You have not described the appearance of the "crystals" contained within
the area of the hemorrhage. But, I will go out on a limb here and guess
that they are very dark (brown-black) and "spikey" with sharp points.
If this is the case, what you are looking at is so called "formalin
pigment". not hemoglobin pigment. This is an artefactual pigment
produced by non-buffered (or inadequately buffered) formalin interacting
with the hemoglobin in red cells. It is frequently seen in areas of
hemorrhage or within any other red cell masses. You can confirm its
identity by examining it using crossed polarizers; formalin pigment
(acid formol hematin) will rotate the plane of polarized light and will
appear bright white on a black background. It does not react with any
other demonstration technique. It can be removed by soaking the dewaxed
sections in saturated alcoholic picric acid for 30-60 minutes.
If it is formalin pigment, your next step is to avoid it in future
specimens. This can be easily done by using only buffered formalin
solutions, pH 7.2 (either commercially prepared or following the
directions found in any standard histology text). Prolonged exposure to
formalin fixatives will increase the tendency to produce the pigment,
even when the solutions are buffered. So, keep the fixation time to no
more than 2-3 days. With rat brain (pretty small) this should be
adequate time for thorough fixation.
Jim Manavis wrote:
> Dear Sir or Madam:
> I am a PhD student studying intracerebral haemorrhage in the rat and have
> found numerous crystals contained within the haemorrhage, which I presume
> are haemoglobin crystals, although it would be nice to demonstrate this.
> Pearl's stain doesn't work, presumably because the iron is ferrous and bound
> tightly, and Lillie's method with ferricyanide and HCL likewise doesn't
> work, presumably because the iron is bound. Our standard DAB technique for
> immunohistochemistry doesn't seem to react either. How might I best
> determine whether or not the crystals are haemoglobin crystals?
> Yours Sincerely,
> Tim Kleinig
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