peripheral smear
TRANSCRIPT
Peripheral blood smear examination
DR. MITHILA MODERATOR : DR MANJUNATH
INTRODUCTION• Peripheral blood smear is a very important tool in the
hematology lab
• It provides rapid, reliable access to information about a variety of hematologic disorders
• Examination of the peripheral blood smear is an inexpensive but powerful diagnostic tool in both children and adults
• The smear offers a window into the functional status of the bone marrow
• Review of the smear is an important adjunct to other clinical data; in some cases, the peripheral smear alone is sufficient to establish a diagnosis
INDICATION FOR PERIPHERAL SMEAR
Features suggestive of anemia, unexplained jaundice, or both .
Features suggestive of thrombocytopenia (e.g., petechiae or abnormal bruising) or neutropenia (e.g., unexpected or severe infection).
Features suggestive of a lymphoma or leukaemia lymphadenopathy, splenomegaly, bone pain, and systemic symptoms such as fever, sweating, and weight loss .
Features suggestive of a myeloproliferative disease — splenomegaly, plethora, itching, or weight loss .
General ill health, often with malaise and fever, suggesting infectious mononucleosis or other viral infection or inflammatory or malignant disease .
Suspicion of a bacterial or parasitic disease that can be diagnosed from a blood smear
Peripheral Smear Preparation Staining of Peripheral Blood Smear Peripheral Smear Examination
OBJECTIVES
SMEAR PREPARATION1. Place a drop of blood, about 2-3 mm in
diameter approximately 1 cm from one end of slide.
2. Place the slide on a flat surface, and hold the other end between your left thumb and forefinger.
3. With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop.
4. Hold the spreader slide at a 30°- 45 angle, and draw it back against the drop of blood
6. Allow the blood to spread almost to the edges of the slide.
7. Push the spread forward with one light, smooth moderate speed. A thin film of blood in the shape of tongue.
8. Label one edge with patient name, lab id and date.
9. The slides should be rapidly air dried by waving the slides or using an electrical fan.
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.
SLIDE FIXATION AND STAINING
ROMANOWSKY STAININGIT INCLUDES:• MAY-GRUNWALD –GEIMSA STAIN,• JENNER’S STAIN,• WRIGHT’S STAIN,• LEISHMAN’S STAIN AND,• FIELD’S STAIN.
COLOR RESPONSES OF BLOOD CELLS TO ROMANOWSKY STAINING
• Cellular component Color• Nuclei Chromatin Purple
• Nucleoli Light blue
• Erythroblast Dark blue
• Erythrocyte Dark pink
• Reticulocyte Grey–blue
CYTOPLASM COLOR
• Lymphocyte Blue
• Metamyelocyte Pink
• Monocyte Grey–blue
• Myelocyte Pink
• Neutrophil Pink/orange
• Promyelocyte Blue
• Basophil Blue
• 1. Macroscopic view : quality of the smear • 2.The microscopic analysis
• begins on lower power (10x), • Determine good distribution of the cells• Scans the edges for abnormal cells• Find a optimal area in the smear for detailed
examination.
PBS examination - preliminary
Hi-power (40x) :• To obtain a WBC estimate. • All of the detailed analysis of the cellular elements using
high power or oil immersion.• Evaluate the morphology of the WBC and record any
abnormalities such as toxic granulation or Dohle bodies.• The WBC estimate can be performed using a factor which
is based on the fact that WBC seen in 40x is approx equivalent to 2000 cells /micro litre.
• For example if the average number of WBC counted per high power field is 5, the estimate WBC is 5 x 2000 = 10000
OIL IMMERSION
• Perform a 100 WBC differential count , counting is done in zig zag motion.
• All WBC have to included until a total of 100 have been counted
• Evaluate RBC for anisocytosis , poikilocytosis , hypochromasia , polychromasia, and inclusions.
• Perform platelet estimate and platelet morphology• Count the number of platelets in 10 OIF.• Divide by 10
(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• Abnormal cells
2. WBC• Total counts• Differential counts• Abnormal WBC
3. Platelets • Counts • Abnormality
4. Parasites
Evaluation of PBS
RED CELL MORPHOLOGY
Morphology of Normal Red Blood Cells
Biconcave disc Diameter : 7 ~ 8 μm Average volume : 90 fl. Central pallor occupy 1/3 rd of total size Approx same as nucleus of mature lymphocyte
RED CELL ABNORMALITY
• Normal MCV is -80-100 fl• Microcytes –MCV<80 fl• Macrocytes – MCV> 100 fl• Anisocytosis - variation in the size of the
RBC• Poikilocytosis – Variation in the shape of
RBC
VARIATION IN SIZE • Anisocytosis- Variation in size of the red blood
cells• The severity of the variation corresponds to increased
RDW.• Anisocytosis results from the abnormal cell
development ( deficiency of iron , B12, Folic acid)• Normal MCV is -80-100 fl• Microcytes ( MCV <80 fl)• Macrocytes (MCV >100fl)
MICROCYTES
• A Microcyte is a small cell having a diameter less <7 & MCV < 80fl.
• Anemia associated with microcytes is said to microcytic
• Expanded central zone of pallor anemia
• Decreased or defective globin synthesis also presents as Microcytic hypochromic anemia.
MORPHOLOGY - MICROCYTE
IRIRON DEFICIENCY THALASSEMIA SIDEROBLASTIC
LEAD POISONING
CHRONIC DIESEASE
POPOSSIBLE PATHOLOGY
MACROCYTES
• When MCV of RBC is Increased(>100fl)• The common cause of macrocytes is due
to the impaired DNA synthesis, RNA synthesis is unaffected resulting in the asynchrony between the cytoplasmic and nuclear maturation .
• Neutrophillic hypersegmentation is typically seen.
MORPHOLOGY - MACROCYTE
MEGALOBLASTICPROCESS
HIGH RETICULOCYTE
COUNT
LIVER DIESEASE
HYPOTHYROIDISMPOST SPLENECTOMY
OVAL MACROCYRTES
HEMOLYTIC ANEMIA / ACUTE
BLOOD LOSS
CHRONIC ALCOHOLISM
FOLATE AND B12 DEFICIENCY
NORMOCYTES
• The average size of the erythrocyte is indicated by the measurement of the MCV
• A Normal MCV would corresponds to the MCV reference range ( 80 -100 )fl
• Subsequent review of the peripheral smear reveals no abnormality in the size variation.
• This scenario is referred to as NORMOCYTIC and red cells are referred as normocytes.
HEMOGLOBIN CONTENT – COLOR VARIATION
• NORMOCHROMIA • HYPOCHROMIA • HYPERCHROMIA • POLYCHROMASIA
NORMOCHROMIA• The term Normochromic indicates the red cell is essentially high
in color
• A normochromic erythrocyte has a well hemoglobinized cytoplasm with a small but distinct zone of central pallor.
• The central pallor does not exceed 3µm .
• The term normochromic is used to describe the anemia with a normal MCHC, and MCH and when used in conjunction with MCV the anemia is described as NORMOCYTIC / NORMOCHROMIC anemia .
HYPOCHROMIA
• Any RBC having a central area of pallor of greater than 3µm is said to be hypochromic
• There is a direct relationship between the amount of hemoglobin deposited in the RBC and the appearance of red cell when stained.
• The term Hypochromia indicates low color and indicates that the cells have less hemoglobin.
• MCHC < 32% the anemic process is described as hypochromic.
HYPOCHROMIA GRADING
1 + AREA OF CENTRAL PALLOR IS ONE HALF OF CELL DIAMETER 2 + AREA OF CENTRAL PALLOR IS TWO THIRDS OF CELL DIAMETER 3 + AREA OF CENTRAL PALLOR IS OF THREE QUARTERS 4 + THIN RIM OF HEMOGLOBIN
POLYCHROMASIA
When RBC are delivered to the peripheral circulation prematurely appearing diffusely basophilic and are gray blue in color and usually larger than normal red cell.
The basophilic color is due to the RNA residue involved in hemoglobin synthesis.
Polychromatic cells are actually reticulocytes.Any clinical condition in which marrow is stimulated
particularly RBC regeneration will produce a polychromatic blood picture .
The degree of polychromasia is a excellent indicator of therapeutic effectiveness when patient is given iron or vitamin therapy as treatment of anemia
POIKILOCYTOSIS• Variation In shape is called Poikilocytosis.• It is of following types-
• Spherocytes• Elliotocytes• Target cells• Schistocytes• Acanthocytes• Keratocytes• Echinocytes• Bite cells • Howel jolly bodies
Spherocytosis • Spherocytes are small dense spheroidal RBC with absence of central pallor .• Because of their density they are easily seen in the peripheral smear.• This abnormality is due to the abnormality of the red cell membrane .• The detailed mechanism for sphering is the congenital condition known as
hereditary spherocytosis.• This is an inherited , autosomal dominant condition and is due to the deficiency
of the membrane proteins , spectrin and ankyrin .• Acquired causes of spherocytes are ABO incompatibility Autoimmune hemolytic anemia (warm antibody type) Infections (e.g., EBV, CMV, E. coli, Sepsis/ sepsis) Severe burns DIC and HUS
Elliptocytes or ovalocytes
Ovalocytes / elliptocytes are due to the result of morphological abnormality due to the result of mechanical weakness or fragility of the membrane skeleton that may be acquired or hereditary.
STOMATOCYTES Red cells with central
biconcave area appears slit like in dried film.
Wet film it appears as cup-shaped.
The abnormal morphology is due to the Membrane defect.
Seen in ArtifactHereditary
stomatocytosis liver disease,Alcoholic cirrhosisHemolytic anemia
Tear drop cells / dacrocytes Tear drop cells appear in
the peripheral circulation as tear drop or pear shaped red cells.
Exact mechanism not known.
It is seen in : Myelofibrosis Bone marrow infiltrated
with hematological or non-hematological malignancies
Iron deficiency anemia megaloblastic anemia
TARGET CELLSCells in which central
round stained area and peripheral rim of cytoplasm.
Seen in ThalassaemiaChronic liver diseaseHereditary hypo-
betalipoproteinemia Iron deficiency anemiaHemoglobinopathies
(Hb C, Hb H, Sickel cell anemia
Post splenectomy
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 Micro angiopathic hemolytic anemia Malnutrition Post splenectomy it is the hallmark in the diagnosis of
the neuro acanthocytosis syndrome such as
Chorea-acanthosis and Mcleod syndrome
SCHISTOCYTES These are fragmented erythrocytes. Smaller than normal red cells and of varying shape
resulting from some trauma to the cell membrane. Triggering events within the circulation leading to
fragmentation of RBC. Fluid alteration results in development of fibrin strands,
damaged endothelium. The flow of the blood in the circulation sweep the RBC
through the fibrin strands splitting the red cells Acquired disorder of RBC formation
Megaloblastic
Dyserythropoietic
Mechanical stress MAHA DIC Heart valve surgery HUS / renal graft rejection Direct thermal injury / Severe burns/
SICKLE CELL
• Cells are sickle (boat shape) or crescent shape
• Present in film of patient with homozygosity for Hb S.
• Usually absent in neonates and rare in patients with high Hb F percentage
RED CELL INCLUSION
• Basophilic stippling (Punctate basophilia)• Howell – jolly Bodies• Heinz body• Cabot Rings• Protozoan inclusions• Rouleaux formation
ROULEAUX • Rouleaux is a condition in which red
cells appear as stacks of coins on the peripheral smear .
• The stacks of RBC are evenly distributed through out the smear , rouleaux formation is the result of elevated globulins or fibrinogens in the plasma where the RBC has been “bathed “ in the abnormal plasma giving sticky consistency.
• It is seen in multiple myeloma and Waldenstroms macroglobulinemia, intra venous administration of plasma volume expanders like dextran.
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
• Presence of irregular basophilic granules with in Rbc which are variable in size .
• Stain deep blue with Wright’s stain
• Fine stippling seen with • Increased polychromatophilia
• Increased production of red cells.
• Coarse stippling• Lead and heavy metal poisoning
• Disturbed erythropoiesis
• Megaloblastic anemia
• Thalassaemia
• infection
• liver disease
• Unstable Hb
• Pyrimidine-5’-nucleotidase defiency
BASOPHILIC STIPPLING
HEINZ BODIES• Seen on supravital stains• Not seen on Romanowsky stain.• Purple, blue, large, single or multiple
inclusions attached to the inner surface of the red blood cell.
• Represent precipitated normal or unstable hemoglobins.
• seen – Post splenectomy• Oxidative stress
• Glucose-6-phosphate dehydrogenase deficiency,
• Glutathione synthetase deficiency• Drugs• Toxins• Unstable hemoglobins
CABOT RINGS
• These are Ring shaped figure of eight or loop shaped
• Red or Reddish purple with Wright’s stain and have no internal structure
• Observed rarely in• Pernicious anemia,• Lead poisoning,
WHITE BLOOD CELLS
TYPES Granulocyte (polymorphonuclear) Agranulocyte (mononuclear)
Contain membrane bound granules, which stains differently with stains
Apparently absent granules, but contain non specific azurophilic granules
E.g.NeutrophilsBasophilEosionophil
E.g.LymphocyteMonocyteMacrophage
POLYMORPHONUCLEARNEUTROPHILS• 40 to 80 percent of total WBC
count(2.0–7.0 ×109/l )
• Diameter - 13 µm
• segmented nucleus and pink/orange cytoplasm with fine granulation(0.2-0.3µm) stain tan to pink with Wright’s
• Lobes -2-5
• Neutrophils usually have trilobed nucleus.
• small percent has four lobes and occasionally five lobes.
BAND FORMS
• neutrophils has either a strand of nuclear material thicker than a filament connecting the lobes, or a U-shaped nucleus of uniform thickness.
• Up to 8% of circulating neutrophils are unsegmented orpartly segmented (‘band’ forms)
• Band cells constitute <5-10% of white blood cells• An increase in number of band cell and other
immature neutrophils is called a “ shift to left” can be seen in
• Severe infections, sepsis• Non infectious inflammatory disease • Pregnancy
GRANULES
• Toxic granulation- increase in staining density and number of granules
• Seen with Bacterial infections and other inflammation
• Administration of G-CSF• Anaplastic anemia
DOHLE BODIES• Small, round or oval, pale blue-grey
structure
• Found at periphery of neutrophil.
• Contains Ribosomes and Endoplasmic reticulum
• Seen in – Bacterial infection
• inflammation
• administration of G-CSF
• during pregnancy
• Pernicious anemia
• Myeloproliferative disorders
• Myelodysplastic disorders
• Cancer chemothrapy
EOSINOPHILS• Normally 1-6( 0.02–0.5 ×
109/l)• Size- 12–17 µm• Nucleus- Bilobed
(spectacle shaped)• Cytoplasm- Pale blue• Granules - Coarse
spherical gold/orange
Eosinopenia- seen with prolonged steroid administration.• Eosinophilia- allergic conditions hay fever,
asthma• severe eosinophilia- parasitic infection
• reactive eosinophilia• Eosinophilic leukaemia• Idiopathic hypereosinophilic syndrome• T-cell lymphoma, B-cell lymphoma
and acute lymphoblastic leukaemia.
BASOPHILS• Rarest <1• Nucleus segments fold up on each other
resulting compact irregular dense nucleus(closed lotus flower like)
• Granules-large, variable size dark blue or purple often obscure the nucleus
• Granules are rich in histamine, serotonin and heparin
• Increase in myeloproliferative disorder-CML
MONOCYTES• 2-10% of total wbc count
• Size- largest circulating leucocyte, 15–18µm in diameter
• Cytoplasm- grey blue
• Nucleus- large , curved , horse shoe shape
• No segmentation occur
• Chromatin- fine evenly distributed
• Increase in chronic infections and inflammatory conditions such as
• Tuberculosis and Crohn’s disease,
• Chronic myeloid leukaemias
• Acute leukaemias with a monocytic component
• Infectious mononucleosis
LYMPHOCYTES• 20-40% of total WBC count
• It is of two types
1. Small lymphocyte(6-10µm)
2. Large lymphocyte(12-15µm)
• Nucleus-single, sharplydefined, stain dark blue on Wright’s stain
• Cytoplasm- Pale blue
• Large lymphocytes less densely stain nuclei & abundant cytoplasm
• Few round purple(azure) granules are present
• Lymphocytes predominate in the blood films of infants and young children.
REACTIVE LYMPHOCYTES
• Have slightly larger nuclei with more open chromatin
• Abundant cytoplasm that may be irregular.
• Seen in infectious mononucleosis
• viral infections
PLATELETS MORPHOLOGY
PLATELETS
• Thrombopoiesis take place in bone marrow
• 1 megakaryocyte produce 4000 platelets
• Normal platelet are about 1.3 micron, blue grey, contain fine, purple to pink granules
• Red cell to platelet ratio : 10-40:1
• Life span 9-12 days
• Range : 1.5-4.5 lakhs/microL
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
EXAMINATION OF BLOOD FILMS FOR PARASITES
MALARIA
• Giemsa stain are used, identifies species and life cycle stages
• Parasitemia is quantifiable• Threshold of detection thin film: 100
parasites/ L, thick film: 2-20 parasite/LThick film Thin film
• Lysed RBCs• Larger volume• 0.25microliter / 100 fields blood
element more concentrated• Good screening for positive or
negative parasitemia and parasite density difficult to diagnose species
• Fixed RBCs• Single layer• Smaller volume• 0.005 microliter blood required• Good species differentiation• Requires more time to ready
A. Peripheral smear
APPEARANCE OF P FALCIPARUM IN THE BLOOD FILMS
Ring or trophozoite
• Many cells infected – same with more than one parasite
• Red cell size unaltered
• Parasite is often attatch to the margin of the host cell: called as accole form (arrow)
Schizont Very rarely seem
except in cerebral malaria
A single brown pigment dot along with 18-32 merozoites
Gamatocyte Sickle shape “cresent” Matuer gametocyte is
about 1.5 times larger than RBC harbouring it
Microgamatocyte: Broader, shorter, blunt ends. Cytoplasm light blue
Macrogamatocytes: Longer, narrower, pointed ends. Cytoplasm deep blue
APPEARANCE OF P VIVEX IN FILM
Ring or trophozoite
• Many cells infected – same with more than one parasite
• Unoccupied portion by parasite shows a dotted or stripped appearance “Schuffner’s dot”
Schizont Represent the full
grown trophozoite Contain 12-24
merozoits Arranged in the form
of rosette with yellow brown pigment at the center
Gamatocyte Certain schizont get
modified and result in sexual forms. Merozoite arising from single schizont are either all males or females
Microgamatocyte: Spherical. Cytoplasm light blue
Macrogamatocytes: spherical. Cytoplasm deep blue
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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