Haematopoiesis PDF

Haematopoiesis Matthew LAU (Scientific Officer) MLS 3009SEF (2025 Spring term)  Area to cover Ontogeny and development of blood forming tissues Functions of the bone marrow Maturation sequence of normal blood cells and influencing factors Structure of n...

Haematopoiesis Matthew LAU (Scientific Officer) MLS 3009SEF (2025 Spring term)  Area to cover 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 (pronounced “heh-ma-tuh-poy-EE-sus”) is blood cell production The body continually makes new blood cells to replace old blood cells Erythrocytes – 120 days Thrombocytes – 10 to 12 days Leukocytes – few days to over a year Begins before birth and continues as a cycle throughout life  Why is haematopoiesis important? In the average adult, 200 to 400 billion blood cells are destroyed and replaced each day Maintain the continuous blood production Give rise to multipotential haematopoietic stem cells (HSC) Also called “haemocytoblasts” By A. Rad and M. Häggström. CC-BY-SA 3.0 license. https://upload.wikimedia.org/wikipedia/commons/f/f0/Hematopoiesis_simple.svg  Fetal Development (You Love a Smart Bunny) Mesoblastic phase (Yolk sac) Hepatic phase (Liver and Spleen) Medullary phase (Bone marrow) https://oncohemakey.com/hematopoiesis-2/  Orelio, C., & Dzierzak, E. (2007). Bcl-2 expression and apoptosis in the regulation of hematopoietic stem cells. Leukemia & lymphoma, 48(1), 16-24. Mesoblastic Phase Take place first in the yolk sac and then the aorta-gonad-mesonephros (AGM) Begins as early as 19th days after fertilization in embryonic life Primitive erythroblasts formed in the mesenchyme of the yolk sac Provides oxygen to fetus Angioblasts surround the mesoderm which later becomes the blood vessel  Hepatic Phase Begins at 4 to 5 gestation weeks Mainly in the liver Produces clusters of developing erythroid, granulocyte, monocyte, megakaryocyte and lymphoid cells Known as blood islands In the mid fetal life , spleen and lymph nodes begin a limited role as secondary lymphoid organs  Medullary Phase Begins at around the 5th month HSC and mesenchymal stem cells migrate towards the BM Production in liver begins to diminish Myeloid production is very active Myeloid 3:1 erythroid ratio Detectable amounts of growth and stimulating factors EPO, G-CSF, GM-CSF, Hf and Ha  Adult haematopoiesis Bone marrow, liver, spleen, lymph nodes, and thymus are involved in the proliferation and maturation of blood cells Bone marrow Erythroid, myeloid, megakaryocytic, and early stages of lymphoid cells development Lymphoid cells development Primary lymphoid tissue: bone marrow and thymus T and B cells develop into immuno-competent cells Secondary lymphoid tissue: spleen and lymph nodes Immuno-competent cells further divide and differentiate into effector and memory cells  Bone Marrow One of the organs in the body and located within the cavities of the cortical bones. These cavities consist of trabecular bone resembling a honeycomb Primary site of hematopoiesis after birth and throughout the adult life Differentiates into myeloid and lymphoid lineages under the influence of growth factors. Function to supply mature and functional blood cells in the circulation Two major components: Red Marrow – heamatopoietically active Yellow Marrow – heamatopoietically inactive – composed primarily of adipocytes (fat cells) Normal adults contain approximately equal amounts of Red and Yellow marrow Mesenchymal cells migrate into a central space created within the bone cavities which differentiates and give rise to blood and BM matrix cells https://oncohemakey.com/hematopoiesis-2/#bib1 Retrogression During infancy and early childhood, BM consists primarily of 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 – process of replacing the active marrow by adipose tissues Eventually restricts RM to the flat bones, sternum, vertebrae, pelvis, ribs, skull and proximal portion of the long bones Areas within the BM cavity where RM has been replaced by YM consist of an mixture of adipocytes, undifferentiated mesenchymal cells and macrophages. Inactive YM is also scattered throughout active RM and is capable of reverting back to active marrow in cases of increased demand on the bone marrow. Such as excessive blood loss or increased erythrocyte destruction in the BM by toxic chemicals or irradiation.  Red Marrow Composed of extravascular cords that contain all of 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 and are located between the trabeculae of spongy bone. The haematopoietic cells tend to develop in H&E x400 BM specimen specific niches within the cords https://oncohemakey.com/hematopoiesis-2/#bib1 H&E x400 BM specimen Hematopoietic cells in Red Marrow 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, which facilitates the release of platelets 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 W-G x500 BM aspirate  Advential cells Mature blood cells move from the BM into the bloodstream. They pass 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. It is responsible for supplying a semifluid matrix, which serves as an anchor for the developing haematopoietic cells to self-renewal, proliferate and differentiate. Stromal cells in the matrix include endothelial cells, adipocytes, macrophages, osteoblasts, osteoclasts, and fibroblasts which support and regulate hematopoietic stem cell survival and differentiation.  Types of stromal cells Endothelial cells – form a single continuous layer along the inner surface and regulates the flow of particles and produces cytokines Adipocytes – are large cells with a single fat vacuole and secretes steroids to influence erythropoiesis and maintain bone integrity Fibroblasts (reticular cells) – associated with the formation of reticular fibers that form a lattice to support the vascular sinuses and developing hematopoietic cells Osteoblasts – bone forming cells Osteoclasts – bone resorbing cells Macrophages – function in phagocytosis and secretion of cytokines that regulate hematopoiesis and are located throughout the marrow space  Liver and Spleen Liver Function in Hematopoiesis: During fetal development, the liver is a primary site for blood cell production until the bone marrow takes over. Other Functions: The liver also helps in the production of certain proteins necessary for blood clotting and the breakdown of old or damaged blood cells. Spleen Function in Hematopoiesis: In the fetus, the spleen produces RBCs. In adults, it can resume this function if the bone marrow is damaged (extramedullary hematopoiesis). Other Functions: The spleen filters blood, removing old or damaged RBCs, and recycles iron. It also plays a role in the immune response by producing antibodies and storing white blood cells.  Lymph and Thymus Lymphatic System Function in Hematopoiesis: The lymphatic system, including lymph nodes and lymphoid tissues, produces lymphocytes (a type of WBC) which are crucial for the immune response. Other Functions: It helps in the removal of toxins and waste from the body, and transports lymph, a fluid containing infection-fighting WBCs. Thymus Function in Hematopoiesis: The thymus is essential for the maturation of T-lymphocytes (T- cells), which are critical for the adaptive immune response. Other Functions: The thymus produces hormones like thymosin that promote the development of T-cells and maintain the immune system.  Haematopoietic Stem Cells (HSC) HSC are actively dividing cells that is capable of self renewal and of differentiation into any cell lineages Give rise to haematopoietic progenitor cells (HPC) which are actively dividing cell that is committed to a single blood cell lineage and is not capable of self renewal Self renewal involvies proliferations accompanied by maintenance of both multipotency and tissue regenerative potential. To achieve: Cell must enter the cell cycle and divide and at least one of the progenies must be an undifferentiated cell Stem cells can differentiate into progenitor cells committed to: 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 Decrease in cell size Decrease in the ratio of nucleus to cytoplasm Changes in the nucleus Decrease in the size of the nucleus Change in the shape of the nucleus Condensation of nucleus chromatin, loss of nucleoli Possible loss of the nucleus Changes in the cytoplasm 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 Cytokines are soluble proteins Draw out biological effects at a low concentration Stimulate or inhibit blood cells production, differentiation and trafficking Suppresses apoptosis Positive influence: KIT ligand, FLT3 ligand, GM-CSF, IL-1, IL-3, IL-6, IL-11… Negative influence: transforming growth factor-, tumor necrosis factor- interferons… Cytokines include Colony-stimulating factors (CSFs), interleukins (ILs), interferons, lymphokines, monokines, chemokines…  Colony-Stimulating Factors (CSF) Function: CSFs are cytokines that stimulate the production, differentiation, and function of blood cells, particularly WBCs. Types: 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 Role in Hematopoiesis: CSFs are crucial for the proliferation and differentiation of hematopoietic stem cells into various blood cell lineages.  Interleukins (ILs) Function: ILs are a group of cytokines that play a significant role in the immune system by regulating the 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 the growth of megakaryocytes, which are precursors to platelets. Role in Hematopoiesis: Interleukins act as signaling molecules that help regulate the balance and production of different blood cell types.  Clinical Lab Hematology, 3rd ed. Mechanisms of Cytokine Regulation Endocrine signal : act over a fairly long distance Autocrine signal : produced by and act on the same cell Paracrine signal : produced by one cell and act on an adjacent cell over a short distances Juxtacrine signal : specialized type of paracrine, direct cell-cell contact to achieve the desired effect by membrane bound cytokines  Lineage Specific Haematopoiesis Myeloid Lineage 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 Lymphoid Lineage 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 An invasive procedure performed by a haematologist (clinician) Preferred collection site in adult is anterior or posterior iliac crest Indications for bone marrow examination: Unexplained anemia, abnormal red cell indices, cytopenia, or cytoses Abnormal peripheral blood smear morphology Diagnosis, staging, and follow-up of malignant hematological disorders Monitoring of treatment Suspected bone marrow metastases Should be evaluated together with a CBC count and peripheral smear examination within 24 hours https://eurjmedres.biomedcentral.com/articles/10.1186/s40001-023-01167-7 https://www.youtube.com/watch?v=3hzVvCl8UkM https://www.youtube.com/watch?v=EYd7OnCt7ug https://www.youtube.com/watch?v=0ZTGoPmCV1w  Bone Marrow Collection BM 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 Squash (Thick) and wedge (Thin) smears Particle clot Trephine biopsy Touch imprint Histology study  Dry Tap Occurs when no bone marrow is obtained during the procedure Due to either one or more of the following reasons: Bone Marrow Pathology: Often, a dry tap indicates an underlying issue such as marrow fibrosis, hypercellularity, hypocellularity with increased adipose tissue, neoplastic infiltration or primary bone disorders. Haematological Malignancies: Conditions like leukemia, myelofibrosis, and lymphoproliferative disorders are common causes. Technical Issues: Occasionally, a dry tap can result from faulty technique during the aspiration https://eurjmedres.biomedcentral.com/articles/10.1186/s40001-023-01167-7  Bone Marrow Processing Bone Marrow aspirate are collected in EDTA bottles Thick smears – May-Grunwald-Giemsa (MGG) or Wright stain and Prussian Blue (Perls’ reaction / iron) stain Thin smears – MGG or Wright stain and cytochemistry Flow cytometry or molecular study Bone marrow aspirate in culture bottle for cytogenetic study Trephine imprints – MGG or Wright stain Trephine biopsy – Histology staining Particle clot – FISH study https://www.researchgate.net/publication/296478669_Simultaneous_deletion_of_3%27ETV6_and_5%27_EWSR1_genes_in_blastic_plasmacytoid_dendritic_cell_neoplasm_Case_report_and_litera ture_review  Peripheral Blood Average 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: albumins, globulins, fibrinogen,… Cells: RBCs (erythrocytes), WBCs (leukocytes) and platelets (thrombocytes) Leukocytes: neutrophils, lymphocytes, monocytes, eosinophils, basophils  RBC (Erythrocyte) Structure: RBC are anucleate (lack a nucleus) and have a biconcave shape, which increases their surface area for gas exchange. They are filled with hemoglobin, a protein that binds oxygen and carbon dioxide. Production: Erythrocytes are produced in the red bone marrow through a process called erythropoiesis. This involves several stages, starting from stem cells and ending with mature erythrocytes. Life Cycle: They have a lifespan of about 100 to 120 days. After this period, old erythrocytes are recycled by macrophages in the spleen, liver, and bone marrow. Function: Their primary function is to transport oxygen from the lungs to body tissues and return carbon dioxide from the tissues to the lungs for exhalation  WBC (Leukocyte) Structure: WBC are a diverse group of immune cells with varying structures. They include granulocytes (neutrophils, eosinophils, basophils) with granules in their cytoplasm, and agranulocytes (lymphocytes, monocytes) without granule. Production: Leukocytes are produced in the bone marrow from hematopoietic stem cells. They undergo differentiation into various types based on their functions Life Cycle: The lifespan of WBCs varies widely. Neutrophils live for a few hours to days, while lymphocytes can live for years. Monocytes circulate for a few days before differentiating into macrophages or dendritic cells. Function: WBCs are essential for the immune system. They protect the body against infections, remove dead or damaged cells, and play roles in inflammation and immune responses.  Neutrophil Structure: Neutrophils are granulocytes with a multi-lobed nucleus and cytoplasmic granules containing antimicrobial substances. Production: Produced in the bone marrow, they mature over about 14 days before entering the bloodstream Life Cycle: They have a short lifespan of less than 24 hours in the bloodstream. Function: Neutrophils are the first responders to bacterial infections, performing phagocytosis to engulf and destroy pathogens.  Eosinophil Structure: Eosinophils are granulocytes with bi-lobed nuclei and cytoplasmic granules that stain red with eosin. Production: Produced in the bone marrow, they migrate to tissues after maturing. Life Cycle: They circulate in the blood for 8-12 hours and can survive in tissues for several days. Function: Eosinophils combat parasitic infections and are involved in allergic reactions and asthma by releasing toxic granules.  Basophil Structure: Basophils are granulocytes with large cytoplasmic granules that stain blue with basic dyes. Production: They are produced in the bone marrow and released into the bloodstream. Life Cycle: Basophils have a short lifespan, ranging from a few hours to a few days. Function: They release histamine and heparin during allergic reactions and inflammation, playing a role in immune responses to parasites.  Lymphocytes Structure: Lymphocytes are agranulocytes with a large, round nucleus and minimal cytoplasm. Production: They are produced in the bone marrow and mature in lymphoid organs like the thymus and spleen. Life Cycle: They can live from several weeks to years, depending on their type. Function: Lymphocytes are crucial for adaptive immunity. T cells attack infected cells and regulate immune responses, while B cells produce 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) Structure: Platelets are small, disc-shaped cell fragments without a nucleus. They are derived from the cytoplasm of megakaryocytes in the bone marrow Production: Platelets are produced in the bone marrow through a process called thrombopoiesis. Megakaryocytes release platelets into the bloodstream. Life Cycle: Platelets have a lifespan of about 7 to 10 days. Old or damaged platelets are removed from the circulation by the spleen. Function: Platelets play a crucial role in haemostasis (process of blood clotting). They adhere to damaged blood vessels, aggregate to form a platelet plug, and release chemicals that activate the clotting cascade.  Complete Blood Count (CBC) Most commonly ordered test in haematology. Provides information to review overall health of bone marrow, diagnosis a medical condition, monitor a medical condition and medical treatment Usually performed with EDTA whole blood (WB) 1.5mg EDTA/ml WB Measures the following: Leukocytes: WBC count, differential count Erythrocytes: RBC count, haemoglobin and RBC indices Platelets: Platelet count Follow with Blood smear examination Can be conducted manually or automatically  CBC – Manual WBC, RBC, PLT count Haemacytometer / Improved Neubauer counting chamber Dilute WBC with 3% acetic acid at 1:20. Count 4 large squares (4 mm2). Expressed in n x 109/L. Dilute RBC with 3.2% sodium citrate at 1:200. Count 5 small squares (0.2 mm2). Expressed in n x 1012/L. Dilute PLT with 1% ammonium oxalate at 1:20. Count 4 large squares (4 mm2) or 25 small squares (1 mm2). Expressed in n x 109/L. Total cells/uL = (Cells counted x dilution factor) / (area in (mm2) x depth (0.1)) Use average numbers of two sides. Multiply by 106 to get count per L.  CBC –Haemoglobin (Hb) concentration Visual: Sahli’s Method: Involves mixing blood with hydrochloric acid to form acid haematin, then comparing the color to a standard using a haemoglobinometer. Dare Method: Similar to Sahli’s, but uses a different type of comparator for color matching. Haden Method: Uses a color scale to visually estimate haemoglobin concentration. Wintrobe Method: Measures packed cell volume (PCV) after centrifugation to estimate haemoglobin. Tallqvist Method: Uses a color chart to visually estimate haemoglobin levels. Spectrophotometric: Oxyhaemoglobin Method: Measures the absorbance of oxyhaemoglobin at specific wavelengths to determine haemoglobin concentration. Cyanmethaemoglobin Method: Converts haemoglobin to cyanmethaemoglobin using a potassium cyanide and ferricyanide, then measures absorbance. This is the most widely used method. Srivastava, T., Negandhi, H., Neogi, S. B., Sharma, J., & Saxena, R. (2014). Methods for hemoglobin estimation: A review of" what works. J Hematol Transfus, 2(3), 1028.  CBC – RBC indices (HCT) Determine haematocrit (HCT) by using microhaematocrit tubes. Expressed in % or L/L Also known as packed cell volume (PCV) Use to calculate the RBC indicies Rule of three (applies to normal samples): RBC x 3 = Hb (± 0.5) Hb x 3 = HCT (± 2)  PCV (%) x 10 CBC – RBC indices MCV = RBC count (x1012/L) (MCV) Mean corpuscular volume Shows the average volume of red cell Expressed in femtoliters (fL) (10-15 L) High in macrocytic anaemia, liver diseases and alcoholism Typical causes are Vit B12 or Folate deficiency Usually macrocytes are present in blood smear with high MCV Low in microcytic anaemia and thalassemia Typical causes are Iron deficiency Usually microcytes are present in blood smear with low MCV MCH = Hb (g/dL) x 10 CBC – RBC indices RBC count (x1012/L) (MCH) Mean corpuscular haemoglobin Shows average weight content of Hb in a red cell Expressed in pictogram (pg) (10-12 g) High in macrocytic anaemia and hyperthyroidism Over reactive thyroid may increase MCH Red blood cells may appear hyperchromic in blood smear with high MCH Low in microcytic anaemia and thalassemia Over reactive thyroid may increase MCH Red blood cells may appear hypochromic in blood smear with high MCH MCHC = Hb (g/dL) x 100 CBC – RBC indices HCT (%) (MCHC) Mean corpuscular haemoglobin concentration Shows average concentration of Hb in a red cell Expressed in gram per deciliter (g/dL) High in hereditary spherocytosis and autoimmune haemolytic anaemia Conditions causing densely packed haemoglobin leading to higher concentrations Increase spherocytes can be observed in hereditary spherocytosis with high MCHC Low in Iron deficiency anaemia and chronic inflammatory or infection anaemia Lack of iron results in lower Hb production and reduced concentrations Target cells may be observed in iron deficiency anaemia with low MCHC RDW = SD of MCV x 100 RBC Distribution Mean MCV Width (RDW) Provided by automated haematology cell counter Represents the coefficient of variation of the red cell volume Not derived from the width of the RBC, but rather from the width of the distribution curve of the corpuscular volume Ref range: 11.0 – 14.0 % High in red cell anisocytosis and nutritional deficiencies (eg. Vit B12, Iron) Can assist in differentiating anaemia types Eg: High = Iron deficiency anaemia. Low = thalassemia % RET = A x 100 Reticulocyte Count Bx9 Miller Disc (RET) Reticulocyte contains remnant cytoplasmic RNA and organelles such as mitochondria and ribosomes. Is the last immature red cell stage and normally spends 2 days in BM and 1 day in peripheral blood before developing into a mature RBC. EDTA WB is stained with supravital stain (new methylene blue or brilliant cresyl blue) RNA and organelles precipitate, forming filamentous network of reticulum Any non-NRBC that contains two or more blue-stained materials is defined as a reticulocyte Clinically assess the erythropoietic activity of the BM Ref range: 0.5 – 2.0% Can be performed manually or by haematology analyzer % RET = A x 100 Reticulocyte Count Bx9 Miller Disc (RET) Reticulocyte contains remnant cytoplasmic RNA and organelles such as mitochondria and ribosomes. Is the last immature red cell stage and normally spends 2 days in BM and 1 day in peripheral blood before developing into a mature RBC. EDTA WB is stained with supravital stain (new methylene blue or brilliant cresyl blue) RNA and organelles precipitate, forming filamentous network of reticulum Any non-NRBC that contains two or more blue-stained materials is defined as a reticulocyte Clinically assess the erythropoietic activity of the BM Ref range: 0.5 – 2.0% Can be performed manually or by haematology analyzer  Interferences of CBC Conditions Parameters Affected Corrective Actions Cold agglutinins RBC, MCV, MCH, MCHC Warm sample to 37oC Haemolysis RBC, HCT, MCHC Check diagnosis, request a new sample Lipemia, icterus Hb, MCH, MCHC Saline replacement Microcytes, fragmented RBCs RBC, PLT Smear review, optical method Nucleated RBCs WBC Smear review, repeat with CBC + nRBCs mode WBC, RBC or PLT too high WBC, RBC, Hb, PLT Repeat with dilution Giant platelet PLT Smear review, optical method Platelet clumps WBC, PLT Smear review, repeat with citrate blood Platelet satellitism PLT Smear review, repeat with citrate blood Sample clot All parameters Request a new sample  Erythrocyte Sedimentation Rate (ESR) Can be performed manually or automated. Measures the distance (mm) of red cells descended in diluted human whole blood in 1 hour Sedimentation consists of three phases: lag , decantation and packing Depends on the ability of erythrocytes to form rouleaux Not used as a screening test because it elevates in many other conditions Such as multiple myeloma, pregnancy, anaemia etc Useful in diagnosis of temporal arteritis and polymyalgia rheumatic High in accelerating proteins (eg. CRP), anaemia, macrocytosis and multiple myeloma Decreases in retarding proteins (eg. Albumin), microcytosis and spherocytosis  Automated CBC (based on Sysmex XN-1000) Haematology analysis is performed according: Hydrodynamically focused DC detection method Flow cytometry method (using a semiconductor laser) SLS-haemoglobin method  Hydrodynamically focused DC detection method The RBC detector counts the RBC and PLT via the Hydrodynamically focused DC detection method. At the same time, HCT is calculated via the RBC pulse height detection method. Inside the detector, the sample nozzle is positioned in front of the aperture and in line with the center. After diluted sample is forced from the sample nozzle into the conical chamber, it is surrounded by front sheath reagent and passes through the aperture center. After passing through the aperture, the diluted sample is sent to the catcher tube. This prevents the blood cells in this area from drifting back and generation of false platelet pulses. The Hydro Dynamic Focusing method improves blood count accuracy and repeatability. Since the blood cells pass through the aperture in a line, it also prevents the generation of abnormal blood cell pulses.  Flow cytometry method using semiconductor laser Cytometry is used to analyze physiological and chemical characteristics of cells and other biological particles. Flow cytometry is used to analyze those cells and particles as they are passed through extremely small flow cells. A blood sample is aspirated and measured, diluted to the specified ratio, and stained. The sample is then fed into the flow cells. This Hydro Dynamic Focusing mechanism improves cell count accuracy and repeatability. Since the blood cell particles pass in a line through the center of the flow cell, the generation of abnormal blood pulses is prevented, reducing flow cell contamination. A semiconductor laser beam (wavelength: 633 nm) is emitted to the blood cells passing through the flow cell. The light is captured and converted into electrical pulses to obtain blood cell information. The forward scattered light and side scattered light is captured by the photodiode. The side fluorescent light is captured by the avalanche photodiode.  Forward Scatter and Side Scatter Light When obstacles pass through a light path, the light beam scatters from each obstacle in various directions. This phenomenon is called light scattering. By detecting the scattered light, it is possible to obtain informationon cell size and material properties. Likewise, when a laser beam is emitted to blood cell particles, light scattering occurs. The intensity of the scattered light depends on factors such as the particle diameter and viewing angle. Forward Scatter provides information on blood cell size Side scattered light provides information on the cell interior (such as the size of the nucleus).  Side Fluorescent Light When light is emitted to fluorescent material, such as stained blood cells, light of longer wavelength than the original light is produced. The intensity of the fluorescent light increases as the concentration of the stain becomes higher. By measuring the intensity of the fluorescence emitted, specific information on the degree of blood cell staining can be obtained. Fluorescent light is emitted in all directions and XN-1000 detects the fluorescent light that is emitted sideways.  SLS – Haemoglobin Method In the past, the mainstream methods for automatically measuring haemoglobin were the cyanmethaemoglobin method and oxyhaemoglobin method. Several disadvantages limits their use on large, fully automatic instrument. Cyanmethaemoglobin method: Pros: Recommended by International Committee for Standardization in Haematology (ICSH) in 1966 Cons: Slow conversion speed, poisonous reagents (cyanide) require treatment of liquid wastes Oxyhaemoglobin method: Pros: Faster conversion speed and no poisonous substances, Cons: cannot convert methaemoglobin resulting in lower true values for samples containing large amounts of methaemoglobin (eg. Control samples) The SLS-hemoglobin method makes use of the advantages of the two aforementioned methods. Fast conversion, no poisonous substances and can accurately analyze methaemoglobin  References and further reading Hoffbrand AV and Moss PAH. (2016). Hoffbrand’s Essential Hematology, 7th ed. Chichester: Wiley-Blackwell. Turgeon ML. (2018). Clinical Hematology: theory & procedures, 6th ed. Philadelphia: Wolters Kluwer. Keohane EM, Otto CN, & Walenga JM. (2020). Rodak’s Hematology: Clinical Principles and Applications. 6th Edition. St Louis: Elsevier. Harmening, D (2024). Clinical hematology and fundamentals of hemostasis, 6th ed. Philadelphia: FA Davis.