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 !