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INTERPRETATION OF CBC

DR. N. BAJAJ

TERMS Anisopokilocytosis: variation in size and shape

Cytometry: measurment of the cell either visual or automated

Cluster analysis: analysis that is based upon the instrument’s ability to cluster different populations, together based upon size, staining, absorption or other parameter

Contour grating: analysis where information is plotted three dimentionally, that can be separate subpopulation of cells

Coulter principle (electrical impedence): sizing and counting cells by detecting and measuring changes in electrical resistance when cell passes through small aperture.

Dimorphic : two population of cells in single blood sample

Forward angle light scatter: light from laser source is scatter in forward direction (0 degree) when it strike a cell or particle, larger object more forward light scatter

Forward high angle light scatter: similar to forward angle light scatter, but angle is 5 to 15 degree variation

Forward low angle light scatter: similar to forward angle light scatter, but angle is 2 to 3 degree variation

Introduction

Haematology comprise of

‘Haima’ = blood in Greek

‘Logos’ = study

Haematology is a unique super speciality in medicine which encompasses the fields of pathology, physiology, biochemistry, molecular biology, obstetrics and gynecology, medicine and paediatrics

CBC

A complete blood count is a series of tests used to evaluate the composition and concentration of the various cellular component of the blood.

Is a basic test Most informative single investigation Tests consists of

1. Counts of RBC, WBC, Platelets

2. Haemoglobin, haematocrit, and red cell indices

3. TLC, DLC

4. Platelet count, mean platelet volume, plateletcrit, PDW

5. Histogram of RBC, WBC, Platelets

How important is CBC ?

To know the importance of CBC we need to know…..

What is CBC?

Why CBC?

What are various parameter of CBC?

What are variation in parameter of CBC?

What these variation can tell us?

How these variations affect the assessesment and care of patients?

Why CBC?

CBC is an inexpensive tool and powerful tool which provide information about

Blood, also about Marrow, Health or disease state of other

organ of body

CBC USES

To diagnose

1. Anemia

2. Haemoglobinopathies

3. Bone marrow aplasia

4. Nutritional deficiencies

5. Thrombocytopenia

6. Autoimmune conditions

7. Infections and Parasitemia

8. Malignancies, response to drug, chemotherapy etc.

Red blood cells

RBC produced in marrow and requires

Iron, copper, magnease, cobalt

Vitamins; especially B12, folic acid

Regulated by erythropoietin, thyroid hormone, androgens

Counts depend upon age, sex, altitude, exercise, drug, tobacco use etc.

Life span - 120 days

Clinical importance of assessment of RBC is to: measures oxygen carrying capacity of blood

Normal values

Newborn 4.1-6.1 million/mm3

Children 3.6-5.5 million/mm3

Adult (M) 4.6-6.0 million/mm3

Adult (F) 4.2-5.0 million/mm3

Decreased RBCsBlood loss Impaired production Increased destruction

• Trauma • Surgery• GI bleed• Gynecologic

al disturbance

• Pure red cell aplasia• Pernicious anemia• Megaloblastic anemia• Iron deficiency

anemia• Thalassemia• Anemia of

prematurity• Anemia of chronic

disorder

Intra-corpuscular• Hereditary spherocytosis• Sickle cell anemia• Abetalipoprotienimia• G6PD • Pyruvate kinase deficiency• PNHExtra-corpuscular• Autoimmune• Haemolytic disease of

newborn• Mismatch transfusion• Microangiopathic haemolytic

anemia TTP, HUS• DIC• infections

• Increased RBCs

• Polycethemia vera • High altitude• chronic obstructive pulmonary disease(COPD,

emphysema, chronic bronchitis),• pulmonary hypertension,• Hypoventilation syndrome,• congestive heart failure• obstructive sleep apnea,• poor blood flow to the kidneys, and

Haemoglobin

Oxygen carrying component of blood

Synthesize in polychromatic normoblast stage of red cell development

Values

Newborn 15.5-24.5 g/L

Adult male 13.5-16.5 g/L

Adult female 12.0-15.5 g/L

Hb estimation

Cynemethamoglobin Method: Recommended 20 microL blood + diluent (potassium cynide

and potassium fericynide) Mixed and read in photo colorimeter

Photo colorimeter is used to determine the concentration Hb% =(test sample absorbance/ standard sample

absorbance)x concentration of standard x dilution factor

Advantage – haemoglobin, methamoglobin and carboxyhaemoglobin are used in measurment.

Disadvantage – sulphamethamoglobin cannot be included in measurment, takes more time for estimation

Hb estimation: other method

Sahli acid haematin

Alkaline haematin method

Sulphahaemoglobin method

Oxyhaemoglobin method

Reticulocyte

Normal value 0.5% - 1.5%.

Hence 0.5% - 1.5% RBCs are replaced per day

Uses

To evaluate anemia

Response to treatment of anemia

Note

If the disease causing the anemia is inside the marrow, the reticulocyte count is decreased

If the disease causing the anemia is outside the marrow, the reticulocyte count is increased

Methods

Manual reticulocyte count using supravital stain Automated reticulocyte count by flouroscent

method - gives immature reticulocyte fraction (IRF) and removes errors like Howell-Jolly bodies, pappenheimer bodies

Reticulocyte production index or corrected reticulocyte count: an index corrected according to level of anemia Reticulocyte index = reticulocyte count x

patient’s haematocrit/ normal haematocrit

Reticulocyte proliferation index: Index is used to determine if a person's bone marrow is properly responding to the body's need for red blood cells

Shift correction factor: normal reticulocyte count survive 3.5 days in marrow and 1 day in peripheral circulation at normal PCV. In case of variation in PCV the survival time is increased which is termed as shift correction factor Reticulocyte proliferation index =

reticulocyte index/ shift correction factor

Shift correlation factor

PCV% Maturation days = shift correction factor

45 1

35 1.5

25 2

15 2.5

Increased reticulocyte count Haemolytic anemia

Recent haemorrhage

Thalassemia

Pregnancy

Response to treatment

Hypoxia

Leukamia

Decrease reticulocyte count Aplastic anemia

Megaloblastic anemia

Anemia of chronic disease

Cirrhosis

Radiation

Decrease ACTH and pitutary hormones

Reticulocyte haemoglobin measurement (RET-He)

Reticulocyte Hemoglobin (Ret-He) is a direct assessment of the incorporation of iron into erythrocyte hemoglobin.

It is a direct estimate of the recent functional availability of iron (2–3 days).

Traditional chemistry tests used for iron assessment (serum iron, Tsat, ferritin) are indirect measurements.

As a direct measurement, Ret-He may identify iron deficiency earlier than traditional parameters.

It is an established parameter used in KDOQI (Kidney Disease Outcome Quality Initiative) guidelines for assessing iron status

Haematocrit

Ratio of the volume of erythrocytes to that of the whole blood in percentage

Most precise method for determining the degree of anemia or polycythemia i.e. increase or decrease RBC concentration

Normal values

Newborn 42-68%

Upto 1 year age 29-41%

Adult Male 39-47%

Adult female 36-44%

Rule of 3:– RBC x 3 = Hb and Hb x 3 = Hct

High Polycythemia vera

Dehydration

Low oxygen in blood

Congenital heart disease

Cor pulmonale

Smoking

Haemoconcentration (Dengue)

Low Anemia

Blood loss

Haemolysis

Bone marrow aplasia

Leukamia

Malnutrition

An elevated haematocrit may be due to spleen hyper function, and reduced haematocrit may indicate low thymus function

Mean corpuscular volume Measures average volume of RBC

MCV = haematocrit/ red cell count x100

Normal values

Newborn 103-106fL

Child upto 1 year 78 fL

Adult 79-98fL

Classified accordingly as

Microcyte – MCV <79

Macrocytic – MCV >98

Presence of microcytic and macrocytic cells in same sample may result in normal MCV

MCV <72 without heterogeneity, is a sensitive and specific predictor of thalassemia trait

Microcytic MCV Hypochromic

Iron deficiency

Thalessemia

Lead poisoning

Porphyria

Normochromic Anemia of

chronic disease

haemoglobinopathies

Macrocytic MCV


Pernicious anemia

Sprue

Di Gulielmo disease

MDS

Post spleenectomy

Alcoholism

Liver disease

Drugs (anticonvulscents, anticancer etc)

• Normocytic MCV

• Acute haemorrhage

• Diamorphic anemia

• Haemoglobinopathies

• Endocrinopathies

Interference in MCV

Cold and warm antibodies

Marked hyperglycemia

Marked leukocytosis

Marked reticulocytosis

Methanol poisoning

Mean corpuscular haemoglobin

MCH = haemoglobin/ red cell count x 100

Normal range

Newborn 36-38%


Adult 26.7-31.9%

MCH decreased in

Microcytic and normocytic anemias

MCH increased in

Macrocytic anemias

Infants and newborns

Interference in MCH

Lipemia

Marked leukocytosis

Cold agglutinin

Monoclonal protein in blood

Mean corpuscular haemoglobin concentration

MCHC = haemoglobin/ haematocrit x 10

Normal range

Newborn 34-36%


Adult 32-36%

MCHC decreased in

Hypocromic microcytic anemia

MCHC increased in

Heridietery spherocytosis

Infant and newborns

Autoagglutinations

Interference in MCHC

Marked leukocytosis

Haemolysis

Cold aggutinins

Rouleaux

Red cell distribution width (RDW)

Red cell distribution is a quantative measure or numerical expression of anisocytosis. It is a coefficient of variation of the distribution of individual RBC volume

In microcytes, RDW increased in iron deficiency anemia but in thalessemia it is not raised

RDW-CV:It is the ratio of standard deviation to the mean corpuscular volume

RDW-CV = standard deviatiom of RBC volume/ mean MCV x 100

value 11.5%-14.5%

RDW-SD: It is the actual measurnment of the width of the RBCdistribution curve

Values 35-45 fL

RBCs on peripheral smear

Preparation

The wedge slide (push slide) technique was developed by Maxwell Wintrobe as is a standard method

The “zone of morphology” (area of optimal thickness for light microscopy examination) should be at least 2cm in length. The smear should occupy the central area of the slide and be margin free at the edges.

RBCs in peripheral smear

Microcytic hypochromic

Size smaller than the nucleus of small lymphocyte

< 7 micron

Markedly increase central pallor >1/3 of the diameter of RBC

Causes

Iron deficiency anemia

Thalassemia

Sideroblastic anemia

Anemia of chronic disease

Haemoglobinopathies

Macrocytic cells

Size > 8.3 micron kin diameter

Causes

Vitamin B 12 and folic acid deficiency

Alcoholism

Liver disease

Myleodysplastic syndrome

Hypothyroidism

Drug that impair DNA synthesis

Oval macrocytes

Vitamin B 12 and folic acid deficiency

Pernicious anemia

Myleodysplastic syndrome

Hypothyroidism

Drug that impair DNA synthesis

Round hypochromic macrocytes

Alcoholism

Hypothyroidism

Liver disease

Post splenectomy

Blue tinged macrocytes

Neonate

Response to anemic stress

Target or bell cell

They have a characteristic ringed appearance. This is because of the “increase surface area to volume ratio” i.e. increase in red cell membrane which get pooled at the centre of cells

Causes

Thalessemia

Haemoglobinopathies Hb AC or CC, HbSS,SC

Liver disease

Post spleenectomy

Severe iron deficiency anemia

abetalipoprotenimia

Schistocytes ‘Schisto’ = split or cleft

Physical assault to erythrocytes with in the blood stream creates these cells

which include

Helmet cells

Triangles

Crescents

Microspherocytes

Horns

Purse

Causes

DIC

Severe haemolytic anemia

Microangiopathic haemolytic anemia

HUS &TTP

Prosthetic cardiac valves

Connective tissue disorders

Burns

Acute tubular necrosis, glomerulonephritis

Malignant hypertension

Tear drop cells(Dacrocytes)

Pear shape cells, usually microcytic and hypochromic

Seen in

Newborn

Thalassemia major

Myleoproliferative disorder

Leukoerythroblastic reaction

Spherocytes

Ball shaped red cells, decreased surface: volume ratio, hyperdense (> MCHC)

Seen in

Hereditary spherocytes

ABO incompatibility

Autoimmune haemolytic anemia

Microangiopathic haemolytic anemia

SS disease

Hyperspleenism

Burns

Posttransfusion

Elliptocytes

Elliptical and normochromic cells, seen normally in less than 1% of RBCs

Causes

Hereditary elliptocytosis

Iron deficiency anemia (increased with severity)

SS disease and SA trait

Thalassemia Major


Malaria


Burr cells (Echinocytes)

10-30 spicules equal in size and evenly distributed over RBC surface; caused by alteration in extracellular environment

Seen in

Liver disease

Renal failure

Dehydration

Pyruvate kinase deficiency

Storage artefacts

Spur cell - Acanthocyte

Acantho = thorn

Cells with 5-10 specules of varying length, irregular in shape, thickness, with wide bases and appear smaller than normal cell because they assume spheroid shape

Result from changes in membrane lipid content

Seen in Spur cell anemia

Alcoholism

Hypothyroidism

Abetalipoprotinemia

Vitamin E deficiency

Malsbsorption

Postsplenectomy

Bite cell (Degmacyte)

Appear as a cookie with a bite taken out

Seen in G6PD

When spleen removes the Heinz bodies from RBCs

Stomatocyte

When examined on dry smear, it has a central slit or stoma

Seen in

Few may be seen normally

Various cardiovascular

and pulmonary disorders

Hereditary

Alcoholism

Liver disease

Malignancies

Howell – Jolly bodies

Small well defined, rounded, densely stained inclusions, 1 micron in diameter, ecentric, that represent DNA fragments

Associated with rapid or abnormal RBC formation

Seen in

Post spleenectomy

Newborns


Dyserythopoietic anemias

Hereditary spherocyosis

Heinz Bodies

Inclusion of denatured haemoglobin caused by oxidation of globin portion of haemoglobin

Removal of Heinz body leads to formation of ‘bite cells’

Causes

Drugs

Certain foods like fava beans

and onion

Sideroblastic granules/ pappenheimer bodies

Irregular dark blue iron containing granules occuring in small clusters, predominantly in periphery

Seen in

Sideroblastic anemia

Spleenectomy

Haemolytic anemia

Myelodysplastic syndromes

Lead poisoining

Its presence can rule out iron deficiency anemia

Sickle cell

Crescent shape cells develop in

people homozygous for haemoglobin S

Heterozygous HbS and either thallasemia or another Hb like Hb C

Nucleated red cells

Cells have dense dark nucleus in the center of the cell

Results from marked stimulation of the bone marrow

Seen in

New born (first 3-4 days)

Acute bleeding severe haemolytic anemia


Congenital infections (syphilis, CMV, rubella)

Postspleenectomy


Fungal and mycobacterial infections

Dyselectropoeitic anemia

Basophilic stippling

Numerous small, purplish inclusions, which results from RNA and mitochrondrial remenants

Seen in

Lead toxicity

Thalessemia

Haemoglobinopathies

Macrocytic anemia

Cabot ring

These are delicate thread like inclusions, remenants of the nuclear membranes, in the RBC

They can take any shape like purplish ring, figure of eight, incomplete ring

Seen in

Pernicious anemia

Lead poisoning

Alcoholic jaundice

Severe anemia

Leukamia

Roulex formation

A stack like arrengment of red blood cells where the biconcave surdface of RBCs are next to each other.

Seen in

Increase in cathodal protien,

such as immunoglobins

and fibronegen

Multiple myleoma

Macroglobulimias

Acute and cronic infections

Connective tissue disease

Diabetes mellitus

Malignancies

Grading of inclusions

Rare 0-1/hpf

Few 1-2/hpf

Mod 2-4/hpf

Many >5/hpf

Qualitative grading of abnormal RBC morphology

Grade degree of abnormalities

1-5 cells /10fields slight

6-15cells /10fields moderate

>15cells /10fields marked