[Histonet] hb crystals

[Paul Bradbury]

Hi Jim,

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.

Paul Bradbury
Kamloops, Canada





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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