continuation from first part with parasitology in peripheral smear and WBC Studies

1. Interpretation of
CBC
contt…
DR. N. BAJAJ

2. Hemoparasites: in
peripheral smears

3. Malaria
 Giemsa stain are used, identifies species and life cycle
stages
 Paresitemia is quantifiable
 Threshold of detection thin film: 100 parasites/ L, thick
film: 2-20 parasite/L
Thick 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

4. 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

5. 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

6. B. Flouroscien Microscopy
 Dyes detect RNA and DNA contents of parasite
 Nucleic material not normally seen in RBCs without
parasitemia
C. Quantative Buffy Coat (QBC)(Becton
Dickenson)
 Flouroscien microscopy after centrifuge
 More sensitive than light microscopy
 Useful for screening large number of samples
 Quick and saves times

7. D. Malarial serology – antibody detection
 Antibodies to asexual parasite appears some days to invasion of
RBCs and may persists for months
 Positive test indicate past infection
 Not useful for treatment decisions
 Valuable epidemiological tools
E. Malarial serology – antigen
detection
 Immunological assey to detect specific antigens
 Monoclonal and polyclonal antibodies used in antigen
capture tests
 Specific species and pan specific antibody
 Cross reactivity with other immunological conditions

8. Malarial antigen detection - RDTs
Feature PfHRP 2 PLDH
Principle Use monoclonal antibodies
Detect HRP of Pf
Use monoclonal and polyclonal
antibodies
Advantage • Threshold for parasite detection
ad low as 10 parasites/ microliter
• Does not react with other species
• Threshold for parasite detection ≥
100 parasite/ microliter
• Can detect all parasite
Disadvantages • Sensitivity and specificity
decreases as low as 10 parasite /
microL
• May remain positive upto 14 days
posttreatment
• Cannot detect mixed infection
• Sensitivity and specificity decreases
< 100 parasite/ microliter
Sensitivity and
specificity
• Sensitivity 94-100%
• Specificity 88-100%
• Sensitivity pf 88-98%, pv 84-94%
• Specificity pf 93-99%,pv 99-100%

9. G. Real time PCR
 Molecular technique to identify parasite genetic materials
 Threshold for detection 1.1 parasite/µL if whole blood is used, if filter
paper used it is 2 parasite/ parasite/µL
 Species diagnosis present
F. Polymerase chain reaction

10. H. Automation based malaria technique
 Hematological parameter and their different combination predict
presence of malaria
 Low platelet count as strongest predictors, variable MPW, normal to low
Plateletcrit and PDW
 TLC can be increase or decrease. Leukopenia more seen
 Normocytic normochromic anemia, low Hb, decrease RBC count raised
ESR, low MCV, MCH, MCHC
 Intracellular pigments can also be detected

12. Filaria
 Lymphatic – wucheria, Brugia
 Subcutaneous: Loa loa
 Sereous: Mansonella
 Sample collection between 10 pm to 4 am
 Appearance of microfilaria
 Measurement 290x7micron
 Covered with sheath
 Nuclei present all over but not at the tip of tail
 Neclei are brolen at different point serving as
lamdmark for identification

13. Babesia
 Parasite are intracellular amastigote form. Essential
parasite of RE System
 Amastigote form are seen in monocytes, less
commonly neutrophils
Leishmania
 Infect mice. Transmitted in between host by
ticks
 Infected humans may be asymptomatic, but in
asplenic host fever, myalgia, haemolysis can be
seen
 Maltase cross seen in PS

14. Trypanosoma Cruzi- Chagas disease
 Trypanomastigote in peripheral blood
 Amastigote in striated muscles

15. White blood cells

16. White blood cells
 The term leuckocyte is derived from Greek word leukos = white and
cyte = cells.
 However blood plasma appears green if there is large amounts of
neutrophils in the sample, due to haem containing enzyme
Myleoperoxidase
Normal counts
Age Count
Birth 4-40 x 109/L
4 years 5-15 x 109/L
Adult 4-11 x 109/L

17. 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.
Neutrophils
Basophil
Esionophil
E.g.
Lymphocyte
Monocyte
Macrophage

18. Leukocytosis
 High count usually indicate
1. Increase production of WBC to fight infection
2. Reaction to drug that enhance WBC production
3. Disease of marrow, causing high production of WBC
4. An immune system disorder that increase WBC

19. Leukocytosis Leukopenia
Acute and chronic infections
Polycythemia vera
Rheumatoid arthritis
Drugs
Allergy
ALL
AML
CLL
CML
Hairy cell leukamia
Smoking
Stress
Tissue damage such as burns
Lymphoma Spillage
Measles
Myleofibrosis
Chemotherapy or radiotherapy
Sepsis
Typhoid
Malaria
Tuberculosis
Dengue
Folate deficiency
Drugs like antipsychotic
Aplastic anemia
HIV and AIDS
SLE
Hodkins lymphoma
Rickettsial infections
Pseudo-leucopenia Seen during the onset of infections due to
marginated WBC

21. Band cells
 Usually 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

22. Causes of increased neutrophil: ANC >75000/cumm
1. Physiologic increase (Demargination)
• Release of cell in marginal pool
• Stress leukocytosis
• Exercise, Seizure
• Anxiety, Epinephrine
2. Acute infections
3. Tissue injury and inflammation
• Collagen vascular disease
• Hypersensitivity
• Burns
4. Myeloproliferative disorders: myeloid leukemia, polycythemia vera
5. Medications: Corticosteroid, lithium
6. Misc.: Sickle cell anemia, acute hemorrhage
Causes of Neutopenia
1. Decrease or ineffective production
• Aplastic anemia
• Drug
• Deficiency – vitamin B12, Folic acid
• Myelodysplastic syndrome
• Inherited disorder – Kostamann
synd.
2. Increased removal from circulation
• Immunological – SLE, Drugs
• Hypersplenism

23. Hematological Scoring System
(HSS): Neonatal Sepsis
 HSS can be very useful to differentiate the infected from non-infected infants
 It has high sensitivity and specificity
 An immature to total neutrophil ratio [I:T] along with degenerative changes >
immature to mature [I:M] is the most sensitive indicator of sepsis in infant
 Immature include: promyleocyte, myleocyte, metamyleocyte and band cells
 Degenerative changes: vacuolization, toxic granules and Dohle bodies.
 Confirmation by Blood Culture

24. Hematological Scoring System (HSS)
Criteria Abnormality Score
WBC <5000/microL
>25000 at birth
>30000-(12-24h)
>21000 day 2 onwards
1
1
1
1
Total PMN count No mature PMN seen
Increase/ destruction
2
1
Immature PMN count Increased 1
Immature: total PMN ratio Increased 1
Immature: mature PMN ratio >0.3 1
Degenerative changes in PMN Toxic granules/ Vacuoles 1
Platelet count <150000 microL 1
Score Interpretation
<2 Sepsis is unlikely
3-4 Sepsis is possible
>5 Sepsis or infrction is very likely

25. Cytokinin
Myeloid precursor in bone marrow
Cell
Cell is altered
White cells more
readly exits marrow
Increase
phagocytic activity
Cytoplasmic
inclusions may
appear
Enhanced enzyme
production and
packing resulting in
large granules
Toxic vacoulisation
Left
shift
Dohle
bodies
Increase
phagocytic activity
Cytoplasmic
inclusions may
appear
Left
shift
Toxic vacoulisation

26. Toxic granulation and vacuolization
 Indicate the presence of increased granules that are larger and
more basophilic in normal
 Seen in
 Severe infection
 Aplastic anemia
 Burns
 Malignancy
 Treatment with CSF
 Pregnancy

27. Dohle bodies
 Composed of rough endoplasmic reticulum and glycogen granules
 Small blue grey inclusion seen in neutrophil usually in periphery
 Seen in
 Infections
 Inflammatory disorders
 Pernicious anemia
 Myeloproliferative disorders
 Myelodysplastic disorders
 Cancer chemothrapy

28. Hypersegmentation
 Exists when > 5% of neutrophils have 5 or more lobes
 Seen in folate and vitamin B12 deficiency
 Myeloproliferative disorders
 Myelodysplastic disorders

29. Pelger Huet anamoly
 70-90% neutrophils have
hypolobulated, rounded nuclei, with
condense chromatine
 A thin strand connect the lobes giving
rise to pince-nez (spectacle) shape, or
a larger bridge give rise to peanut
shape.
 Heridetery hypolobulation has no
significance
 Acquired (Pseudo Pelger Huet)
anamoly, common in myelodysplastic
and myeloproliferative disorders

30. Auer Rods
 Seen in myeloid blast of acute leukemia
 They are fused lysosomes and contain lysosomal
enzyme and large crystalline inclusions, seen in
the cytoplasm of leukemic blast
 They are virtually pathognomic of myeloid
leukemia

31. Leukamoid reactions
 Leukamoid reaction is a haematological
disorder that simulates leukemia due to high
WBC counts and presence of some immature
leukocytes. In leukamoid reaction the cells
are not clonally derived.
 Persistant neutrophilia with cell count of
>30000-50000/microL is called myeloid
leukamoid reaction
 Leukamoid alkaline phosphate score (LAP-
score) can differentiate leukamoid reaction
from CML. LAP score is raised in leukamoid
reaction whereas decreased in CML

32. Some causes of leukamoid reaction
Causes Myelocytic Lymphocytic Monocytic
Infections Endocarditis
Pneumonia
septicemia
Leptospirosis etc.
Infectious mononucleosis
Pertusis
Varicella
Tuberculosis
Tuberculosis
Toxic conditions Burns
Poisoning -mercury
Eclampsia
Neoplasia Ca Colon
Embryonal carcinoma of
kidney
Carcinoma of stomach
Carcinoma of breast
Others Acute haemorrhage
Acute haemolysis
Dermatitis herpitiformis

33. Eosinophils
 Cells having large dinstintive red orange specific
granules in cytoplasm, which contain histamine
and other substances
 Lives 6-12 hours in circulation, migrate into tissues
 Normal range: 1-4% of total WBCs
 Absolute count: 12-500cells/ microliter
 Diurnal variation – related to cortisol level: lowest
in morning, highest in evening

34. Eosinophilia Eosinopenia
Mild 700-1500
• Allergic rhinitis
• Extrinsic asthma
• Mild drug reaction
• immunodeficiency
Usually related to increased steroids
Cushing syndrome
Drugs
ACTH, epinephrine, thyroxine
Acute bacterial infections
Moderate 1500-5000
Parasitic disease
Intrinsic asthma
Pulmonary Eosinophilia syndrome
Marked >5000
Trichinella
Hookworm
Toxocara canis
Severe drug reaction
Eosinophilic leukamia

35. Basophils
 Contain large purplish granules, granules obscuring the nucleus
 Releases bradykinin, heparin, serotonin, histamine
 Mediates allergic reactions
 Circulate for few hours(6-12) then migrates into tissue
 Range 0.5-2%, absolute count 6-200 /microloiter
 Basophilia - causes
 Hypothyroidsm
 CML
 Ulcerative colitis
 Polycythemia vera
 Uticaria
 Chickenpox
 Splenectomy

36. Monocytes
 Agranulocytes, contain greyblue granules
 Life span 8hrs-30days, migrate in tissue and became macrophage
Monocytosis Monocytopenia
>700 /mcL or >12% WBC
• Viral infections
• Tuberculosis
• Sub acute bacterial endocarditis
• Collagen disease
• Chronic inflammation
• Stress
• Infectious mononucleosis
• Sarcoidosis
• Autoimmune
• SLE
• Rheumatoid disease
Hairy cell leukemia
Aplastic anemia

37. Lymphocytes
Lymphocytosis Lymphocytopenia
Infectious mononucleosis
Tuberculosis
Brucellosis
Cytomegalvirus
Rubella toxoplasma
Hepatitis A,B
Wooping cough
ALL
Burkitt lymphoma
CLL
Hairycell leukemia
Non Hodgkin's lymphoma
X- linked lymphoproliferative disorders
Viral infections
HIV
SARS
Marrow suppression
Pancytopenia
Drugs: Vinblastin, Doxorubicin,
Chromphenicol

38. 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

39. Thrombocytopenia
 Grade 1- counts is between 75,000 -150,000
 Grade 2- counts is 50,000 < 75,000
 Grade 3 – 25,000 to < 50,000
 Grade 4 - < 25,000

40. Thrombocytopenia
Decrease production Increase destruction Abnormal distribution
• TAR syndrome
• Amegakaryocytic
thrombocytopenia
• Aplastic anemia
• Myelodysplatic synd
• Bome marrow
hypoplasia or
infiltration
• Ineffective
thrombopoisis due to
folate deficiency
• Heridietery
• May Hegglin
anamoly
• Wiskott Aldrich
Syndome
• Immune mediated
• SLE
• ITP
• Drugs like heparin
• HIV
• Posttransfudion
purpura
• Non immune
• Severe bleeding
• DIC
• Vasculitis
• vWD
• TTP
• HUS
Hypersplenism
Dilutional, due to
massive transfusion

41. Pseudo- thrombocytopenia
(artifactual)
A. EDTA induced platelet agglutination
 This is invitro phenomenon due to presence if auto antibodies against a
crypt antigen on the GP IIb/ IIIa receptor, when calcium is chelated by
EDTA, the GPIIb/IIIa get exposed and causes agglutination of platelets
 Occurs in 1% of hospitalized patients
 No evidence of abnormal haemostasis
 Confirmed by sampling on citrated blood
B. Platelet satellitsm: platelet rossete formed around the neutrophil or
any other cells. These satellite platelets are not counted by
counter. It is caused by EDTA dependent antiplated and
antineutrophil IgG antibodies. It is not associated with any disease

42. C. Cold agglutinin: temperature dependent phenomenon. Sample has to
be warm to 37 degree C to get accurate platelet count
D. Giant platelet or Megakaryocyte: platelet larger than 36fl is counted
as red cell in counter, resulting in low platelet count
 Mean platelet volume is increase in giant platelets
 Young platelets are usually larger
 Causes of large platelets include:
 Hereditary – Bernard Soulier Syndrome, Benign Mediterranean
macrothrombocytopenia
 Acquired – immune thrombocytopenia purpura
 Myeloproliferative syndrome
 Myleodysplasia
 DIC
 TTP
 Partially clotted specimen: some platelets get consumed

43. Thromboasthenia: Platelets with normal count but abnormal
function, leading to episodes of bleeding
(A) Inherited : (B) Acquired
1. Aggregation defect: Glanzmann
thrombosthenia, congenital
afibrinogenemia
2. Platelet adhesion defect: Bernard Soulier
syndrome, vBD
3. Signaling pathway defect: defect in
calcium mobilization, thromboxane
synthetase deficiency,cyclooxygenase and
lipoxygenase deficiency
4. Agonist receptor defect: thromboxane
receptor deficiency
5. Secretion defects: Chediak Higasi synd,
storage pool disease, Wiskott Aldrich synd,
Grey platelet syndrome
1. Essential thrombocytopenia
2. Uremia
3. Antiplatelet antibodies
4. Myeloproliferative disorders
5. Polycythemia vera
6. CML
7. Acute leukemia
8. Myleodysplastic syndrome
9. vWD
10.Liver disorders

44. Thrombocytosis
Myleoproliferative disorders Transfer from extravascular pool Thrombocytosis secondary to
Essential thrombocytosis
Idiopathic myleofibrosis
Polycythemia vera
Chronic granulomatous
leukemia
Splenectomy
Exercise
Epinephrine
Iron deficiency
Infections
Hemolysis
Malignancy
Acute blood loss

45. Mean platelet volume - MPV
 Measurment that describe the average size of the platelet in the
blood.
 It is indicator weather bonemarrow is manufacturing platelets
normally or there is some kind of production pressure
 MPV has inverse relation with platelet count
 change in mean platelet volume without any change in platelet
count may be early indicator of bone marrow problem
 Platelet are considered large when 49-8 micron diameter and giant
when equals RBCs
Normal range – 7.4-10.4fL

46. Increase MPV (megathrombocytes) Decrease MPV
ITP
TTP
Bernard Soulier synd
May Hagglin disease
Sepsis - recovery phase
Heart valve prosthesis
Myelodysplasia
Sickle cell anemia
Hyperthyroidsm
Aplastic anemia
Wiskott Aldrich syndrome
TAR synd
Storage pool disease
Megaloblastic anemia
Hypersplenism
Note: in general platelets are large when thrombocytopenia results from increased
destruction and small with disorders of diminished production.
If platelet count is low and MPH is high the risk of bleeding is comparatively less as
larger platelets have multifold better hemostatic capacity than normal size
platelet.

47. Platelet distribution width (PDW)
 Compares uniformity and heterogeneity of platelet size; as RDW
 Increased in
 Essential thrombocytopenia
 Aplastic anemia
 Megaloblastic anemia
 CML
 Chemotherapy
 Fragmented RBCs
 PDW is a relative good tool to distinguishessential thrombocythemia
(PDW increase) from reactive thrombocytosis (PDW normal)

48. Plateletcrit
 It is the volume percentage that platelets match on total blood
volume of blood, and it is directly related to the total volume of the
platelets and MPV
 Normal Range 0.110-0.280

49. Peripheral smear in thrombocytopenia
RBC lineage
• Schistocytes Microangiopathic haemolytic anemia, DIC, HUS
• Malaria parasite Thrombocytopenia, pf
• Spherocytes AIHA+thrombocytopenia (Evan Syndrome)
• Normoblast and polychromasia HELLP
• Autoagglutination Cold antibodies
WBC lineage
Increase polymorphs Infection/ septicima
Toxic granules band cells Septicimia
Precursor cells, blast cells Leukaemia
Dysplastic cells MDS
Platelet lineage
Giant platelets ITP,BSS, May Heglin, grey platelet synd,
montreal platel,sebstian syndrome
Scattered platelet in direct smear Glanzmann synd

50. Manual vs Automation hematology
 Manual with Neubauer chamber are used mainly used
where there is economic considerations and non
availability of automation
 Disadvantages of manual counting
 Cell identification:
 mostly between lymphocyte, monocytes, band cells
 Segmented form and abnormal cells
 Lymphocytes may be over estimated and monocytes may be
underestimated
 Cell distribution error : increased cell concentration along
edges and also bigger cells
 Statistical sampling errors

51. Automated counters provides a 3
or 5 or 7 part differential count
3 part differentiation 5 part differentiation 7 part differentiation
1. Granulocytes or larger cells
2. Lymphocyte or smaller cells
3. Monocytes or mid cell
population
1. Neutrophils
2. Eisonophils
3. Basophils
4. Lymphocytes
5. Monocytes
6. A sixth category “large”
unstained cells, include
cells larger than normal
and lacks peroxidase
activity- atypical
lymphocytes and other
abnormal cells
Include 5 part
• Large immature cells- blast
and immature granulocytes
• Atypical lymphocytes

52. Cell counter – basic principle
 Inventor - Wallace Coulter
 Electrical impedence principle of cell counting: The cell size are
counted by detecting and measuring changes in the electrical
resistance when a particle passes through a small aperture.
 Mathematically
V=RxC V-voltage, C – current, R= resistance

53.  The electrical system : circuitry, sequence controls, transformers
 The hydraulic system : aspirating unit, dispenser, diluents, mixing
chambers, flow cells, aperture bathes and haemoglobinometre
 Pneumatic system : vacuum and pressure devices
 Computer system
Cell counter – basic components

54. Radiofrequency principle of cell counter
 This employ high voltage electromagnetic currents, which can estimate the cell
size based on cellular density and nuclear volume
 It measures the conductivity and the conductivity is altered by nuclear to
cytoplasmic ratio, nuclear density, granulation.
VCS principle of cell counter
 VCS= volume, conductivity, scatter
 Direct current – measures the size of the leukocytes based upon its volume
 Conductivity – HF radiowaves measures conductivity of the cells
 Scatter – laser light beam evaluates the surface feature, structure, shape,
granularity and reflectivity

55. Coincidental or Recirculation errors
 If more than 1 cell passes through the counting aperture at the
same time and is counted as one cell, this is called coincidental
error

56. Advantages of automated cell
counters
 No inter-observer variability
 No slide distribution errors
 Eliminate statistical error
 Many parameter are available e.g. RDW, histogram
 More efficient and time effective
 High level of precision and accuracy

57. Thanks
and
give blood