MORPHOLOGY OF HUMAN NEUTROPHILS - A COMPARISON OF CRYOFIXATION, ROUTINE GLUTERALDEHYDE FIXATION, AND THE EFFECTS OF DIMETHYL-SULFOXIDE

Cell shape and density are critical to the evaluation of neutrophil fu nction and/or activation. Dimethyl sulfoxide-cryofixation-freeze-subst itution processing (DCF) instantly preserves cell processes and ultras tructural elements with fewer artifacts than routine chemical fixation with glutaraldehyde and postfixation osmium tetroxide (GO). This stud y morphometrically examined density-separated neutrophils to assess di fferences in DCF and GO processing procedures and studied the effect o f dimethyl sulfoxide followed by GO fixation (DGO) on morphology. Fift een consecutive neutrophils were analyzed using computerized planimetr y for differences in DCF v. GO treatments (n = 4) and DGO v. GO treatm ents (n = 4). Cryofixed and DGO-fixed cells were significantly rounder than GO cells which had a more irregular surface with membrane projec tions. The cell volume of GO cells was 27-30% smaller than in DCF or D GO processing, while the surface area was similar. The increased volum e in DCF and DGO cells did not appear to be due to abnormal cell swell ing, since membranes, nuclear envelope, and mitochondrial cristae were more intact than in GO cells. Preservation of mitochondria as well as endocytic caveolae with a subplasmalemmal coating was best in DCF sam ples, moderate in DGO, and poorest in GO. Morphometric data showed tha t the nuclear compartment was 22% smaller, while the cytoplasm land it s associated compartments) was 29% smaller in GO compared to DCF-proce ssed neutrophils. This was consistent with the more dense cytoplasm in GO cells. Pretreatment of neutrophils with dimethyl sulfoxide (DMSO) resulted in volume preservation and improved the morphology of GO fixa tion. In summary, DCF appears to be an excellent method for preserving neutrophil membranes and cytoplasmic organelles (particularly mitocho ndria), and prevents a number of artifacts caused by routine GO fixati on. Morphology can also be improved by using DMSO in conjunction with GO. Anat. Rec. 252:254-263, 1998. (C) 1998 Wiley-Liss, Inc.