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Peripheral blood smear examination

Dr Shanaz Khodaiji Consultant Hematopathologist P.D. Hinduja National Hospital & Medical Research Centre

CBC on automated analyzers

Hemogram: measured and calculated parameters

Histograms: size distribution of WBC, RBC and Plt

Cytogram: WBC differential

Flagging for abnormalities necessitates a manual PBS review

A well made peripheral smear is thick at one end and progressively thinner at the opposite end. The "zone of morphology" (area of optimal thickness for light microscopic examination) should be at least 2 cm in length. The smear should occupy the central area of the slide and be margin-free at the edges

PBS examination requires a systematic approach in order to gather all possible information. In addition, all specimens must be evaluated in the same manner, to assure that consistent information is obtained.

PBS examination - preliminary


The examination starts with Macroscopic view to evaluate the quality of the smear The microscopic analysis begins on lower power (10x), primarily to assess cellular distribution, staining quality, and to select an area where the RBCs are barely touching each other. This area is used to assess the cellular elements on higher magnification. On hi-dry (40x), the slide is principally scanned to obtain a WBC estimate. All of the detailed analysis of the cellular elements is performed using high power or oil immersion.

Scanning technique for WBC differential count and morphologic evaluation

(a) Ten microscopic fields are examined in a vertical direction from bottom to top or top to bottom (b) The slide is horizontally moved to the next field (c) Ten microscopic fields are counted vertically. (d) The procedure is repeated until 100 leukocytes have been counted

PBS examination - final


This final microscopic examination is performed at 50x or 100x oil immersion and includes:

A WBC differential The identification of abnormal leukocytes Assessment of RBC morphology The number and morphology of the platelets The identification of intra- and extra-cellular elements Assessment of any organisms present

Hematology analyzer and PBS

Microscopic examination of the peripheral blood is used to Hematology analyzers provide accurate quantitative In addition to providing cell counts and graphical displays The instrument operator reviews the information from each

supplement information provided by CBC analyzers.

information about blood cells and can identify abn cells

these instruments also provide a warning flags

specimen and decides if smear preparation and light microscopy are necessary.

If not, the information is released to the clinician.

WBC estimation on peripheral smear

A fairly accurate estimate of the WBC count (cells/mL)

can be obtained by counting the total number of leukocytes in ten 50X microscopic fields, dividing the total by 10, and multiplying by 3000. These estimates should approximate that obtained by the cell analyzer. If the estimate does not match the automated cell count, obtain the original blood specimen, confirm patient identity, repeat the automated analysis, and prepare a new smear.

Morphologic Evaluation of Red Blood Cells


Round to very slightly ovoid cells with a mean diameter of approximately 7 A central pale area - central pallor approximately 1/3 the diameter of the cell It is approximately the same size as the nucleus of a mature lymphocyte. Any deviation in size, volume, or shape represents an abnormal red blood cell.

Microcytic hypochromic red cells


Decreased size and Hb content (MCH) and conc (MCHC). Expanded central zone of pallor Iron deficiency, thal trait ? Anemia of chronic disease, sideroblastic an

Iron deficiency anemia

Tanja Tornow

Thalassemia trait

CBC + reticulocyte count in ACD

Elliptocytes or ovalocytes
Ovalocytes are due to abnormal membrane cytoskeleton found in hereditary elliptocytoisis

Megaloblastic anemia (PS)


Macrocyte Large RBCs (> 8.5 mm, MCV > 95 fL). Normal MCH Accelerated erythrocytosis. Macrocytic anemia (B12/folate def) (oval Increased diameter, macrocytes) normal MCV. Usually Liver disease, hypochromic postsplenectomy

"Thin" macrocyte

Tear drop cells / dacrocytes

Tear drop shaped red cells or dacryocytes are seen when there is extramedullary erythropoiesis or with marrow disorders or marrow infiltration, such as myelofibrosis or metastatic carcinoma.

Polychromasia

Blue-gray coloration of RBCS. Due RNA remnants

Increased - Increased erythropoietic activity. Decreased Hypoproliferative states.

Sickle cell anemia

Irregular, curved Hb S hemoglobinopathies (sickle cell cells with pointed anemia, hb SC disease, hb S-betaends thalassemia, hb SD disease, hb Memphis /S disease), other hemoglobinopathies (especially Hb I, Hb CHarlem, HbCCapetown).

Spherocytosis

Acanthocytes
Acanthocytes or spur cells, are spherical cells with blunt-tipped or club-shaped spicules of different lengths projecting from their surface at irregular intervals. (Echinocytes, or crenated red cells, in contrast, have shorter, sharp to blunt spicules of uniform length which are more evenly spaced around their periphery).

Acanthocytes or spur cells


Spheroid RBCs Abetalipoproteinemia, with few large postsplenectomy, alcoholic spiny projections. cirrhosis and hemolytic anemia, 5-10 spicules, microangiopathic hemolytic irregular spacing anemia, autoimmune hemolytic and thickness anemia, sideroblastic anemia, (must be thalassemia, severe burns, renal differentiated from disease, pyruvate kinase echinocytes). deficiency, McLeod phenotype, infantile pyknocytosis,

Crenated / Burr cells / Echinocytes

Echinocytes "Sea urchin cells, crenated cells, burr cells"

RBC with many tiny spicules (1030) evenly distributed over cell

Post-splenectomy, uremia, hepatitis of the newborn, malabsorption states, after administration of heparin, pyruvate kinase def phosphoglycerate kinase deficiency, uremia, HUS.

Schistocyte fragmented RBC


Mechanical damage to RBCs from fibrin deposits

normal newborns bleeding peptic ulcer Aplastic Anemia pyruvate kinase def Vasculitis Glomerulonephritis renal graft rejection severe burns iron deficiency, thalassemia

DIC MAHA TP prosthetic heart valves severe valvular stenosis malignant hypertension march hemoglobinuria myelofibrosis hypersplenism

Some specific hemolyic anemias


Mechanical HAs: RBC are injured by excess physical trauma as they circulate in the blood vessels Cardiac hemolytic anemias artificial heart valve MAHA - due to thrombosed vessels or fibrin strands as in DIC, TTP, malignancy Hallmark: Presence of schistocytes in the PB

Stomatocyte fish mouth cell

Uniconcave RBC, Hereditary or acquired hemolysis. slitlike area of central Hereditary stomatocytosis, alcoholic pallor cirrhosis, acute alcoholism, obstructive liver disease, malignancy, severe infection, treated acute leukemia, artifact.

HA due to red cell enzyme defects bite or blister cells

G6PD deficiency Sex linked transmission Presents as hemolysis after drug intake, infections Common drugs antimalarials, acetanilide dapsone furantoin Lab diagnosis screening, quantitative tests Pyruvate kinase deficiency uncommon Lab diagnosis fluorescent spot test

RBC autoagglutination
Irregular RBC agglutination/ clumping Anti-RBC antibody, paraprotein. Cold agglutinin disease, autoimmune hemolytic anemia, macroglobulinemia, hypergammaglobinemia

Howell Jolly bodies

Small (1 mm), round, dense, basophilic bodies in RBCs.

Splenectomized patients, megaloblastic anema, severe hemolytic processes, hyposplenism, myelophthistic anemia.

WBC Morphology

Alder-Reilly anomaly
Large, coarse, dark purple, azurophilic granules that occur in the cytoplasm of most granulocytes. These are characteristically found in the Alder-Reilly anomaly and in patients with mucopolysaccharidoses

Chdiak-Higashi

Chdiak-Higashi granules are very large red or blue granules that appear in the cytoplasm of granulocytes, lymphocytes, or monocytes in patients with the ChdiakSteinbrinck-Higashi syndrome. It is a rare autosomal recessive disorder

Dhle bodies

Variably sized (0.1 to 2.0 um) and shaped, blue or grayishblue cytoplasmic inclusions usually found near the periphery of the cell. Dohle bodies are lamellar aggregates of rough endoplasmic reticulum, which appear in the neutrophils, bands, and metamyelocytes of patients with infection, burns, uncomplicated pregnancy, toxic states, or during treatment with hematologic growth factors - G-CSF.

May-Hegglin anomaly
Neutrophils contain small basophilic cytoplasmic granules which represent aggregated ribosomes. Leukopenia and large platelets are also found. An autosomal dominant trait, the May-Hegglin anomaly is associated with a mild bleeding tendency, but not by an increased susceptibility to infection

Neutrophilic toxic granulation


Small dark blue to purple granules resembling primary granules in the cytoplasm of metamyelocytes, bands, and segmented neutrophils during inflammatory states, burns, and trauma, and upon exposure to hematopoietic growth factors. It is usually accompanied by a shift to the left and vacuolations in the cytoplasm (toxic vacuolations) and Dohle bodies.

Platelet morphology Macroplatelets

Platelet satellitism

Macrocytosis with giant platelets (MDS, 5q- syndrome)

Disadvantages of the Peripheral Blood Smear


Provides information that cannot be obtained from automated cell counting. However, some limitations are:

Experience is required to make technically adequate smears. There is a non-uniform distribution of white blood cells over

the smear, with larger leukocytes concentrated near the edges and lymphocytes scattered throughout.

There is a non-uniform distribution of RBCs over the smear,

with small crowded red blood cells at the thick edge and large flat red blood cells without central pallor at the feathered edge

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