15.3 Bone Marrow & Hematopoietic Specimen Preparation
Key Takeaways
- A complete bone marrow examination comprises aspirate smears, touch imprints of the core, a clot section, and a decalcified trephine core biopsy, each answering a different question.
- Storage iron is assessed by Perls Prussian blue on aspirate smears or the clot section, because acid decalcification of the trephine core leaches iron and produces a false-negative result.
- B-5 was the historic marrow fixative but contains mercuric chloride, so zinc formalin and 10 percent neutral buffered formalin have replaced it where immunohistochemistry and molecular testing are needed.
- EDTA at neutral pH is the preferred marrow decalcifier because it preserves immunohistochemical epitopes, fluorescence in situ hybridization signal, and nucleic acid integrity.
- Marrow fibrosis is graded on a reticulin stain from MF-0 to MF-3, with collagen confirmed by trichrome at the highest grade.
1. The Four Marrow Preparations
The ASCP BOC content outline lists bone marrow explicitly among Staining procedures, and marrow work touches Fixation, Processing, and Embedding and Microtomy as well. A complete examination usually yields four different preparations, and the histotechnologist handles all of them.
| Preparation | What It Answers | Handling |
|---|---|---|
| Aspirate smears | Cytologic detail, differential count, storage iron, ring sideroblasts | Air-dried, Wright-Giemsa or Romanowsky; a separate slide for Perls |
| Touch (imprint) preparations | Cytology when the aspirate is dry or diluted | Core rolled or touched gently onto a slide before fixation, air-dried, Romanowsky |
| Clot section | Architecture and cellularity from aspirated particles; iron without decalcification | Plasma-thrombin or agar cell block, formalin-fixed, paraffin, no decalcification needed |
| Trephine core biopsy | Cellularity, architecture, fibrosis, focal lesions, staging | Fixed, decalcified, paraffin, sectioned thin with step levels |
[!IMPORTANT] Touch preparations are made before the core is placed into fixative. Once the core has been in formalin, imprints are useless. If the technologist receives an unfixed core, this is the moment to make the imprints.
2. Fixation Choices and Their Consequences
| Fixative | Advantage | Cost |
|---|---|---|
| B-5 (mercuric chloride, sodium acetate, formaldehyde added just before use) | Historic gold standard for lymphoid and marrow nuclear detail | Mercury pigment requires dezenkerization; mercury waste disposal; acidic and hostile to molecular testing |
| Zinc formalin | Nuclear crispness approaching B-5, no mercury, better antigen retention | Slightly less crisp than B-5 |
| Acid zinc formalin | Combines fixation with mild decalcification of small trabeculae | Acidity can compromise sensitive epitopes and nucleic acid |
| 10 percent neutral buffered formalin | Universal compatibility with immunohistochemistry, in situ hybridization, and sequencing | Less nuclear crispness than zinc or mercuric formulations |
Because marrow biopsies increasingly drive flow-adjunct immunohistochemistry, fluorescence in situ hybridization, and next-generation sequencing panels, most laboratories have moved to zinc formalin or neutral buffered formalin. Acidic mercuric fixation followed by acid decalcification is the worst possible combination for downstream molecular work.
3. Decalcification of the Trephine Core
The core contains bony trabeculae that must be demineralized before sectioning, but marrow is small and easy to over-decalcify.
| Agent | Time Scale | Suitability |
|---|---|---|
| Neutral-buffered EDTA, 0.5 M, pH 7.0 to 7.4 | Hours to a day for a small core | Preferred; preserves epitopes, FISH signal, and nucleic acid |
| Buffered formic acid | 1 to 4 hours | Acceptable for morphology and many antibodies; monitor closely |
| Formic acid-sodium citrate | 1 to 4 hours | Common commercial marrow decalcifier |
| Strong mineral acids (HCl, nitric) | Under an hour | Avoid: destroys nuclear basophilia, iron, epitopes, and DNA |
Over-decalcification produces pale, washed-out nuclei that will not take hematoxylin, loss of Perls reactivity, and failed fluorescence in situ hybridization. The endpoint is monitored, not assumed, and the core is rinsed thoroughly before processing.
4. Processing, Embedding and Microtomy
- Orient the core along its long axis so the full length of marrow between trabeculae is presented; a core embedded on end wastes most of the specimen.
- Embed flat and press gently to the mold floor so the entire length lies in one plane.
- Section at 3 to 4 micrometers. Marrow is a cell-dense tissue, and thick sections make overlapping nuclei uninterpretable.
- Cut step levels and save unstained slides at the time of initial sectioning, because refacing later sacrifices tissue and a marrow core cannot be replaced.
- Handle with care. Crush artifact from forceps at the gross bench or embedding station destroys the cytology that the biopsy exists to show.
5. The Marrow Stain Battery
| Stain | Target | Technical Note |
|---|---|---|
| Hematoxylin and eosin | Cellularity, architecture, focal lesions | Baseline for every core and clot |
| Wright-Giemsa / Romanowsky | Cytologic detail on smears and imprints | Requires air-dried preparations |
| Giemsa on sections | Mast cells, plasma cells, cytoplasmic granularity | Complements hematoxylin and eosin on the core |
| Reticulin silver | Type III collagen fiber network; fibrosis grading | The single most important special stain on a core |
| Masson trichrome | Mature type I collagen | Confirms the highest grade of fibrosis |
| Perls Prussian blue | Storage iron, ring sideroblasts | Best on aspirate smears or the clot section |
| Periodic acid-Schiff | Megakaryocytes, glycogen, some parasites | Highlights megakaryocyte cytoplasm |
| Chloroacetate esterase (Leder) | Granulocytic differentiation, mast cells | Enzyme method; sensitive to over-decalcification |
| Congo red | Amyloid | Requires 8 to 10 micrometer sections |
| Immunohistochemistry | CD34 blasts, CD61 or CD42b megakaryocytes, CD117 and tryptase mast cells, CD138 plasma cells, kappa and lambda light chains | Epitope survival depends on the fixation and decalcification history |
5.1 Why iron is read on the aspirate
Acid decalcification leaches iron out of the trephine core, so a Perls stain on a decalcified core can be falsely negative even in a patient with iron overload. Storage iron is therefore graded on aspirate smears or on the clot section, neither of which is decalcified. Ring sideroblasts — erythroid precursors with a perinuclear ring of iron-laden mitochondria — can only be identified on aspirate smears, never on a section.
5.2 Reticulin fibrosis grading
Marrow fibrosis is graded on the reticulin stain using a widely adopted four-tier consensus scheme:
| Grade | Reticulin Pattern |
|---|---|
| MF-0 | Scattered linear reticulin with no intersections, corresponding to normal marrow |
| MF-1 | Loose network of reticulin with many intersections, especially in perivascular areas |
| MF-2 | Diffuse and dense increase in reticulin with extensive intersections, occasionally with focal bundles of collagen and/or focal osteosclerosis |
| MF-3 | Diffuse and dense reticulin with extensive intersections and coarse bundles of collagen, usually with osteosclerosis |
Because collagen bundles distinguish MF-2 from MF-3, a trichrome stain is run alongside reticulin when fibrosis is advanced. The grading is only as good as the silver technique, which is why reticulin control tissue and careful gold toning matter on marrow cases.
6. Common Technical Failures on Marrow
| Problem | Cause | Prevention |
|---|---|---|
| Pale nuclei that will not take hematoxylin | Over-decalcification | Monitor the endpoint; prefer EDTA |
| Negative iron stain in a transfused patient | Perls performed on the decalcified core | Stain aspirate smears or the clot section |
| Failed FISH or sequencing | Acidic fixative plus acid decalcification | Zinc or neutral buffered formalin, EDTA decalcification |
| Crushed, smeared marrow cells at the core edge | Forceps pressure during grossing or embedding | Handle by the cassette, never squeeze the core |
| Only a short segment of marrow on the slide | Core embedded on end or not pressed flat | Orient along the long axis, press to the mold floor |
| Uninterpretable overlapping nuclei | Sections cut too thick | Cut at 3 to 4 micrometers |
A hematologist questions a bone marrow report stating that storage iron is absent in a patient who has received twenty units of packed red cells. Review shows that the Perls Prussian blue stain was performed on the formic acid-decalcified trephine core. What is the explanation and the correct action?
A reticulin stain on a trephine core shows a diffuse, dense reticulin network with extensive intersections plus coarse bundles of collagen confirmed by trichrome, accompanied by osteosclerosis. Which fibrosis grade does this represent?
An unfixed trephine core arrives in the histology laboratory with a request for touch preparations. What must the technologist do, and why is the timing critical?