intermittent staining of blood and bone marrow slides

1 post / 0 new
Nic
Nic's picture
intermittent staining of blood and bone marrow slides

We are intermittently seeing some strange staining happening.
 
We make routine smears of (human) bone marrow (in EDTA) and peripheral bloods (EDTA). Nothing unusual about this, like any ordinary major hospital laboratory stuff.
We use clean commercial slides (like everyone else). We do not wipe them or wash them. Straight out of the packet.
 
The stain used is the Romanowsky stain. Home brew (made for literally centuries now so its not reagent based).
 
Simplified procedure:
Smear slides
Let air dry for 1 hour
Stain.
 
 
 
The problem we are experiencing is that after staining we see the red cells appear to have been lysed. This is particularly evident in the ideal region of the smear.   White cells and platelets look unchanged.
 
 
We have tried looking at different slides (manufacturers and types) and being so intermittent that nothing stands out.
We have tried wiping the slides with lint free tissue before smearing and still see this phenomenon.
We are going to try to wash the slides with methanol prior to smearing.   (personally I doubt this will work)
 
The thought is that the slides are dirty – which does not bode well with me as they are leaving the manufacturers supposedly clean. Also then why not more samples showing this.
 
 
We could spend hundreds of man hours trying to figure this out but because its so intermittent its difficult to reproduce at times.
 
I can list the make of slides used/tested if that helps
 
I hope somebody here has some thoughts.
 
 
 
 
Ideas that come to mind:

  1. Lab humidity – could it be a humidity thing. 
  2. Smears drying too rapidly? – what would it look like morphologically after staining??
  3. Smears not dry sufficiently?
  4. EDTA ? doubt it – same batch of tubes.

5.     Most likely exclude the pyrogens argument.
 

Apologies for being long winded... thought I try to make it as informative as possible 
 
It may just be one of those things.
Thanks
Nic