Comments to the clinical cytometry questions
ctnebe at t-online.de
Wed Nov 6 18:01:00 EST 1996
1) Cytoplasmic CD3 and CD22
There are several techniques to stain cytoplasmic antigens and fix and
perm originally from Walter Knapp from Vienna works well for most of them.
There are some few acute T cell leukemias where cy CD3 is essential to ensure
the diagnosis of T-ALL. I have not seen a pro B-ALL with cyCD22 which has not
been taken by CD19. It may help sometimes to resolve coexpression of CD19 in
AML. There are only two clones for CD22 and they behave slighly different as
discussed in our leukemia working group (Bernd Woehrmann, Goettingen).
CD19 and CD79a have been used in parallel in the German reference lab for acute
leukemias (Prof. Ludwig, Berlin, 60-70 AL per week). He hasn´t seen a
difference between CD19 and CD79a despite the higher specificity of CD79a for
the B lineage. I have heard that this single (!) CD79a clone will be available
from DAKO soon.
High IgE density as described by Leon in Cytometry is also in our hands the best
discriminator for basophils using a monoclonal with high specificity for IgE
(e.g. anti-IgE-PE from ORPEGEN). There are other CDs on basophils best decribed
by Peter Valent from Vienna (eg. CD17...) which are either difficult to obtain
in a directly conjugated form or not specific for basos.
Their low 90 degree light scatter signal (SSC) holds true for the BD lyse, Bob
Hofmans NH4Cl-Lyse, Ficoll isolated ones and unlysed diluted whole blood. This
behaviour might be explained by their granules that absorb light as their SSC
signal increases upon degranulation.
Their detection by low CD45 density and lymphocyte light scatter properties
is a good approach for peripheral blood in healthy normals. Progenitor cells,
makrothrombocytes, dendritic cells and other cells, esp. in the marrow share
similar cytometric properties. We use it as a screening approach but be careful
in sic people!
The story of increased autofluorescence has already been explained by my
brother. Eos are MPox neg and CD16 neg. We use CD45/14/16 for five part diff as
it also gives the left shift of neutrophils. CD16 is polymorphic on neutrophils
and GPI anchored (cave PNH) what needs to be reflected by the clone used (eg.
3FG8). The CD16/CD14 combination also resolves the two major monocyte
subpopulations first described by Ziegler-Heitbrock (the phagocytic CD14++/CD16+
and the AG presenting CD16++/CD14+ and there are even more).
5) Left shift
CD16 is the best marker for maturity of neutrophils. It is a little bit lower on
bands and significantly lower on myelocytes. This has been validated with blood
films and marrow by my group and will hopefully appear in CCC.
6) Acridine Orange
I appreciate the great work of Darzynkiewicz and we used the dye for a long time
but the staining is not stable over time (best after 10-15 min) either in flow
or in the fluorescence microscope. (And it stains best the tubings of flow
cytometers and pipets).
7) Vital stains for cells and Argon excitation
Thiazole orange and rhodamin 123 are two good examples for vital stains and
green fluorescence. Their formulas, binding sites and properties are well known
in opposite to miracle names from Molecular Probes (excuse me, Dick Haughland).
Thomas Nebe, Klinikum Mannheim
Faculty for Clinical Medicine of the University of Heidelberg
D-68135 Mannheim, GERMANY
More information about the Cytometry