PERIPHERAL BLOOD SMEAR

PERIPHERAL BLOOD SMEAR
Peripheral blood smear is a microscope slide made from a drop of blood, that allows the cells to be examined ultramicroscopically(under oil immersion mx)
Blood films are made by placing a drop of blood on one end of a slide, and using a spreader slide to disperse the blood over the slide's length. Ideal PBS should be tongue shaped without any windows in it.
The slide is left to air dry, after which the blood is fixed to the slide by immersing it briefly in methanol. The fixative is essential for good staining and presentation of cellular detail. After fixation, the slide is stained to distinguish the cells from each other.
Common blood film staining methods
* Romanowsky stain * Giemsa stain * Wright's stain * Jenner's stai * Leishman stain * Field's stain

RED BLOOD CELLS

• Best place to examine blood cell morphology is the feathered edge of the blood smear where red cells lie in a single layer, side by side, just barely touching each other but not overlapping
RBC SIZE :
• One can gauge their size by comparing the red cell to the nucleus of a small lymphocyte. Both are about 8 µm wide. Red cells that are smaller than the small lymphocyte nucleus may be microcytic; those larger than the small lymphocyte nucleus may be macrocytic. .
• When the red cells vary greatly in size, anisocytosis is said to be present.
Hb CONTENT :
• They are either normal in color (normochromic) or they are pale in color (hypochromic). They are never "hyperchromic.".


RED CELL INCLUSIONS :
1. Basophilic stippling—diffuse fine or coarse blue dots in the red cell representing usually RNA residue—especially common in lead poisoning
2. Howell-Jolly bodies—dense blue circular inclusions that represent nuclear remnants—their presence implies defective splenic function
3. Nuclei—red cells may be released or pushed out of the marrow prematurely before nuclear extrusion—often implies a myelophthisic process
4. Parasites—red cell parasites include malaria and babesia (Chap. e18)
5. Polychromatophilia—the red cell cytoplasm has a bluish hue, reflecting the persistence of ribosomes still actively making hemoglobin in a young red cell


RBC SHAPE :
Red cells can take on a variety of different shapes. . When the red cells vary greatly in shape, poikilocytosis is said to be present.
• Small red cells without the central pallor are spherocytes; they can be seen in hereditary spherocytosis and clostridial sepsis.
• Dacrocytes are teardrop-shaped cells that can be seen in hemolytic anemias, severe iron deficiency, thalassemias, myelofibrosis, and myelodysplastic syndromes.
• Schistocytes are helmet-shaped cells that reflect microangiopathic hemolytic anemia or fragmentation on an artificial heart valve.
• Echinocytes are spiculated red cells with the spikes evenly spaced; they can represent an artifact of abnormal drying of the blood smear or reflect changes in stored blood. They can also be seen in renal failure and malnutrition and are often reversible.
• Acanthocytes are spiculated red cells with the spikes irregularly distributed. This process tends to be irreversible and reflects underlying renal disease, abetalipoproteinemia, or splenectomy.
• Elliptocytes are elliptical-shaped red cells that can reflect an inherited defect in the red cell membrane, but they are also seen in iron deficiency, myelodysplastic syndrome, megaloblastic anemia, and thalassemias.
• Stomatocytes are red cells in which the area of central pallor takes on the morphology of a slit instead of the usual round shape. Stomatocytes can indicate an inherited red cell membrane defect and can also be seen in alcoholism.
• Target cells have an area of central pallor that contains a dense center, or bull's eye. These cells are seen classically in thalassemia, but they are also present in iron deficiency, cholestatic liver disease, and some hemoglobinopathies. They can also be generated artifactually by improper slide making.
DISTRIBUTION:
• AGGLUTINATION of red cells  pile upon one another; it is seen in certain paraproteinemias and autoimmune hemolytic anemias.
• Red cells lying in single cell rows on top of one another like stacks of coins. This is called rouleaux formation and reflects abnormal serum protein levels.
WHITE BLOOD CELLS :
Three types of granulocytes are usually present; neutrophils, eosinophils, and basophils, in decreasing frequency.
Neutrophils
• They are round, 10–14 µm wide, and contain a lobulated nucleus with two to five lobes connected by a thin chromatin thread.
• Bands are immature neutrophils that have not yet completed nuclear condensation and have a U-shaped nucleus. Bands reflect a left shift in neutrophil maturation in an effort to make more cells more rapidly.
• Vacuolated neutrophils may be a sign of bacterial sepsis.
• Dohle bodies - 1- to 2-µm blue cytoplasmic inclusions can reflect infections, burns, or other inflammatory states.
• "Toxic granulations” - neutrophil granules are larger than normal and stain a darker blue- systemic inflammation.
• Hypersegmented neutrophils - The presence of neutrophils with more than five nuclear lobes suggests megaloblastic anemia.
• Large misshapen granules may reflect the inherited Chédiak-Higashi syndrome.
Eosinophils are slightly
• Larger than neutrophils, have bilobed nuclei, and contain large red granules.
• Diseases of eosinophils are associated with too many of them rather than any morphologic or qualitative change.


Basophils
• They have large dark-blue granules
• Increased as part of chronic myeloid leukemia,
Lymphocytes - Cells with a small dark nucleus and scarce cytoplasm.
• In the presence of viral infections, more of the lymphocytes are larger, about the size of neutrophils, with abundant cytoplasm and a less condensed nuclear chromatin. These are called reactive lymphocytes.
• About 1% of the lymphocytes are larger and contain blue granules in a light blue cytoplasm; these are called large granular lymphocytes.
• In CLL, the small lymphocytes are increased in number, and many of them are ruptured in making the blood smear, leaving a smudge of nuclear material without a surrounding cytoplasm or cell membrane; these are called smudge cells
Monocytes are the largest white blood cells, ranging from 15–22 µm in diameter. The nucleus can take on a variety of shapes but usually appears to be folded; the cytoplasm is gray.
Most often the abnormal cells originate from neoplasms of bone marrow–derived cells including lymphoid cells, myeloid cells, and occasionally red cells. The chances of seeing such abnormal cells is increased by examining blood smears made from buffy coats, the layer of cells that is visible on top of sedimenting red cells when blood is left in the test tube for an hour.
PLATELETS :
• First counts the platelets in five to six fields, averages the number per field, and multiply by 20,000 to get a rough estimate of the platelet count.
• The platelets are usually 1–2 µm in diameter and have a blue granulated appearance. There is usually 1 platelet for every 20 or so red cells
• Large platelets may be a sign of rapid platelet turnover, as young platelets are often larger than old platelets; alternatively, certain rare inherited syndromes can produce large platelets.
• Platelet clumping visible on the smear can be associated with falsely low automated platelet counts.









BLOOD GROUPS
The blood groups are determined by antigens on the surface of red cells; more than 400 blood groups have been found. The ABO and Rh systems are the two major blood groups.
ABO Antigens and Antibodies
The first blood group antigen system, recognized in 1900, was ABO. Major blood groups of this system are A, B, AB, and O. O type RBCs lack A or B antigens.
• The antigens are carbohydrates attached to a precursor backbone, may be found on the cellular membrane either as glycosphingolipids or glycoproteins, and are secreted into plasma and body fluids as glycoproteins.
• H substance is the immediate precursor on which the A and B antigens are added. H substance - addition of fucose to the glycolipid or glycoprotein backbone.
• The subsequent addition of N-acetylgalactosamine creates the A antigen, while the addition of galactose produces the B antigen.
GENETICS :
• The genes that determine the A and B phenotypes are found on chromosome 9p and are expressed in a Mendelian codominant manner.
• The gene products are glycosyl transferases, which confer the enzymatic capability of attaching the specific antigenic carbohydrate. Individuals who lack the "A" and "B" transferases are phenotypically type "O," while those who inherit both transferases are type "AB."
• Rare individuals lack the H gene, which codes for fucose transferase, and cannot form H substance. These individuals are homozygous for the silent h allele (hh) and have Bombay phenotype (Oh).
Phenotype Genotype Antigens Antibodies
O OO None Anti-A and anti-B
A AA or AO A Anti-B
B BB or BO B Anti-A
AB AB A and B None

AB blood group – “Universal recipients” O group – “Universal donor”
Importance : A and B antigens are secreted by the cells and are present in the circulation. Nonsecretors are susceptible to a variety of infections (e.g., Candida albicans, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae) as many organisms may bind to polysaccharides on cells. Soluble blood group antigens may block this binding.
Rh System
• The Rh antigens are found on a 30- to 32-kDa RBC membrane protein that has no defined function.
• The three Rh genes, E/e, D, and C/c, are arranged in tandem on chromosome 1 and inherited as a haplotype, i.e., cDE or Cde.
• The presence of the D antigen confers Rh "positivity," while persons who lack the D antigen are Rh negative.
• The D antigen is a potent alloantigen. About 15% of individuals lack this antigen. Exposure of these Rh-negative people to even small amounts of Rh-positive cells, by either transfusion or pregnancy, can result in the production of anti-D alloantibody.
Other blood groups :
1. The Kell - large protein with different antigenic epitopes. The Kx gene is linked to the 91-kDa component of the NADPH-oxidase on the X chromosome. The immunogenicity of Kell is third behind the ABO and Rh systems. The absence of the Kell precursor protein (controlled by a gene on X) is associated with acanthocytosis, shortened RBC survival, and a progressive form of muscular dystrophy that includes cardiac defects. This rare condition is called the McLeod phenotype.
2. The Duffy antigens are codominant alleles, Fya and Fyb, that also serve as receptors for Plasmodium vivax. More than 70% of persons in malaria-endemic areas lack these antigens, probably from selective influences of the infection on the population.
3. The Kidd antigens, Jka and Jkb, may elicit antibodies transiently. A delayed hemolytic transfusion reaction that occurs with blood tested as compatible is often related to delayed appearance of anti-Jka. T
4. he P system controlled by specific glycosyltransferases. Its clinical significance is in rare cases of syphilis and viral infection that lead to paroxysmal cold hemoglobinuria. In these cases, an unusual autoantibody to P (Donath-Landsteiner antibodies) is produced that binds to RBCs in the cold and fixes complement upon warming. The P antigen is the cellular receptor of parvovirus B19 and also may be a receptor for Escherichia coli binding to urothelial cells.
5. The MNSsU system is regulated by genes on chromosome 4. M and N are determinants on glycophorin A, an RBC membrane protein, and S and s are determinants on glycophorin B. Anti-S and anti-s IgG antibodies may develop after pregnancy or transfusion and lead to hemolysis. Anti-U antibodies are rare but problematic; virtually every donor is incompatible because nearly all persons express U.
6. Antibodies to Lewis system carbohydrate antigens are the most common cause of incompatibility during pretransfusion screening. The Lewis gene product is a fucosyl transferase and maps to chromosome 19. The antigen is not an integral membrane structure but is adsorbed to the RBC membrane from the plasma.