Haematopoiesis

• Haematopoiesis refers to blood cell production
• The areas covered include:
  • Ontogeny and development of blood forming tissues
  • Functions of the bone marrow
  • Maturation sequence of normal blood cells and influencing factors
  • Structure of normal cells and their precursors
  • Production, structure and function of platelets
  • Basic haematological investigations

What is Haematopoiesis?

• Haematopoiesis is pronounced "heh-ma-tuh-poy-EE-sus"
• The body continually makes new blood cells to replace old blood cells
  • Erythrocytes last about 120 days
  • Thrombocytes last about 10 to 12 days
  • Leukocytes last from a few days to over a year
• Haematopoiesis begins before birth and continues as a cycle throughout life
• 200 to 400 billion blood cells are destroyed and replaced each day in the average adult
• It maintains continuous blood production
• Haematopoiesis gives rise to multipotential haematopoietic stem cells (HSC), also known as haemocytoblasts

Fetal Development

Three phases of fetal development include:

• Mesoblastic phase (Yolk sac)
• Hepatic phase (Liver and Spleen)
• Medullary phase (Bone marrow)

Mesoblastic Phase

• Takes place first in the yolk sac and then the aorta-gonad-mesonephros (AGM)
• Begins as early as the 19th day after fertilization in embryonic life
• Primitive erythroblasts are formed in the mesenchyme of the yolk sac, providing oxygen to the fetus
• Angioblasts surround the mesoderm, which later becomes the blood vessel

Hepatic Phase

• Begins at 4 to 5 gestation weeks and mainly occurs in the liver
• Produces clusters of developing erythroid, granulocyte, monocyte, megakaryocyte, and lymphoid cells
• Blood islands are known to form during this phase
• In mid-fetal life, the spleen and lymph nodes begin a limited role as secondary lymphoid organs

Medullary Phase

• Begins around the 5th month of gestation
• HSC and mesenchymal stem cells migrate towards the bone marrow (BM)
• Production in the liver begins to diminish
• Myeloid production increases, with a myeloid to erythroid ratio of 3:1
• Detectable amounts of growth and stimulating factors such as EPO, G-CSF, GM-CSF, Hf, and Ha are present

Adult Haematopoiesis

• Bone marrow, liver, spleen, lymph nodes, and thymus are involved in the proliferation and maturation of blood cells
• Bone marrow supports erythroid, myeloid, megakaryocytic, and early stages of lymphoid cell development
• Primary lymphoid tissue include bone marrow and thymus where T and B cells develop into immuno-competent cells
• Secondary lymphoid tissues include spleen and lymph nodes are where immuno-competent cells further divide and differentiate into effector and memory cells

Bone Marrow

• Located within the trabecular bone cavities of cortical bones, resembling a honeycomb structure
• Primary site of hematopoiesis after birth and throughout adult life
• Differentiates into myeloid and lymphoid lineages under the influence of growth factors
• The function of bone marrow is to supply mature and functional blood cells into the circulation
• Two major components:
  • Red Marrow: Heamatopoietically active
  • Yellow Marrow: Heamatopoietically inactive and composed primarily of adipocytes (fat cells)
• Normal adults contain approximately equal amounts of red and and yellow marrow
• Mesenchymal cells migrate into the bone cavities, differentiate, and give rise to blood and BM matrix cells

Retrogression

• During infancy and early childhood, bone marrow consists primarily of red marrow (RM)
• Between 5 and 7 years of age, adipocytes become more abundant and begin to occupy the spaces in the long bones previously dominated by active marrow
• Retrogression is the process of replacing the active marrow by adipose tissues Restrict RM to the flat bones, sternum, vertebrae, pelvis, ribs, skull, and proximal portion of the long bones
• Areas within BM that replaced by yellow marrow consist of mixture of adipocytes, undifferentiated mesenchymal cells, and macrophages
• inactive yellow marrow is also scattered throughout active red marrow and is capable of reverting back to active marrow, for example in cases of increased demand
• such as after excessive blood loss or increased erythrocyte destruction by toxic chemicals or irradiation

Red Marrow

• Composed of extravascular cords containing all the developing blood cell lineages, stem and progenitor cells, adventitial cells, and macrophages
• The cords are separated from the lumen of the sinusoids by endothelial and adventitial cells
• They are located between the trabeculae of spongy bone
• Haematopoietic cells develop in specific niches within the cords
• Normoblasts develop in small clusters adjacent to the outer surfaces of the vascular sinuses
• Some normoblasts are found surrounding iron-laden macrophages
• Megakaryocytes are located close to the vascular walls of the sinuses to facilitate platelet release into the lumen of the sinusoids
• Immature myeloid (granulocytic) cells through the metamyelocyte stage are located deep within the cords
• As these maturing granulocytes proceed along their differentiation pathway, they move closer to the vascular sinuses

Adventitial Cells

• Mature blood cells move from the BM into the bloodstream by passing through layers of adventitial cells, which form a discontinuous layer on the outer side of the bone marrow sinus
• Next to these adventitial cells is a basement membrane, followed by a continuous layer of endothelial cells on the inner side of the sinus
• The adventitial cells (also called reticular cells) extend long processes into the marrow, forming a supportive mesh for developing blood cells
• These cells can contract, allowing mature blood cells to pass through the basement membrane and interact with the endothelial layer to enter the bloodstream
• As blood cells come in contact with endothelial cells, they bind to the surface via a receptor-mediated process, passing through pores in the endothelial cytoplasm and released into the circulation

Haematopoietic Micro-environment

• Plays an important role in stem cell differentiation and proliferation
• Responsible for supplying a semifluid matrix, serving as an anchor for developing haematopoietic cells to self-renew, proliferate, and differentiate
• Stromal cells in the matrix (endothelial cells, adipocytes, macrophages, osteoblasts, osteoclasts, and fibroblasts) support and regulate HSC survival and differentiation

Types of Stromal Cells

• Endothelial cells form a single continuous layer along the inner surface, regulating the flow of particles and producing cytokines
• Adipocytes are large cells with a single fat vacuole, secreting steroids to influence erythropoiesis and maintain bone integrity
• Fibroblasts (reticular cells) assist the formation of reticular fibers to support vascular sinuses and developing haematopoietic cells
• Osteoblasts assist bone formation
• Osteoclasts assist bone resorption
• Macrophages function in phagocytosis and secretion of cytokines that regulate haematopoiesis and are located throughout the marrow space

Liver

• During fetal development, the liver is a primary site for blood cell production
• Other functions of the liver include: production of certain proteins necessary for blood clotting and the breakdown of old or damaged blood cells

Spleen

• In the fetus, the spleen produces red blood cells
• In adults, the spleen can resume red blood cell production if the bone marrow is damaged (extramedullary hematopoiesis)
• Other functions of the spleen include: filtering blood, removing old or damaged RBCs, recycling iron, and producing antibodies and storing white blood cells

Lymphatic System

• Lymphatic system including lymph nodes and lymphoid tissues produces lymphocytes which are crucial for the immune response
• Assists removal of toxins and waste from the body, that contains infection-fighting WBCs

Thymus

• Thymus is essential for maturation of T-lymphocytes are critical for the adaptive immune response
• The thymus produces hormones such as thymosin to promote the T-cell development

Haematopoietic Stem Cells (HSC)

• Actively dividing, capable of self-renewal and differentiation into any cell lineage
• Give rise to haematopoietic progenitor cells (HPC),which are actively dividing but committed to a single blood cell lineage
• Self renewal involves maintenance of both multipotency and tissue regenerative potential which can produce undifferentiated cells
• Stem cells differentiate into progenitor cells with the following specifications:
  • Common myeloid progenitor proliferates and differentiates into granulocytic, erythrocytic, monocytic, or megakaryocytic lineages
  • Common lymphoid progenitor proliferates and differentiates into T lymphocytes, B lymphocytes, or NK lineages

Morphologic features of blood cell during maturation

• Overall changes include:
  • Decrease in cell size
  • Decrease in the ratio of nucleus to cytoplasm
• Changes in the nucleus include:
  • Decrease in the size of the nucleus
  • Change in the shape of the nucleus
  • Condensation of nucleus chromatin and loss of nucleoli
  • Possible loss of the nucleus
• Changes in the cytoplasm include:
  • Decrease in basophilia
  • Increase in the proportion of cytoplasm
  • Possible appearance of granules in the cytoplasm

Regulation of Haematopoiesis

• Regulated by haematopoietic growth factors or cytokines (soluble proteins)
• Biological effects include:
  • Draw out biological effects at a low concentration
  • Stimulate or inhibit blood cell production, differentiation and trafficking
  • Suppresses apoptosis
  • Positive or negative influence
• Cytokines include:
  • Colony-stimulating factors (CSFs)
  • Interleukins (ILs)
  • Interferons, lymphokines, monokines, or chemokines

Colony-Stimulating Factors (CSF)

• CSFs are cytokines that stimulate the production, differentiation, and function of blood cells, particularly WBCs
• Types include:
  • Granulocyte-CSF (G-CSF): Promotes the production of granulocytes
  • Macrophage-CSF (M-CSF): Stimulates the production of macrophages
  • Granulocyte-Macrophage CSF (GM-CSF): Encourages the production of both granulocytes and macrophages
  • Erythropoietin (EPO): Produces erythrocytes
• CSFs are crucial for the proliferation and differentiation of haematopoietic stem cells into different blood cell lineages

Interleukins (ILs)

• Play a significant role in immune system
• Regulate growth, differentiation, and activation of hematopoietic and immune cells

Key Interleukins in Hematopoiesis:

• IL-3: supports the growth and differentiation of multipotent hematopoietic stem cells
• IL-6: involved in the stimulation of immune responses and hematopoiesis
• IL-7: essential for the development of T and B lymphocytes
• IL-11: promotes megakaryocytes growth, precursors to platelets
• Interleukins act as signaling molecules that help regulate the balance and production of different blood cell types

Lineage Specific Haematopoiesis

• Erythropoiesis: Production of RBC from common myeloid progenitors.
• Thrombopoiesis: Production of platelets (thrombocytes) from megakaryocytes.
• Granulopoiesis: Production of granulocytes (neutrophils, eosinophils, and basophils).
• Monocytopoiesis: Production of monocytes, which can differentiate into macrophages and dendritic cells
• Lymphopoiesis: Production of lymphocytes, including:
  • B-cells: Responsible for antibody production.
  • T-cells: Involved in cell-mediated immunity.
  • Natural Killer (NK) cells: Play a role in the innate immune response
• Dendritic Cells: Both lineages can give rise to dendritic cells, which are crucial for antigen presentation and initiating immune responses

Bone Marrow Examination

• Invasive procedure performed by a haematologist (clinician), preferred collection site in adult is anterior or posterior iliac crest
• Indications for bone marrow examination include:
  • Unexplained anemia, abnormal red cell indices, cytopenia, or cytoses + Abnormal peripheral blood smear morphology
  • Diagnosis, staging, and follow-up of malignant haematological disorders + Monitoring of treatment
  • Suspected bone marrow metastases
• Bone marrow examination should be evaluated together with a CBC count and peripheral smear examination within 24 hours

Bone Marrow Collection

• Removes marrow fluid and cells and Trephine biopsy from the posterior iliac crest
• Collected aspirate (flakes) can be used to make marrow smears by MLTs
• Bone marrow aspirate can be Squash and or wedge Thin smears and or particle clot

Trephine Biopsy needs to be touch imprint and or for histology study

Dry Tap in Bone Marrow Examination

Occurs when no bone marrow is obtained during the procedure due to:

• Bone Marrow Pathology often, this indicates an underlying issue like marrow fibrosis, or hyper/hypo-cellularity with adipose tissue
• Haematological Malignancies: Conditions like leukaemia, or myelofibrosis, and lymphoproliferative disorders are common causes
• Technical Issues: Can occasionally result from faulty technique during the aspiration

Bone Marrow Processing

Processes include:

• Collection of sample in EDTA bottles
• Creation of thick and thin smears and or perform flow cytometry and or place sample in culture (for diagnostic and further analysis)
• Trephine imprints, for histology staining and FISH study

Peripheral Blood

• Average circulating blood volume in an adult is 4 to 6 liters, around 8% of total body weight
• Blood is composed of 55% plasma and 45% cells
  • Plasma includes: albumins, globulins, fibrinogen,...
  • Cells can be RBCs (erythrocytes), WBCs (leukocytes) and platelets (thrombocytes)

Leukocytes: neutrophils, lymphocytes, monocytes, eosinophils, and basophils

Characteristics of RBCs (Erythrocytes)

• Are anucleate cells (lacking a nucleus) with a biconcave shape, which increases their surface area for gas exchange
• Filled with hemoglobin, a protein that binds oxygen and carbon dioxide
• Produced in the red bone marrow through erythropoieses (several stages, starting from stem cells and ending with mature erythrocytes)
• Have a lifespan of about 100 to 120 days
• Old erythrocytes are recycled by macrophages in the spleen, liver, & bone marrow
• Function in transporting oxygen from the lungs to body tissues and return carbon dioxide from the tissues to the lungs for exhalation

WBCs (Leukocytes)

• Structure: diverse group of immune cells with varying structures
• Include granulocytes [neutrophils, eosinophils, basophils with granules in cytoplasm], and agranulocytes lymphocytes, monocytes without granules)
• Produced bone marrow from hematopoietic stem cells
• Can differinitiate into various types based on functions
• The lifespan of WBCs varies widely: Neutrophiles live for hours to days, lymphocytes live for years.
• Monocytes circulate for days before differentiating into macrophages or dendritic cells
• Essential to immune system that protect the body against infections, remove dead of damaged cells and play inflammation

Neutrophils

• Are granulocytes with a multi-lobed nucleus and cytoplasmic granules containing antimicrobial substances
• Are produced in the bone marrow, mature over about 14 days before entering the bloodstream
• Have a short lifespan of less than 24 hours in the bloodstream
• Are first responders that travel to bacterial infections and proceed by performing phagocytosis to engulf and destroy pathogens

Eosinophils

• Structure: granulocytes with bi-lobed nuclei and red cytoplasmic granules that stain with Eosin
• Produced in the bone marrow, migrate to tissues after maturing
• Circulates in the blood for 8-12 hours and survive in tissues for days
• Combat blood parasitic infections and are involved in allergic reactions and asthma by releasing toxic granules

Basophils

• Are granulocytes with large cytoplasmic granules stain blue with Basic dyes
• Are produced in the bone marrow then released
• have a short lifespan ranging from a few hours to a few days
• Releases histamine and heparin during allergic reactions and inflammation, while playing a role in immune responses to parasites

Lymphocytes

• (Structure and Production):
• These are large a-granulocytes which have round nucleus and small minimal cytoplasm
• Produced in the bone marrow and lymph organs like the thymus and spleen. the lifetime of the blood is very diverse ( weeks and to years.) and this is the core immune response for adaptive immunity
• T cells attack infected cells and regulate immune responses, while B cells make the antibodies

Monocytes

• Structure: Monocytes are the largest white blood cells with a kidney-shaped nucleus
• Production: Produced in the bone marrow, they circulate in the blood before migrating to tissues
• Life Cycle: They circulate in the blood for 1-3 days before differentiating into macrophages or dendritic cells in tissues
• Function: Monocytes perform phagocytosis, ingesting pathogens and dead cells, and play a role in immune regulation and tissue repair

PLTs (Thrombocytes)

• Small, disc-shaped cell fragments without a nucleus
• Derived from the cytoplasm of Megakaryocytes found in the bone marrow (produced in the medula)
• produced by process called : thrombopoiesis
• The life cyle is short (7-10 days)
• Old PLT are removed from spleen

Function:

• Blood Vessel haemostasis
• They help blood by adhering/aggregating to the damanged vessel and releasing chemicals to the area for clot formation

Complete Blood Count (CBC)

• Most commonly ordered test in haematology that diagnoses medical reviews and monitors marrow functions
• It is performed with EDTA whole blood
• Measures Leukocytes (WBC count of differential count)
• The examination includes Erythrocytes the RBC count, that shows the indices platelet is included
• It is followed with blood smear exam
• Conducted automatic/manually

CBC manual

• Hemacytometer / improved chamber
• Dilute WBC with Acetic (3%) at 1:10.Count 4 big Squares expressed 10^9/L
• Dilute RBC with Citrate (3.2) % at 1:20.Count 5 small Squares expressed 10^12/L
• Dilute PLT with ammonium at 1:20 Squares expressed 10^9/L
• Total count/uL=(CellXfactor)/(Area Xdepth)
• Average number of Sides and X10^6

Cbs -haemoglobin

• Can be performed visually or by spectrophotometer that absorbs oxygen at wavelengths to determine haemoglobin concentration
• A method the converts haemoglobin with cyanide was the most wildly used due to accuracy and reliability

Cbc and red blood cells

• The red count by HCT (microhaematocrit tubed) expressed/L Also is PCV the Rule/3 applies for normals
  • RBCX3=hb
    • the hbX3=hst

CBC (RBC indices)

MCV (Mean Corpuscular volume) (fl average per RBC)

• MCV (Mean Corpuscular volume) average of RED cells, expressed in 10-15 L
• MCV high in macrocytic anamia/liver
• typical B12 def (macrocytic in blood) If low means microcytic anema/althesesmia

RBC

• Hb / dl then average weight average
• overreact increased then cells appear high Hb/dl and pictogram 10'2 g of blood cells

microlyctic /thalassemia, cells with hypercrome and lower amounts of hB/dl

Mean Corpuscular Hemoglobin Concentration (MCHC).

• average level (g/dL) , High in hereditary conditions. Spherocytosis (dense packed hemoglobin higher levels with a sphere. can have a Hb/dl
• low count iron. Hb concentration. result to anemia

RBC Distribution Width (RDW)

• It is performed for analysis of cell counter and represents the width of the blood
• 11-40% if RDW is higher , cell anisocytosis is present and nutritional defieciens are shown . Also differtiates anemia that could e g,
  • High = Iron deficiency anaemia
  • low = thallasemia

RET

• Remnant count
• • count the cytoplasmic organs , the stain last 2 days normally, can asses the erythropoetic by performing a a clinical test

Interferences of CBC

• Cold Agglutinin: Red blood cells (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) will affect the corrective Actions ,the warm for sample is C37' • Haemolysis - RBC, haematocrit (HCT), MCHC the check requests for new samples

• In blood smear, there are optical methods in order is performed by a microscope
• if levels were increased in high red cell count, smear review for NRBCS is performed
• Review smear for platelet reviews and sample with citrate and follow with saline repacment

Erythrocyte Sedimentation Rate (ESR)

• Test measures the distance that red blood cells fall within a blood sample over an hour(mm)
• Consist for three phases lag, decantation and packing that depends on on three ability to rouaeax
• This elevation indicate multiple myeloma to test conditions of preganncy and other and anaemia
• To test for diagnoses test with CRP and micro cyrosis

automated Cbs that focus on flow chemistry

• hydro- focus-d c detection method

• cdf-method (semi laser) • Sls. hema method

hydro focusing Dfc detection method

• the rbc detector counts the rbc and put to hydro by a focus focus a method that cacultes a pulse hict from detection inside the the detector the samples nozzle position. After the sampl eis placed the tube prevents in and generate platelets a tubes

Side fluorescent

• Whem Fluo, and then are stained into longs produced . then is to obtained in the staining

cbc

• calculated as a parcle , by in is checked if has a cd calculate it that have been as assumed area the plcr is is calculated by ratio

blood light

• measure blood size and measure side , the information can be then detected.