Labeled polymorphonuclear leukocytes: a comparison of methodology.
Polymorphonuclear neutrophilic granulocytes were separated from anti-coagulated whole blood using three techniques. The methods employed included volex sedimentation (VS), volex sedimentation with hypotonic lysis (VSHL), and Ficoll-Hypaque gradient separation (FH). The cells were labeled with 111In-oxine and 111In-tropolone. Studies were done with both blood from normal human volunteers and with canine blood. From the cell counts and differential, the harvested granulocytes, platelets, and red blood cells per milliliter of whole blood were calculated. Using the granulocyte chemotactic response to E. coli in agarose plates, the ratio of chemotactic migration to random migration (c.m./r.m.) was determined. Survival time for 111In labeled granulocytes were also determined in a canine model. The studies demonstrated that all procedures yielded 100% viability by the Trypan blue exclusion test. Chemotactic migration and leukocyte survival times were similar amongst all techniques. With the VSHL technique, there were significantly fewer red blood cells and platelets in the final preparation approaching the results of FH separation. The results suggest that for a relatively pure granulocyte preparation VSHL is an acceptable alternative to FH.
Chowdhury
S
S
Mayo Clinic and Foundation, Rochester, MN 55905.
Brown
M L
ML
Dewanjee
M K
MK
Forstrom
L A
LA
Katzmann
J A
JA
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D004285:Dogs; D006801:Humans; D066298:In Vitro Techniques; D007205:Indium Radioisotopes; D007553:Isotope Labeling; D009504:Neutrophils; D009942:Organometallic Compounds; D015125:Oxyquinoline / Q000031:analogs & derivatives; D014334:Tropolone / Q000031:analogs & derivatives
D007205:Indium Radioisotopes; D009942:Organometallic Compounds; C043895:indium tris(tropolonate); C032950:indium oxine; D015125:Oxyquinoline; D014334:Tropolone