05_Peripheral Blood Smear Examination

8/3/2019 05_Peripheral Blood Smear Examination

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Peripheral blood smear examination

Dr Shanaz KhodaijiConsultant Hematopathologist

P.D. Hinduja National Hospital & Medical

Research Centre


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Hemogram:

measured and

calculated

parameters

Histograms:

size distribution

of WBC, RBCand Plt

Cytogram:

WBC differential

CBC on automated analyzers

Flagging for abnormalitiesnecessitates a manual PBS

review


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

the edges


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PBS examination requires a systematic approach in

order to gather all possible information. In addition, allspecimens must be evaluated in the same manner, toassure that consistent information is obtained.


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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.

PBS examination - preliminary


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(a) Ten microscopic fields are examined in a vertical directionfrom bottom to top or top to bottom (b) The slide ishorizontally moved to the next field (c) Ten microscopic fieldsare counted vertically. (d) The procedure is repeated until100 leukocytes have been counted

Scanning technique for WBC differentialcount and morphologic evaluation


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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

PBS examination - final


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Microscopic examination of the peripheral blood is used to

supplement information provided by CBC analyzers.

Hematology analyzers provide accurate quantitative

information about blood cells and can identify abn cells

In addition to providing cell counts and graphical displays

these instruments also provide a warning flags

The instrument operator reviews the information from each

specimen and decides if smear preparation and light

microscopy are necessary.

If not, the information is released to the clinician.

Hematology analyzer and PBS


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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, confirmpatient identity, repeat the automated analysis, and

prepare a new smear.

WBC estimation on peripheral smear


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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 thediameter 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.


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Microcytic hypochromic red cells

Decreased size and Hbcontent (MCH) and conc

(MCHC). Expanded

central zone of pallor

Iron deficiency, thal trait? Anemia of chronic

disease, sideroblastic

an


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Iron deficiency anemia

Tanja Tornow


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CBC + reticulocyte count in ACD


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Elliptocytes or ovalocytes

Ovalocytes are due to abnormal membranecytoskeleton found in hereditary elliptocytoisis


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Megaloblastic anemia (PS)

Macrocyte

"Thin"

macrocyte

Large RBCs (> 8.5

mm, MCV > 95 fL).Normal MCH

Increased diameter,

normal MCV. Usuallyhypochromic

Accelerated

erythrocytosis.Macrocytic anemia

(B12/folate def) (oval

macrocytes)

Liver disease,postsplenectomy


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

Tear drop cells / dacrocytes


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Polychromasia

Blue-gray coloration

of RBCS. Due RNA

remnants

Increased - Increased

erythropoietic activity.

Decreased -

Hypoproliferative states.


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Sickle cell anemia

Irregular, curved

cells with pointed

ends

Hb S hemoglobinopathies (sickle cell

anemia, hb SC disease, hb S-beta-

thalassemia, hb SD disease, hb

Memphis /S disease), other

hemoglobinopathies (especially Hb I, Hb

CHarlem, HbCCapetown).


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Spherocytosis


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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


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Spheroid RBCswith few large

spiny projections.

5-10 spicules,

irregular spacing

and thickness

(must be

differentiated fromechinocytes).

Abetalipoproteinemia,postsplenectomy, alcoholic

cirrhosis and hemolytic anemia,

microangiopathic hemolytic

anemia, autoimmune hemolytic

anemia, sideroblastic anemia,

thalassemia, severe burns, renal

disease, pyruvate kinasedeficiency, McLeod phenotype,

infantile pyknocytosis,

Acanthocytes or spur cells


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Echinocytes

"Sea urchin

cells,

crenated cells,burr cells"

RBC with

many tiny

spicules (10-

30) evenlydistributed

over cell

Post-splenectomy, uremia,

hepatitis of the newborn,

malabsorption states, after

administration of heparin,pyruvate kinase def

phosphoglycerate kinase

deficiency, uremia, HUS.

Crenated / Burr cells / Echinocytes


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Mechanical damage to

RBCs from fibrin deposits

DIC

MAHA

TP prosthetic heart valves

severe valvular stenosis

malignant hypertension march hemoglobinuria

myelofibrosis hypersplenism

Schistocyte fragmented RBC

normal newborns

bleeding peptic ulcer

Aplastic Anemia

pyruvate kinase def

VasculitisGlomerulonephritis

renal graft rejection

severe burnsiron deficiency, thalassemia


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Some specific hemolyic anemias

Mechanical HAs: RBC are injured by excessphysical trauma as they circulate in the bloodvessels

Cardiac hemolytic anemias artificial heart

valveMAHA - due to thrombosed vessels or fibrin

strands

as in DIC, TTP, malignancy

Hallmark: Presence of schistocytes in the PB


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Uniconcave RBC,

slitlike area of central

pallor

Hereditary or acquired hemolysis.

Hereditary stomatocytosis, alcoholic

cirrhosis, acute alcoholism,

obstructive liver disease,

malignancy, severe infection, treated

acute leukemia, artifact.

Stomatocyte fish mouth cell


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HA due to red cell enzymedefects bite or blister cells

G6PD deficiency Sex linked transmission Presents as

hemolysis after drug intake, infections

Common drugs antimalarials, acetanilide dapsone furantoinLab diagnosis screening, quantitative tests

Pyruvate kinase deficiency uncommon

Lab diagnosis fluorescent spot test


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Irregular RBC

agglutination/

clumping

Anti-RBC antibody, paraprotein. Cold

agglutinin disease, autoimmune

hemolytic anemia,

macroglobulinemia,

hypergammaglobinemia

RBC autoagglutination


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Small (1 mm),

round, dense,

basophilic bodiesin RBCs.

Splenectomized patients,

megaloblastic anema, severe

hemolytic processes,hyposplenism, myelophthistic

anemia.

Howell Jolly bodies


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WBC Morphology


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Large, coarse, dark purple, azurophilic granules that occur in

the cytoplasm of most granulocytes. These arecharacteristically found in the Alder-Reilly anomaly and inpatients with mucopolysaccharidoses

Alder-Reilly anomaly


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

Chdiak-Higashi


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Variably sized (0.1 to 2.0 um) and shaped, blue or grayish-blue cytoplasmic inclusions usually found near the periphery

of the cell. Dohle bodies are lamellar aggregates of roughendoplasmic reticulum, which appear in the neutrophils,bands, and metamyelocytes of patients with infection,burns, uncomplicated pregnancy, toxic states, or duringtreatment with hematologic growth factors - G-CSF.

Dhle bodies


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May-Hegglin anomaly

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


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Neutrophilic toxic granulation

Small dark blue to purple granules resembling primary

granules in the cytoplasm of metamyelocytes, bands, andsegmented 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) andDohle bodies.


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Macroplatelets

Platelet morphology


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Platelet satellitism

M t i ith i t l t l t (MDS


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


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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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05_Peripheral Blood Smear Examination

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