Pbs

1. Peripheral blood smear examination
DrHemang Mendpara
DNB pediatrics
ChoithramHospital & Research Centre
Indore
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2. •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
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3. 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
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4. Slide fixation and staining
1. Romanowskystaining
Leishman'sstain : a polychromatic stain
•Methanol : fixes cells to slide
•methylene blue stains RNA,DNA
blue-grey color
•Eosin stainshemoglobin, eosin granules
orange-red color
•pH value of phosphate buffer is very important
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5. 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.
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6. •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
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7. (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 zagmotion)
Scanning technique for WBC differential
count and morphologic evaluation
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8. 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
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9. •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
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10. Morphologic Evaluation of Red Blood Cells
Biconcave disc
Diameter : 7 ~ 8 μm
Central pallor occupy 1/3 rdof total
Size : approx. same as nucleus of
mature lymphocyte
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11. Microcytichypochromicred cells
Decreased size and Hb
content (MCH) and conc
(MCHC). Expanded
central zone of pallor
Iron deficiency,
thalasemiatrait
Anemia of chronic
disease
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12. Iron deficiency anemia
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13. Thalassemia trait
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14. Megaloblasticanemia(PS)
Macrocyte
Large 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
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15. Elliptocytes or ovalocytes
Ovalocytes are due to abnormal membrane
cytoskeleton found in hereditary elliptocytoisis
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16. seen when there is extramedullaryerythropoiesis
Tear drop cells / dacrocytes
•Osteopetrosis
• Myelofibrosis
• Bone marrow infiltrated with
hematological or
non-hematological malignancies
• Iron deficiency anemia
• Pernicious anemia
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17. Polychromasia
Blue-gray coloration of RBCS.
Due RNA remnants
Increased -Increased erythropoietic
activity. Decreased -Hypoproliferative
states.
Hemolytic anemias
•Blood loss anemias
•Recovering anemia
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18. Sickle cell anemia
Irregular, curved cells
with pointed ends
HbS hemoglobinopathies(sickle cell anemia, HbSC
disease, HbS-beta-thalassemia, HbSD disease, hb
Memphis /S disease)
*Don’t be confused with fragmented RBC
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19. 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
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20. Acanthocytesor spur cells, are spherical cells with blunt-tipped
or club-shaped spicules of different lengths projecting from their surface at
irregular intervals.
Acanthocytes
Acanthocytesare seen in
•Hereditary abetalipoproteinemia
•Hereditary acanthocytosis
•End stage liver disease
•Anorexia nervosa
•Malnutrition
•Post splenectomy
•Intravenous hyperalimentation
particularly with intralipidinfusion
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21. Echinocytes
"Sea urchin cells,
crenatedcells, burr cells"
Post-splenectomy,
uremia, hepatitis of the newborn, malabsorption
states, after administration of heparin, pyruvate
kinase defphosphoglyceratekinase deficiency,
uremia, HUS.
Crenated / Burr cells / Echinocytes
(Echinocytes, or burr cells or
crenatedred cells, in contrast, have
shorter, sharp to blunt spicules of uniform
length which are more evenly spaced
around their periphery).
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22. Mechanical damage to RBCs from fibrin deposits
DIC
MicroAngiopathicHA
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
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23. hemolyicanemias
Hallmark: Presence of schistocytes, fragmented RBC
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24. UniconcaveRBC,
slitlikearea 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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25. 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
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26. 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
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27. Irregular RBC
agglutination/
clumping
Anti-RBC antibody, paraprotein. Cold
agglutinin disease, autoimmune hemolytic
anemia, macroglobulinemia,
hypergammaglobinemia
RBC autoagglutination
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28. Roulexformation
Seen in case of high level of fibrinogen, immunoglobulins,
intra venous administration of plasma volume expanders like dextran
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29. •multiple blue-purple inclusions attached to the inner surface of the red cell membrane.
visible in supravitallystained smears.
•are precipitated normal or unstable hemoglobin usually secondary to oxidant stress.
•G6PD deficiency
•Unstable
hemoglobinopathy
•Cong. Bite cell
anemia
Heinz body
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30. Small (1 mm), round,
dense, basophilic
bodies in RBCs.
Splenectomizedpatients,
Functionalasplenia,
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
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31. Basophillicstrippling
•Lead poisoning
•Iron deficiency anemia
•Thalassemia
Are abnormal aggregratesof ribosome and polyribosomes
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32. •Smaller then Howell jolly body
•Stain with Prussian blue stain
•Suggest iron over load
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33. WBC Morphology
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35. 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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36. •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 5,000100/110 = 4545.50
•The corrected white count is 4545.50
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37. •Left-shift:non-segmented neutrophil > 5%
–Increased bandsMeans acute infection, usually
bacterial
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38. •Basophils are increased in the blood in
–Myeloproliferativedisorders (e.g., chronic myelogenousleukemia)
–Hypersensitivity reactions
–Mastocytosis
–Xerodermapigmentosa
–Hypothyroidism
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39. •Morphologically abnormal eosinophilsare seen in
–Myelodysplasticsyndrome
–Megaloblasticanemias
•Eosinophilsare increasedin the following conditions:
Allergies
Parasitic infestations
Infections
Acute leukemia
Myeloproliferativediseases
Hypereosinophilicsyndrome
Drug-associated
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40. Band cells
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41. Leukemic myeloblast
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42. Leukemic myeloblaststained with peroxidase
Note the AUER ROD
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43. Burkittlymphoma
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44. 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
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45. 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
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46. Variably sized (0.1 to 2.0 um) and shaped, blue or grayish-
blue cytoplasmic inclusions usually found near the
periphery of the cell. Dohlebodies are lamellar aggregates
of rough endoplasmic reticulum, which appear in the
neutrophils, bands, and metamyelocytesof patients with
infection, burns, uncomplicated pregnancy, toxic states, or
during treatment with hematologic growth factors -G-CSF.
Döhle bodies
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47. 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
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48. 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.
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49. Platelets
Neubarschamber : 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
−Amegakaryocyticthrombocytopenia (AMT)
•Increased destruction
−Immune thrombocytopenia
*Idiopathic thrombocytopenic purpura(ITP)
*Neonatal alloimmunethrombocytopenia
(NAITP)
−Disseminated intravascular coagulation (DIC)
−Hypersplenism
Thrombocytosis
•Reactive thrombocytosis
Post infection
Inflammation
Juvenile rheumatoid arthritis
Collagen vasvulardisease
•Essential thrombocythemia
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51. :Giant platelets Platelet morphology
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52. Platelet satellitism
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53. Macrocytosiswith giant platelets (MDS,
5q-syndrome)
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54. 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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55. Merozoits
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56. 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
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57. Schuffer’sdots seen in plasmodium vivex
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58. Cresentshaped gametocyte charectaristicllyseen in p.falciparum
malaria
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61. Eucheriabancrofti
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64. OSMOTIC FRAGILITY
TEST
•Defination:
•it is a testthat 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
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65. •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.
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66. •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
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67. -Hereditary spherocytosis
-Acquired spherocytosis
-Hemolytic anemia (HDN)
-Malaria
-Severe pyruvate kinase deficiency
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69. •Thalassemia
•Sickle cell anemia (hemoglobinopathy)
•Iron deficiency anemia
•Asplenia
•Liver disease
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72. introduction
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73. Principle of test
•Deoxygenated Hb-S is insolublein the presence of a
concentrated phosphate buffer solution and forms a turbid
suspension that can be easily visualized.
•Normal Hemoglobin A and other hemoglobinsremain in
solution under these conditions. These different qualitative
outcomes allow for the detection of sickle cell disease and its
traits.
•SICKLEDEX® uses Saponinto lysethe 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.
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74. Procedure
•1. sodium diethanoid200mg+10 ml distilled
water
•2. sickling buffer solutions
•Take 2 part of 1
st
solution and 3
part
of 2
nd
solution
•Have one drop of blood on slide and put single
drop of mixed solution
•Wait for 30 mins
•Watch under microscope
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75. Result
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