Bone Marrow

Abstract

The bone marrow is the primary site of hematopoiesis. Its dynamic environment can make interpretation of immunohistochemistry problematic. Furthermore, decalcification of the bone can alter antigen expression in the tissue, adding another layer of complexity to immunohistochemistry interpretation. Several variables should be considered during interpretation including processing techniques, antigen distribution, and expression levels. Therefore, this chapter was written taking into consideration these factors. This chapter begins with an extensive immunohistochemical stain list of both frequent and infrequently used markers for bone marrow specimens that includes the cellular location of antigen expression, typical distribution in normal bone marrow, the physiologic function associated with the targeted antigen, and the main use(s) of the marker for bone marrow evaluation. It also provides an overview of immunohistochemical markers, new and old, used most frequently in assessing diseases involving the bone marrow encompassing acute and chronic lymphoid and myeloid leukemias, myeloproliferative neoplasms, myelodysplastic syndromes, mast cell disease, plasma cell dyscrasias, T-and B-cell lymphoproliferative disorders, histiocytic lesions, and metastatic disease.

Frequently Asked Questions

• 32.1 What is the expression pattern of common immunohistochemical stains in normal bone marrow (Table 32.1)?

• 32.2 What factors should be considered when developing a bone marrow processing protocol (Table 32.2)?

• 32.3 How is immunohistochemistry different from flow cytometric immunophenotyping? (Table 32.3)

• 32.4 What is the distribution and immunophenotype of hematopoietic elements in normal bone marrow (Table 32.4)?

• 32.5 What is the distribution and immunophenotype of nonhematopoietic elements in normal bone marrow (Table 32.5)?

• 32.6 How is immunohistochemistry utilized to assess acute leukemia/immature morphology (Table 32.6)?

• 32.7 How to approach immunohistochemical staining for an acute leukemia (Fig. 32.1)?

• 32.8 How sensitive are specific immunohistochemical markers for acute leukemia (Table 32.7)?

• 32.9 What immunohistochemical clues suggest recurrent genetic changes in acute lymphoblastic leukemia (Table 32.8)

• 32.10 What markers suggest acute myeloid leukemia with genetic changes (Table 32.9)?

• 32.11 What markers are used to differentiate B-cell acute lymphoblastic leukemia from B-cell non-Hodgkin lymphoma (Table 32.10)?

• 32.12 What markers are used to differentiate T-cell lymphoblastic leukemia from T-cell non-Hodgkin lymphoma and thymoma (Table 32.11)?

• 32.13 What immunohistochemical stains are used for bone marrow assessment in patients with cytopenia(s) (Table 32.12)?

• 32.14 What are the markers for myelodysplastic syndrome (Table 32.13)?

• 32.15 What are the markers for myeloproliferative disorders (Table 32.14)?

• 32.16 What are the markers for mast cell disease (Table 32.15)?

• 32.17 What markers are used for differentiating benign from malignant lymphoid aggregates in bone marrow biopsies (Table 32.16)?

• 32.18 What are the common immunohistochemical markers for mature B-and T-cell neoplasms (Tables 32.17, 32.18, and 32.19)?

• 32.19 What markers are useful in hairy cell leukemia (Table 32.20)?

• 32.20 What markers are used to differentiate reactive versus malignant plasmacytosis (Table 32.21)?

• 32.21 What immunohistochemical stains are used for plasma cell myeloma and aggressive plasmacytoid neoplasms (Table 32.22)?

• 32.22 What markers are used for histiocytic tumors in the bone marrow (Table 32.23)?

• 32.23 What markers are used for metastatic tumors to the bone marrow (similar to the workup of unknown primary in Chap. 12 (Table 32.24)?

Table 32.1 Summary of distribution, applications, and limitations of useful markers in normal bone marrow

32.2 What factors should be considered when developing a bone marrow processing protocol?

• Preanalytical handling of bone marrow specimens is different from tissue specimens and affects antigen retrieval.

• Optimal histologic processing of the bone marrow biopsy requires proper fixation and either decalcification of the bone biopsy or tungsten-carbide knives for sectioning. Decalcification remains the most widely utilized method despite its adverse consequences to epitope and nucleic acids. A general rule when using decalcification is, “the faster the biopsy is decalcified the faster the epitopes and nucleic acids are destroyed.”

• Numerous processing protocols exist and use different fixatives and decalcification solutions (Table 32.2) and each has its own unique advantages and disadvantage. The Geisinger protocol uses B-plus fixative for a minimum of 3 hours and Decalcifier B for 30 minutes to allow for next-day interpretation.

• Immunohistochemical protocols may not work on tissue from an outside institution.

Table 32.2 Different protocols for bone marrow fixation and decalcification

32.3 How is immunohistochemistry different from flow cytometric immunophenotyping (Table 32.3)?

Table 32.3 Comparison of immunophenotyping techniques

• Bone marrow immunophenotyping is primarily performed using flow cytometry and fresh bone marrow aspirate or disaggregated cells from a fresh tissue biopsy.

• Peripheral blood containing abnormal cells can be used. Caution is advised as different phenotypes can be observed between the blood and bone marrow specimens, especially in immature cell populations. When peripheral blasts are ≥30%, the phenotype is usually considered equivalent to bone marrow phenotype and further workup on the bone marrow is not typically required.

• Instances when IHC should be utilized include the following:

  • Flow cytometry phenotype is ambiguous/undifferentiated or insufficient for lineage subtyping.

  • Abnormal cells are unevenly distributed.

  • Bone marrow morphology does not match flow cytometry involvement and/or phenotype.

  • Antigen of interest is only available by immunohistochemistry.

32.4 What is the distribution and immunophenotype of normal hematopoietic elements in bone marrow?

• Bone marrow biopsies are small in size (1–2 cm ideally) yet typically represent overall marrow cellularity and distribution of hematopoietic elements.

• Immunohistochemistry can help assess cellular distribution and enumeration, but if abnormal cell population is admixed normal hematopoietic elements, it could also be a potential pitfall if positive staining is of normal cells is misinterpreted.

• Table 32.4 describe normal bone marrow elements and key immunohistochemical features.

Table 32.4 Common IHC stains used to identify hematopoietic elements

32.5 What is the distribution and immunophenotype of nonhematopoietic elements in normal bone marrow (Table 32.5)?

Table 32.5 Immunohistochemical staining of nonhematopoietic elements in normal bone marrow

32.6 How is immunohistochemistry utilized to assess acute leukemia/immature morphology?

• If immunohistochemistry is utilized for initial diagnosis and classification recommend a judicious panel based on morphologic findings (Table 32.6).

• Misclassification is more likely if only a few stains are utilized. For example, myeloperoxidase (MPO) a myeloid lineage-specific marker, can be observed in B-cell acute lymphoblastic leukemia (especially if the polyclonal antibody is utilized). If only MPO is used, misclassification is possible.

• Olsen et al. proposed a stepwise approach for working up acute leukemia (Fig. 32.1), which minimizes cost and unexpected findings associated with larger immunohistochemical panels. A panel is recommended over individual lineage-specific markers since many antigens are aberrantly expressed in various leukemias (Table 32.7).

• Immunohistochemical staining patterns also may suggest various cytogenetic and molecular findings (Tables 32.8 and 32.9). However, it should not be solely utilized for genetic subtyping, and if tissue is negative, it does not exclude those abnormalities as processing may have affect antigen retrieval.

• Differential diagnosis of immature cells should include lymphomas with blastoid morphology (mantle cell lymphoma, high-grade B-cell lymphoma) and blastic plasmacytoid dendritic cell tumor (Tables 32.10 and 32.11).

Table 32.6 Assessment of acute leukemia by morphology and immunohistochemistry

32.7 How to approach immunohistochemical staining for an acute leukemia?

Fig. 32.1

Stepwise approach to IHC acute leukemia workup

32.8 How sensitive are specific immunohistochemical markers for acute leukemia?

Table 32.7 Immunohistochemical stains and percent positivity in acute leukemias

32.9 What immunohistochemical clues suggest recurrent genetic changes in acute lymphoblastic leukemia?

Table 32.8 IHC clues suggesting recurring genetics changes in acute lymphoblastic leukemia

32.10 What markers suggest an acute myeloid leukemia (AML) with genetic changes?

Table 32.9 Immunohistochemistry in acute myeloid leukemia (AML) with genetic changes

32.11 What markers are used to differentiate B-cell acute lymphoblastic leukemia (B-ALL) from B-cell non-Hodgkin lymphoma (B-NHL)?

Table 32.10 Immunohistochemistry to differentiate B-cell acute lymphoblastic leukemia (B ALL) from B-cell non-Hodgkin lymphoma (B-NHL)

32.12 What markers are used to differentiate T-cell lymphoblastic leukemia from T-cell non-Hodgkin lymphoma and thymoma?

Table 32.11 Immunohistochemistry to distinguish T-cell lymphoblastic leukemia (T ALL) from T-cell non-Hodgkin lymphoma (T-NHL) and thymoma

32.13. What immunohistochemical stains are used for bone marrow assessment in patients with cytopenia(s)?

Table 32.12 Immunohistochemical stains used for bone marrow assessment in patients with cytopenia(s)?

32.14 What are the marker for myelodysplastic syndrome?

Table 32.13 Markers for myelodysplastic syndrome (MDS)

32.15 What immunohistochemical stains are used for the evaluation of common myeloproliferative neoplasms and chronic myelomonocytic leukemia?

• The most common myeloproliferative neoplasms (MPNs) are chronic myelogenous leukemia (CML), polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocytosis (ET) and these disorders are assessed by morphology predominately.

• Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative disorder also predominately assessed by morphology.

• The common MPNs and CMML have characteristic features that overlap with each other.

• Immunohistochemistry can help highlight morphologic features when needed.

• Cytogenetic and molecular mutational studies are used to categorize these neoplasms and surrogate immunohistochemical markers are limited.

Table 32.14 Markers for common myeloproliferative neoplasms (chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocytosis) and chronic myelomonocytic leukemia

32.16 What are the markers for mast cell disease?

Table 32.15 Markers for mast cell disease

32.17 What markers are used for differentiating benign from malignant lymphoid aggregates in marrow biopsies?

Table 32.16 Markers for differentiating benign from malignant lymphoid aggregates in bone marrow biopsies

32.18 What are the common immunohistochemical markers for mature B-and T-cell neoplasms?

• Bone marrow examination for lymphoma is performed in several different clinical scenarios:

  • Establish a primary diagnosis

  • Extramedullary lymphoma staging workup

  • Leukemic manifestation of lymphoma

• Immunohistochemical stains may not work as well on decalcified tissue creating a potential pitfall, especially when establishing a new diagnosis.

• Several tables show immunohistochemical stains for lymphomas that most commonly involve the bone marrow (Table 32.17).

• More extensive immunohistochemical evaluations for lymphomas are available in Chap. 31.

Table 32.17 Immunophenotype of small cell B-cell lymphomas

Table 32.18 Immunohistochemistry of large B-cell lymphomas frequently involving bone marrow

Table 32.19 Immunohistochemistry for T-cell lymphomas frequently involving bone marrow

32.19 What markers are useful in hairy cell leukemia?

Table 32.20 Useful markers in hairy cell leukemia

32.20 What markers are used to differentiate reactive versus malignant plasmacytosis?

Table 32.21 Immunohistochemistry in reactive versus malignant plasmacytosis

32.21 What immunohistochemical stains are used for plasma cell myeloma and aggressive plasmacytoid neoplasms?

Table 32.22 Immunohistochemical stains for plasma cell myeloma and aggressive plasmacytoid neoplasms

32.22 What markers are used for histiocytic tumors in the bone marrow?

Table 32.23 Immunohistochemistry of common histiocytic lesions involving bone marrow

32.23 What markers are used for metastatic tumors to the bone marrow (similar to workup of unknown primary in Chap. 12)?

Table 32.24 Markers for metastatic tumors to the bone marrow