ctnebe ctnebe at
Sun Feb 23 15:46:00 EST 1997

Dear Collegues:
there was some discussion on phagocytosis that I would like to comment.
1. The important principle is to use whole blood. Ficoll disturbs the test and 
cultered cells are much slower.
2. The next is to freeze the result and lyse.
3. Quenching is done well with FITC labeled organisms.
4. Cell aggregates should be avoided as the quenching dye doesn´t reach the 
5. The pH dependent flurescence intensity of fluorescein does not seem to play a 
major role (acidification in the phagosome) because BODIPY (pH independent) 
stained E.coli give the same time course.
6. The bacteria should be clean (no debris), of similar size (small CV), sterile 
(no other bacteria), fixed without disturbing the surface and occur as singlets.
7. Opsonization is done by the plasma in the whole blood unless you are dealing 
with immunodeficient patients or little children.  Preopsonized bacteria test 
the phagocytosis more directly independent of the patients specific Ig and 
complement levels.
8. E.coli is opsonized more by complement, S. aureus more by immunoglobulin.
9. Consider the different principles at various size ranges: Immune complexes, 
bacteria, yeast and tumor cells.
These were some of the points we considered when I developed the test at ORPEGEN 
years ago for clinical studies in immunopharmacology. A good day to day 
reproducibility over long times was a prerequisite for clinical trials and 
requires a serious batch control. We also used it for animal studies and I know 
from collegues that it works well for pigs, rabbits, rats and mice.
Those who have further questions may contact me directly as we have no internet 
access at our hospital or Werner Hirt at ORPEGEN (via the purdue site).

Thomas Nebe
Klinikum Mannheim
Faculty for Clinical Medicine, Univ. of Heidelberg
Inst. f. Clinical Chemistry
Theodor-Kutzer-Ufer 1-3
D-68167 Mannheim, GERMANY
Phone: +49 621 383-3485
FAX:   +49 621 383-3819

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