the formaldehyde-based rapid leucocyte fixation and preparation technique (4,8) is probably the best currently available procedure for quantitating the expression of adhesion molecules and function-associated antigens on peripheral blood granulocytes, because it prevents changes due to activation in vitro. However, it must be remembered that storing anticoagulated blood and fixation per se can also induce artefactual changes.…”

Section: Discussionmentioning

confidence: 99%

“…When investigating whether blood could be kept with formaldehyde for longer than the time originally used (4 min) (4, 8), freshly drawn unanticoagulated blood was mixed with formaldehyde and kept a t 37°C for 4,20, or 55 min before processing (see below).…”

Section: Experimental Protocols Effects Of Anticoagulants On Antigen mentioning

confidence: 99%

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Quantitation of adhesion molecules and other function‐associated antigens on human peripheral blood leucocytes

Macey

McCarthy

1993

Cytometry

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Quantitating satisfactorily the expression of adhesion molecules and other functional antigens on the surface of human peripheral blood granulocytes by flow cytometry is difficult. Artefactual (in vitro) changes, which occur in the expression of these molecules during conventional cell preparation and labelling procedures, led to the development of a leucocyte preparation technique that prevents upregulation of the leucocyte integrins and other antigens. The technique, as originally evaluated, involves fixing freshly drawn blood with formaldehyde (final concentration 0.2%) for 4 min at 37°C before adding ammonium chloride to lyse the erythrocytes; procedures which are inconvenient when blood samples are taken in wards or clinics that are some distance from a laboratory. We therefore investigated (i) whether blood could be kept with commonly used anticoagulants for up to 1 h before fixation, and (ii) whether unantico agulated blood could be kept with formaldehyde for a similar period before processing was continued. The results showed that when quantitating those granulocyte surface antigens which can be rapidly upregulated: (i) that it is preferable to mix freshly drawn unanticoagulated blood directly with formaldehyde; (ii) that if blood must be collected into anticoagulants, it should be kept for only the briefest possible time before processing; and (iii) that the time for which cells are exposed to 0.2% formaldehyde must be carefully controlled and limited to 4 min. A corollary of the results with anticoagulants is that it is probably impossible to isolate live granulocytes by currently available procedures without artefactually altering the expression of these antigens in vitro. © 1993 Wiley‐Liss, Inc.

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mentioning

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Experimental Protocols Effects Of Anticoagulants On Antigen mentioning

confidence: 99%

See 3 more Smart Citations