The different laboratory
examinations used in the investigation of blood disorders may be grouped as:
A. Basic Hematological Tests
1. Measurement of Hemoglobin
2. Enumeration of Blood cells (cell
counting)
3. Examination of blood cell
morphology
4. Measurement and calculation of
absolute values and blood cell indices
5. Determination of Erythrocyte
Sedimentation Rate
6. Determination of Hematocrit value
or Packed Cell Volume
B. Test for blood cell morphology
C. Examination of blood cell morphology
D. Diagnostic and Ancillary Tests for the Investigation of Hemostatic
Disorders
E. Diagnostic and Ancillary Tests for the Investigation of Hemolytic
Disorders
F. Diagnostic and Ancillary Tests for the Investigation of Red Cell
Pathology
G. Miscellaneous Tests for the Investigation of White Cell Pathology
H. Test for Miscellaneous Blood Diseases
This chapter deals with
the second basic hematological test included in the above mentioned list. It
includes the enumeration of erythrocytes, leukocytes, eosinophils, basophils
reticulocytes and platelets. Enumeration of blood cells should be distinguished
from the term Complete Blood Count which does not only involve blood cell count
but rather include the following basic tests:
1.
Hemoglobin determination
2.
Leukocyte count
3.
Erythrocyte count
4.
Differential Leukocyte count
5.
Stained Red Cell Examinatio
6.
Hematocrit determination
Differential leukocyte
count and stained red cell examination are included under the examination of
blood cell morphology
LEUKOCYTE COUNT –
LEUKOCYTE NUMBER CONCENTRATION
The number of leukocyte
contained in 1 liter of blood is called the leukocyte number concentration. In
traditional units, it is expressed as the number of cells per cubic millimeter
and is called the leukocyte or white cell count.
In the total leukocyte
count, no distinction is made among the six normal cell types (neutrophils and
bands, lymphocytes, monocytes, eosinophils and basophils). Although each cell
type has its particular function in defining the body against foreign threats,
here we are concerned with the total leukocyte concentration in the blood.
MICROSCOPIC OR MANUAL
METHOD
This method consists of
diluting the blood and dissolving the red cell, transferring a small portion of
the solution to a counting chamber, counting the white cells with a microscope
and making the calculations.
Equipments:
1. White blood cell diluting pipette
2. Rubber sucking tube
3. White blood cell diluting fluid
4. Hemocytometer (counting chamber) and coverglass
5. Cotton balls and alcohol
6. Puncturing device (skin or venipuncture)
7. Microscope
8. If venipuncture is to be employed, blood should be collected with
anticoagulant, preferably EDTA should be used.
White Blood Cell
Pipette
The Thoma type of
glass pipette is most widely used. This consists of graduated capillary tube
having a volume of one unit with calibrated markings at unit. Above the
capillary tube is a mixing bulb, containing a glass bead (white). Above the
bulb is a capillary tube with an engraved mark “11.” The bulb has a volume of
10 units.
In making a white cell
count, blood is drawn up to 0.5 mark and diluting fluid to the 11 mark. Since
the last diluting fluid in the graduated capillary stem does not mix with the
blood in the bulb, it does not enter into the dilution of the blood and is
therefore discarded before the count is made. The actual dilution then is 0.5
to 10, or 1:20.
If very low counts are
suspected, the blood may be drawn the 1 mark instead of the 0.5 mark, thus
giving a 1 to 10 dilution. If there is insufficient blood to reach the 0.5
mark, the sample can be taken at any mark below 0.5 and suitable correction mad
for the increased dilution. For example, if blood is drawn to the 0.4 mark the
dilution is 1:25 in a white cell pipette.
Rubber sucking tube
The rubber tubing attached
to the pipette should be at least 10 inches long, so that the pipette is far
enough from the eye to enable one to read the graduations.
WBC diluting fluids:
1. 2% Acetic acid with Gentian violet
Glacial
acetic acid – 2 ml
Gentian
violet – 1 ml
Distilled
water – 100 ml
2. Turk’s diluting fluid
Acetic
acid – 2 ml
Methyl
violet – 1 gtt
Distilled
water – 100 ml
3. 1% HCl
Hydrochloric
acid – 1 ml
Distilled
water – 100 ml
Note:
Gentian or methyl violet is used to
stain the nuclei of the cells and thereby aids
in the identification of cells.
The
leukocyte diluting fluid is a hypotonic solution which lakes the red cells but
preserve the white cells.
Hemocytometer or
Counting chamber
Numerous types of counting
chambers with various types of markings have been designed. The type which is
most widely used at present is one made of a single piece of colorless glass
with two separate elevated counting platforms, each having the Improved
Neubauer ruling. The platform of which the engraved lines are placed is
surrounded by a moat. On each side of the platform there is an elevated glass
on which the coverglass is placed. The distance between the counting surface
and the bottom of the coverglass is 0.1 mm. This is sometimes referred to as
the depth of the chamber. Diluted blood is introduced by capillary attraction
between the counting platform and the coverglass.
The improved Neubauer type
of ruling consists of a system of squares. There is a large square measuring 3
x 3 mm (9 sq.mm.) divided into 9 intermediate squares, each 1 x 1 mm (1
sq.mm.). The four corner squares which are used for WBC count, are divided into
16 medium squares, measuring 0.25 x 0.25 mm. The central sq.mm is redivided
into 25 medium squares, each one of which measuring 0.2 x 0.2 mm. Each one of
these medium squares is subdivided 16 small squares which measure 0.05 x 0.05
mm. The total number of small squares in the central area is 400. As a rule, 5
of the medium squares are used for erythrocyte count.
The ruling in most
counting chambers is so designed that the medium squares or 16 small or tiny
squares each are enclosed by triple lines.
Procedure
1. Diluting the blood
The
dilution of the blood for white cell count facilitates the counting process by
suspending and dispersing the white cells. It dissolves the mature red cells,
since the erythrocytes greatly outnumber the white cells and would therefore
interfere in the count.
The
standard procedure is to draw blood to mark 0.5 of the leukocyte pipette and
the WBC diluting fluid to mark 11. This gives a 1:20 dilution.
The
ratio of dilution may vary.
2. Charging the counting chamber
A
representative sample of the diluted mixture is transferred to a counting
chamber. Prior to charging the mixture in the pipette should be well mixed by
shaking the pipette in any manner except along the longitudinal axis. The first
few drops coming from the capillary stem should be discarded because these
drops are cell–free. Shaking along the longitudinal axis has to be avoided
because it causes the admixture of the cell–free fluid in the stem with the
diluted blood inside the bulb.
3. Counting the cells
Before
counting the cells, students are advised to make a study of the uncharged
counting chamber to familiarize themselves with the ruling of the chamber
After
charging the counting chamber, let the cells settle for 1 to 2 minutes so that
all cells are in the same plane. Using the low power objective, locate and scan
the ruled area. If there is a good distribution of cells, start counting. If
not, clean the counting chamber and recharge. Count the cells within the 4
corner squares (4 sq.mm.) with 16 medium square each.
4. Making the calculations
Since
the cells in 4 sq.mm. have been counted and the dilution is 1:20 and the
distance between the counting chamber and the cover glass is 0.1 mm (depth).
The number of white cells per cu.mm. or per microliter is calculated by the
following formula:
#
of WBC counted in x Area
c.f. x depth c.f x dilution c.f.
4 sq.mm.
= number of WBC/cu.mm or microliter
(conventional unit)
Number
of WBC/cu.mm x 0.001 = x 109/liter
Area used in white cell count – 4
sq.mm
Area correction factor – ¼
Depth of counting chamber – 0.1 or 1/10 mm
Depth correction factor – 10
Dilution correction factor – 20
Variation in technique:
1. In leukopenia, draw blood to mark 1 and the diluent to 11; the ratio of
dilution then is 1:10. In the above formula, instead of using 20 as the
dilution factor, use 10.
2. In leukocytosis, draw blood to mark 1 or 0.5 of the RBC pipette and the
diluent to 101; and the ratio of dilution is 1:200 (if blood is up to 0.5) or
1:100 (if blood is up to 1). In the above formula, change the dilution factor
accordingly.
3. If big 8 squares were used (4 in each ruled platform), that means the
cells were counted in 8 sq.mm and the corresponding area correction factor is
1/8 and in the above formula, change the area correction factor accordingly.
Note: The following is suggested to avoid
confusion in counting the cells that lie on
borderlines; cells that touch
any of the three lines or the single line on the left
and the top borders of the
small squares should be counted as though they were
within the squares, but those
that touch any of the lines on the right and the
bottom
borders of the small squares should not be counted. In this way, no cell is
counted twice.
Normal values
Adults 5
to 10 x 109/liter
Infants at birth 10 to 25 x 109/liter
1 year 8
to 15 x 109/liter
Correction of leukocyte
count in the presence of predominating number of nucleated or immature
erythrocytes
On a stained blood smear
examination, one may sometimes see predominating number of immature or
nucleated erythrocytes. If this is so, the leukocyte count computed as
previously described should be corrected because it is erroneously high. WBC
diluting fluid causes hemolysis or mature red cells only and nucleated or
immature RBC are not destroyed by it. It is possible then that these nucleated
red cells could have been counted as leukocytes and included in the total
leukocytes count.
To correct the total
leukocyte count, determine the number of nucleated red blood cells (NRBC) per
100 WBC on the blood smear and proceed with the computation using the following
formula:
No. of NRBC x Leukocyte
count = Absolute number of NRBC
100 + No. of NRBC (uncorrected)
Uncorrected WBC count –
Absolute no. of NRBC = corrected
WBC count
Electronic method of
leukocyte count
1. Coulter counter method
The
white cells are suspended in a solution which is capable of conducting an
electric current. This solution is then made to pass through a narrow opening
between two electrodes. Since white cells do not conduct electricity, each
white cell passing momentarily decreases a flow of current between the two
electrodes. The decrease in current causes a voltage to drop in the line. The
number of voltage drop in the line is the key to the count.
2. Sequential Multiple Analysis (SMA) method
In
this method, the white cells are suspended in a solution. The solution is made
to pass through an optical system which causes each white cell to generate an
impulse. Each light impulse is then transformed into an electrical impulse. The
number of electrical impulses is recorded and become the key to count.
3. Fisher Autocytometer Method
This
method utilizes an optical counting system, in which each cell or other
particle in the diluted sample appears as a bright spot of light against a dark
field, as it passes through a “sensing zone” only 15 microns high. The high
flashes of light are picked up by photo multiplier and converted to electrical
impulses, which are totaled electronically and shown on the meter as the cell
count in the original blood sample.
Diluents
used in electronic counting (Leukocyte count)
1. 3% saponin solution
2. Zaponin or zap–oglobin (Coulter
counter)
3. Cetrimide–citrate–saline solution
Ratio
of dilution: 1:500; mixing 20
microliters of blood and 10 ml diluent
Advantages
of electronic cell counter over the microscopic method
1.
Speed of performance
2.
Elimination of visual fatigue of the operator
3.
Improve precision
Leukcytosis
– refers to an increase in the total white cell count above the upper limit of
normal for age and sex.
a. Physiologic factors causing leukocytosis
Strenuous
exercise
Epinephrine
injections
Anxious
emotional reactions
Pain
Anoxia
After
meals
During
pregnancy
Diurnal
variations being lowest in the morning and rising gradually in the course of
the day
b. Pathologic leukocytosis is caused by disease. An increase in
circulating leukocytes is rarely due to a proportional increase in leukocytes
of all types. This is usually caused by hemoconcentration and may be called
balance leukocytosis. Leukocytosis is usually due to an increase of only one
type of cell and is given the name of the principal type of cell increased,
such as neutrophilic leukocytosis (neutrophilia), monocytic leukocytosis
(monocytosis), lymphocytic leukocytosis (lymphocytosis), eosinophilic leukocytosis
(eosinophilia) and basophilic leukocytosis (basophilia)
Absolute and relative
counts
Example: A patient’s differential count shows 30%
monocytes and 70% neutrophils; and
his total Leukocyte count is 10,000/mm3
His relative monocytes count is 30% and his absolute
monocyte count is 30% x 10,000
= 3 x 109/liter
His relative neutrophil count is 70% and his absolute
neutrophil count is 70% x 10,000
= 7 x 109/liter
Absolute
leukocytosis – if the total number of any cell per cu.mm. is increased or if
the leukocyte number concentration is increased.
Relative
leukocytosis – if only the percentage of one type of cell increased because the
other types of cells have decreased.
Neutrophilia
or neutrophilic leukocytosis – refers to an absolute concentration or increase
of neutrophils in the blood above normal for age. Causes:
1. Systemic infections due to various bacteria, fungi, spirochetes and
fungi
2. Metabolic disturbances – uremia, diabetes
3. Drug intoxication
4. Physical and emotional stimuli – heat, cold, pain, fear, anger,
muscular activity
5. Tissue destruction or necrosis
6. Hemorrhage
7. Hematologic disorders
8. Hemolysis
Eosinophilia
or eosinophilic leukocytosis – refers to an absolute concentration or increase
in the number of eosinophils in the blood. Causes:
1.
Allergic disease – bronchial asthma and seasonal rhinitis
2.
Skin disorders – atopic dermatitis and eczema
3.
Parasitic infestations
4.
Infectious diseases – scarlet fever, pneumonia
5.
Hemopoietic disorders
Lymphocytic
leukocytosis or lymphocytosis – refers to an increase in the number of
lymphocytes in the blood. Causes:
1. Chronic infections
2. Viral infections – measles, infectious mononucleosis, mumps, viral
pneumonia
3. Bacterial infections – tuberculosis, syphilis
4. Radiation
5. Blood diseases
Monocytosis
or monocytic leukocytosis – refers to an increase in the number of monocytes in
the blood. Causes:
1.
Viral infections
2.
Bacterial infections
3.
Rickettsial infections
4.
Protozoal infections
Basophilia
or basophilic leukocytosis – refers to an increase in the number of basophils
in the blood. Causes:
1. Myeloproliferative disorders
2. Allergic reactions
3. Hypothyroidism
4. Granulocytic and basophilic
leukemias
Plasmacytosis
– plasma cells are not normally present in the circulating blood, but may be
increased in:
1. Chronic infections
2. Allergic states
3. Presence of neoplasms
4. In other conditions in which serum gamma globulins is increased
5. Moderately increased in cutaneous exanthemas, syphilis
The
increase in plasma cells is usually linked with an increase in lymphocytes,
monocytes and eosinophils. These four cells form the antigen–antibody quartet.
Leukopenia
means reduction in the number of leukocytes in the peripheral blood below
normal.
1. According to etiology
a. Diminished formation of leukocytes due to bone marrow defect, radiation
effect, effects of drugs.
b. Maturation arrest of white blood cells
c. Excessive peripheral destruction of leukocytes
2. According to cell type involved
a. Neutropenia
(1) Drug intoxication
(2) Ineffective granulocytopoiesis
(3) Bacterial infections –
brucellosis, salmonella infections
(4) Viral infections – measles,
rubella
b. Eosinopenia
(1) Stress
(2) ACTH administration
(3) Acute inflammatory states
(4) Hyperadrenalism
c. Basopenia – decrease in basophils
(1)
Acute infections
(2)
Stress
(3)
Following treatment with adrenal glucocorticoids
d. Lymphocytopenia
(1) Impaired lymphoiesis
(2) Acute pyogenic infections
(3) After ACTH administration
(4) Irradiation
e. Monocytopenia – decrease in monocytes
(1)
During therapy with prednisone
(2)
Hairy cell leukemia
Sources of error in WBC
counting
1.
Error due to nature of the sample
2.
Operator’s error
3.
Error due to equipment
4.
Inherent field of error
ERYTHROCYTE COUNT OR
ERYTHROCYTE NUMBER CONCENTRATION
The number of erythrocytes
contained in 1 liter of blood is called erythrocyte number concentration. In
traditional units, it is expressed as the number of cells per cubic millimeter
and is called the erythrocyte or red cell count.
Microscopic method or manual
or hemocytometer method
This method consists of
diluting the blood, transferring a small portion of the solution to a counting
chamber, counting the red cells with a microscope and making the calculations.
Equipment
Same as those employed in
leukocyte count with the exception of the pipette and diluting fluid. In the
red cell count, the red blood cell pipette and isotonic diluting fluid are
employed.
Red Blood Cell Pipette
Just like the white cell
pipette, it consists of graduated capillary pipette or tube having a volume of
one unit with calibrated markings at 0.1 unit. Above the capillary tube is a
mixing bulb, containing a red glass bead. Above the bulb is a capillary tube
with mark “101.” The bulb has a volume of 100 units.
In making a red cell
count, blood is drawn up to 0.5 mark and the diluting fluid to the 101 mark.
Since the last diluting fluid in the capillary stem does not mix with the blood
in the bulb, it does not take part in the dilution and is therefore discarded
before the count is made. The actual dilution then is 0.5 to 100 or 1 to 200.
If every low counts are
suspected as in anemia, the blood may be drawn to the 1 mark, thus giving a 1
to 100 dilution. If there is insufficient blood to reach the 0.5 mark, the
sample can be taken at any mark below 0.5 and suitable concentration made for
the increased dilution.
RBC Diluting fluid
1. Hayem’s diluting fluid
Bichloride
of mercury – 0.5 grams
Sodium
sulfate – 5 grams
Sodium
chloride – 1 gram
Distilled
water – 200 ml
2. Gower’s solution
Sodium
chloride – 0.85 grams
Sodium
sulfate – 12.5 grams
Glycerin
– 33.3 grams
Distilled
water – 200 ml
3. Toisson’s fluid
This
solution has a high specific gravity and stains the WBC
Sodium
chloride – 1 gram
Sodium
sulfate – 8 grams
Glycerin
– 30 grams
Methyl
violet – 0.025 grams
Distilled
water – 180 ml
4. Dacie’s or formol citrate solution
This
is considered the best diluent. It keeps for a long time and does not alter the
shape of cells.
40%
solution of formaldehyde – 10 ml
3%
v/v trisodium citrate – 990 ml
5. Bethell’s fluid
Sodium
sulfate – 5 grams
Sodium
chloride – 1 gram
Glycerin
– 20 ml
Sodium
merthiolate – 2 ml
Distilled
water – 200 ml
6. Normal saline solution or Physiologic Salt Solution
This
is used in emergency case, in the presence of rouleaux formation and
autoagglutinins of cells
Sodium
chloride – 0.85 grams
Distilled
water – 100 ml
7. 3.8% Sodium citrate
Sodium
citrate – 3.8 grams
Distilled
water – 100 ml
A good red cell
diluting fluid should be:
1.
An isotonic solution
2.
A good preservative
3.
One that does not initiate the growth of molds and yeasts
4.
One with a high specific gravity
5.
One with buffer action
6.
Cheap and easy to prepare
Hemocytometer or
Counting Chamber
1. Diluting the blood
The
dilution of the blood for red cell count facilitates the counting process by
suspending and dispersing the red cells. The standard procedure is to draw
blood to mark 0.5 of the RBC pipette and the red cell diluent to mark 101. This
gives a 1:200 dilution. The ratio of dilution may vary as the need arises.
2. Charging the counting chamber
Same
as leukocyte count
3. Counting the cells
The
same precaution in leukocyte count should be observed.
For
the standard erythrocyte counts, using the high power objective of the
microscope, count the red cell seen in 5 medium squares in the central ruled
area. These 5 medium squares with 16 small squares each make a total of 1/5 of
a square mm.
4. Making the calculations
Since
the cells in 1/5 sq.mm are counted, the dilution is 1:200, and the depth of the
counting chamber 1/10 mm, the number of erythrocytes/cu.mm. or microliter is
calculated with the following formula:
#of
RBC counted x area c.f. x depth
c.f. x dilution c.f.
in
1/5 sq.mm
=
counts in millions /cu.mm. or 1012/liter
= 5.4 x 1012/liter
Area
used in actual cell count = 1/5 sq.mm
Area
correction factor = 5
Depth
of counting chamber = 0.1 or 1/10 mm
Depth
correction factor = 10
Ratio
of dilution = 1:200 or 1/200
Variation in technique
1. In polycythemia or erythremia, when the number of RBC is markedly
increased, the blood is drawn to 0.3 mark of the RBC pipette and the diluent to
101. This gives a ratio of dilution of 1:333.33 and in the formula the dilution
factor varies accordingly.
2. In case the number of cells is markedly decrease as in anemia, the
procedure may be modified by :
a. Sucking blood to mark 1 and the diluent to 101; the dilution is 1:100
and in the formula the dilution varies accordingly.
b. Sucking the blood in the usual standard manner but counting more
squares other than the standard 5 medium squares and in the formula, the area
factor varies accordingly.
If
10 medium squares are used = area factor is 2.5
If
15 medium squares are used = area factor is 1.67
If
20 medium squares are used = area factor is 1.25
If
25 medium squares are used = area factor is 1.0
Sources of error in RBC
counting (microscopic method)
1.
Error due to the nature of the sample
2.
Operator’s error
3.
Error due to equipment
4.
Inherent error or field error
Electronic method of
erythrocyte count
1.
Coulter counter
2.
Sequential multiple analysis
3.
Fisher autocytometer method
Normal values
Male 5 – 6 x 1012/liter
Female 4.5
– 5 x 1012/ liter
Late pregnancy 3
– 5 x 1012/liter
Birth 7 x 1012/liter
Physiologic variation
1. Increase count in case of dehydration
2. Increase count in exercise or excitement
3. Newborns have higher count than adult
4. Women have lower count than men
5. Individuals living at higher altitude have higher count than those
living at sea level
Pathologic variation
1.
Increase in polycythemia vera
2.
Increase in pulmonary tuberculosis
3.
Increase in acute poisoning
4.
Decrease in anemia, after hemorrhage
5.
Increase in pulmonary fibrosis
Polycythemia – refers to
an increase in the concentration of erythrocytes in the blood that is above the
normal for age and sex
Oligocythemia – refers to
an increase in the concentration of erythrocytes in the blood that is below the
normal for age and sex
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