In other series in which both aspirates and sections of bone marrow biopsy specimens were examined for the presence of haemosiderin, discrepancies in the assessment of iron stores were evident. Fong et al
reported the absence of stainable iron in 8% of sections in contrast to definite deposits seen in the corresponding aspirate film, and 6% of sections had significantly less stainable iron than the corresponding films3
; this American study used decalcification in Zenker’s acetic acid solution, a technique not commonly used in the UK or the rest of Europe. Contrary findings were reported by Krause and colleagues, who studied 1000 simultaneously collected aspirate smears and needle biopsies, with the biopsy specimens being placed in Zenker’s acetic acid for 15–18 hours and washed for at least eight hours before processing.4
They found that when storage iron was present in aspirates, it was always present in the corresponding needle biopsy specimen; the grade of iron stores tended to be lower in films than in sections, leading them to conclude that bone marrow sections were preferable for the evaluation of iron stores.4
Neither of these studies specified their criteria for adequacy for assessment of an aspirate film. The series by Krause et al
only included specimens in which an aspirate “contained several spicules” (spicule being a term sometimes used to describe a fragment or particle of bone marrow). They did not state the exact number of spicules required. Fong et al
stated in their methods that only “technically satisfactory specimens” were used, but did not further define this. We have defined an assessable aspirate precisely as one having either a minimum of seven bone marrow fragments, all of which are negative, or at least one iron positive fragment.1
From our study, it is apparent that aspirate films are more sensitive than trephine biopsy sections for the detection of haemosiderin when the biopsy specimens are decalcified in formic acid. They also provide a more accurate reflection of bone marrow iron stores, because decalcification leads to an unquantifiable loss of iron. It is possible that the findings would be different if decalcification were with ethylenediaminetetraacetic acid, which is a less powerful decalcifying agent. For those aspirates that were not evaluable, two thirds of cases had detectable haemosiderin on a Perls’ stained biopsy section. There are many reasons for the lack of an assessable aspirate; there may be a total failure to obtain an aspirate in patients with bone marrow fibrosis, or the aspirate may be composed of sinusoidal blood, may clot before films can be made, may be aparticulate, or may contain fewer than seven negative particles. With the increasing use of special tests such as immunophenotyping and cytogenetic and molecular analysis, larger volumes of bone marrow are aspirated. This may contribute to the increased incidence of haemodilute pauciparticulate samples received. Care should be taken to ensure that only a small volume of bone marrow is aspirated into a separate syringe at the start of the procedure for film preparation, before taking a larger volume of marrow for special tests. This would probably lead to a reduction in the number of trephine biopsy specimens requiring an iron stain.
“Aspirate films are more sensitive than trephine biopsy sections for the detection of haemosiderin when the biopsy specimens are decalcified in formic acid”
The scoring of iron status in trephine sections was more reproducible with Perls’ staining than with H&E staining and was also considerably less tedious. Therefore, the examination of H&E sections to assess haemosiderin cannot replace the Perls’ stain, although if plentiful haemosiderin is present a Perls’ stain is not needed. Because of the loss of iron during decalcification, pathologists need to use appropriate wording when reporting trephine biopsy sections from specimens that have been decalcified. Haemosiderin can be reported as present or increased but not as normal or decreased. A negative Perls’ stain should merely be reported as “negative” rather than as showing “absent iron” because this last phrase would be misleading unless further qualified. Similarly, if a Perls’ stain is not performed routinely it is useful to report that an H&E stained section shows haemosiderin to be present or increased. Because of the lack of sensitivity, it is not helpful to comment on the absence of haemosiderin in an H&E stained section.
Take home messages
- Perls’ staining of trephine biopsy sections is worthwhile when the bone marrow aspirate is unassessable and there is no obvious haemosiderin in the haematoxylin and eosin stained section
- However, routine Perls’ staining of trephine biopsy sections is not necessary
- Not performing a Perls’ stain as routine would produce savings in terms of staff time and reagent costs
We conclude that an iron stain is worthwhile when the aspirate is unassessable and there is no obvious haemosiderin in the H&E stained section, but that a routine Perls’ stain on a trephine biopsy section is not necessary. Not performing a Perls’ stain as routine would produce savings in terms of staff time and reagent costs.