AUTOMATED CELL COUNTING

The principle, advantages and limitations of the Automated cell counters.

Automated Blood Cell Counters

Automated blood cell counters have come to stay in a modern Haematology laboratory because
they provide data with increased reliability, precision and accuracy.

The Automated blood cell counters available today are based on two principles

A-Electrical Impedance

B-Light Scatter

Electrical Impedence

Cells passing through an aperture through which a current is flowing cause changes in electrical
resistance that are counted as voltage pulses. This principle is illustrated in Figure 5.4.

Fig. 5.4: Schematic diagram of particle counter in which changes in electrical resistance are
counted as voltage pulses. CS = cell suspension,
GC = glass cylinder, A = aperture, E1 and E2 = platinum electrodes, V = valve, M =
mercury column, EC1 and EC2 = electrical contacts VP = vacuum pump.

Each cell that passes through the aperture displaces an equal volume of conductive fluid,
increasing the electrical resistance and creating the voltage pulse, because its resistance is much
greater than that of the conductive solution. The pulses, which are proportional in height to the
volume of the cells are counted. This is the “Coulter principle”.

In the simplest system, the counting mechanism is started when the mercury contacts EC1 and
stopped when it contacts EC2; during this time the cells are counted in a volume of suspension
exactly equal to the volume of glass tubing between contact wires EC1 and EC2. If two or more
cells enter the aperture simultaneously, they will be counted as one pulse; this produces a
coincidence error that must be corrected. The size of the coincidence error can be diminished by
decreasing the concentration of cells and decreasing the size of the aperture.

Light Scatter

In the electro-optical analysers (Fig. 5.5), a light sensitive detector measures light scattering.
Light is focused on the flow cell. Only light scattered by a cell reaches the photomultiplier tube
(PMT), which converts it to an electrical pulse. The size of the pulse detected is proportional to
the size of the particle.

Fig. 5.5: Schematic diagram of the electro-optical cell counter. Light is focused on the flow
cell. Only light scattered by a cell reaches the photomultiplier tube (PMT), which converts
it to an electrical pulse.

The erythrocyte / platelet channel determines RBC and Platelet counts by the analysis of light
scattering measurements obtained as the diluted cells pass singly through a helium-neon laser
beam. The RBCs are counted and sized by both high-angle and low-angle light scattering
measurements and the MCV is thus determined.

Within the hemoglobin channel, a portion of EDTA anticoagulated blood is mixed with the
hemoglobin diluent. The RBCs are lysed and free hemoglobin is converted to
cyanmethemoglobin. The concentration of hemoglobin is determined photometrically at 546 nm.

The leucocyte count and five – part differential count are obtained using two different methods
and two separate channels, the peroxidase channel and the basophil / lobularity channel. In the
peroxidase channel, neutrophils, monocytes, and eosinophils are identified by the degree of
peroxidase positivity and the amount of forward light scatter. Lymphocytes and the large
unstained cells (LUCs) are identified by the amount of forward light scatter and the fact that they
remain unstained by this peroxidase cytochemical staining method. RBCs are removed before
peroxidase staining by lytic action. The amount of forward scatter and degree of positivity are
detected as the cells pass through a tungsten - based flow cell. Within the basophil / lobularity
channel, a fourth portion of EDTA – anticoagulated blood is mixed with basophil diluent. The
basophil diluent lyses RBCs and platelets and strips all WBCs except basophils of their
cytoplasm. This dilution is measured by the helium – neon laser flow cell. Basophils will have
large, low angle scatter and the remaining cell nuclei will be classified as mononuclear or
polymorphonuclear based on their high angle scatter.
BLOOD FILM MORPHOLOGICAL EXAMINATION
Erythrocyte Morphology:
(1) Study the erythrocytes and report any evidence of rouleaux formation or
signs of immaturity.
(2) Report the erythrocyte morphology with reference to size, shape,
staining characteristics, and inclusions. Report the degree of the specific morphological
characteristic (for example, moderate hypochromia).
(3) If nucleated erythrocytes are found (usually these are megaloblasts),
report the number per 100 leukocytes counted.

Qualitative Platelet Evaluation:

(1) Observe the thrombocytes in several oil immersion fields to obtain a
rough estimation of their numbers (normal, increased, or decreased). Normal is an
average of 8 to 10 per oil immersion field.
(2) Note any abnormality in morphology (giant platelets, etc.) If the
thrombocytes appear to be significantly decreased, a thrombocyte count and/or a clot
retraction test may be indicated.
Discussion.
(1) All abnormal white cells (for example, immature, hypersegmented, toxic,
atypical lymphocytes, etc.) should be classified or described and reported in percent,
separately. Cells that are ruptured, fragmented, or degenerated are not included in the
differential count, but should be noted separately and reported as the number seen per
100 leukocytes.
(2) In view of the gradual transition from the metamyelocyte to the banded
neutrophil and then to the segmented neutrophil, exact classification is sometimes
difficult. In such cases, classify the cell according to the more mature form.
(3) Size considerations in differentiating blood cells require a defined linear
standard. The micron (.001 mm) is usually used in reference to microscopic
dimensions. Ocular micrometers are available through Federal medical supply
channels and are easily calibrated, using a hemacytometer that has standardized
dimensions. In routine screening of blood smears, an experienced technician relates
the size of a normocytic erythrocyte (seven to eight microns) to the size of the white cell
to be differentiated, since erythrocytes are usually present throughout the microscopic
field. Finally, it should be understood that personal visual discrimination is an
inaccurate gauge of linear measure. Some reference measure should be employed.
(4) The shape of blood cells often depends upon the smear and staining
technique. Variations that have no clinical significance occur from physical and
chemical distortions that result from technical error. These variations are avoided with
careful technique. Each routine smear should be scanned initially to evaluate the smear
and stain quality before differential analysis.
H/W: Define these terms
Normocytic/normochromic
Anisocytosis
Poikilocytosis
Hyperchromasia/hypochromasia
Erythrocytosis/erythrocytopaenia
Leucophilia/leucocytopaenia
Thrombocytosis/thrombocytopaenia
HEAMOGLOBIN FORMS + VARIANTS
Haemoglobin (Hb) is the main content of the red cell, which is a conjugated protein that gives
red colour to blood. Its main function is to carry oxygen to the tissues and carbon dioxide away
from tissues.
 Each haemoglobin molecule carries one molecule of oxygen and each gram of
haemoglobin can carry 1.34 ml of oxygen.
 Haemoglobin occupies approximately 33% of volume of the erythrocyte and accounts for
90% of its dry weight.
 Each RBC contains between 27 to 32 pico grams of Hb.
 About 6.25 grams of haemoglobin is synthesized each day to replace the haemoglobin
lost due normal destruction of erythrocytes.
 The main function of haemoglobin is to transport oxygen from the lungs where oxygen
tension is high to the tissues where oxygen tension is low. The oxygen carrying capacity
of blood depends to a large extent on the amount of haemoglobin it contains.
 A molecule of Hb consists of two pairs of polypeptide chains (globins) and 4 ring like
structures, each with one central atom of ferrous iron called haeme. Each polypeptide
chain of the globin chain is linked with one heme group. Depending on the composition
of the globin chain tetrad, there are three types of haemoglobin that occur in human
infants and adults.

Normal Haemoglobins

1. Haemoglobin A (adult haemoglobin) – contains 2  and 2  chains.

2. Haemoglobin A 2 – 2  and 2  chains.

3. Haemoglobin F (foetal haemoglobin) – 2  and 2  chains.

4. Gower 1 and Gower 2 Embryonic Hb

Abnormal Haemoglobins

These are formed from the abnormal polypepetide chain synthesis of the haemoglobin molecular
structure. They result from either switches, deletions or spontaneous mutations of the amino acid
sequences making up the polypeptide chain. Consequently, they result to abnormal hemoglobin
structures and hence a deranged haemoglobin molecule, which is impaired in its function, a term
to describe them is haemoglobinopathies. The type of Hb resulting from this complications
include:
Hb S, Hb Barts, Hb Heinz, Hb Thellasaemia

Acquired haemoglobin derivatives

a) Methaemoglobin – Here the iron is in the ferric state.
b) Sulphaemoglobin – It is formed when sulphur combines with the heme.
c) Carboxyhemoglobin – It is formed by the exposure of normal haemoglobin to carbon
 monoxide. It imparts a cherry red color to blood.
d) Glycosylated haemoglobin – It is a minor component of adult haemoglobin in which glucose
is irreversibly attached to the  chain.
e) Haptoglobulins-

Methods of Estimation of Haemoglobin

Haemoglobin can be measured by any of the following methods,

utilizing different principles.

a) Calorimetric method – based on color

b) Physical method – based on specific gravity

c) Chemical method – based on iron content of Hb.

d) Gasometric method – based on oxygen combining capacity of Hb.

a) Calorimetric Methods

These are based on measuring the color of haemoglobin or a derivative of haemoglobin in blood.
The estimation is based on Beer’s law i.e. the optical density (OD) of a colored solution is
directly proportional to the concentration of the colored material in the solution.

The common colorimetric methods are:

i-Sahli’s method or Acid hematin method

ii-Alkaline Hematin method

iii-Photoelectric method

1) Cyanmethemoglobin method
2) Oxyhemoglobin method

i-Sahli’s Method or Acid Hematin method

Principle

Haemoglobin is converted to acid hematin by N/10 HCl, the resulting brown color is
compared with standard brown glass.

Instruments
  Sahli’s Hemoglobinometer – the main parts of which are a graduated glass tube, color
comparators, glass stirrer and Sahli’s pipette to measure 20 cu mm of blood.
 The tubes commonly used are square with graduations in percent on one side and grams
per 100 ml on the other.
 The color comparators are made of brown colored glass

Requirements

 N/10 Hydrochloric acid (HCl)

 Distilled water for dilution.
 Blood anticoagulated with EDTA

Procedure

 Place N/10 HCl in the tube up to the lowest mark.

 Draw blood up to 20 cu mm mark in the pipette and transfer it to the acid in the tube.
 Rinse the pipette well by drawing up the acid re-expressing it.
 Allow it to stand for at least 10 minutes - to allow brown color to develop due to the
formation of acid hematin.
 95% of Hb is converted at the end of 10 minutes, 98% of the color develop at the end of
20 minutes, and the maximum color is reached after about 1 hour.
 Now dilute the solution with distilled water adding a few drops.
 Mix the solution well after each addition of water using the glass rod provided.
 Match the color with that of the glass plates in the comparator.
 While comparing take care not to leave the glass rod inside the glass tube.
 Reading is taken when the color of the solution in the tube exactly matches the
comparator.
 The level of the fluid at its lower meniscus is noted.

Sources of Error

 Technical errors – Most of the errors described under Cyanmeth Hb method discussed later
also occur in this method.
o E.g. Improper mixing of blood, errors in pipetting, tissue fluid contaminating
capillary blood.
 Visual errors – Taking the reading is very subjective.
 Quality of the color comparators can affect the reading
  Insufficient time allowed for the conversion of Hb to acid hematin.
 Other forms of hemoglobin: Carboxyhaemoglobin, methaemoglobin and
sulphhemoglobin are not converted to acid hematin.
 Time delay – The brown colour of acid hematin is not stable, so undue delay in reading
the test result is not allowed.

ii-Alkaline Hematin method

In this method, the Hb is converted to alkali hematin by the addition of a strong alkali (N/10
NaOH).

iii-Photoelectric method

1- Cyanmethaemoglobin Method

This is the prefered and the most accurate method for determining the maemoglobin
concentration. It is the standard method used in most of the countries.

In this method, Blood is diluted in a solution of potassium ferricyanide and potassium cyanide.
The ferricyanide oxidizes haemoglobin to hemiglobin (Hi,) or methaemoglobin. Potassium
Cyanide provides cyanide ions (CN–) to form hemiglobincyanide (HiCN) or Cyanmethaemo-
globin. The absorbance of the solution is then measured in a spectrophotometer at a wavelength
of 540 nm.

B) Specific gravity method (Physical method)

Haemoglobin being the largest single constituent, affects the specific gravity of blood more than
other substances. Serum proteins are the next heaviest constituents of blood. It is assumed that
(which is not always true) the level of serum proteins and other smaller constituents remain the
same, so any change in the specific gravity of blood is mainly due to change in concentration of
haemoglobin.

Allow a drop of blood to fall into a series of solutions of copper sulphate of varying specific
gravity and note the behaviour of the drop and estimate the specific gravity of blood. If the drop
sinks to the bottom, its specific gravity is more than that of the copper sulphate solution. If the
drop rises after its initial fall then its specific gravity is less than that of the copper sulphate
solution.

C) Chemical methods (estimation of the iron content)

The principle is based on the fact that each molecule of hemoglobin contains 4 atoms of iron or
0.347 grams of iron per 100 grams of hemoglobin. The iron present is detached from the
hemoglobin and measured. The hemoglobin is calculated by using the formula.
Hemoglobin (gm/dl) =

Though this is very accurate, it is a complex and very time-consuming test. So it is not done
as a routine procedure. It is used only as a reference method i.e. a method used to check the
accuracy of the other methods.

D) Gasometric methods (Measurement of Oxygen combining capacity)

This is a reference method as it is very accurate, however it is not used for routine laboratory
work. The principle is based on the fact that one molecule of O2 binds to each iron atom. So one
molecule of haemoglobin binds 4 molecules of oxygen. Thus oxygen combining capacity thus
indirectly measures the amount of Hb.

It is estimated that 1 gram of haemoglobin binds about 1.34 ml of oxygen. From this the
haemoglobin concentration is calculated by using the following formula

Hb in gm/dl =

Errors in Haemoglobin Estimation

1-Improper sampling

Improper venipuncture or capillary sampling produce, hemodilution or hemoconcentration.

2-Method used

Sahli’s method has an error of 5% to 20%, CUSO4 method is inaccurate while the
cyanmethhemoglobin method is the most accurate.

3-Equipment / Apparatus

The pipette, cuvette and photometer need proper calibration and appropriate checking
periodically to eliminate the errors due to them.

4-Human errors

Human errors can be reduced by good training, understanding the clinical significance of the test
and the necessity for a dependable method, adherence to oral and written instructions and
familiarity with the equipment and with sources of errors. Automated instruments are widely
used now-a-days and this eliminates most of these errors.
Quality Control

The important aspect of quality control is to identify those steps in which the likelihood of error
is high and to consider ways to minimize that likelihood. Some of the measures followed are:

1. Duplicating samples.
2. Haemolysate of known value are run with batches of tests.
3. Haemoglobin values are compared with other values.

Normal Values

The normal value depends on the age and sex of the individual

Men – 15.5  2.5 gm/dl

Women – 14  2.5 gm/dl

Full term / cord blood – 16.5  3 gm/dl

Children 1 year – 12  1 gm/dl

Children 10 - 12 years – 13 1.5gm/dl

Significance of haemoglobin estimation

a) Decrease in the haemoglobin below the normal range is an indication of anemia.

b) Causes for increase in the haemoglobin concentration

1. Hypoxic states – high altitudes, emphysema, congenital heart disease.

2. Increased secretion of erythropoetin – some lung cancers, renal cancers.
3. Polycythemia vera – It is a myeloproliferative disorder.