Reviewer ( Hema Lab )

Topic 6: RBC COUNT

Red Blood Cell

Compromises almost 45% of the human blood

It is responsible for the transport of oxygen and nutrients throughout the body

Principle

To dilute the blood in an isotonic fluid without damaging or lysing the cells

Computation

# of cells counted x Correction for dilution x correction of volume x 10^6

Where in:

Dilution: 1 : 200

Correction for volume: 50

Normal Values

Male

4.2 - 5.4 x 10^12 / L

Female

3.6 - 5.0 x 10^12 / L

Newborn

5.5 - 6.5 x 10^12 / L

Topic 7: Hemoglobinometry

Hemoglobin

Is an iron containing protein molecule found in Red Blood Cells which transport
oxygen from the lungs to the body’s tissue

In Adult Human Body there are 3 kinds of normal hemoglobin molecules

HbA1 ( A2 B2 )

96 - 98%

HbA2 ( A2 D2 )

1.5 - 3.2%
 1.5 - 3.2%

Hb F ( A2 Y2 )

0.5 - 0.8%

Can be tested manually or using n automated machine

It is important to diagnose the patient for possible anemia or polycythemia

Hemoglobincyanide Method

Is the internationally recommended for determining the hemoglobin

concentration of the blood

Reference method for Hb estimation cause:

All Hb forms except sulphaemoglobin are estimated

Highly reliable and stable reagents are available

The reagent is sensitive to light

It contains cyanide, making it highly toxin

The method can be easily standardized

It takes an hour to convert in heavy smokers

However, the value increased is not clinically significant

Principle

Blood is diluted in Drabkin’s Solution…

Test Sample

Venous or capillary blood collected into an EDTA tube

Sources of Error

Turbid samples due to increased wbc or platelet may cause false increase result

This can be corrected by centrifugation of the samples and testing the

supernatant

Another reason for turbidity is lipemic specimen

To correct this, add 0.1 of Patient’s plasma to 5 ml of reagent and use the
solution as blank

HbS and HbC can be resistant to hemolysis and cause turbidity

To correct this, add 1:2 solution of diluted sample water, then multiply the
 standard cure by 2

Addition of potassium carbonate are used to correct abnormal globulins

Abnormal Hemoglobin in Adults

Carboxylhemoglobin

Hemoglobin + carbon monoxide

Methemoglobin

Hemoglobin in which ferrous iron are oxidize to ferric states

Sulfhemoglobin

Irreversible formation of from sulphonamides and aromatic

amines

Normal Ranges

Male

13 - 17 g / dl

Female

11.5 - 16.5 g / dl

Newborns and infant

14 - 22 g / dl

Topic 8: Hematocrit Determination

Hematocrit

The packed red cells of red blood cells

Constitute to the volume of red blood cells over the volume of whole blood

Principle

Constitute of the use of centrifuge o separate red blood cells from whole blood
to its maximum packaging to measure the percentage expressed red blood cells
over the whole blood

Normal Values

Neonates

45 - 60%

1 year old above

24 - 44%
 24 - 44%

Women

36 - 48%

Men

40 - 55%

Methods

Micro hematocrit

Macro hematocrit

Rarely used for it is time and sample consuming

Make used of wintrobe tube nd centrifuged at the 2000 -

2300 from 30 minutes

Topic 9: Peripheral Blood Smear

Blood Smear

Can be a key to different picture in the blood film of the patient

Types of Blood Smear

Cover Glass smear

More even distribution of WBC but they are harder to master, too small for
automated strainers, and easily broken

Wedge Smear

Most commonly used blood smear in the laboratory

Components

The feathered Edge ( Head )

This is the end of the blood smear

Monolayer ( Body )

This is the optimal area for examination of cells

Base of the smear ( Tall )

These areas are usually too thick to evaluate cells

under high power

Spun Smear

More easily handled and labelled with properly distributed WBC and RBC
 More easily handled and labelled with properly distributed WBC and RBC

Buffy Coat Smear

Buffy coat is used instead of whole blood in smear preparation

This is done to verify if there is leukopenia or thrombocytopenia

Thick Blood Smear

This is usually done in identification of blood parasites such as malaria,

babesia..

Stains used in hematology laboratory

Romanovsky Stains

Contains methylene blue and eosin

Wright’s Stain

Methylene Blue and eosin azure

Giemsa Stain

Eosin Y and azure B

Wright’s - Giemsa Stain

Leishman Stain

Jenner Stain

May - Grunwald Stain

Methanol

Fixative or stabilizer

Counting Method

Cross Sectional / crenellation

Longitudinal Method

Battlement Method

Things to Examine in a Blood Smear

Check first the condition of blood smear if this is well - made

Perform WBC differential count

Scan for abnormal blood cells

Count 100 WBC in the smear, categorizing each based on its identification
 Count 100 WBC in the smear, categorizing each based on its identification

If there are presence of nRBC, do not include them in the 100 WBC’s that you
counted, instead, take note of how many nRBC’s you encountered while
completing he 100 WBC, then report it as numbers of nRBC’s / 100 WBC

Examine the RBC morphology

Presence of poikilocytes

Presence of inclusions and abnormalities

Presence of immature RBC’s

Take note the size and pallor of RBC’s

Rouleaux and Agglutination

Polychromatophilia

Examine the morphology and number of platelets

Computation for absolute differential count

Absolute # cells / L = % of cell type in differential x WBC / L

Topic 10: Erythrocyte Sedimentation Rate

Erthrocyte Sedimentation Rate

Is a non specific test that indicates changes In plasma protein concentrations

due to infection or injury

It is the rate at which the red blood cells settles in a tube over a given amount of
time

Factors affecting ESR

Plasma Protein

Changes in plasma protein occur rapidly following tissue injury or in

response of inflammation

RBC Size and Number

The size and number of RBC’s that show alterations in their biconcavity,
usually do not increase the ESR rate

Technical Factors

Tilted ESR tube

ESR tube should always be in perpendicular position

Temperature
 Temperature

Temperature higher than room temperature accelerates

sedimentation

Vibration

Can reduce the rate of erythrocyte sedimentation

Methods of Measuring ESR

Westergren method

Recommended

Principles

It measures the sedimentation of red cells after 1 hour in an

open ended tube mounted vertically on a stand

Wintrobe and Landsberg Method