Histology of Blood

Blood—specialized connective tissue

consists of cellular elements (45%) and aqueous fluid matrix, plasma (55%)

cellular elements—RBC (44%, called hematocrit or packed cell volume (PCV)) and WBC (1%)

plasma—salts, nutrients, metabolites, gases, proteins (albumin, globulin, fibrinogen, enzymes, hormones)

Cells Observed in Blood Smear – stained by a mixture of acid and basic dyes called Romanovsky type stain

Erythrocyte—biconcave circular disk, 7.5um in diameter, stains orange-pink with pale center

non-motile but flexible and can change shape to pass through narrow capillaries
no nucleus or cellular organelles—essentially a 33% solution of hemoglobin (Hb) within cell membrane
Hb: conjugated protein, MW=68 kD, 4 polypeptide chains each with iron containing heme group

heme group (O₂ binding) formed by mitochondria, and globin chains synthesized on polysomes
anemias—insufficient Hb concentration in the blood, either too few cells or too little Hb
sickle cell anemia—genetic disorder; Hb insoluble when deoxygenated, rigidity shortens life of cell

Platelet—protoplasmic convex disks about 2-4um in diameter (or length)

originated by fragmentation of multinucleated giant cell (megakaryocyte) in bone marrow
central zone: granulomere—contains azure granules (EM: dense granules, alpha granules, lysosomes)

granules contain serotonin, fibrinogen and clotting factors

outer pale zone: hyalomere—contains microtubules which maintain the disc shape of the platelet
help repair damaged areas of blood vessels, to arrest bleeding and initiate the blood clotting process.

Neutrophil (polymorphonuclear neutrophilic leukocyte)—2/3 of WBC, 12um

nucleus 3-5 lobed, dense chromatin
cytoplasm contains azure granules (primary lysosomes) and specific granules (smaller, not visible here)
highly motile—after a few hours in the blood, they migrate into tissue and survive 2-4 days
phagocytosis and destruction of microorganisms (esp. bacteria) by granule enzymes—acute inflammation

Eosinophil—2-4% of WBC, 10-14um

nucleus 2 lobed
cytoplasm contains specific granules (orange-red colored), contain lysosomal enzymes (EM: crystalloid)
phagocytize antigen/antibody complexes (esp. parasites)
reduce allergic inflam. by producing enzymes that inactivate vasoactive subst.from basophils and mast cells

Basophils—less than 1% of WBC, 12um

nucleus 2-lobed or irregular, occupies a large portion of the cell
cytoplasm contains specific granules (histamine and other vasoactive substances) dark-purple with basic dye
similar but not identical to mast cells (in tissue)—both produce allergic inflammatory response
immunoglobin E binding to surface + interaction with specific antigen Þ release vasoactive mediators

Lymphocytes—20-40% of WBC, small (6-10um), medium (10-12um), large (12-15um) (most small or medium)

nucleus round or oval with slight indentation, occupies a large part of the cell—moderately dense chomatin
cytoplasm sparse (more in larger) with free ribosomes (basophilic) and occasional azure granules
immunological defense mechanism—circulate between lymphoid organs and body tissues
two types (indistinguishable here, but can be distinguished with immunocytochemistry)

T-lymphocytes—cell mediated immunity—kill foreign and virus infected cells, regulate other WBC

proliferate then differentiate—cytotoxic (cell killer), helper and suppressor (regulate other WBC)

B-lymphocytes—humoral immunity—production of antibody

proliferate then differentiate into plasma cells which synthesize and secrete antibody

Monocytes—4-8% of WBC, 12-25um

nucleus kidney or horseshoe shaped, chromatin is less dense than other mature leukocytes
cytoplasm is extensive and contains azure granules (lysosomes), vacuoles and residual bodies
highly motile—after 1-2 days in blood, they migrate into tissue and differentiate into macrophages

unlike neutrophils, they may undergo further division and enzyme synthesis, survive for months

mononuclear phagocytic system—monocytes + macrophages dispersed throughout the body

phagocytosis and digestion of foreign organisms and senescent cells, also present antigen to lymphocytes

Slides

blood/bone marrow smear—drop of blood spread into thin film on a glass slide, air dred/MeOH fixed

stained with a mixture of dyes originally developed by Romanovsky
Wright's stain—modified Romanovsky—methylene blue (basic), azure n (basic), and eosin (acidic)

Blood Smear—human peripheral blood—can identify RBC, platelets, small and medium lymphocytes, monocytes, neutrophils, eosinophils, and basophils (rare, less than 1% in peripheral blood)

The nuclei of the leukocytes are stained with azure component (purple-pink)
lymphocytes - round, oval, with dense chromatin, occupying most part of the cell
monocytes - kidney-shaped chromatin pale to moderately dense
neutrophils - 2 - 5 lobes, chromatin coarse and clumpy
basophils - 2 lobes or S-shaped, chromatin coarse, usually obscured by dark cytoplasmic granules
eosinophils - 2 lobes, coarse chromatin

Cytoplasm is usually not stained except in:

erythrocyte - orange-red (eosinophilic)
monocyte - slight gray-blue
lymphocyte - light blue

Many blood cell types azurophilic granules (red-purple) in cytoplasm:

neutrophils
platelets
some lymphocytes and monocytes

Granulocytes contain specific granules

neutrophils - not easily resolved at light microscopic level, give salmon-pink color to the cytoplasm
eosinophils - orange-red large refractile granules
basophils - dark purple large granules

Red Marrow Smear: Hematopoiesis—continuum, classification is arbitrary

Erythropoiesis

Pronormoblast—proerythroblast, rubriblast
Basophilic normoblast—basophilic erythroblast, prorubricyte
Polychromatic normoblast—polychromatophilic erythroblast, rubricyte
Orthochromatic normoblast—orthochromatophilic erythroblast, normoblast, metarubricyte
Reticulocyte—polychromatophilic erythrocyte
Erythrocyte

Granulopoiesis (identify stages promyelocyte to mature form for neutrophils)

Myeloblast
Promyelocyte
Myelocyte
Metamyelocyte
band form
mature granulocyte (segmented granulocyte)

Non-granular leukocyte developmental stages

Monocyte precursors: monoblasts and promonocytes
Lymphocyte precursors: lymphoblasts and prelymphocytes—difficult to distinguish
small lymphocyte
plasma cell - antigen stimulated B - lymphocyte, large amount of basophilic cytoplasm - with eccentric, dense, clock-faced nucleus. Compare with basophilic normoblast and polychromatic normoblast and myelocyte.
Megakaryocyte - the largest cell with the largest nucleus - azure granules in cytoplasm