P1 - Identification of Immune Cells, Lymphoid Organs etc

• Preparation of Peripheral Blood Smear


  1. Place a drop of blood about 2-3 mm in diameter approximately 1cm from one end of the slide
  2. Place the slide on a flat surface, and hold the other end between your left thumb and forefinger.
  3. With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop.
  4. Hold the spreader slide at a 30°. 45 angle, and draw it back against the drop of blood
  5. Allow the blood to spread almost to the edges of the slide.
• Preparation of Blood Smear cont.

  6. Push the spread forward with one light, smooth moderate speed. A thin film of blood in the shape of tongue.

  7. Label one edge with patient name, lab id and date.

  8. The slides should be rapidly air dried by waving slides or using an electrical fan
• What stain is used to stain blood film

  Giemsa stain
• Identification of WBCs/ Immune Cells is done by noting: 


  a)    Size  of  the  cell

  b)    Shape  of  the  nucleus  and  the  number  of  lobes

  c)    Staining  of  the  nucleus,  its  intensity  and  uniformity

  d)    Presence of granules, their size, number and staining reaction.
• Depending on the presence of granules or not, WBCs are classified into: 


  Granulocytes and Agranulocytes
• What stain are granules stained with

  Leishman stain
• Granulocyets are


  1. Neutrophils
  2. Eosinophils
  3. Basophils
• Agranulocytes are


  1. Lymphocytes
  2. Monocytes
• Salient Features of Eosinophils 


  
  

  • Diameter is 10-14 microns
  • The nucleus is bilobed
  • The granules are in the cytoplasm - they are coarse and red/pink as they take both acidic stain (eosin)

  
• Salient Features of Neutrophils 



  • Diameter is 10-14 microns
  • Multi-lobed nucleus (2-5 lobes)
  • Granules in the cytoplasm are very fine and violet in colour as they take both basic and acid stains

  
• Salient Features of Basophils

  • Diameter is 10-14 microns
  • The nucleus is bilobed
  • Granules in the cytoplasm are coarse and blue in colour as the take up the basic stain, methylene blue

  
• Salient Features of Lymphocytes 


  • Diameter of a large lymphocyte = 10-12 microns and smaller ones = 7-9 microns
  • The nucleus is round or slightly bean shaped and it is comparatively larger and the cytoplasm is less
  • The cytoplasm is clear - no granules

  
• Salient Features of Monocytes 


  • Largest of all leukocytes with a diameter of 16-22 microns
  • The nucleus is kidney bean shaped and it is mostly pushed to one side
  • The cytoplasm is clear without granules

  
• Blood Cells 


  Blood Cells are:

  
• Plasma Cells 


  • A type of immune cell that makes large amounts of specific antibodies
  • Plasma cells develop from B cells that have been activated
  • Plasma cell neoplasms like multiple myeloma occur when abnormal plasmas cells from cancerous tumours are in the bone or soft tissue

  
• Erythrocytes


  a
• Neutrophil


  • 3000 - 7000 cells per mm3 of blood
  • Development takes 14 days and lifespan is 6 hours to a few days
  • Function = phagocytose bacteria
• Eosinophil


  • 100 - 400 cells per mm3 of blood
  • Development takes 14 days and lifespan is about 5 days
  • Function = kill parasitic worms; complex role in allergy and asthma
• Basophils 


  • 20-50 cells per mm3 of blood
  • Development takes 1-7 days and lifespan is a few hours to a few days
  • Function = release histamine and other mediators of inflammation ; contains heparin which is an anticoagulant
• Lymphocytes


  • 1500-300 cells per mm3 of blood
  • Development takes days to weeks and lifespan is hours to years
  • Function = mount an immune response by direct cell attack or via antibodies
• Monocytes 


  • 100-700 cells per mm3 of blood
  • Development takes 2-3 days and lifespan is months
  • Function = phagocytosis; develop into macrophages in tissues
• What are the central lymphoid organs

  Thymus

  Bone marrow
• Function of Thymus


  Site of proliferation and maturation of T cells
• Structure of thymus


  2 lobes surrounded by a fibrous capsule

  Septa/trabeculae arising from the capsule divide the thymus into lobules and each lobule is differentiated into an outer cortex and inner medulla
• Thymus - cortex


  Dense populated and contains:

  1. thymocytes - lymphocytes of thymus, the cortical thymocytes are immature and many in number
  2. cortical epithelial cells
  3. Nurse cells - specialised epithelial cells with long membrane extensions that surround many thymocytes
• Thymus - medulla 


  Sparsely populated and contains

  1. thymocytes - medullary thymocytes are more mature and fewer in number
  2. Medullary epithelial cells
  3. Interdigitating dendritic cells
  4. Hassall's corpuscles = concentric layers of degenerating epithelial cells
• What are the peripheral lymphoid organs 


  lymph nodes, spleen, tonsils, and mucosal-associated lymphoid tissues 
• Function of Lymph Node


  It is a small bean-shaped organ, that occurs in clusters or chains, along the length of the lymphatic vessel

  Function = acts as a physiological barrier - filter the microbial antigens carried to lymph node by activating T and B cells
• Structure of Lymph Node

  Divided into 3 parts:
• Structure of Lymph Node (part 1)


  1. Cortex - contains lymphoid follicles that are composed mainly of B cells and follicular dendritic cells. Lymphoid follicles are of 2 types:
  2. Primary lymphoid follicles - before antigenic stimulation, smaller, contain resting B cells
  3. Secondary lymphoid follicles - following contact with an antigen, resting B cells become activated and differentiate into plasma cells and memory B cells. So, it is larger in size and has 2 areas
  4. Germinal centre,central area - contains dividing B cells of various stages - site of activation
  5. Mantle area - activated B cells
• Structure of Lymph Node (part 2 and 3) 


  2. Paracortex - present between cortex and medulla that are rich in naive T cells, macrophages and interdigitating dendritic cells - trap antigens and present to T cells

  3. Medulla - innermost area of lymph node, rich in B cells; mainly plasma cells
• Spleen 


  Largest secondary lymphoid organ

  Function = acts as a physiological barrier - to clear out microbial antigens through stimulation of T and B cells
• Structure of spleen 


  surrounded by capsule and intervened by trabeculae - divided into 2 compartments

  1. white pulp = central densely populated area with T and B cells
  2. Periarteriolar lymphoid sheath (PALS) = T cell rich area, surrounds branches of the splenic artery
  3. Marginal zone = located peripheral to the PALS and is populated by B cells and macrophages
  4. 2. red pulp = area that surrounds the sinusoids and is filled with RBCs
• Lymphocyte Isolation and Phenotyping - step 1

  1. Sample time and Blood Collection.

  Blood samples from volunteers are collected after the completion of two doses of Sinopharm vaccine.

  • Approximately 10mL of the peripheral venous blood sample is taken in an EDTA vacutainer by a licensed phlebotomist in the Thumbay Laboratory.
• Lymphocyte Isolation and Phenotyping - step 1

  2. Ficoll density gradient centrifugation.(HISTOPAQUE)

  • After collection, the blood sample is processed by the Ficoll density gradient centrifugation for blood plasma separation.
  • Blood sample is diluted at a 1:1 ratio with an appropriate medium.ie, Phosphate Buffer Solution (PBS) here.
• Lymphocyte Isolation and Phenotyping - Step 3a


  Ficoll density gradient centrifugation - Histopaque-1077 Cell Separation Medium.

  • Histopaque medium facilitates rapid recovery of viable lymphocytes and other mononuclear cells from small volumes of whole blood.
• Lymphocyte Isolation and Phenotyping - Step 3b


  1. Add 3 mL of Histopaque-1077 to a 15-mL conical centrifugetube and bring to room temperature.
  2. Carefully layer 3mL of the diluted blood on the Histopaque layerwithout mixing the two.
• Lymphocyte Isolation and Phenotyping - Step 3c


  3. At room temperature, centrifuge at 400 g for exactly 30

  minutes. Cell clumping and poor recovery may occur when centrifugation is performed at lower temperatures, such as 4°C.

  4. Using a Pasteur pipette, carefully aspirate the upper layer to within 0.5 cm of the opaque interface containing mononuclear cells after centrifugation. Remove the top layer and discard it.
• Lymphocyte Isolation and Phenotyping - Step 3d

  contd

  
• Collecting Peripheral Blood Mononuclear Cells (PBMCs)

  cont.