Re: blood stain; DNA vs cytoplasm

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From:Paul Millikin <> (by way of histonet)
To:histonet <>
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Philip Oshel wrote:
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> Jim,
> This question would be best asked on Histonet--you'd get lots of quick
> replies from folks who do blood smears and any other histo work for living.
> I've included the Histonet listserver information below. Including some who
> teach histotech and write texts on it.
> If you can find a copy of "Staining Procedures" (Biological Stain
> Commission), there are several stains given. Any histo text ought to have
> these as well. The usual stains last time I did this were Wright's and
> Giemsa for thin film. If you receipes, let me know--I can send you these
> and a few others. Mind, mine are older ones.
> Phil

Paul Ehrlich pioneered the air-dried blood smear before the turn of the
century.  Air-dried because wet fixatives washed the specimen off of the
slide.  Various stains were tried at first, but eventually the Romanowski
stains (Wright's Giemsa, etc.) prevailed.  When a smear is air-dried, the
spherical white cells flatten out to become discs, introducing a number
of artefacts, notably loss of nucleolar staining.

For some artefactual reason, nucleolar staining is retained in many
leukemic cells, leading to the term "blast."  However, with wet fixation
or with supravital staining of live cells, even normal, mature small
lymphocytes contain nucleoli.

So information obtainable from blood staining depends more on the mode of
fixation than on the stain.

Wet fixation of a blood clot is almost useless because of the
overwhelming preponderance of red blood cells.  Wet fixation of a buffy
coat is possible if it is treated as a cell block, but centrifugation
introduces a few relatively minor artefacts of its own. Any specimen that
is embedded in paraffin undergoes considerable shrinkage during alcoholic

Supravital staining means putting a small drop of blood on a stained
slide and adding a coverslip, then waiting for a few minutes for the
living white cells to take up the stain.  Unfortunately these slides are
not permanent, although their significant information can be archived

I use slides dipped in 0.25-0.5% toluidine blue (in absolute ethanol) and
then dried on end at 60 degrees C.  They keep for months. After a few
minutes, a complete cell differential can be done.  Each kind of
granulocyte is readily identified, and the basophils are often
spectacular, with bright, purple granules.  Mature monocytes usually have
an elongated nucleus with a tiny nucleolus at each end, and small
lymphocytes have either 1 or 2 somewhat purple nucleoli.  Large
lymphocytes have more than one nucleolus, up to 5 or 6.

Incidentally, when this stain is used for pleural or peritoneal fluid,
any cancer cells may appear spectacularly large because they are not
shrunken by fixation or other processing.

In the final analysis, the stain you use depends on the information you
Paul Millikin
Peoria, IL

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