MLAB Hematology Keri Brophy-Martinez Unit 2 : Hematopoiesis Unit 3 : Hematopoietic Organs

Chapter 2: Terms Homeostasis  Maintenance of an adequate number of cells to carry out the functions of the organism. Hematopoiesis  Dynamic formation and development of blood cells, normally in the bone marrow  Process responsible for the replacement of circulating blood cells.

Review of Cell Morphology

Cell Morphology Cell Membrane  Outer boundary of the cell  Allows passage of nutrients, ions and information between cytoplasm and the exterior  Phospholipid bilayer

Cell Membrane: Phospholipid Bi-layer

Cell Morphology Cytoplasm: Location of metabolic activities  Golgi complex: involved in formation of gamma globulins, lysosomes, specific granules and other cellular components.  Ribosomes: RNA, synthesize proteins  Endoplasmic reticulum: Communication system with the nucleus, smooth or rough  Mitochrondria: furnish cell with energy  Lysosomes: granules with hydrolytic enzymes.  Plasma membrane: 2 layers of phospholipids, surrounding cytoplasm

Cell Morphology Nucleus:  Chromatin Dark staining chromosomal material composed of DNA and proteins. DNA regulates all cellular functions  Nucleolus/Nucleoli Pale staining patches rich in RNA  Parachromatin Zones of pale staining material between chromatin clusters  Nuclear membrane Double membrane which surrounds the nuclear contents.

Maturation Characteristics of Blood Cells Cytoplasm  Immature: stains deep blue due to abundant RNA, scant amount of cytoplasm, no granules  More mature: blue color decreases as RNA decreases, granulocytes acquire non-specific granules, cytoplasm amount decreases  Mature - granules (if present) become more specific

Maturation Characteristics of Blood Cells Nucleus  Immature - round or oval, very large occupying most of cell, active DNA and RNA replication (dividing), fine chromatin pattern, nucleoli present  More mature - size decreases, chromatin (DNA) becomes more coarse and clumped, # of nucleoli decreases  Mature - nucleus assumes final shape, no nucleoli, chromatin is clumped, no division, in RBC’s nucleus has been extruded

Overall Changes in Cell N/C ratio: nuclear to cytoplasmic ratio  Immature “ blast” cell has a large nucleus, small amount of cytoplasm. N/C ratio is high  N/C ratio decreases with maturity as the nucleus decreases in size and the cytoplasm becomes more abundant. Size  Immature cells are larger than mature cells  The size of almost all cell lines decreases with maturity Exception is thrombocyte/ platelet precursor in the bone marrow increase in size with maturation

Lab Method: Wright’s Stain Stain used to identify cells on blood smear Eosin is acid stain - stains red Methylene is basic stain - stains shades of blue

Cell Growth Factors Cytokines  Growth factors secreted by cells for the purpose of cell-to-cell communication. They stimulate the multipotential stem cells to proliferate and differentiate. Types  Colony stimulating factors (CSF): These regulate blood cell development. CSF’s are being synthetically manufactured to treat diseases. G-CSF : Stimulate granulocyte production, Used to treat cancer and AIDS patients with low WBC. Trade name is Neupogen GM-CSF - Stimulate granulocyte-macrophage production, Used to treat cancer patients with low WBC. Trade name is Leukine. EPO - Stimulate erythrocyte production, Used to treat chronic anemia caused by renal failure, increase RBC prior to surgery which may cause blood loss, illegally by athletes to boost performance. Trade name is Epogen or Procrit.

Cell Growth Factors Interleukins  Wound healing, activating lymphocytes, assisting in the growth of transplanted or damaged bone marrow.

Cluster Designation (CD) nomenclature  Using monoclonal antibodies and flow cytometry techniques, it is possible to identify specific populations of hematopoietic cells.

Hematopoietic Organs

Hematopoiesis: blood formation Intramedullary  Origin of blood cells and sequential sites of normal blood production within the bone marrow Fetus  Yolk sac  Liver and spleen  Bone marrow (all bones) Adult  Child up to teen years - all bones  18 years and up - flat bones (sternum, ribs, pelvis, vertebra, skull)  Adults- bone marrow

Hematopoiesis Extramedullary hematopoiesis  Blood is produced in the spleen, liver and other tissues when demand is increased.

Storage Pools Stem cell pool  Multipotential stem cell  Unipotential (committed) stem cell Bone marrow pool  Granulocytes - proliferation & maturation/storage  Thrombocytes - proliferation & maturation  Erythrocytes - proliferation & maturation Peripheral blood pool  Granulocytes - 50% storage, 50% functional  Thrombocytes - 30% storage, 70% functional  Erythrocytes - 100% functional

Bone Marrow Bone marrow differentiates into myeloid, erythroid and lymphoid cell lineages under the influence of cytokines or growth factors. Organized around bone vasculature Function: supply mature hematopoietic cells into peripheral blood and respond to demands

Indication for Bone Marrow Studies Hematologic diseases  Anemia, erythrocytosis, polycythemia  Leukopenia and unexplained leukocytosis  Appearance of abnormal or immature cells in the peripheral blood circulation  Thrombocytopenia or thrombocytosis Systemic disease  Solid malignant tumors elsewhere in the body - done at initial diagnosis to determine the degree of tumor spread and to stage the malignancy  Infections known as “fever of unknown origin” (FUO) in which organisms may be within the marrow.  Certain histiocytic diseases

Procedure: Bone marrow Bones which have active hematopoiesis in adults in order of preference for performing bone marrow procedure:  Posterior superior iliac crest - biopsy and aspiration almost always done at this site  Sternum - used for aspiration only  Anterior superior iliac crest  Spinal processes  Anterior tibia used for infants and small children

Procedure Needles used for procedure  Biopsy - Jamshidi trephine needle (8 or 11 gauge)  Aspiration - University of Illinois (U of I) needle

Laboratorian’s role in bone marrow procedure Provide the bedside equipment necessary for the procedure. Be sure you know what tests the physician wants so you have the proper specimen containers and all necessary equipment. Sometimes the physician will perform bilateral biopsies for disease staging. Practice sterile procedure in handing the physician items needed for the aspiration and biopsy. Receive the aspirate syringe from the physician and quickly prepare smears at bedside and place in appropriate anticoagulated tubes  Direct smear straight from syringe  Crush prep from marrow spicules Receive other syringes of marrow if tests are requested such as Leukemia/Lymphoma Profile (LLP or Flow Cytometry), Immunophenotyping (chromosome studies), cultures (routine, viral or fungal). Transfer into appropriate tubes. Receive the biopsy specimen from the physician, prepare trephine imprints and place in fixative

Processing the specimen in the lab Place EDTA specimen (liquid aspirate) in Wintrobe tube and centrifuge at low speed. Measure layers formed by centrifugation  FPV - fat and perivascular (used for iron stain)  Plasma  Buffy coat (myeloid:erythroid cells- M:E) Normal is 4:1. M:E is the ratio between all granulocytes and their precursors and all nucleated red cell precursors.  RBC’s Prepare and stain ME smears. Perform special stains if requested (further discussion later). Deliver clot remaining in syringe and biopsy to histology for processing. Deliver other specimens obtained such as viral, fungal or routine culture specimens to microbiology. Complete paperwork and package specimens to be sent out to reference lab.

Information derived from specimens Direct smear from syringe tip - evaluation of cellular morphology with Wright’s stain Particle (crush) smear - evaluation of cellularity and the relationship of cells to each other M:E smear - evaluation of hematopoietic cells and M:E ratio FPV smear - evaluation of iron (use Prussian blue stain for iron) Biopsy  If marrow cannot be aspirated (“dry tap”), this is the only specimen for examination  Examination for malignancy for clinical staging of lymphomas and cancers  Examination of the architecture of the bone marrow and the cells in their natural relationship to each other  Trephine imprint (touch prep) - examination of cells with Wright’s stain; may be the only source to study cellular detail if an aspirate is not obtained