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258
is the clear plasma, in the middle is the thin layer of the buy coat (whitish) and at the
boom is the column of red cells (Figure 10.7).
8. Use the Hct reader for nding out the value of Hct for each sample (Figure 10.7). Note
the value and immediately write it in the register. Make it a habit to handle specimens
in number sequence to read and register results accordingly. This avoids many clerical
mistakes. The register must show the accession number, the assigned number for the
day and the Hct value.
Medical Laboratory Technology: Volume 1
Use of microhaematocrit card reader
If a manufacturer supplies the microhaematocrit card reader, the instructions for using it
come along with. The card reader can also be made in the laboratory as shown in Figure 10.7.
It is used in the following way:
1. Hold the tube against the scale so that the boom of the column of red cells (not the
boom of the tube which includes the height of the clay) is aligned with the horizontal
zero line.
2. Move the capillary tube across the scale until the line marked 1.0 passes through the
top of the plasma column. Check to make sure that the boom of the red cell column is
still on line 0; also check (by means of heavy vertical lines) to make sure that the tube
is vertical.
3. The line that passes through the top of the column of red cells gives the value of
haematocrit. Depending on the scale, the value can be in percentage or as a fraction
of 1. If the top of the column of red cells is not on a line, approximate the value to the
nearest 1% (do not use decimals).
Sources of error
• Determine the Hct within 6 h after the collection of blood.
• Mix the blood thoroughly before taking the sample for Hct determination. This should
be performed more carefully for the microhaematocrit method. Poor mixing is one of
the most common causes of erroneous results.
• Do not use a haemolysed specimen; it yields falsely low values.
• The anticoagulant used must be of recommended concentration; use of higher
concentrations of anticoagulant leads to false low values of haematocrit.
• Leakage of blood, poor sealing, reading of Hct with the buy coat, error in reading,
error in recording, specimen mix-up (dangerous!), use of a non-standardized
centrifuge and improper timing of centrifugaţi on are some common technical errors.
• Always be careful to follow the accession number of the specimen with the tubes in
use; failure to do this often leads to a frustrating situation for the technician and may
hurt the patient by incorrect reporting.
• Always re-check the nal reading and nal reporting.
Additional Information Along with results of Hct mentioned in the report, note any
abnormal ndings like colour of the plasma (deep yellow-green for jaundiced plasma, reddish
for haemolysis) or increased buy coat.
reD blooD Cell inDiCes
The quantitative measurements of the average size by volume (V), haemoglobin content by
weight (W) and Hb concentration (w/v) of RBCs are of substantial aid to the physician in
diagnosing various types of anaemia. Manually, these numerical values are calculated from
the total number of red cells, the Hb concentration (per unit volume of whole blood) and
Routine Haematological Tests
259
the haematocrit. With the discontinuation of manual red cell count by manual procedure,
reporting of red cell indices is only possible if an automated particle counter is available
to provide the value of red cell count. The only exception to this is MCHC, which does not
require the RBC count.
Three indices, expressing the average Hb weight and red cell volume, are commonly used:
mean cell volume (MCV), mean cell haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC). Another quantitative measurement of the red cells, the mean
cell diameter (MCD), is made directly under the microscope. In case of several automated
systems (e.g., model S of the Coulter Counter), some of the indices are actually determined
(e.g., MCV), while the result of manually determined tests (e.g., haematocrit) are calculated.
The indices, calculated from results of manual procedures, are questionable because of a high
percentage of error in the red cell count. This is overcome in the automated systems. It is important to check all indices against observations of red cell morphology on the blood smear
prepared for the dierential count.
You must be acquainted with the clinical classication of anaemias: microcytic, normocytic,
macrocytic, hypochromic, normochromic, hyperchromic (an erroneous term but used fre-
quently) and their combinations. These are based on values of indices and morphological ob-
servation of red cells. Thus, microcytosis (increased number of small red cells and decreased
MCV) and macrocytosis (increased number of bigger red cells and increased MCV) are relat-
ed to iron-deciency and megaloblastic (macrocytic) anaemia, respectively. Other laboratory
tests, such as the reticulocyte count and the osmotic fragility test, in combination with red
cell indices provide useful information and may enable the physician to reach a diagnosis of
anaemia, for example, aplastic anaemia, thalassemia, spherocytosis (haemolytic) and others.
(a) Enlarged view of haematocrit reader. A laminated copy of this can be used in the
laboratory, (b) Method of measuring haematocrit from the card reader.
Haeatocrit (g/dL) 10
RBC count in millions
43
Medical Laboratory Technology: Volume 1
Clinical significance
Results of red cell indices and reports of abnormalities in red cell morphology are helpful in
the diagnosis of several types of anaemia.
Normal values
MCV = 86 +/− 10 fL
MCH = 29.5 +/− 2.5 pg
MCHC = 32.5 +/− 2 g/dL (or %)
Mean Cell Volume (MCV)
This is the average volume of red cells. Since the size of the cell is very small, the volume is
expressed in femtolitres (1 femtolitre or fL is equivalent to 10
Mean Cell Volume (MCV) is calculated by the following formula:
MCV=
Haeatocrit (%) 10
RBC count in millions
In an automated system, cells cannot be centrifuged and yet Hct value is reported. This
is performed by the measurement of the average volume of red cells (MCV), which is per-
formed by the automated counter and along with the counting of the red cells. These values
are reproducible under automated conditions. As a result, the machine calculates the value of
Hct without spinning the sample.
-15
L) or cubic micrometers (μm3).
×
fL
Mean Cell Haemoglobin (MCH)
The Mean Cell Haemoglobin (MCH) is the average haemoglobin content (by weight, W) of a
red cell. Since the amount is very small, the weight is expressed in picograms (pg), which is
equivalent to 10
-12
g. Mean cell haemoglobin is calculated by the following formula:
MCH=
×
pg
Mean Cellular (Corpuscular) Haemoglobin Concentration (MCHC)
The mean erythrocyte haemoglobin concentration, which is also called ‘Mean Corpuscular
Haemoglobin Concentration (MCHC)’, is a measure of the average Hb content of erythrocytes.
It is calculated as Hb concentration per unit volume of packed red cells (%). The alternative
expression of mmoles of haemoglobin (Fe) per litre has is not yet been adopted in regular
practice. MCHC is calculated by dividing the Hb concentration of the blood by the erythrocyte
volume fraction or haematocrit.
MCHC =
Example If the Hb concentration is expressed in gram of Hb per dL and the Hct in per cent,
the calculation goes as follows:
Haemoglobin concentration = 15 g/dL
Haematocrit = 43%
Haemoglobin conc. (g/dL)
Haematocrit (%)
MCHC =
15
100= 34.9%×
100×
The normal range of this value is 32–37% and is referred as normochromic (normal colour).
Values lower than the above range is referred as hypochromic (less colour than normal). If the
value is higher than the upper limit of the reference range, then MCHC should be determined
again. Haemoglobin forms about 95% of the erythrocyte mass. Therefore, erythrocytes are
Routine Haematological Tests
5
..
..
5
never ‘hyperchromic’ (more colour than normal); however, they may increase in volume and
thus be capable of containing more haemoglobin than normal; in this case the MCHC may be
as high as 38%, but never exceeds this value.
interpretation of abnormal finDings
If the volume of the red cells is within the range of MCV, they will be termed normocytic. If the
RBC morphology is observed they will look normal (about 7 μM in diameter). Increased MCV
is a sign of macrocytosis, which may be related to anaemia due to deciency of vitamin В and/
or folic acid with the occurrence of megaloblasts in the bone marrow. Deciency of vitamin
В and/or folic acid deciency hampers the division of red cell precursors. Nucleated red cells
with megaloblastic morphology may appear in the peripheral blood smear. Microcytosis
(cells smaller than 7 μM in diameter and MCV lower than the normal range), on the other
hand, is associated with iron deciency anaemia and thalassemia.
A low MCH is found in anaemias associated with microcytic and/or hypochromic red cells.
Increased MCH is found with macrocytic red cell morphology. Decreased MCH is recognized
by the pale colour of the red cells, as seen in the peripheral blood smear. Increased MCH
appear darker.
The value of MCHC is normal or slightly decreased in macrocytic anaemia, while a severe
fall (below 25%) is seen in microcytic hypochromic anaemia.
Hints on checking results of red cell studies
Manual methods are subject to high error in the enumeration of red cells. Before the results
are nally communicated, you must make the following quick estimates and if results do
not tally, there may be two reasons—technical error or an abnormal pathologic state. The
following formulae are applicable to only normal conditions and re-checking of results
eliminates ‘technical error’.
Normal values
RBC count = 5 × 106/μL
Hb = 14.5 g/dL
Hct = 42%
Haemoglobin and Red Blood Cell
Haemoglobin (Hb) concentration × Factor (0.35)* = Expected value of RBC/μL
If the Hb value is 14.2 g/dl, it represents 5 × 106 RBC/μL.
Example If the Hb value of the specimen is 11.6 g/dL, the expected RBC count should be
11.6 × 0.35 = 4 × 106/μL.
Haematocrit and Red Blood Cell
Haematocrit (Hct) value (%) × Factor (0.12)** = Expected RBC/μL
From the standard values, 42% Hct represents 5 × 106 RBC/μL.
Example If the Hct value is 35%, the expected RBC count should be: 35 × 0.12 = 4.2 × 106 RBC/
μL
Caution As mentioned earlier, the aforesaid formulae should not be applied to calculate
and report red cell counts from Hb and Hct values. In pathological states, these equations do
not work. The formulae are used only as a check on the manual method described earlier. If
accurate red cell count is not possible, this should not be reported.
*Therefore the factor for Hb/RBC conversion is
**
Therefore the factor for Hct/RBC conversion is
14 2
=
035
.
012=
42
262
Medical Laboratory Technology: Volume 1
erytHroCyte seDimentation rate (esr)
When anticoagulated whole blood is left undisturbed, the column of red cells, i.e., the heavier
cellular component, seles down to the boom of the container leaving the layer of plasma
above. The rate at which erythrocytes sele or fall under controlled laboratory conditions is
the erythrocyte sedimentation rate (ESR).
Clinical significance
Erythrocyte sedimentation rate or ESR is a non-specic test that reects changes in plasma
protein that accompany most acute and chronic infections. In blood samples from most
healthy person, ESR occurs slowly. In many disorders, particularly inammatory diseases,
the rate of sedimentation is rapid. In some cases, the rate is proportional to the severity of
the disease. Some of the other pathologic conditions like lymphoproliferative disorders
(e.g., multiple myeloma), the presence of abnormal protein in plasma accelerates Rouleaux
formation of red cells (Figure 10.9), whereby the red cells ‘stack up’ but do not agglutinate as
in the antigen-antibody reactions seen in the blood bank. When a lile saline is added to red
cells in Rouleaux formation (seen under a microscope) they get separated. This dierentiates
Rouleaux formation from the agglutination reaction. As a result of Rouleaux formation, ESR
increases which suggests a possible pathologic condition. Conversely, normalization of the
ESR indicates possible recovery from the diseased state. The most important use of ESR is to
follow the progress of certain diseases such as tuberculosis and rheumatism.
Principle
To perform the ESR test, the anticoagulated blood specimen is placed in a calibrated tube of
standard dimensions and set at a vertical position in a rack for an exact time of one hour. At
the end of the time, the distance the erythrocytes fallen from the plasma meniscus (at the zero
mark) is measured in millimetres (mm) and reported as the ESR.
Sources of error
• Sedimentation tubes must be kept exactly vertical during the test. Even minor tilting
can greatly increase the ESR.
• The test must be set up in a quiet area, free from vibration and away from
draft. Temperature has considerable eect on the ESR. It slows down with low
temperatures.
• Set up the ESR within two hours after the blood collection. However, blood collected
in EDTA can be stored at 4°C for up to 6 h; however, it must be brought to room
temperature before the test is performed.
• Only standardized tubes must be used.
• Anticoagulated blood specimen must be well mixed before seing it up for the ESR
test.
• Carefully ll the sedimentation tube without inserting air bubbles. Air bubbles in the
tube interfere with the test’s accuracy.
• Accurately time the test. Erythrocyte sedimentation rate increases with time. The rst
one hour reading is considered as standard. Do not measure half-hour reading and
multiply by two in order to report for one hour. Take the reading only after one hour.
There are several methods of ESR determination. Each has advantages and disadvantages.
Considering the laboratory conditions of developing countries, only two of these methods
will be described here in detail—Westergren method and Wintrobe method. The Westergren
Routine Haematological Tests
sedimentation method gives more accurate results than Wintrobe, but the laer determines
both ESR and haematocrit.
263
Westergren Method
The method described here is a modied version of the original method. In the original
Westergren method (Figure 10.8), citrated blood was used. Here we shall use EDTAanticoagulated blood, which is the specimen of choice in the haematology laboratory.
Citrated blood has limited use in blood bank and coagulation studies. The results of ESR with
EDTA-anticoagulated blood are not signicantly dierent from citrated blood after four-fold
dilution. This avoids an extra step of specially preparing a citrated specimen.
Normal values
Westergren method (mm) Male: 5–15 mm/h (<50 years); 5–20 (>50 years)
Female: 5–20 mm/h (<50 years); 5–30 (>50 years)
Wintrobe method (mm) Male: 0–9 (all ages)
Female: 0–20 (all ages)
ESR (%) All ages: 40–51 normal; 51–54 borderline; >55 elevated
Specimen
EDTA-anticoagulated blood is used and its ESR is determined within 2 h of blood collection.
Equipment and supplies
• Westergren tubes calibrated in millimetres.
• Suction device to ll.
• Westergren stand, preferably provided with a spirit level.
• Timer.
• Test tubes.
• Graduated syringe, 5 mL.
• Graduated pipee, 5 mL.
Reagents
Sodium chloride aqueous solution (0.85%)
Sodium chloride (NaCl) 8.5 g
Distilled water (q.s.) 1000 mL
Transfer the salt in a 1-L volumetric ask, dissolve in 500 mL of distilled water and then
make it to volume (1000 mL). Transfer the salt solution into a bole, label the bole and put
the date.
Procedure
1. Deliver 0.5 mL of 0.85% sodium chloride into a plain test tube (15-mL).
2. Add 2 mL of well-mixed EDTA-anticoagulated blood to the test tube and gently mix
by swirling. Mix thoroughly for 2 min.
3. Check the Westergren tube rack. It musl be exactly levelled resting on a plain surface
and away from air draft. Variation in temperature aects the ESR value.
4. Fill the Westergren tube exactly to the 0 mark, making certain that there is no air
bubble in the blood column drawn through the tube. Filling of the Westergren tube
should be done by means of a rubber ‘bulb. Do not use mouth suction.
5. Place the Westergren tube in the stand, making sure that the tube is upright and ts
snugly and evenly into the groove provided for it.
6. Allow the tube to stand for exactly 60 min (set the timer to ring). Note the level to
which the red cell column has fallen at the end of one hour.
7. Report the result in mm/rst hour.
264
Medical Laboratory Technology: Volume 1
Determination of ESR by the Westergren method: (a) Fill the Westergren ESR tube, (b) by
mouth (not recommended) or special suction devices, (c) Make sure that the level of blood
is at ‘0’, (d) Put the tubes on the levelled stand, (e) Keep in a quiet place in the laboratory
and read the upper level of red cell column after one hour, (f) Cutler tube is used when the
amount of specimen is limited
Additional information
• After the day’s use, soak Westergren tubes in clean water for 4–5 h, wash thoroughly
in running tap water, rinse in deionized water and dry completely in the incubator
(37°C). Do not use washing powder, acid or alcohol.
• Bubbles and brin clot invalidate the results and haemolysis aects the results. Do not
use haemolysed blood.
• Cleanliness of the tube is important. While cleaning tubes used for determining
sedimentation rate, rst soak in water (at least 4–5 h, preferably overnight) followed
by thorough washing with tap water and rinsing with deionized water. Completely
dry the tubes in an oven (37°C). Do not use acid or detergent for cleaning and do not
use ether, ethanol or acetone for drying.
• Wear gloves while handling blood.
• If the upper level of the blood is above the 0 mark (e.g., 2 mm), care must be taken to
add the additional distance travelled by the blood column (2 mm). For example, if the
reading is 12 mm after 1 h, report it as 14 mm/h.
Routine Haematological Tests
265
Wintrobe Method
The Wintrobe method (Figure 10.9) is still used in many laboratories in developing countries
because it provides two test-results simultaneously (ESR and haematocrit) and is economical.
Erythrocyte sedimentation rate reading is rst taken after one hour and then the tube is
centrifuged for the Hct value (described earlier). The tubes used are reusable after cleaning.
The results of Wintrobe method, however, are not as accurate as the Westergren method.
Specimen
Fresh EDTA-anticoagulated blood is used.
Note The blood is not diluted in this method.
Equipment
• Wintrobe tube: Note the calibration on the tube. The scale with 0 marked at the top is
used for ESR determination.
• Rack for holding Wintrobe tube: Make sure that the rack has arrangements to hold
the tube in a perpendicular position.
Determination of ESR by Wintrobe method: (a and b) Fill the Wintrobe tube with anticoag-
ulated blood, (c) Bring to the '0’ mark and place vertically in the stand, (d and e) Take the
reading of the upper level of blood column after 1 h, (f) Rouleaux formation increases ESR.
Note: If the initial level is above the ‘0’ mark, add the additional divisions to the nal reading.
• Pasteur pipee with long neck or special syringes for lling Wintrobe tubes.
• Watch or timer.
266
Procedure
1. Mix the blood thoroughly by inversion or swirling for at least 2 min. Use of a rotor is
recommended. The blood must be at room temperature.
2. With a long-necked Pasteur pipee or with a special syringe, ll the Wintrobe tube to
the 0 mark. While lling, draw out the pipee tip as the tube is lled with blood. This
avoids air bubble formation.
3. Place the Wintrobe tube in an exactly vertical position in the rack, and set the timer for
60 min.
4. At the end of one hour record the level of the erythrocyte column.
5. Report the ESR as mm/rst hour.
Medical Laboratory Technology: Volume 1
Other Methods
Sediplast ESR System This is a modied Westergren method that uses a disposable kit with
a closed system. These kits eliminate the biohazard risks present in the original Westergren
method and also provide accurate lling of the tube.
Automated Methods Three automated methods are available in advanced laboratories—
Sedimat, Ves-matic and Zeta sedimentation ratio.
Sedimat (Polymedco) In this system the lled Sediplast Westergren tube is placed into the
Sedimat automated ESR reader. The reader displays the results of each sample on an LCD
display. The results are also stored in memory and can be printed out using the aached
thermal printer. The system eliminates technician bias and variability in technique among
technicians.
Ves-Matic The Ves-Matic ESR system is an automated walk away analyzer. A venepuncture
is performed using a special vacuum tube that draws 1 mL of blood into a solution of sodium
citrate. The tube is placed directly into the analyzer; the ESR is determined by infrared light
and the results are available in approximately 22 min.
Zeta Sedimentation Ratio The Zeta Sedimentation Ratio (ZSR) is performed using a special,
small-bore capillary tube that is lled with blood and spun for 3–4 min in a special centrifuge,
called the Zetafuge (Beckman Coulter). This centrifuge alternately compacts and disperses
RBCs under standardized centrifugal force. The tube is then read on a special reader to obtain
a value called Zetacrit, which represents the percentage of sedimented erythrocytes. The
Zetacrit value is divided into the patient’s Hct (also a percentage) and the result is the ZSR,
expressed as a percentage.
The ZSR advantages are that it is rapid, corrects for anaemia and requires only a small
blood sample, which is desirable for paediatric patients. However, a special centrifuge and
reader are required to perform the test.
enumeration of formeD elements
Enumeration of formed elements (blood cells) is a quantitative measure of the population
of blood cells in circulation. The counting of cells can be done manually with the help of a
microscope after diluting the blood and making a special type of wet mount. The technique
is known as haemocytometry (blood cell measurement). This method is able to give the
absolute count of cells under study as they are in circulation. The cells most often counted by
this procedure are the white cells. Manual red cell count has now been discontinued due to
high error rates. The values of Hct and Hb, however, provide sucient information for the
routine diagnosis of anaemia, which along with morphological studies of the blood smear,
help in determining the severity and type of anaemia.
Routine Haematological Tests
In recent years automated systems of blood cell counting by electrometric and photometric
(including laser technology) methods have been introduced in developing countries which
are now quickly switching to these systems as the price of the equipment is geing cheaper
and automation is proving to be protable over the manual procedure. The automated system is also more accurate, reliable and provides more information than is possible by manual
methods (e.g., average cell diameter). The laboratory should try to switch to the automated
systems when possible. As the automated systems are varied and fast changing, textbooks
emphasizing a particular model will soon be outdated. Furthermore, each manufacturer provides complete information regarding their product in their procedure manual, which makes
the discussion of the automated systems in greater detail in this book redundant.
The haemocytometric technique cannot dierentiate between various types of white cells
(neutrophils, eosinophils, basophils, lymphocytes and monocytes) and reticulocytes from
mature red cells. Study of the dry blood smear under high power of the microscope gives val-
ues of the relative distribution of various types of cells in the circulating blood. Dierential
count of white cells and reticulocyte count are done by this procedure. The absolute count of
these cells can, however, be indirectly determined from the values of the relative distribution
and the total count of the reference cell. For example, the total number of eosinophils can be
calculated if the percentage of eosinophils (dierential count) and the total leukocyte count
(TLC) are known. Study of the blood smear provides another piece of information—the mor-
phology of the blood cells. This is extremely important in certain types of diagnoses. The
specic techniques used to study the blood smear will be discussed separately in another
section.
267
Clinical significance
A transient increase in total leukocyte count (leukocytosis) is common in bacterial infections
and some other conditions; however, a progressive increase suggests the possibility of
leukaemia. Increase in eosinophils (eosinophilia) is common in parasitic infections and
allergic reactions.
Total Leukocyte Count by Haemocytometry
Total leukocyte count (TLC) is most commonly asked for by the physician and provides a
useful piece of information for the diagnosis of several pathological conditions including
infections, leukemias and leukopenias.
Blood is diluted with an acid solution, which removes the red cells by haemolysis and
also accentuates the nuclei of the white cells; thus making the counting of white cells easier.
Counting is done with a microscope under low power (100x magnication). Knowing the
volume of uid examined and the dilution of the blood, the number of white cells per cu.mm*
(or μL) in undiluted whole blood is calculated.
Normal values
White cell count: 4.5–11.0 × 103cells/μL
In order to express these values (μL) in SIU, which is in litre (L), multiply them by 106.
Note 1 L is equivalent to 106 μL. In other words, 4.5 × 106 (non-SIU) = 4.5 × 1012 (SIU). SIU is
the standard international unit.
Principle
Whole blood (anticoagulated) is diluted with appropriate diluting uid which is held inside
a microchamber made by a slide with a grid at the boom (haemocytometer) and a standard
* cu.mm is the older expression of μL.