HC24: Diagnostics in hematology

Case

A 40-year-old man visits the GP with complaints of fatigue and pallor. The GP thinks the most likely clinical problem is anemia.

Automatic hematology analyzer

The GP requests a complete blood count (CBC) → a differential count of Hb, leukocytes, and thrombocytes. Blood goes through an automatic hematology analyzer, a laser which gets scattered when it encounters a blood cell:

• Forward scatter/low angle: shows the volume of the cell
• Side scatters/high angle: shows the complexity of the cell (granules, MPC, etc)

The machine is able to identify the cells based on size and complexity. The results are shown in a diagram, which makes it possible to determine how many and what kind of cells there are.

Results

Results of the CBC show:

• Hb: 4,3 mmol/L → too low
• Leukocytes: 40 x 10⁹/L → too high
• Platelets: 70 x 10⁹/L → too low

Acute leukemia is suspected. In leukemia, there are too many leukocytes compared to other cells. The diversity also is less.

Microscopic leukocyte differentiation

Microscopic leukocyte differentiation consists of a blood smear stained with May Grunwald Giemsa. In case of acute leukemia, differentiation of leukocytes is lost → many strange looking leukocytes which all look the same are present.

Levels of diagnostics

There are several levels of diagnostics which make it possible to examine different structures:

• Resections/biopsies: tissue
• Cytology: cells
• Molecular pathology: molecules

Bone marrow examination

The bone marrow is the principle site of hematopoiesis. Examination of the bone marrow provides additional information for diagnostic clues seen in the peripheral blood:

• Increased or decreased leukocytes
• Increased or decreased Hb
• Increased or decreased platelets
• Presence of abnormal or immature cells

Diagnostic tests:

Several diagnostic tests on the bone marrow tissue can be done:

• Standard histology: on tissue level
  • H&E: hematoxylin (nucleus) and eosin (cytoplasm)
  • Histochemical stainings (PAS, alcian blue, etc.)
• Immunohistochemistry: on tissue/cell level
• Molecular assays: on molecular level

Bone marrow aspirates are liquid components of bone marrow. On bone marrow aspirates, tests can be done as well:

• Hematomorphology
  • Distinguishes cell types based on morphological criteria
    • Blasts
    • Mature and immature cells
      • Myeloid
      • Erythroid
      • Megakaryocytic
    • Lymphocytes
    • Plasma cells
    • Macrophages
    • Mast cells
  • Count: 2 x 500 cells
  • Sensitivity: 1%
    • 1 abnormal cell in background of 100 normal cells
  • Expertise, experience and pattern recognition is necessary
  • Acute leukemia: there is way less diversity → everything looks the same
• Immunophenotyping (cytology)
  • Distinguishes cell types based on presence or absence of proteins or antigens
    • Blasts
    • Mature and immature cells
      • Myeloid
      • Erythroid
      • Megakaryocytic
    • Lymphocytes
    • Plasma cells
    • Macrophages
    • Mast cells
  • Count: 100.000-1.000.000 cells
  • Sensitivity: 0,0001-0,001%
  • Expertise, experience and pattern recognition is necessary
  • Antigens called clusters of differentiation markers (CD markers) are used to identify cells → specific combinations of CD markers on the cell surface are used to identify different cells
    • CD markers are attached to fluorescent markers → show which antigen is present
      • Monoclonal antibodies are labeled to fluorochromes
    • Can be used to determine malignancy → low differentiation indicates malignancy
      • CD markers can differentiate between different types of leukemia
    • Flowcytometry can be used
      1. Cells pass a laser beam
      2. Detection of emitted light takes place → data processing
      3. The data is analyzed
• Cytogenetics
  • A microscope is used to identify stained DNA or chromosomes
  • Chromosomes in the metaphase and interphase are stained
    • Metaphase: cell division must occur
      • R-banding: karyotyping
    • Interphase: no cell division is necessary
      • Fluorescent in situ hybridization (FISH)
    • 30 metaphases or 200 interphases are counted
  • Cell types cannot be distinguished
    • Fluorescent in situ hybridization is used to further distinguish cytogenetic abnormalities
  • Sensitivity: 10% or 1-2%
  • Expertise, experience and pattern recognition is necessary
• Molecular diagnostics
  • Focusses on specific parts of whole DNA/RNA content of cells
  • No distinction between different cell types
  • Starting with >5 million cells
  • 2 techniques can be used:
    • Distinction due to DNA/RNA probes → biased approach
    • Next generation sequencing (NGS) → unbiased approach

Diagnostic methods in leukemia:

Different diagnostic methods are necessary because different types of leukemia require different treatments. Not all types of leukemia behave similarly → depending on the characteristics of the leukemic cells, the prognosis is different.

Allogeneic hematopoietic stem cell transplantation

The patient is treated with 2 courses of high dose chemotherapy. Subsequently, hematopoietic stem cell mobilization is done in a donor with G-CSF, making allogeneic hematopoietic stem cell transplantation possible:

1. The patient’s leukemia cells and immune system are destroyed
2. Donor stem cells are infused in the patient, who still doesn’t have an immune system
3. The immune system is rebuilt with donor cells → the patient has a new hematopoietic system