Peripheral blood smear examination

Dr Hemang MendparaDNB pediatrics

Choithram Hospital & Research CentreIndore

• Hemogram:

measured and

calculated

parameters

• Histograms:

size distribution of

WBC, RBC and Plt

• Cytogram: WBC

differential

CBC on automated analyzers

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

Slide fixation and staining

1. Romanowsky stainingLeishman's stain : a polychromatic stain• Methanol : fixes cells to slide• methylene blue stains RNA,DNAblue-grey color • Eosin stains hemoglobin, eosin granulesorange-red color • pH value of phosphate buffer is very important

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.

• 1. Macroscopic view : quality of the smear

• 2.The microscopic analysis

• begins on lower power (10x),

• to assess cellular distribution, staining quality,

and to select an area where the RBCs are barely

touching each other.

• On hi-dry (40x), to obtain a WBC estimate. All of the

detailed analysis of the cellular elements using high

power or oil immersion.

PBS examination - preliminary

(a) Ten microscopic fields are examined in a vertical direction from bottom to top or top to bottom

(b) slide is horizontally moved to the next field(c) Ten microscopic fields are counted vertically.(d) procedure is repeated until 100 WBCS have been

counted (zig zag motion)

Scanning technique for WBC differential count and morphologic evaluation

1. RBC • Size• Shape • Color • Arrangement • Inclusions• Young RBCs

2. WBC• Total counts• Differential counts• I:T ratio• Abnormal WBC

3. Platelets • Counts • Abnormality

4. Parasites

Evaluation of PBS

• A fairly accurate estimate of the WBC count

(cells/mL) can be obtained by counting the total

number of leukocytes in ten 40X microscopic fields,

dividing the total by 10, and multiplying by 3000.

These estimates should approximate that obtained

by the cell analyzer.

WBC estimation on peripheral smear

Morphologic Evaluation of Red Blood Cells

Biconcave disc Diameter : 7 ~ 8 μmCentral pallor occupy 1/3 rd of totalSize : approx. same as nucleus of mature lymphocyte

Microcytic hypochromic red cells

Decreased size and Hb content (MCH) and conc (MCHC). Expanded central zone of pallor

Iron deficiency, thalasemia trait Anemia of chronic disease

Iron deficiency anemia

Thalassemia trait

Megaloblastic anemia (PS)

MacrocyteLarge RBCs• size > 8.5 mm, • MCV > 95 fL• Normal MCH

•Normal newborn •Chromosomal disorders (e.g., Trisomy 21) •Drug associated anticonvulsants, antidepressants, sulpha, chemotherapeutic agents, estrogen and antiretroviral agents)

•Dyserythropoiesis •Myelodysplasia •Preleukemia •Hypothyroidism •Liver disease•Folate deficiency •BI2 deficiency

Elliptocytes or ovalocytes

Ovalocytes are due to abnormal membrane cytoskeleton found in hereditary elliptocytoisis

seen when there is extramedullary erythropoiesis

Tear drop cells / dacrocytes

• Osteopetrosis • Myelofibrosis • Bone marrow infiltrated with hematological or non-hematological malignancies • Iron deficiency anemia • Pernicious anemia

Polychromasia

Blue-gray coloration of RBCS. Due RNA remnants

Increased - Increased erythropoietic activity. Decreased - Hypoproliferative states.

Hemolytic anemias •Blood loss anemias •Recovering anemia

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)* Don’t be confused with fragmented RBC

Spherocytosis

Hereditary spherocytosis •ABO incompatibility •Autoimmune hemolytic anemia (warm antibody type) •Infections (e.g., EBV, CMV, E. coli, Sepsis/Urosepsis) •Severe burns •DIC and HUS

Acanthocytes or spur cells, are spherical cells with blunt-tipped

or club-shaped spicules of different lengths projecting from their surface at

irregular intervals.

Acanthocytes

Acanthocytes are seen in •Hereditary abetalipoproteinemia •Hereditary acanthocytosis •End stage liver disease •Anorexia nervosa •Malnutrition •Post splenectomy •Intravenous hyperalimentation particularly with intralipid infusion

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

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

(Echinocytes, or burr cells or

crenated red cells, in contrast, have

shorter, sharp to blunt spicules of uniform

length which are more evenly spaced

around their periphery).

Mechanical damage to RBCs from fibrin deposits

DIC

MicroAngiopathic HA

TTP/HUS

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

Vasculitis

Glomerulonephritis

renal graft rejection

severe burns

iron deficiency, thalassemia

hemolyic anemias

Hallmark: Presence of schistocytes , fragmented RBC

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

HA due to red cell enzyme defects – bite or blister cells

• Glucose 6 phosphate dehydrogenase (G-6-PD) deficiency

• Unstable hemoglobin variants • Congenital Heinz body anemia

Suggest oxidative stress

Target cell

Peripheral rim of pallor surrounding central hyperchromia

Target cells are commonly seen in •Hemoglobin C •Sickle cell disease •Hemoglobin E •Hemoglobin H disease •Thalassemias •Iron deficiency anemia •Liver disease •Target cells are seen with most of the hemoglobinopathies

Irregular RBC agglutination/ clumping

Anti-RBC antibody, paraprotein. Cold agglutinin disease, autoimmune hemolytic anemia, macroglobulinemia, hypergammaglobinemia

RBC autoagglutination

Roulex formation

Seen in case of high level of fibrinogen, immunoglobulins, intra venous administration of plasma volume expanders like dextran

• multiple blue-purple inclusions attached to the inner surface of the red cell membrane. visible in supravitally stained smears.

• are precipitated normal or unstable hemoglobin usually secondary to oxidant stress.

• G6PD deficiency• Unstable

hemoglobinopathy• Cong. Bite cell

anemia

Heinz body

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

Splenectomized patients, Functional asplenia,Anatomical absence of spleen

Howell Jolly bodies

Howell-Jolly bodies are small round bodies composed of DNA, about 1 µm in diameter, usually single and in the periphery of a red cell. They are readily visible on the Wright-Giemsa-stained smear. The spleen is responsible for the removal of nuclear material in the red cells, so in absence of a functional spleen, nuclear material is removed ineffectively. Howell-Jolly bodies are seen in •Post splenectomy •Functional asplenia •Anatomical absence of spleen

Basophillic strippling• Lead poisoning • Iron deficiency anemia • Thalassemia

Are abnormal aggregrates of ribosome and polyribosomes

• Smaller then Howell jolly body• Stain with Prussian blue stain• Suggest iron over load

WBC Morphology

Manual differential counts• These counts are done in the same area as

WBC and platelet estimates with the red cells barely touching.

• This takes place under × 100 (oil) using the zigzag method.

• Count 100 WBCs including all cell lines from immature to mature.

Reporting results• Absolute number of cells/µl = % of cell type in

differential x white cell count

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• If 10 or more nucleated RBC's (NRBC) are seen, correct the

• White Count using this formula:• Corrected WBC Count = WBC x 100/( NRBC + 100)• Example : If WBC = 5000 and 10 NRBCs have

been counted• Then 5,000× 100/110 = 4545.50• The corrected white count is 4545.50

• Left-shift: non-segmented neutrophil > 5%– Increased bands Means acute infection, usually

bacterial

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• Basophils are increased in the blood in– Myeloproliferative disorders (e.g., chronic myelogenous leukemia)– Hypersensitivity reactions – Mastocytosis – Xeroderma pigmentosa – Hypothyroidism

• Morphologically abnormal eosinophils are seen in – Myelodysplastic syndrome – Megaloblastic anemias

• Eosinophils are increased in the following conditions: Allergies Parasitic infestations Infections Acute leukemia Myeloproliferative diseases Hypereosinophilic syndrome Drug-associated

Band cells

Leukemic myeloblast

Leukemic myeloblast stained with peroxidase

Note the AUER ROD

Burkitt lymphoma

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

Alder-Reilly anomaly

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

Chédiak-Higashi

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 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.

Döhle bodies

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.

Platelets Neubars chamber : count platelets in 64 small squares

Counts * 250 = total platelets

Normal counts 4.5 to 5.5 lakh

Common Causes of Thrombocytopenia •Decreased production

−Aplastic anemia −Acute leukemia −Viral infections *Parvovirus *CMV −Amegakaryocytic thrombocytopenia (AMT) •Increased destruction −Immune thrombocytopenia *Idiopathic thrombocytopenic purpura (ITP) *Neonatal alloimmune thrombocytopenia (NAITP) −Disseminated intravascular coagulation (DIC) −Hypersplenism

Thrombocytosis• Reactive thrombocytosis Post infection Inflammation Juvenile rheumatoid arthritis Collagen vasvular disease• Essential thrombocythemia

:Giant platelets Platelet morphology

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

Merozoits

Schizonts are commonly seen in P. vivax infection and appear as large bodies containing 12 to 24 nuclei and a loose pigmented body. This photograph shows an early schizont of P. vivax on the left and mature schizonts

Schuffer’s dots seen in plasmodium vivex

Cresent shaped gametocyte charectaristiclly seen in p.falciparum malaria

Eucheria bancrofti

Osmotic fragility of RBC

OSMOTIC FRAGILITY TEST• Defination:

• it is a test that measures the resistance to hemolysis of red blood cells (RBC) by osmotic stress created by hypotonic solutions

• RBC are exposed to a series of saline (NaCl) solutions with increasing dilution

• The sooner hemolysis occurs, the greater is osmotic fragility of RBC

• Isotonic (physiological) solution – 0.9 % NaCl

• RBC burst in hypotonic (< 0.9 % NaCl), and shrink (crenate) in hypertonic solutions (> 0.9 % NaCl)

• Red cells are suspended in a series of tubes containing hypotonic solutions from 0.9 to 0 % NaCl. Degree of hemolysis measured for each NaCl concentration.

• NORMAL RANGE:• - hemolysis onset at: 0.45-0.5 % NaCl• - hemolysis complete at: 0.3-0.33 % NaCl

• FACTORS AFFECTING OSMOTIC FRAGILITY• - cell membrane permeability• - surface-to-volume ratio

increased osmotic fragility

- Hereditary spherocytosis- Acquired spherocytosis- Hemolytic anemia (HDN)- Malaria- Severe pyruvate kinase deficiency

• Thalassemia• Sickle cell anemia (hemoglobinopathy)• Iron deficiency anemia• Asplenia• Liver disease

Decreased osmotic fragility

Sickling TestSickle Cell Screening Test

introduction

Principle of test

• Deoxygenated Hb-S is insoluble in the presence of a concentrated phosphate buffer solution and forms a turbid suspension that can be easily visualized.

• Normal Hemoglobin A and other hemoglobins remain in solution under these conditions. These different qualitative outcomes allow for the detection of sickle cell disease and its traits.

• SICKLEDEX® uses Saponin to lyse the red blood cells. Sodium Hydrosulfite then reduces the released hemoglobin. Reduced Hb-S is insoluble in the concentrated phosphate buffer and forms a cloudy, turbid suspension. Thus give a positive result.

Procedure

• 1. sodium diethanoid 200mg+10 ml distilled water

• 2. sickling buffer solutions• Take 2 part of 1st solution and 3 part of 2nd

solution• Have one drop of blood on slide and put single

drop of mixed solution• Wait for 30 mins• Watch under microscope

Result

“ Thank you !