Introduction

Clinical laboratory in the 21st generation has evolved from simple observation and
description of blood and its components to a highly automated and extremely technical
science. However, some of the more basic tests have not changed dramatically over the
years. This research presents the significance of manual and semi automated methods that
can be used in lieu of automated instrumentation.
Although most routine cell-counting procedures in the laboratory are automated,
it may be necessary to use manual methods when counts exceed the linearity of an
instrument, when an instrument is nonfunctional and there is no backup, in remote
laboratories, or in a disaster situation when testing is done in the field.
The used of automated cell-counting analyzers has directly affected the
availability, accuracy, and clinical usefulness of the CBC and WBC differential count.
Some parameters that are available on laboratory instrumentation, but cannot be derived
manually, have provided further insight into various clinical conditions.
This research emphasizes the procedures for manual counting of leukocytes.
Manual cell counts and differentiation were originally the only means of enumerating and
classifying cellular elements in blood and body fluids. To a large extent, the laboratory
evaluation of leukocytes, erythrocytes, and platelets has now been automated. If
automated leukocyte is available, why must laboratory scientist learn manual techniques
for evaluating them? There are several reasons.

Thesis statement
Manual computation is relevant in counting white blood cell in patient.

Objectives
1. Emphasize how and why manual and semi-automated method would be
convenient in a laboratory in counting white blood cells in patients blood.
2. Ensure that the use of manual and semi-automated method in WBC count is
accurate.

Overview of the white blood cells

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Importance of white blood cells

The primary function of leukocytes as a group is defense against any invader into
the body that may be recognized as foreign. Each cell type has a specific function and
behaves as a separate but related system. Five principal types of leukocytes normally
circulate in peripheral blood: neutrophils, eosinophils, basophils, monocytes and
lymphocytes. Several different criteria may be used to classify leukocytes. According to
granularity leukocytes may be classified as granulocytes (neutrophils, eosinophils,
basophils) and nongranulocytes (monocytes and lymphocytes). The granulocytes contain
distinct granules in their cytoplasm when stained and examine on a routine smear,
whereas nongranulocytes have no cytoplasmic granules and have as small, rounded
nuclei.
Neutrophils are the most common PMN and are produced in 7 to 14 days and
remain in the circulation for 6 hours. The primary function of the neutrophil is
phagocytosis, killing and digestion of bacterial microorganisms. Acute bacterial
infections and trauma stimulate neutrophil production, resulting in an increased WBC
count. Basophils also called as mast cells and especially eosinophils are involved in the
allergic reaction. They are capable of phagocytosis of antigen-antibody complexes. As the
allergic response diminishes the eosinophil counts decreases. Lymphocytes are divided
into two types: T cells which mature in thymus and B cells which mature in bone marrow.
T cells involved primarily with cellular-type immune reactions, whereas B cells
participates in humoral immunity. The primary function of lymphocytes is to fight
chronic bacterial infection and acute viral infections.

Interfering factors that may lead to increase and decrease of white blood cell in patient

Factors that may lead to increase and decrease of white blood cells
Eating, physical activity and stress may cause an increased WBC count and alter
the different values. Final months in pregnancy and labor may be associated with
increased WBC levels. Patients who have had a splenectomy have a persistent wild to
moderate elevation of WBC counts. The WBC count tends to be lower in the morning
and higher in the late afternoon. The WBC count tends to be age-related. Normal
newborns and infants tend to have higher WBC count than adult. Drugs that may cause
increased WBC levels include adrenaline, allopurinol, aspirin, chloroform, epinephrine,
heparin, quinine, steroids and triamterene. Drugs that may decrease WBC levels include
antibiotics, anticonvulsants, antihistamines, antimetabolites, antithyroid drugs, arsenicals,
barbiturates, chemotherapeutic agents, diuretics, and sulfonamides.

Procedures that may take place in drawing white blood cell count in patients
Before the test is taken, explain the procedure to the patient and tell the patient
that no fasting is required. During the actual test is taken, Collect approximately 5 to 7 ml
of venous blood in a lavender-top tube. After the test is taken, apply pressure to the
venipuncture site.

Meaning of an increased WBC count

Infection: WBCs are integral initiating and maintaining the bodys defense
mechanism against infection.
Leukemia neoplasia or other myeloproliferative disorders: These neoplastic cells
are produced by the marrow and are released into blood stream.
Other malignancy: Advanced non-marrow cancers (e.g. lung) are associated with
leukocytosis.
Trauma, stress, or hemorrhage: the WBC count is probably under hormonal
influence (e.g. epinephrine).
Inflammation: the pathophysiology of this observation is complex, including
recognition of neurotic or normal tissue as foreign so that a WBC response is instituted.
Dehydration: not only is dehydration a stress that, itself, increases the WBC
count, but also by virtue of hemoconcentration, the WBC count increases.
Thyroid storm: the WBC is probably influence by thyroid hormones.
Steroid use: glucocorticosteriods stimulate WBC production.

Meaning of a decreased WBC count

Drug toxicity

Bone marrow failure

Overwhelming infections
Dietary deficiency
Congenital marrow aplasia
Bone marrow infiltration
Autoimmune disease
Hypersplenism

Manual method of white cell count

Principle
Whole blood is mixed with a weak acid solution to dilute the blood and hemolyze
the red blood cells. Then loaded into a Hemacytometer and counted.
Specimen
Whole blood may be obtained from a venous EDTA sample or a free flowing
capillary puncture and diluted 1:100 with a Unopette.
Reagents
A prepared kit is available that uses the Unopette system for white blood cell
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counts. The reservoir contains 3% glacial acetic acid. A 25 ul capillary pipette is used to
aspirate the blood sample and make a 1:20 dilution in the reservoir. (Alternatively, the
Unopette containing ammonium oxalate and result in a 1:100 dilution may be used).

Procedure
(1) Dilute the specimen - let stand for 10 minutes to allow red cells to hemolyzed.
(2) Expel first 3 to 4 drops of diluted specimen to clean capillary bore.
(3) Charge the Hemacytometer (both sides) with the diluted specimen.
(4) Cells must settle for a minimum 3 to 5 minutes after placing Hemacytometer
in moist chamber.
(5) Count white blood cells. Use low power objective and low light. Viewed under
low power, leukocyte nuclei appear slightly iridescent but not retractile; cells should have
a visible cell wall and nucleus; use fine focus to differentiate them from artifacts. Count
all WBCs within the 9 large squares (1:100) and those WBCs touching upper and righthand perimeter lines. Count second side of Hemacytometer in the same manner.
Validation that each side of the chamber was charged equally - Total number of cells
counted on each side of the counting chamber should agree within 10 percent of each
other - calculate acceptable range using lower count.
Calculation

The depth of the counting chamber is 0.1 mm and the area counted is 4 sq mm (4
squares are counted, each with an area of 1.0 sq mm therefore, 4 X 1.0 sq mm = a total of
4 sq mm). The volume counted is: area X depth = volume. 4 mm2 X 0.1 mm = 0.4
mm3 (cubic millimeters).
Here is the formula:

Sources of Error
Improper collection of blood specimens causes variable results.
Wet or dirty pipets.
Not allowing cells to settle for an adequate amount of time.
Poor pipetting technique causes high or low counts. Poor pipetting technique
includes:
Undershooting Unopette with blood.
Overfilling Unopette with blood.
Air bubbles in the shaft.
Not mixing the blood specimen thoroughly.
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Failure to expel 3 or 4 drops in the pipet tips before charging the Hemacytometer.
Overfilling the chamber of the hemacytometer, which causes erroneously high counts.
Not mixing the diluted specimen prior to filling the Hemacytometer.
Uneven distribution of cells in the counting chamber causes erroneous results.
Counting artifacts.
Dirty or scratched Hemacytometer.
Failure to mix anticoagulated blood thoroughly before use.

Evolution of automated cell counters

Traditionally, the blood counts were performed manually using the
hemocytometers and the differential leukocyte counts by studying the peripheral blood
smears (also referred to as the 100-cell slide differential, eye count leukocyte differential
or manual counts). The Coulter principle led to the availability of the Coulter counters
and thereafter, the development of sophisticated automated blood-cell analyzers. The
level of sophistication has been rising ever since. Technological advancements have made
it possible to incorporate increasingly more analysis parameters as possible into single
instrument platforms, in order to minimize the need to run a single sample on multiple
instruments. The modern versions of analyzers are capable of measuring white blood
cells (WBC), WBC differentials (5 part differentials), red blood cells (RBC), hemoglobin
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(HGB), platelets, mean corpuscular volume (MCV), and mean platelet volume, and
automatically calculating hematocrit (HCT), mean corpuscular hemoglobin
(MCH), MCH concentration (MCHC), RBC distribution width, plateletcrit, and platelet
(PLT) distribution width. The other crucial considerations in automatic analyzers are the
speed with which they perform and the number of specimens they can process per batch
(reduction in turnaround time due to high throughput).

Limitations in manual method of white blood cell count

Experience is needed to make technically adequate smears consistently.
Non-uniform distribution of WBCs over the smear, with larger leukocytes
concentrated near the edges and lymphocytes scattered throughout.
There is a non-uniform distribution of red blood cells as well, with small crowded
red blood cells at the thick edge and large flat red blood cells without central pallor at the
feathered edge of the smear.
It is subjective, labor-intensive, and statistically unreliable (only 100-200 cells are
counted).
It is imprecise with reported Coefficients of Variation (CV) ranging from 30 110
%.
Cell identification errors in manual counting: This is mostly associated with
distinguishing lymphocytes from monocytes, bands from segmented forms and abnormal
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cells (variant lymphocytes from blasts). The monocytes tend to be underestimated and the
Lymphocytes tend to be overestimated.

Advantages of manual method in WBC count

Not all samples can be evaluated by automated methods for one reason or another.
For example, when a leukocyte count is extremely low or high, it may be necessary to
perform manual count because of loss of instrument linearity (capability to count cells
accurately) at the extreme ends of the spectrum.
Other samples may require manual cell counting including those with abnormal
proteins, clumped platelets, or antibody elements in the plasma that interfere with an
instruments ability to count leukocytes.
For some laboratories automated method may be evaluated by manual methods,
although automated leukocyte and erythrocyte counts should be evaluated with singlechannel automated cell counter as a backup rather than a manual method.
Extremely abnormal leukocytes, such as those seen in leukemia, may not, in some
cases, be accurately differentiated by automated methods.

Conclusion

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Using manual method would be definitely more time consuming but to

consider the linearity of the computation of the specimens white blood cell count, it is
needed to use the manual method for back up purpose and variety of any laboratory use.

Creating these automated analyzers we may lessen the number of laboratory

technologists but considering the extent of how far can these analyzers give an exact
result and if ever there would be an emergency cases and there is no supply of electricity,
would question that we need to learn the manual techniques in many laboratories.

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