Introduction to Haematology and Blood Transfusion

Centrifugation

• Preparation of a buffy coat
  • 2 glass test tubes are filled with EDTA anticoagulated blood
  • The test tubes are centrifuged at RCF 1000g for 15 minutes
  • The supernatant plasma above the buffy coat is removed and discarded
  • The buffy coat layer, red cells immediately below, and small amount of supernatant plasma are transferred on a slide and mixed using an applicator stick
  • A thin preparation is made
  • The preparation is air-dry
  • Once dry it is fixed with alcohol for 2 minutes
  • The slide is then stained using Giemsa staining method
  • The preparation is observed at 40X objective and under oil immersion at 100X
• Uses of a buffy coat preparation
  • DNA isolation from blood samples
  • Purification of large amounts of DNA from relatively small sample sizes
  • Differential counting of white blood cells
  • Quantitative Buffy coat (QBC) to detect blood parasites: plasmosium spp, Leishmania donovani, trypanosomes, microfilariae and Histoplasma capsulatum infection

Complete Blood Count

• Components and utility of a complete blood count
  • Components
    • Total WBC count
    • WBC differential count
    • Total RBC count
    • Hematocrit
    • Hemoglobin
    • Red cell indices (MCV, MCH, MCHC, RDW)
    • Total platelet count
    • Platelet indices (MPV, PDW)
  • Uses
    • Diagnosis of anemia
    • Diagnosis of myeloproliferative disorders
    • Diagnosis of platelet abnormalities
• Principles employed by a coulter counter
  • Electrical impedance
  • Photometry
  • Light scatter

Peripheral blood film

A PBF is a thin layer of blood smeared on a glass slide and stained to allow microscopic examination of blood cells

• Preparation ****
  • Blood is spread on a glass slide
  • Air dried on the glass slide
  • Stained using a Romanowsky stain i.e. Leishman stain
• Clinical utility of a peripheral blood film
  • Morphological classification of Anaemia
  • Screening and diagnosis of malignancies with possible bon
  • Screening and diagnosis of acute and chronic myeloproliferative diseases
  • Diagnosis of infection
  • Evaluation of therapeutic response to RBC and WBC disorders

Bone Marrow Aspirate

A bone marrow aspirate is a procedure used to aspirate the cellular components, tissue fragments, or both, from a bone marrow for cytological assessment and analysis directed toward assessing the morphology and obtaining a differential cell count.

• Sites
  • PSIS, ASIS, Sternum, Anterior tibia of neonate
• Procedure
  • Site is prepared with antiseptic
  • The skin and underlying tissue to the periosteum is infiltrated with a local anesthetic
  • A skin incision is made with a surgical blade
  • A bone marrow aspiration, with a stylet locked in place, needle is passed through the incision
  • Once it contacts bone it is rotated clockwise and counter clockwise until it penetrated into the marrow cavity
  • A 2mL syringe is used to aspirate approximately 0.3mL of bone marrow
• Slide preparation
  • Squash preparation
• Stains
  • Wright and May-Grunwald-Giemsa stains
  • Prussian blue for iron study
  • Myeloperoxidase, Sudan Black B and LAP for AML
  • PAS for glycogen storage diseases
• Clinical Utility of Bone Marrow Aspirate
  • Diagnosis, staging and monitoring of lymphoproliferative disorders
  • Evaluation of cytopenia, thrombocytosis, leukocytosis, anaemia and iron status
  • Rule out infiltrative infectious disease (fungal infections, tuberculosis and other granulomatoses
  • Diagnosis of storage diseases (Niemann-Pick disease and Gaucher disease)
  • Assessment of metastatic carcinoma and granulomatous disease (Sarcoidosis)
  • Reveal toxic effects of certain offending medications or substances e.g. alcohol or nutritional deficiencies e.g. copper, zinc, B12, folate

Trephine Biopsy

A biopsy of bone marrow yielding a cylindrical-shaped solid piece of BM

• Sites
  • PSIS
  • ASIS
  • Sternum
  • Anterior tibia of neonate
• Procedure
  • Site is prepared with antiseptic
  • The skin and underlying tissue to the periosteum is infiltrated with a local anaesthetic
  • A skin incision is made with a surgical blade
  • A trephine needle is advanced with a twisting motion and rotated to obtain a solid piece of bone marrow
• Slide preparation
  • Squash preparation
• Stains
  • Wright and May-Grunwald-Giemsa stains
  • Prussian blue for iron study
  • Myeloperoxidase, Sudan Black B and LAP for AML
  • PAS for glycogen storage diseases
• Clinical utility of bone marrow biopsy
  • Diagnosis staging and monitoring of lymphoproliferative disorders
  • Evaluation of cytopenia, thrombocytosis, leukocytosis , anemia and iron status
  • Rule out infiltrative infectious disease (fungal infections, tuberculosis and other granulomatoses
  • Diagnosis of storage diseases (Niemann-Pick disease and Gaucher disease)
  • Assessment of metastatic carcinoma and granulomatous disease (Sarcoidosis)
  • Reveal toxic effects of certain offending medications or substances e.g. alcohol or nutritional deficiencies e.g. copper, zinc, B12, folate
• Components of bone marrow micro-environment and their role in hematopoiesis
  • Bone marrow stroma
    • Allows attachment of Stem Cells
    • Contributes signals controlling proliferation and differentiation
    • Regulates HSC localization, facilitating interaction with stromal cells
    • fibronectin, laminin, collagen
  • Bone marrow stromal cells
    • Physical support for HSCs
    • Produce growth factors and cell surface proteins that influence differentiation
    • Regulate hemopoiesis via direct contact and soluble mediators (adhesive ligands, synthesis of ECM, production of signaling molecules, cytokines)
    • Fibroblast, osteoblast (maintain HSC in a quiescent state), macrophage (Phagocytosis), endothelial cells, adipocytes, mesenchymal stem cells (0.01% diff to mesenchymal tissue)