Interpretation of

CBC

contt…

DR. N. BAJAJ

Hemoparasites: in

peripheral smears

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

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. Merozoitearising from single schizontare either all males or females

Microgamatocyte: Spherical. Cytoplasm light blue

Macrogamatocytes: spherical. Cytoplasm deep blue

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

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

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%

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

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

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

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

Trypanosoma Cruzi- Chagas disease

Trypanomastigote in peripheral blood

Amastigote in striated muscles

White blood cells

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

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

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

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

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

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

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

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

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

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

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

Hypersegmentation

Exists when > 5% of neutrophils have 5 or more lobes

Seen in folate and vitamin B12 deficiency

Myeloproliferative disorders

Myelodysplastic disorders

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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)

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

Peripheral smear in thrombocytopeniaRBC 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

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

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

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

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

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

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

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

Thanks

and give blood