MLT 2002 Lecture 1 Notes 2013

PRACTICAL ASPECTS OF HAEMATOLOGY

Hematology, also spelled haematology, is the branch of internal medicine, physiology, pathology,
clinical laboratory work, and pediatrics that is concerned with the study of blood, the blood-forming
organs, and blood diseases. Hematology includes the study of etiology, diagnosis, treatment, prognosis,
and prevention of blood diseases.
The laboratology work that goes into the study of blood is frequently performed by a medical
technologist. Hematologists physicians also very frequently do further study in oncology - the medical
treatment of cancer.
In a clinical laboratory the hematology department performs numerous different tests on blood. The most
commonly performed test is the complete blood count (CBC) also called full blood count (FBC). Studies
of blood coagulation is a sub-specialty of hematology; basic general coagulation tests are the prothrombin
time (PT) and partial thromboplastin time (PTT). Another common hematology test in the erythrocyte
sedimentation rate (ESR).

Hematology Tests
Changes in time of day and fasting state may alter some values. Blood consists of red cells (erythrocytes),
white cells (leukocytes), and platelets (thrombocytes), suspended in a liquid called plasma.
A CBC usually includes white blood cell count (WBC), red blood cell count (RBC), hemoglobin,
hematocrit, red cell indices (MCV, MCH, MCHC), and platelet count. Some others tests listed under the
CBC include red cell distribution width (RDW), mean platelet volume (MPV) and a differential
examination of the quality and quantity of various white cells reported either in percent or absolute terms.
Reference values for the different parts of the CBC are difficult to list as some vary by age, sex, and
altitude. Also different instruments will have different principles of measurement that can impact on the
final values.

A complete blood count (CBC), also known as full blood count (FBC) or full blood exam (FBE) or
blood panel, is a test requested by a doctor or other medical professional that gives information about
the cells in a patient's blood. A scientist or lab technician performs the requested testing and provides the
requesting medical professional with the results of the CBC. The cells that circulate in the bloodstream
are generally divided into three types: white blood cells (leukocytes), red blood cells (erythrocytes), and
platelets (thrombocytes). Abnormally high or low counts may indicate the presence of many forms of
disease, and hence blood counts are amongst the most commonly performed blood tests in medicine, as
they can provide an overview of a patient's general health status. A CBC is routinely performed during
annual physical examinations in some jurisdictions.

Manual blood count

Counting chambers that hold a specified volume of diluted blood (as there are far too many cells if it is
not diluted) are used to calculate the number of red and white cells per litre of blood. To identify the
numbers of different white cells, a blood film is made, and a large number of white cells (at least 100) are
counted. This gives the percentage of cells that are of each type. By multiplying the percentage with the
total number of white blood cells, the absolute number of each type of white cell can be obtained. 30%
of CBCs have medical scientists manually looking at a blood film down the microscope, not only to find
abnormal white cells, but also because variation in the shape of red cells is an important diagnostic tool.
While automated analysers give fast, reliable results regarding how many red cells, the average size of the

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red cell and the variation in size of the red cells, they don't tell us anything about the shape. Also, a
percentage of normal patient's platelets will clump in EDTA anticoagulated blood. In these cases the
automatic analysers will give a falsely lower platelet count. On looking manually at the slide in these
cases, clumps of high numbers of platelets but an absolute number cannot be reported.

A complete blood count will normally include:

Red cells
 Total red blood cells (RBC)- The number of red cells is given as an absolute number per litre.
 Hemoglobin (Hb)- The amount of hemoglobin in the blood, expressed in grams per decilitre.
(Low hemoglobin is called anemia.)
 Hematocrit or packed cell volume (PCV) - This is the fraction of whole blood volume that
consists of red blood cells.
 Red blood cell indices
 Mean corpuscular volume (MCV) - the average volume of the red cells, measured in
femtolitres. Anemia is classified as microcytic or macrocytic based on whether this value
is above or below the expected normal range. Other conditions that can affect MCV
include thalassemia and reticulocytosis.
 Mean corpuscular hemoglobin (MCH) - the average weight of Hgb in a given amount
of red cells, in picograms.
 Mean corpuscular hemoglobin concentration (MCHC) – the average concentration of
hemoglobin per red cell
 Red blood cell distribution width (RDW) - a measure of the variation of the RBC
population

HEMOCYTOMETRY: Please my sons and daughters, see handout and endeavor to acquaint
yourself 100% to it. Thanks….

RED BLOOD CELL (RBC’s) COUNT

According to the Merck Manual, normal values of RBC/cmm for males is 5.4 + 0.8 million, and 4.8 +
0.6 for females. Anemic levels for adult males are below 4.5 million, for females below 4.0 million. We
will perform red and white blood cell counts on your blood in the lab using a hemocytometer and
appropriately diluted blood.
Blood cell counts can be performed using the hemacytometer. This precision instrument possesses a
platform with microscopic grid scoring. Rails on either side hold up a cover slip so that a specified
quantity of fluid is held. By properly diluting blood, counting all cells in specified squares, and
multiplying by the proper conversion factor, the number of cells per cubic millimeter can be determined.

Requirements and procedure

1. Swab towards the side of the tip of a little-used finger with 70% EtOH.
 Hemacytometer
 Coverslip
 Rbc pipette with hose and Mouthpiece
 Autolet

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  Lancet needless (for drawing blood)
 Platforms (for drawing blood)
 Microscope
 Squirt bottle with 70% ethanol
 Kimwipes (to be soaked in etoh)
 Bottle ringer's solution or formol citrate (a clear diluent for rbc)
 Paper towel
2. Lance by placing the platform of the autolet against the finger tip and pressing the trigger.
3. Using the dilution pipette with RED mixer from hemacytometer kit, draw blood up to the 0.5
mark. (Do not allow air to be drawn into the pipette or it will not draw the correct volume of
blood. Do not allow blood to congeal in pipette! Immediately proceed to the next step:
4. Continuing to hold the pipette as horizontal as possible, draw Ringer's solution or formol citrate
diluent up to the 101 mark. (Dilution of 1 to 200.) or simply transfer 20µL of anticoagulated blood
to 4mL of diluent (1:201 dilution).
5. Seal the tip with your finger and shake well to mix.
6. Empty ~1/2 of pipette into waste container add a small amount of the diluted blood to one
chamber of the hemacytometer to just fill the chamber of the hemacytometer. (Do not over fill).
7. Let the preparation sit for a minute (for cells to settle).
8. Center the grid at 100x, switch to 400x and count and record the RBCs in each of five fields ( each
with 16 smallest squares of the 25 centered squares) with a clicker (fields: top R & L, bottom R
& L, center). Include in the count all cells touching left and bottom sides, ignore cells touching top
and right sides. Calculate the RBCs/cmm by adding the cells in the 5 groups and multiplying by
10,000 (i.e., add four zeros) or use the formula below. Enter your RBCs/mm in the class data
table.
9. Wash out the pipette thoroughly with soap and water, rinse well, finish with distilled H2O rinse,
replace in case.

Calculation:
(1) Routinely, blood is drawn to the 0.5 mark and diluted to the 101 mark with RBC diluting fluid. All
the blood is washed into the bulb of the pipette (which has a volume of 100). Therefore, 0.5
volumes of blood are contained in 100 volumes of diluting fluid. The resulting dilution is 1:200.
(These figures are arbitrary and refer strictly to dilution and not to specific volumetric
measurements). Remainder, cells must be counted in a total of 80 small squares (16x5 = 80).

RBC count = (N x DF x 106) ÷ A (mm ²) x D (mm) = C/mm³

Where:
C/mm³ = number of cells/ mm³
N= Total number of cells counted in the counting chamber.
D (mm) = Depth factor in mm
DF = Dilution Factor
E- A (mm²) = Area counted (mm²)

(2) Sample:
EDTA anticoagulated whole venous blood.

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(3) Diluting Fluid:
Isotonic saline:0.85% sodium chloride (NaCl) in distilled water.
(4) Apparatus and Equipment:
1-Micropipette – 20 ml is the desired volume.
2-Serological Pipette, 5ml.
3-Handy Tally counter.
4- Improved Neubauer counting chamber with the cover slips.
5-Conventional light microscope.
(5) Normal Reference Range:
Males : 4.6 – 6.2 x 1012/L
Females : 4.2 – 5.4 x 1012/L
Children: 4.5 – 5.1 x 1012/L

Courtesy: Mbukam ED

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 MLT 2002 Lecture 2 Notes 2013

HEMOGLOBINOMETRY (HEMOGLOBIN DETERMINATION)

Decrease in hemoglobin concentration beyond established normal ranges for age and sex is called “
anemia”, whereas increase in hemoglobin concentration beyond established normal ranges for age , sex,
and geographical distribution is called “polycythemia”. So that, for correct diagnosis it is important to
determine accurately and precisely hemoglobin concentration. Haemoglobin is therefore measured to
detect anaemia or polycythemia and its severity, monitor an anaemic patient’s response to treatment and
to investigate the functioning of the reticulo-endothelial system. Monitoring the haemoglobin level (or
PCV) is also required when patients with HIV disease are being treated with drugs such as AZT. The test
is also performed to check the haemoglobin level of a blood donor prior to donating blood.
Many methods are available for the determination of hemoglobin, but among them the relevant, and the
recommended one is the Modified Drabkin’s Method. ICSH (International Committee for
Standardization in Hematology) consider this method as the reference method for hemoglobin
determination.

Drabkin’s solution contains the following:-

 Potassium Ferricyanide
 Potassium Cyanide.
 Non- ionic Detergent
 Dihydrogen Potassium Phosphate.

Well mixed EDTA anticoagulated blood is diluted in Drabkin’s solution; nonionic detergent will lyse
the red cells to
(1) liberate hemoglobin, and to
(2)decrease the turbidity caused by red cell membrane fragments by dissolving them.
Then, hemoglobin is oxidized and converted to methemoglobin (Hi) by potassium ferricyanide, this
step is accelerated by the dihydrogen potassium phosphate, and requires approximately 3 minutes for
total conversion. Potassium cyanide will provide cyanide ions to form cyanomethemoglobin (HiCN),
which have a broad spectrum of absorption at 540 nm. The absorption can then be compared with a
hemoglobin standard with a known hemoglobin concentration, and by applying Beer’s law extract the
hemoglobin concentration of the unknown (i.e. the patient).

Hemoglobin + Potassium Ferricyanide →Methemoglobin (Hi)

Methemoglobin + Potassium Cyanide → Cyanomethemoglobin

Hemoglobin Concentration Determination (Modified Drabkin’s Method)

Principle:
Whole blood is diluted in a solution containing Potassium Ferricyanide and Potassium Cyanide.
Hemoglobin will be oxidized by the action of Potassium Ferricyanide to form Methemoglobin
(Hemiglobin, Hi). Potassium Cyanide will provide Cyanide ions to form Cyanomethemoglobin (HiCN).
This solution can be measured spectrophotometrically and compared to known hemoglobin standards.

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This procedure is applicable in diagnosing and monitoring therapy in cases of hemoglobin deficiency
anemia’s.
Sample: EDTA anticoagulated venous whole blood.
Apparatus:
 Brown bottle- 1 liter
 Volumetric flask 1 liter
 Balance
 Spectrophotometer adjusted at 540 nm
 Spectrophotometer Cuvettes
 Glass or plastic centrifuge tubes
 Micropipette (adjusted at 20µl)
 5 ml volumetric pipette or graduated pipette

Procedure:
 Add 20 µl of whole anticoagulated blood to 5 ml of Drabkin’s solution.
 Mix well.
 Allow the mixture to stand at room temperature for at least 3 minutes.
 Measure the absorbance at 540 nm against a diluent Drabkin’s solution (blank).
 Measure the absorbance of the standard HiCN solution in the same manner.
 Extract the unknown hemoglobin concentration, using the following equation:

Unknown Hb concentration = (Abs. of Unknown ÷ Abs. of STD) X Conc. Of STD

Or read directly from the hemoglobin standard curve. See below, how to prepare the hemoglobin standard
curve.

Hemoglobin Standard Curve Preparation:

A standard curve must be made each time new Drabkin’s solution is prepared. A commercially prepared
standard kit with hemoglobin concentrations of 20, 15, 10, 5 g/dl is available, read each corresponding
absorbance, and plot the results on a linear graph paper (absorbance versus Hb concentration). Or, you
can use a stock standard solution of 20 g/d, and dilute it to various Hb concentrations with Drabkin’s
solution.

Note:
 Drabkin’s method is the recommended method by the ICSH.
 Drabkin’s solution should be clear and have a pH of 7.0 to 7.4, discard if turbid.
 The Drabkin’s solution is the blank, and should read zero (0) absorption.
 Take care when preparing the solution, as cyanide is fatal, and toxic, although the amount of
cyanide in the prepared solution is less than the human lethal dose.
 Do not expose the solution to acids, because cyanide will be released.
 Keep the solution in a dark bottle at room temperature, but discard after a month.
 Do not expose Drabkin’s solution and standards to light, and sunlight.
 People living at high attitudes tend to have polycythemic hemoglobin concentrations, because of
the low oxygen tension at high attitudes, causing tissue hypoxia, so that body compensate and
adapt for this by increasing hemoglobin.

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  Pregnant females tend to have lower hemoglobin concentrations than normal for their age,
because their fetuses compete with them for iron, vitamins and other essential substances for
hemoglobin synthesis, this is why pregnant females are of great needs for iron and vitamin
supplements during their pregnancies.

Haemoglobin Colour Scale

This technique for estimating haemoglobin is based on comparing the colour of a drop of blood absorbed
on a particular type of chromatography paper, against a printed scale of colours corresponding to six
different levels of haemoglobin: 4, 6, 8, 10, 12, and 14 g/dl (intermediate readings can be made between
any two adjacent shades of colour). Although similar in principle to the obsolete Tallqvist blotting paper
method (discredited due to inaccuracy and imprecision) the new Haemoglobin Colour Scale uses modern
materials and techniques to provide a simple, inexpensive, method for estimating haemoglobin in
community health care where photometric measurement is not possible. Lewis advizes, that the Colour
Scale is intended as a clinical device for the clinician/health worker at point-of-care so that it is possible
to identify anaemia and judge its severity in the following clinical categories:
Hb ≥12 g/dl Not anaemic
Hb 10–11 g/dl Mild anaemia
Hb 8–9 g/dl Moderate anaemia
Hb 6–7 g/dl Marked anaemia
Hb 4–5 g/dl Severe anaemia
Hb < 4 g/dl Critical

HEMOCUE HAEMOGLOBIN TECHNIQUE

The most recently developed HemoCue haemoglobin meter is the model 201. It differs from previous
models in being smaller, having an internal performance check system (control cuvette is no longer
needed), and a new cuvette holder which prevents contamination of the optronic unit and is easier to
clean. The 201_ meter can also store up to 600 test results. The microcuvettes are specially designed for
use with the model 201_ (cannot be used in previous HemoCue models). They are individually packaged
and available in a smaller pack size (25 per pack).

DHT 523 HAEMOGLOBINOMETER (Direct read-out Hb meter)

This modern electronic direct read-out haemoglobinmeter is precalibrated by the manufacturer and
therefore requires no calibration standard solutions. A glass control standard is provided for checking the
performance of the meter. Haemoglobin values are digitally displayed in g/l. The measuring range of the
meter is 20–300 g/l. The DHT Haemoglobinometer is particularly easy to use. The user simply inserts the
cuvette and removes it. Placing the cuvette in the cuvette holder automatically turns on the electronic
circuitry.

MICROHEMATOCRIT

Principle
Hematocrit is the ratio of the total volume of RBC’s to that of whole blood expressed as percentage(%)
(whole blood = total volume of cells + plasma). The second synonym for hematocrit is PCV (Packed Cell
Volume). The procedure is easy to perform, whole blood is centrifuged in a narrow tube (capillary tube),
cellular elements will be separated from the plasma, after centrifugation blood will be separated into 3

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layers : (1) Bottom layer contains packed RBC’s, (2) Middle layer contains WBC’s and Platelets (on top
of RBC’s), (3) Upper plasma layer. The hematocrit value is determined by comparing the volume of
RBC’s to the total volume of the whole blood sample, it is usually reported as a %.

Reagents and Equipment

1. Microhematocrit centrifuge
2. Microhematocrit reader disk
3. Capillary tubes
a. Plain-blue tip (for EDTA [ethylenediaminetetraacetic acid] tubes)
b. Heparinized-red tip (for Microtainer specimens)
Note: both types of tubes contain self-sealing clay
4. Mechanical rocker

Standard microhematocrit centrifuge. Maximum packing time is dependent on a calibrated centrifuge.

Specimen Collection and Storage

1. Fresh whole blood collected in EDTA in which the patient tube is at least half full.:
2. EDTA anticoagulated whole venous blood (correct volume is highly required. When EDTA is in
excess, cell shrinkage occurs, and as a result a falsely low hematocrit is obtained, with
corresponding increase in MCHC, and decrease in MCV).
3. Heparin
4. Or directly from a finger prick, to a heparin coated capillary tube.
5. Capillary blood collected in an EDTA Microtainer.

Quality Control
Hematocrits are run in duplicate and must agree within ±1%.

Procedure
1. Mix EDTA tube by placing on a mechanical rocker for 3 minutes.
2. After adequate mixing, fill the self-sealing capillary tubes two thirds to three fourths full. Prepare the
tubes in duplicate.
3. Wipe the outside of the capillary tubes with lint-free wipe or gauze.
4. Invert the tube so that the blood runs to sealed end.

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5. Place the tubes directly across from each other in the microhematocrit centrifuge, with the sealed ends
away from the center of centrifuge.
6. Record the identification and the position number of each patient specimen.
7. Place the head cover and hand-tighten only. Close the outer lid.
8. Centrifuge for 5 minutes at 12000g, for maximum packing.
9. Remove the tubes from the centrifuge and place in the microhematocrit reader. Read the hematocrit
according to the manufacturer’s instructions. The results are recorded in percent. The tubes should match
Within ±1%.

Interpretation
The spun capillary tube should have three visible sections: RBCs, buffy coat (contains leukocytes and
platelets), and plasma. Read the hematocrit results by placing the centrifuged capillary tube in the
groove of the plastic indicator reader. The bottom of the red cell column should meet with the black line
on the plastic indicator.

Reference intervals:-
Males : 40 - 53%
Females : 37 - 47%
Newborns: 51 - 60%
Children : 34 - 49%

Notes:
1. Higher values than the reference intervals is called polycythemia.
2. Lower values than the reference intervals is called anemia.
3. In cases of very high HCT, additional centrifugation for 5 minutes is recommended to reduce
plasma trapping. In general the higher the hematocrit, the greater the centrifugal force required.
4. Adequate centrifugation time and speed are important for accurate hematocrit.
5. Cells should be packed so that additional centrifugation does not alter or reduce HCT reading.
6. Plasma trapping is slightly more in macrocytic anemia’s, spherocytosis, hypochromic anemia’s,
and in sickle cell anemia.
7. Errors may occur as a result of:
 Inadequate mixing of the blood.
 Improper reading of the column lengths.
 Inclusion of buffy coat height with RBC column height (in leukocytosis or in
thrombocytosis, the buffy coat column height will be increased).
 Plasma trapping is still one of the causes of erroneously increased HCT results.
 Hemolysis of blood sample (due to improper collection, delay in processing) will cause
erroneously decreased HCT.
8. Increased anticoagulant to red cell ratio (short EDTA sample), will cause red cell shrinkage and
the hematocrit will be erroneously decreased.

Clinical Significance:
HCT is used to detect anemia’s, polycythemias, hemodilution, hemoconcentration, and also is used in the
laboratory to calculate the MCV, and the MCHC manually.

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Remark
 If you direct the capillary tube towards the microhematocrit centrifuge center, the sealed material
will be removed, and at the end of centrifugation you will find an empty capillary tube, blood will
go out from the tube!!
 Nowadays, Hct is supplied by the widely used automated hematology analyzers. But this Hct is
calculated rather than measured, these analyzers are not equipped with centrifuges, Hct is
calculated from the MCV, and RBC count, by using the following formula: Hct= (MCV X RBC)
÷ 10
 The sealed end of the capillary tube should be directed to the outside.
 The microhematocrit should be read at the top of red cell layer – not at the top of the buffy coat.
 There are two types of capillary tubes, red banded and blue banded capillary tubes. The red
banded are heparin coated, and use it when doing finger prick hematocrit. Blue banded capillary
tubes are plain, and use it with EDTA blood samples.
 Manual hematocrit is slightly more than the calculated automated hematocrit, because of the
trapped plasma which will be included with manual hematocrit, and excluded with automated
hematocrit (because it is calculated not measured).

Courtesy: Mbukam ED

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 MLT 2002 Lecture 3 Notes 2013

CALCULATING RED CELL INDICES AND THEIR ROLE IN SAMPLE INTEGRITY

Red Blood Cell Indices

Red blood indices are calculated parameters which determine red blood cell size, hemoglobin content of
red cells, and hemoglobin concentration of red cells. These parameters are useful in classifying
anemia’s into microcytic, normocytic, or macrocytic; and hypochromic or normochromic. These
parameters are calculated from total red cell count, hematocrit and hemoglobin.

MCV

Mean Cell (Corpuscular) Volume, is the average volume of red cells. This parameter is useful in
classifying anemia’s into: Microcytic, normocytic, and macrocytic. MCV is calculated from the
hematocrit (HCT), and the Red Blood Cells Count (RBC count).

MCV = (HCT ÷ RBC) x 10

The results of MCV are expressed in femtoliters (fl). 1 fl = 1 x 10-15 L. In automated hematology,
analyzers measure (not calculating) MCV from the area under the RBC histogram, and then calculating
the HCT from MCV and Total RBC count.

MCV Normal Range: 80 – 96 fl

If results are less than 80 fl, the red cells are said to be Microcytic:
a. Slight Microcytosis 75-79 fl
b. Moderate Microcytosis 70-74 fl
c. Marked Microcytosis <70 fl
If results are within 80-96 fl, the red cells are said to be Normocytic.
If results are higher than 96 fl, the red cells are said to be Macrocytic:
a. Slight Macrocytosis 96-105 fl
b. Moderate Macrocytosis 106-110 fl
c. Marked Macrocytosis > 110 fl

MCH

Mean Cell Hemoglobin, is the hemoglobin content in the average red blood cell, or in other words, the
average weight of hemoglobin per RBC. It is calculated from the hemoglobin concentration (Hb), and the
total RBC count.

MCH = (Hb (g/dl) ÷ RBC) X 10

Results of MCH are expressed in picograms (pg). 1 pg = 1 mmg = 10-12 g.

MCH Normal Range: 27 – 32 pg

· Macrocytic red cells have higher MCH, because they are larger and contain more hemoglobin.

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· Microcytic red cells have lower MCH, because they are smaller and contain less hemoglobin.

MCHC

Mean Cell Hemoglobin Concentration, is the concentration of haemoglobin in g/l in 1 litre of packed red
cells. It indicates the average weight of hemoglobin as compared to the cell size. It correlates with the
degree of hemoglobinization of the red cells on the peripheral blood film. MCHC is calculated from the
hematocrit and hemoglobin.

MCHC = (Hb (g/dl) ÷HCT) X 100

OR

MCHC = (MCH in pictograms ÷ MCV in femtoliters)

Results of MCHC are expressed in percentage (%) or gm/dl.

Normal Range: 32 – 36 g/dl (%)

If results are within this range, it is said that red cells are Normochromic.
If results are less than normal, red cells are said to be Hypochromic, which is seen in microcytic
hypochromic anemias e.g. iron deficiency anemia.

Notes:
The only highly comparable red cell parameter between automated cell counters and manual hematology
tests is the MCHC, because MCHC needs hemoglobin, and hematocrit in order to calculate it, which are
easy to perform manually with high reproducibility and accuracy. Red cells can’t accommodate more than
37 g/dl of hemoglobin, which is seen only in cases associated with spherocytosis. Macrocytic anemias
have normal MCHC. If you have a case with high MCHC, and you checked the blood film and you didn’t
find spherocytes, this may indicate an error in hemoglobin and/or Hct. Since Hct is a calculated
parameter, it is derived from RBC and MCV, so may also indicate an error in RBC count and / or MCV.
Solutions to resolve this error include: retesting the specimen, perform a spun microhematocrit,
performing a manual hemoglobin determination, and checking the quality control and other patients
results.

Hematology Automated Analyzers nowadays can perform all of the following:

 Count RBC
 Measure hemoglobin spectrophotometrically.
 Directly measure MCV, from the area under the RBC histogram.
 Calculate Hematocrit, which is derived from MCV and RBC count.
 Calculate MCH, which is derived from Hb, and RBC count.
 Calculate MCHC, which is derived from Hct, and Hb.

Normal Average Values

Hematocrit
Newborn (1 to 7 days) 56 ± 2%

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Adult (female) 42 ± 2%
Adult (male) 47 ± 2%

Normal Erythrocyte Indices Values

MCV 80 to 96 fL
MCH 27 to 32 pg
MCHC 32% to 36%

Notes
• Buffy coats are not included as part of the red
cell column.
• Repeat procedure if specimen has leaked in
the centrifuge.
• Repeat procedure if centrifuge has been stopped manually.

THE VALUE OF THE RED CELL DISTRIBUTION WIDTH (RDW)

The eighth parameter of the CBC is the RDW, a mathematical calculation that gives insight into the
amount of anisocytosis (variation in size) and, to some degree, poikilocytosis (variation in shape) in a
peripheral smear.
The red blood cell distribution width, or RDW, is a measure of the variation of red blood cell (RBC)
width that is reported as part of a standard complete blood count. Usually red blood cells are a standard
size of about 6-8μm. Certain disorders, however, cause a significant variation in cell size. Higher RDW
values indicate greater variation in size. Normal reference range in human red blood cells is 11 - 14%. If
anemia is observed, RDW test results are often used together with mean corpuscular volume (MCV)
results to figure out what the cause of the anemia might be. It is mainly used to differentiate an anemia of
mixed causes from an anemia of a single cause. Vitamin B12 deficiency produces a macrocytic (large
cell) anemia with a normal RDW. However, iron deficiency anemia initially presents with a varied size
distribution of red blood cells, and as such shows an increased RDW. And in the case of a mixed iron and
B12 deficiency we will have a mix of both large cells and small cells hence the RDW will usually be
elevated. An elevated RDW, i.e. red blood cells of unequal sizes, is known as anisocytosis.
The "width" in RDW is actually misleading, since it in fact is a measure of deviation of the volume of
RBCs, and not directly the diameter. Mathematically the RDW is calculated with the following formula:
RDW = (Standard deviation of MCV ÷ mean MCV) × 100
there is no known cause of decrease red cell distribution width.

WBC (LEUKOCYTE) MANUAL COUNTING

Principle:
Blood sample is mixed and diluted with weak concentration of hydrochloric acid (HCl), or acetic acid (in
specified known volumes). Weak acids will lyse red blood cells, and will darken WBC’s to facilitate
counting by the hemacytometer.
Manual WBC counting is used in cases of very low WBC count (leukopenia) with automated
hematology cell counters, and when automated cell counters are not available.

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Sample:
EDTA anticoagulated whole venous blood.

Reagent and Supplies To Prepare Diluting Fluid:

 Volumetric Flask 100ml.
 Serological pipettes.
 Concentrated HCL
 Glacial Acetic Acid
Preparation of Diluting Fluid:
Diluting fluid is either:
1% hydrochloric acid in distilled water (1 ml Conc. HCL + 99 ml Dist. water).
2% Acetic Acid in distilled water (Turk’s solution) (2 ml glacial acetic acid + 98 ml distilled water).
Glassware, Apparatus, Equipment :
 Neubauer improved hemacytometer.
 Clean cover slip slide (especially made for the hemacytometer).
 Automatic micropipette (20µl, 380µl are the required volumes).
 Gauze 10 x 10 cm
 Glass/Plastic tubes- (12x75 mm).
 Handy tally counter.
 Conventional light microscope.

Procedure:
1. Mix the blood sample gently but thoroughly by inversion, manually or by mechanical rocking
mixer.
2. Pipette 0.38 ml (380µl) of diluting fluid into a 12x75 mm tube.
3. Pipette 0.02 ml (20µl) of well mixed blood to be counted and wipe the tip with gauze into the tube
containing diluting fluid and mix the tube.
4. Let the tube stand for 2-3 minutes to ensure complete RBC lyses, then mix well.
5. Prepare the clean hemacytometer and cover it with the designed coverslip.
6. Load one side of the hemacytometer with the aid of a capillary tube or micropipette, do not
attempt to overload or underload the hemacytometer.
7. Allow the hemacytometer to sit for several minutes to allow the WBC’s to settle in the counting
chamber, to avoid drying effect, place the loaded hemacytometer in a covered Petri dish with a
moist gauze, until counting.
8. Place the hemacytometer in the microscope stage.
9. Focus with x10 objective lens (low power), with lowering the condenser until they appear as small
black dots.
10. The WBC’s are counted in the 4 corner large squares (64 squares) of the 9 large squares, (total
area of 4 mm2) with the aid of hand tally counter. During counting, do not count cells that touch
the right or bottom boundaries to ensure unduplicated counting. The total counted WBC’s in the 4
squares are added together.

Note: If the number of cells in a square varies from any other square by more than 9 cells, the count must
be repeated, because this represents an uneven distribution of cells, which is may be caused by improper
mixing of the dilution or improperly filled hemacytometer.

14
WBC count = (N x DF x 106) ÷ A (mm ²) x D (mm) = C/mm³

Reference Range
Adults : 4.5 – 11.0 x 109 /L
Six years: 4.5 – 12.0 x 109 /L
One year: 6.0 – 14.0 x 109 /L
Newborn: 9.0 – 30.0 x 109 /L
WBC count varies according to age but not to sex.

Sources of Error:
 Contaminated diluting fluid.
 Incorrect dilution.
 Uncalibrated Micropipettes.
 Uneven distribution of WBC’s.
 Presence of clumped WBC’s.
 Unclean hemacytometer or cover slips.
 Presence of air bubbles.
 Incompletely filled hemacytometer.
 Over flow.
 Presence of debris.
 Drying of the dilution in the hemacytometer.

1 ml of gentian violet can be added to the diluent to color the white blood cells, thus counting will be
easier. In leukopenia ( decreased WBC count), with a total WBC count below 2500/cubic mm, the
blood is diluted 1:10, whereas in leukocytosis (increased count), the dilution may be 1:100 or even 1:200.

Quality control of WBC counts

Whenever possible perform WBC counts in duplicate. The difference between the two counts (as a
percentage of the mean) should not be more than 20%.

PLATELETS (PLT) COUNT

Platelets are essential for the clotting of blood. One could think of them as tying together the clotting
mechanism and the damaged area. When platelets are low, it may take longer for the blood to clot. When
platelet counts are too high, unnecessary blood clots may occur. Strangely enough, bleeding can also
happen if the platelets interfere with each other! Platelet function is affected by many drugs; one of the
most well known is aspirin. Easy bruising may be a sign of a decreased platelet count. Bruising like
Goldilocks’ companions comes in three "sizes". The largest is called hematoma; it’s multicolored,
raised, has very well defined edges, and painful. The middle one is flat, usually red/blue and not well
defined edges. Many normal women get these on their thighs from bumping into furniture, etc. The
smallest is called petechiae. These looks something like freckles. They occur in multiples, may be itchy,
and are caused by very small blood vessel damage. Petechiae can be seen in nosebleeds and gum
bleeding.
Value of test: A platelet count may be requested to investigate abnormal skin and mucosal bleeding
which can occur when the platelet count is very low (usually below 20 x 10 9/l). Platelet counts are also
performed when patients are being treated with cytotoxic drugs or other drugs which may cause
thrombocytopenia.

15
Blood sample: Use EDTA anticoagulated venous blood. Capillary blood should not be used because
plateletsclumpas the blood is being collected.

Equipment
An Improved Neubauer ruled Bright-line counting chamber and other equipment as described previously
for WBC counting are required for counting platelets.

Reagent
Ammonium oxalate 10 g/l (1% w/v) diluting fluid.

Test method
Perform a platelet count within 2 hours of collecting the blood.
1. Measure 0.38 ml (380 µl of filtered ammonium oxalate diluting fluid and dispense it into a small
container or tube.
2. Add 20 µl (0.02 ml, 20 cmm) of well-mixed anticoagulated venous blood and mix.
3. Assemble the counting chamber and fill it with well-mixed sample as described previously in steps
3 and 4 of the method for counting white cells.
4. Leave the chamber undisturbed for 20 minutes. To prevent drying of the fluid, place the chamber
in a petri dish or plastic container on dampened tissue or blotting paper and cover with a lid.
5. Dry the underside of the chamber and place it on the microscope stage. Using the 10x objective,
focus the rulings of the grid and bring the central square of the chamber into view. Change to the
40x objective and focus the small platelets. They will be seen as small bright fragments
(refractile).
6. Count the platelets in the small squares as in RBC count.
7. Report the number of platelets in 1 litre of blood.

Quality control
In district laboratories the most feasible quality control of platelet counts is to follow the test procedure
exactly, perform duplicate counts, and examine the platelet fluid microscopically (at the time of
performing the count) to ensure it does not contain refractile particles resembling platelets. The mean of
the two counts should be calculated as described previously for WBC counts.

Platelets count = (N x DF x 106) ÷ A (mm ²) x D (mm) = platelets/litre

Sources of error in counting platelets

Sources of error when counting platelets are similar to those mentioned previously for WBC counts.
Special care must be taken when counting platelets:
Interpretation of platelets counts
In health there are about 150–400 x 109 platelets/litre of blood. Platelet counts are lower in Africans.
Thrombocytopenia
The main causes for a reduction in platelet numbers are:

REDUCED PRODUCTION OF PLATELETS

 Infections, e.g. typhoid, brucellosis
 Deficiency of folate or vitamin B12

16
  Aplastic anaemia
 Drugs (e.g. cytotoxic, quinine, aspirin), chemicals (e.g. benzene), some herbal remedies,
alcoholism
 Leukaemias, lymphoma, myeloma, myelofibrosis, carcinoma
 Hereditary thrombocytopenia (rare condition).

INCREASED DESTRUCTION OR CONSUMPTION OF PLATELETS

 Infections, e.g. acute falciparum malaria, dengue, trypanosomiasis, visceral leishmaniasis
 Disseminated intravascular coagulation (DIC)
 Hypersplenism
 Immune destruction of platelets, e.g. idiopathic thrombocytopenic purpura (ITP), systemic lupus
erythematosus (SLE), other connective tissue disorders, chronic lymphatic leukaemia, lymphomas
and HIV/AIDS. Also, exposure to drugs, e.g. quinine, mefloquine, penicillin, and some herbal
remedies.

Courtesy: Mbukam ED

17
 MLT 2002 Lecture 4 Notes 2013
PREPARATION OF BLOOD FILMS

Principle:
Blood film enables us to evaluate WBC, RBC, and PLT morphology, also, allows us to perform
differential WBC count, furthermore estimation of WBC and platelets counts can be done on blood
films. Blood films are made on glass microscopic slides. Two types of films are thick and thin blood
films. For differential count, a thin film is preferable.

Sample:
Finger stick blood or EDTA anticoagulated venous whole blood may be used. Films of peripheral blood
must be made immediately.
Films may be made from EDTA anticoagulated blood as long as two to three hours after collection. All
specimens should be free of clots.

Procedure for preparing thin films:

1. Use clean standard size glass slides (3 inch x 1 inch = 7.5 cm x 5 cm), wiped from dust just
immediately before use.
2. Place a small drop of well mixed anticoagulated whole blood, in the center line of the slide, about
1.5 to 2 cm from one end, with the aid of a capillary tube.
3. Immediately, without delay, with the aid of a second clean slide with uniform smooth edges
(spreader slide), with a 30 –40 degrees angle, move back so blood drop will spread along the
edge of the spreader slide, when this occurs, spread, or smear the film by a quick, unhazizating,
uniform forward motion of the spreader.

Notes:
 Before preparing the films, you must check that blood samples are free from clots, and this is
done with two wooden applicator sticks. If clots are present the specimen is unsatisfactory.
 Films can be labeled with patient’s name and /or Lab. No. on the thick end of the film itself, after
being dried, by using a pencil.
 With anemia (low Hct, reduced viscosity), the spreading angle should be greater, to avoid
running off the slide.
 With polycythemia (high Hct, increased viscosity), the spreading angle should be less, to avoid
short, too thick films.
 With large blood drops, increase the spreading angle.
 With small blood drops, decrease the spreading angle.
 If the anemia is too severe, let the blood specimen settle, so that blood is divided into two layers,
plasma layer and red cell layer, then discard part of the plasma layer, then mix the blood
specimen, by doing this you have increased the viscosity of blood, by this you will be able to
prepare a nice blood film.

STAINING BLOOD FILMS WITH ROMANOVSKY STAINS

Blood films are stained so that morphology of blood cells become more easily viewed, identified, and
evaluated. In addition, blood films may be examined for the presence of blood parasites (Malaria,

18
Trypanosoma, Babesia). Furthermore, stained blood films can provide important information about a
patients health, they may lead to a diagnosis or verify a diagnosis, or they may rule out a diagnosis.
Evaluation of stained blood films also may lead to the decision of performing other hematology special
blood stain procedures in order to identify specific cell components. As soon blood films are air dried, it
is best to stain them as soon as possible. Blood films are stained with one of the Romanovsky stains,
which are universally used for staining blood films. There remarkable property is creating distinctions in
shades of staining granules differentially and this is dependent on two staining components:
Azure B (the basic dye) and Eosin Y (the acidic dye).
Other factors which affects the staining results include :
1. Staining time,
2. Ratio of Azure B to Eosin Y,
3. pH of the staining solution.
a. Azure B will stain the acidic cell Components (e.g. nucleus, because it contains nucleic acids;
basophilic granules also take the Azure B staining because they contain heparin, which is acidic
in origin),
b. while Eosin Y will stain the alkaline basic components (e.g. Eosinophilic granules in eosinophils,
because these granules contain spermine derivatives, which are basic in origin). Red cells have
affinity for acidic Eosin Y dye, because it contains hemoglobin which is basic in origin.

Romanovsky stains include:

 Giemsa Stain
 Wright’s Stain
 Leishman Stain
 May-Grünwald Stain
The widely and popular used Romanovsky stains are: Leishman Stain and Wright’s Stain

Leishman Stain Procedure:

1. Let the films be air dried.
2. Put the films on a staining trough rack.
3. Flood the slides with the stain.
4. After 2 minutes (or more, if the stain in newly prepared), add double volume of water, and blow to
mix the stain with water, until a shiny layer is seen.
5. After 5-7 minutes, wash with a stream of water.
6. Wipe the back of the slides with gauze.
7. Set the films in upright position on a filter paper to dry.
8. Read the blood films microscopically.
If delay in staining blood films may occur, fix the films in absolute methanol, for 1-2 minutes, but do not
stain the slides until completely dried.

Romanovsky Stain Blood Cell Characteristics

1. Red cells →Red or pinkish red

2. Nuclei of all cell types→ Purple/violet
3. Lymphocyte cytoplasm →Blue
4. Monocyte cytoplasm →Grayish blue

19
 5. Platelets cytoplasm →Light blue
6. Neutrophilic granules →Violet-pink
7. Eosinophilic granules →Orange-red
8. Basophilic granules →Purplish black/ Deep blue
9. Platelets granules →Purple

Sources Of Errors In Staining

1. Stain Precipitate: May obscure cell details, and may cause confusion with inclusion bodies. Filter
the stain before use.
2. pH of the buffer or water: Too acidic pH causes too pinkish slides while too basic pH causes too
bluish slides.
3. Improper stain timing may result in faded staining or altered colors: Too long staining time
causes too blue slides (overstaining) while too short staining time causes too red slides.
4. Forced drying may alter color intensities and/or distort cell morphology.
5. Non-stain related errors:
 1st- EDTA causes crenation of the cells after blood collection.
 2nd- Severely anemic blood samples causes slower drying (before staining) due to
excessive plasma.
 3rd- Old blood specimens may cause disintegration in WBC’s and decrease in their
numbers.
 4th- Collection of blood in heparin causes blue staining of RBC’s with bluish
background, which makes heparin unsatisfactory for routine hematology testing, also
heparin induces platelet aggregation and clumping, with subsequent erroneous platelet
count with automated counters.
Note: Always filter the stain before each use, to eliminate stain precipitates.

Remark: Automatic stainers are available in the market, in which slides are moved automatically and
they are stained as they move.

WBC DIFFERENTIAL COUNT

White blood cells are evaluated by a differential count, which reports percentages of the types of WBCs
present. These are neutrophils which fight infection (also known as polys and bands, polymorphonuclear
leukocytes, PMN’s, grans, segs and nonsegs), lymphocytes which produce antibodies and other immune
system activities (lymphs, ly), monocytes which also fight infection (mono’s), eosinophils (eos) and
basophils (basos) which are involved with allergies. The red cells are also evaluated for size, shape,
color and the presence of any abnormalities.
Manual differentials are performed by taking a drop of blood, spreading it on a slide, staining it, and
evaluating 100 cells individually for quality and changes in morphology. For patients with elevated white
cell counts, differentials of 200 cells or greater might be done. Automated differentials are performed by
either testing for specific compounds within the cells or comparing their size, shape, and content. Most
instruments will count thousands of cells. For both types of differentials, the numbers are reported in
percentages. In some patients percentages might be misleading so absolute values of the types of WBC,
i.e., the number of white blood cells multiplied by the percentage seen are valuable in diagnosing illness
or following therapy. Persons receiving chemotherapy often have decreased WBC. If a patient’s absolute

20
granulocyte count (ANC or AGC) goes below 2,000 cells, then physicians become concerned about the
possibility of infection. A number below 1,000 is cause for greater concern and less than 500 usually
lands the patient in the hospital.

Principle:
Testing a Romanovsky stained blood films in order to determine and assess the percentage of various
classes of WBC’s present, and to assess red blood cells and platelets morphology.
Increased or decreased normal WBC’s class/subpopulation counts or the presence of immature
precursors of WBC’s or RBC’s in the peripheral blood film are of diagnostic importance in various
inflammatory and disease states.
Morphological red blood cells abnormality are important in various anemia’s (spherocytes, sickle cells,
acanthocytes, burr cells, microcytes, macrocytes, target cells, ….. etc).
Platelet morphology, distribution, and size abnormality are suggesting a platelet disorder.

Sample:
EDTA anticoagulated whole venous blood film, bone marrow film, and body fluid sediments (e.g. CSF).
Reagents, Supplies, and Apparatus:
1. Differential Tally Counter.
2. Conventional Binocular Microscope.
3. Oil immersion.
4. Well Stained Blood Film/s.

Procedure:
1. Focus the film under x10 lens, and scan the film to check cell distribution.
2. Add a drop of oil, and move to the x100 oil immersion lens.
3. Choose a suitable area, where cells are evenly distributed without appreciable overlapping- the
monolayer cell zone.
4. Count the WBC’s using tracking pattern.
5. Each cell identified should be immediately tallied as: Neutrophil- segmented, Neutrophil –
band, Lymphocyte, Monocyte, Eosinophil, Basophil, Immature cells: blast, promyelocyte,
myelocyte, metamyelocyte, promonocyte, Variant atypical lymphocyte.
6. Morphological abnormalities of WBC’s, RBC’s and platelets should be noted.
7. Nucleated red blood cell precursors (nucleated red blood cells-NRBC) are not included in the
differential count, but are counted per 100 WBC’s, and if they are more than 10 NRBC/100
WBC’s, a corrected WBC count should be made (because as discussed before that NRBC’s are
counted as WBC’s, and will be included in the total WBC count erroneously) by applying the
following formula:
Corrected WBC count = Uncorrected WBC X 100 ÷ NRBC + 100
NRBC is the number of NRBC seen per 100 WBC during differential process.
8. Express the results as percentage for each cell class/ subpopulation
Blood film made for WBC differential counting should be evaluated for red cells. Red cells are evaluated
for;
1. Variation in red cell volume/size,
2. Variation in shape,
3. Variation in staining properties,
4. Alteration in distribution,

21
 5. Presence of intracellular inclusions and
6. The presence of extracellular or intracellular parasites.
Blood film for WBC differential counting should be evaluated for;
1. Variation in platelet size (large, giant),
2. Presence of megakaryocyte fragments,
3. Dwarf megakaryocytes, or megakaryocytes.
4. Also, platelet distribution (satellitism, aggregation, clumping) which produce erroneous platelets
count results with impedance technology electronic counters, should be noted (causing
thrombocytopenia, and an increase in WBC count and/or RBC count, and affects RBC and WBC
histograms).
Blood film is evaluated for abnormal WBC’s inclusions,
1. Toxic granulation,
2. Alder-Reilly granules,
3. Chediak Higashi granules,
4. Döhle bodies, and 6- toxic vacuolization.
All of the above may indicate specific disease process of acquired or inherited origin.

Normal Values for Differential WBC count in Adults:

Cell Type/ Population %Normal Differential Normal Absolute Count x 109

Neutrophils- Segmented 50-70% 2.0-7.0 x 109/L
Neutrophil- Band 0-10% 0.0-1.0 x 109/L
Lymphocytes 15-45% 0.6-4.0 x 109/L
Monocytes 0-10% 0.0-1.0 x 109/L
Eosinophils 0-6% 0.2-0.7 x 109/L
Basophils 0-1% 0.0-0.2 x 109/L

Automated Differential Counting

Nowadays, hematology laboratory is equipped with automated hematology analyzers capable of

automated differential counting, which are more quicker, precise, and accurate than the manual time
consuming differential count, but this is true when the sample contains normal cell populations. When the
sample contains abnormal or immature cell populations, your eyes are not substituted. Although current
laser cell counters identify abnormal and immature cell populations in a sample, but this does not
substitute looking to a nice looking blood film to identify these abnormal cell populations or
subpopulation.

Clinical Significance Of Increased Normal WBC Counts:

Neutrophilia: Bacterial Infections, Inflammation , Stress, Chronic Myelocytic Leukemia.

Eosinophilia: Invasive parasite, Active Allergy, Myeloproliferative diseases/disorders..
Basophilia: Ulcerative Colitis , Myeloproliferative Diseases, Hyperlipidemia.
Lymphocytosis : Viral Infections, Whooping cough, Chronic Lymphocytic Leukemia.
Monocytosis: Tuberculosis, Rheumatoid Arthritis, Pyrexia of unknown origin.

22
Blood Film WBC and Platelet Quantitative Estimation
Total WBC count can be quantitatively estimated from blood films (under x40 objective lens), also this
estimation may be used for quality control purposes, and as delta check for manual and automated WBC
counts, follow the table below for estimating WBC count in blood films: Also WBC count can be
estimated quantitatively from blood film, by the following formula:
Average number of nucleated cells per field at x100 magnification = nucleated cell count x 109/L.
Platelets can be estimated quantitatively from blood films, each platelet seen under oil immersion lens
approximately equals to 20,000 PLT/cumm.
Normally, blood film from healthy individuals usually shows 7-22 platelets per oil immersion field. When
platelet aggregates or clumps are present in the blood film, then platelet estimation would be absolutely
unreliable.

Differential Tally Counter

Interpretation
Certain disease states are defined by an absolute increase or decrease in the number of a particular type of
cell in the bloodstream. For example:

Type Of Cell Increase Decrease

Red Blood Cells Erythrocytosis or Anemia or
(RBC) Polycythemia Erythroblastopenia
White Blood Cells Leukocytosis Leukopenia
(WBC):
Lymphocytes Lymphocytosis Lymphocytopenia
Granulocytes: Granulocytosis Granulocytopenia or
Agranulocytosis
Neutrophils Neutrophilia Neutropenia
Eosinophils Eosinophilia Eosinopenia
Basophils Basophilia Basopenia
Platelets Thrombocytosis Thrombocytopenia
All Cell Lines - Pancytopenia

23
Granulocytes

Cells that contain large visible granules are sometimes called granulocytes. They can be separated into 3
distinct cell lines, based on the reaction of the granules to the most commonly used stain in Hematology,
the Wright stain. The stain is a pH based stain. Structures that favor the basic stain stain dark blue or
basophilic; while those that favor the acid stain, eosin, stain bright red-orange. Some structures seem
indifferent to the stain and are called neutral.

Neutrophils cells are usually between 50 - 70% of all of the cells seen in a normal differential performed
on an adult.

Cells whose granules stain bright red orange are called eosinophils and are part of the allergic response.
Those granules contain histamine among other proteins. They should constitute between 0 - 4% of a
normal differential for an adult.

24
Cells whose granules stain dark blue are called basophils and are also involved in allergic reactions.
Agranulocytes

Monocytes are a type of cross over cell. They are primarily responsible for removing dead or damaged
cells and constitute less than 10% of the adult differential.

Lymphocytes are cells that do not contain large numbers of any granules. They are responsible for
producing antibodies against foreign material such as antigens found in viruses and some bacteria. They
also are active against malignancy. In the adult, they range between 20 and 45% of the cells seen in the
differential.

25
When a lymphocyte is actively defending against an antigen, there will be changes seen in the cell and
the cell is called a "reactive" lymphocyte.
Red blood cells in the differential

Normal red cells should be monotonous. They should all be about the same size, biconcave in shape, well
filled with hemoglobin, and be about the same color. Changes in these descriptions are clues to many
disease processes. Changes are usually graded as slight/moderate/ marked or 1+, 2+, 3+, and 4+ with 4+
being the most unusual. Terms that are used to describe these changes include: anisocytosis (changes in
size), poikilocytosis (changes in shape), hypochromia (less than adequate hemoglobin) and
polychromasia/polychromatophilia (changes in color).

CRITICAL VALUES
Critical values are those that are outside the reference range and that demand immediate action by the
operator or technologist. If a patient presents with a critical value on a CBC, the physician or unit must be
notified immediately. Records of this communication are essential and are a major part of quality
assurance. All technologists should realize the importance and urgency of acting appropriately once a
critical value has been obtained. Examples of critical values include:
WBC
Low 3.0 X 109/L
High 25.0 X 109/L
Hgb
Low 7.0 g/dL
High 17.0 g/dL
Platelets
Low 20.0 X 109/L
High 1000 X 109/L
Courtesy: Mbukam ED

26
 MLT 2002 Lecture 4 Notes 2013
MODIFIED WESTERGREN SEDIMENTATION RATE

Principle

The erythrocyte sedimentation rate is a non-specific indicator of inflammation. It is measured by the

degree of settling of red blood cells in a specific time period, usually one hour. There are several methods
for determination of the ESR; the most common are the Wintrobe and the Westergren, named for the
developers of the procedure. Blood for these procedures is drawn into either a lavender or blue top tube,
depending on the procedure. There is also an automated method for determining the sedimentation rate.
There is no special patient preparation.
ESR are best used when comparing changes over time. A single test does not give much usable
information. Some things that can cause an elevated ESR include exercise, arthritis, rheumatic fever,
myocardial infarct (MI), infections, some malignancies, menstruation, and normal pregnancy after the
third month. The normal range varies by the method used. The erythrocyte sedimentation rate (ESR),
also called a sedimentation rate or Biernacki Reaction, is the rate at which red blood cells precipitate in
a period of 1 hour. It is a common hematology test which is a non-specific measure of inflammation. To
perform the test, anticoagulated blood is placed in an upright tube, known as a Westergren tube, and the
rate at which the red blood cells fall is measured and reported in mm/h. There is also a wintrobe method.
Since the introduction of automated analyzers into the clinical laboratory, the ESR test has been
automatically performed. It is used as an initial screening tool and also as a follow-up test to monitor
therapy and progression or remission of disease. This test measures the distance that RBCs will fall in a
vertical tube over a given time period. The ESR is directly proportional to red cell mass and inversely
proportional to its surface area. The ESR is reported in millimeters. Any condition that will increase
rouleaux formation will usually increase the settling of red cells. Factors affecting the ESR are as follows:
 Red cell shape and size: Specimens containing sickle cells, acanthocytes, or spherocytes will settle
slowly and give a decreased ESR
 Plasma fibrinogen and globulin levels: Increased fibrinogen or globulin levels will cause
increased settling and give an increased ESR
 Mechanical and technical conditions: Surfaces that are not level will influence red cell settling.
 Specimens that are not properly anticoagulated will also affect red cell settling. EDTA is the
recommended anticoagulant.
The ESR is governed by the balance between pro-sedimentation factors, mainly fibrinogen, and those
factors resisting sedimentation, namely the negative charge of the erythrocytes (zeta potential). When an
inflammatory process is present, the high proportion of fibrinogen in the blood causes red blood cells to
stick to each other. The red cells form stacks called 'rouleaux' which settle faster. Rouleaux formation
can also occur in association with some lymphoproliferative disorders in which one or more
immunoglobulins are secreted in high amounts. The basal ESR is slightly higher in females. In some parts
of the world the test continues to be referred to as the Biernacki Test or Westergren test, which uses
sodium citrate-anticoagulated specimens.
Normal Values
Note: mm/hr. = millimeters per hour.
Westergren's original normal values (men 3mm and women 7mm) made no allowance for a person's age
and in 1967 it was confirmed that ESR values tend to rise with age and to be generally higher in women.
Values are increased in states of anemia, and in black populations.

27
Reagents and Equipment

1. Sediplast Autozero Westergren ESR system

a. Fixed bore pipettes
b. Sedivials filled with 0.2 mL of 3.8% sodium citrate
c. Vial holder (rack)
2. Disposable plastic pipettes
3. Rotator/mixer
4. Timer
Specimen Collection and Storage
Specimens are collected in EDTA. Tubes must be at least half full, well mixed, and free of clots and/or
fibrin. ESR can be set up on specimens at room temperature up to 4 hours old. Refrigerated specimens (2
to 80 C) can be set up until 24 hours old.

Sediplast ESR rack. The sample must be placed on a level surface with no vibration

Quality Control

Commercially prepared controls stored at 2 to 8 0 C are valid until expiration date. Controls are prepared
like patients’ specimens. Controls are run once daily prior to setting up patient tests. Do not report out
patient results unless controls are in reference range.

Procedure

1. Mix the EDTA tube on the rotator/mixer for a minimum of 5 minutes. If the sample has been
refrigerated, allow 30 minutes for the sample to come to room temperature before proceeding.
2. Remove the top of the Sediplast vial, which contains 3.8% sodium citrate. Using a disposable
pipette, add blood to the indicated line, return the top, and mix thoroughly.
3. Insert Westergren tube into Sediplast vial with a slight twist, allowing the blood to rise to the zero
mark.
4. Place the vial in the rack on a level surface.
5. Set a timer for one hour and read at the end of the hour.

28
 6. Record the ESR in millimeters per hour.

Normal Ranges
Men 0 to 15 mm/hr
Women 0 to 20 mm/hr

Limitations

1. Tubes not filled properly will yield erroneous results.

2. Refrigerated specimens must come to room temperature for 30 minutes prior to testing.
3. The ESR rack must be on a level surface and free of vibration. Vibration can cause a falsely
increased ESR.
4. Cold agglutinins can cause a falsely elevated ESR. An ESR can be performed at 37 0C (incubator)
for 60 minutes with no ill effects.
5. Cell size and shape affect ESR, usually resulting in a decreased ESR result.
6. Increased rouleaux formation, excessive globulin, or increased fibrinogen will increase the ESR.
7. Specimen must be free of clots and/or fibrin.
8. A tilted ESR tube gives erroneous results.
9. Hemolyzed samples are not acceptable.
10. Specimens older than 24 hours are not acceptable.

Conditions Associated With…

Increased ESR
1. Kidney disease
2. Pregnancy
3. Rheumatic fever
4. Rheumatoid arthritis
5. Anemia
6. Syphilis
7. Systemic lupus erythematosus
8. Thyroid disease
9. Elevated room temperature
Decreased ESR
1. Congestive heart failure
2. Hyperviscosity
3. Decreased fibrinogen levels
4. Polycythemia
5. Sickle cell anemia
Note: A rapid ESR method (ESR Stat Plus) is now available, which gives a result in 3 minutes.

Courtesy: Mbukam ED

29
MANUAL RETICULOCYTE PROCEDURE

Principle
The reticulocyte count is an index of bone marrow red cell production. The reticulocyte is the cell stage
immediately before the mature erythrocyte. This cell spends 2 to 3 days maturing in the bone marrow
before it is released into the peripheral circulation, where it spends an additional day of maturation. The
reticulocyte count is the most effective measure of erythropoietic activity. Reticulocytes contain RNA and
can be observed using supravital stains such as New Methylene Blue or Brilliant Cresyl Blue. Low
reticulocyte counts indicate decreased erythropoietic activity. Increased reticulocyte counts indicate
increased erythropoietic activity usually as the bone marrow compensates in response to anemic stress.
Therefore, reticulocyte counts are a reflection of bone marrow health or injury. These counts assist
physicians in diagnosis, treatment, or monitoring of patients with various anemias.

Reagents and Equipment

1. New Methylene Blue (Supravital Stain)
2. Test tubes
3. Microscope slides with a frosted end
4. Microscope with X100 (oil immersion objective)
5. Transfer pipettes

Specimen Collection and Storage

1. One EDTA tube or EDTA Microtainer
2. Specimens can be stored at room temperature for 8 hours or refrigerated at 2 to 8 0C for 24 hours.

Quality Control
Commercially prepared controls are performed each day when reticulocytes are reported manually.
Controls are prepared like the patient specimens and follow the procedure below for counting
reticulocytes. Do not report out patient results until quality control results fall within the acceptable
reference range.

Procedure
1. Mix 4 drops of New Methylene Blue with 4 drops of patient’s blood. If the specimen is a small amount
(such as a Microtainer), add an equal amount of stain to the Microtainer after the complete blood count
(CBC) has been completed.
2. Let the specimen mix for 10 to 15 minutes. Make a wedge smear and let it air dry. Label the smears
with the patient’s name, specimen number, and the date.
3. Allow the smear to completely dry and read under the microscope using X100 oil immersion.
a. Count the number of reticulocytes in 1000 cells.
b. Use the following formula to calculate the percentage of reticulocytes

Reticulocyte count (%) = (Number of reticulocytes counted per 1000 cells X 100) ÷ 1000

Normal Values
Adults: 0.5% to 2.0%
Infants: 2.5% to 6.5%

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Conditions Associated With…
Decreased Reticulocyte Count
1. Aplastic anemia
2. Exposure to radiation or radiation therapy
3. Chronic infection
4. Medications such as azathioprine, chloramphenicol, dactinomycin, methotrexate, and other
chemotherapy medications
5. Untreated pernicious anemia/megaloblastic anemia
Increased Reticulocyte Count
1. Rapid blood loss
2. High elevation
3. Hemolytic anemias
4. Medications such as levodopa, malarial medications, corticotrophin, and fever-reducing medications
5. Pregnancy

Limitations
1. Recent blood transfusion can interfere with accurate reticulocyte results.
2. Mishandling, contamination, or inadequate refrigeration of the sample can interfere and cause
inaccurate test results.
3. Red cell inclusions such as Heinz bodies, siderocytes, and Howell-Jolly bodies can be mistaken for
reticulocytes. If these are counted as reticulocytes, they will falsely increase the reticulocyte count.
Inclusions should be confirmed with Wright’s stain.

RETICULOCYTE PROCEDURE WITH MILLER EYE DISC

Principle
A Miller Eye Disc is placed inside the microscope eyepiece as an aid to counting reticulocytes. This
reticule is a large square inside a small square and provides the technologist the ability to isolate the
reticulocytes while counting.

Reagents and Equipment

1. New Methylene Blue (Supravital Stain)
2. Test tubes
3. Slides
4. Microscope with Miller ocular eye disc
5. Transfer pipettes

Specimen Collection and Storage

1. One EDTA tube or EDTA Microtainer
2. Specimens can be stored at room temperature for 8 hours or refrigerated at 2 to 8 0C for 24 hours.

Quality Control
Commercially prepared controls are performed each day when reticulocytes are reported. Controls are
prepared like the patient specimen and follow the procedure below for counting reticulocytes. Do not
report out patient results until quality control results are acceptable.

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Procedure
1. Mix 4 drops of new methylene blue with 4 drops of patient blood in a test tube. If the specimen is a
small amount (such as a Microtainer), add an equal amount of stain to the Microtainer after the CBC has
been completed.
2. Let the specimen mix for 10 to 15 minutes. Make an appropriate smear with feather edges. Label the
slides with the patient’s name, specimen number, and date.
3. Allow the smear to completely dry and read the slides under the microscope with oil immersion using
the Miller Eye Disc.
a. The Miller Eye Disc is a counting aid that provides a standardized area in which to count RBCs. There
are two squares that make up the disc. Square 1 is nine times the area of square 2.
b. To use the disc, all reticulocytes are counted in the large (1) and the small (2) square. The RBCs are
counted only in the small square.
c. Count the number of reticulocytes in 111 red cells in the small square. At this time, the counting is
concluded and the number of reticulocytes is reported.
d. Use this formula for calculating reticulocytes in percentage.
Reticulocyte count (%) = (Total number of reticulocytes counted in large square X 100) ÷ Total RBCs
in small square X 9

PERIPHERAL SMEAR PROCEDURE

Principle
When automated differentials do not meet specified criteria programmed into the automated hematology
instrument, the technologist/technician must perform a manual differential count from a prepared smear.
There are two types of blood smears: the wedge smear and the spun smear. The wedge smear will be
discussed in this procedure. Smears are prepared by placing a drop of blood on a clean glass slide and
spreading the drop using another glass slide at an angle. The slide is then stained and observed
microscopically. A well-stained peripheral smear will show the red cell background as red orange. White
cells will appear with blue purple nuclei with red purple granules throughout the cytoplasm.
A well made, well distributed peripheral smear will have a counting area at the thin portion of the wedge
smear which is approximately 200 red cells not touching. A good counting area is an essential ingredient
in a peripheral smear for evaluating the numbers of and types of white cells present and evaluating red
cell and platelet morphology.

Reagents and Equipment

1. Glass slides (frosted)
2. Wooden applicator sticks
3. DIFF-SAFE (an apparatus designed to avoid removing the tube top)

Specimen Collection and Storage

1. EDTA specimen or EDTA Microtainer
2. Smears are made from EDTA
a. Microtainers within 1 hour of collection
b. EDTA blood within 2 to 3 hours
c. Check all Microtainers for clots with applicator sticks

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Quality Control
A random slide is picked after it has been stained and a technologist/technician checks the quality of the
stain for the WBCs and RBCs, platelets, and the distribution of cells (see Principle)

Procedure

1. Insert the DIFF-SAFE dispenser through the stopper of the tube held in an upright position.
2. Turn the tube upside down and apply pressure at the frosted end of the slide. When the drop of blood
appears, discontinue pressure.
3. Using a second slide (spreader slide), place the edge of the second slide against the surface of the slide
at an angle between 30 and 45
4. Bring the spreader slide back into the blood drop until contact is made with the drop of blood.
5. Move the spreader slide forward on the slide, so a smear is made approximately 3 to 4 cm in length.
The smear should be half the size of the slide, with no ridges, and a “feather edge” should be toward the
end of the smear.
6. Label the frosted end of the slide with the patient’s last name and first initial, specimen number, and
the date.
7. Allow the smear to air dry completely.
8. Proceed with staining. Manual Wright staining is not found often in the clinical laboratory setting.
Most clinical laboratories have an automated staining instrument attached to their automated CBC
analyzer. If there is no automated stainer attached to the analyzer, there still is a separate staining
instrument.

Limitations

1. The angle between the slides is dependent upon the size of the blood drop and viscosity of the blood.
The optimal angle is 45 degrees.
2. The larger the drop of blood and lower the hematocrit, the higher the angle needs to be so the blood
smear is not too long.
3. Blood with a higher hematocrit needs to have a lower angle so the smear is not too short and thick.
4. Glass slides must be clean; otherwise, this results in imperfect distribution of cells and improper
staining.
5. Once the drop of blood has contact on the slide, the smear needs to be made immediately. Otherwise,
the blood will clump and dry, again resulting in uneven distribution of WBC and platelets.

PERFORMING A MANUAL DIFFERENTIAL AND ASSESSING RED BLOOD CELL

MORPHOLOGY

Principle

When blood samples are evaluated by the use of automated hematology analyzers, this analysis includes
automated differentials. Specific criteria pertaining to normal, abnormal, and critical values have been
programmed into the analyzers by the institution, and if the differentials do not meet these criteria,
verification is necessary. This is done by performing manual differentials and further evaluating the
peripheral smear. First, a differential white blood cell (WBC) count is performed to determine the relative

33
number of each type of white cell present. Technologists/technicians must recognize and properly record
the type(s) of white cell observed.
Simultaneously, red cell, white cell, and platelet morphology is noted and recorded. Also, a rough
estimate of platelets and WBC counts is made to determine if these numbers generally correlate with the
automated hematology analyzer. Technologists/technicians must be proficient at recognizing red and
white cell abnormalities, identifying them correctly, and quantifying them.

Reagents and Equipment

1. Microscope
2. Immersion oil
3. Differential cell counter

Specimen Collection and Storage

Well-made stained blood smear obtained from a capillary puncture or an EDTA tube at least three-fourths
full.

Quality Control
The slide should have three zones: head, body, and tail. In the tail area, neutrophils and monocytes
predominate, while red cells lie singly. In the body area, lymphocytes predominate, and red cells overlap
each other to some extent.

1. WBCs should contain a blue nucleus along with a lighter staining cytoplasm.
2. RBCs should have good quality of color ranging from buff pink to orange.
3. Platelets should be blue with granules and no nucleus.

Procedure
Observations Under X10

1. Place a well-stained slide on the stage of the microscope, smear side up, and focus using the low-power
objective (X10).
2. Check to see if there are good counting areas available free of ragged edges and cell clumps.
3. Check the WBC distribution over the smear.
4. Check that the slide is properly stained.
5. Check for the presence of large platelets, platelet clumps, and fibrin strands.

Observations Under X40 -: WBC Estimates

1. Place a drop of immersion oil on the slide and change the objective to X50 oil. (In cases where no X50
is available, use the X40 high dry with no oil.)
2. Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs. Count 10
fields, take the total number of white cells and divide by 10, and refer to the table 1 below to determine
the WBC estimate.
Table 1: WBC estimates

WBC/High power field Estimated WBC count

2 to 4 4.0 to 7.0 X 109/L
4 to 6 7.0 to 10.0 X 109/L

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 6 to 10 10.0 to 13.0 X 109/L
10 to 20 13.0 to 18.0 X 109/L

Table 2: Platelets estimates

Average No of platelets per X100 field Estimate Platelet count

0 to 1 < 20,000
1 to 4 20,000 to 80,000
5 to 8 100,000 to 160,000
10 to 15 200,000 to 300,000
16 to 20 320,000 to 400,000
> 21 > 420,000

4. An alternative technique is to do a WBC estimate by taking the average number of white cells and
multiplying by 2000.

Observations Under X100: Platelet Estimates

1. Platelet estimates are done under X100 with the RBCs barely touching, approximately 200 RBCs. This
takes place under the X100 objective (oil). On average there are 8 to 20 platelets per field. See Table 2
above.
2. Ten fields are counted using the zigzag method. This method of counting is done by going back and
forth lengthwise or sidewise. Platelets per oil immersion field (OIF)
< 8 platelets/OIF = decreased; 8 to 20 platelets/OIF = adequate; > 20 platelets/OIF = increased
3. After the 10 fields are counted, the number of platelets is divided by 10 to get the average. The average
number is now multiplied by a factor of 20,000 for wedge preparations. For monolayer preparations, use a
factor of 15,000.

Courtesy: Mbukam ED

35
 MLT 2002 Lecture 6 Notes 2013

OVERVIEW OF BLOOD COAGULATION

Coagulation is a complex network of interactions involving vessels, platelets, and factors. The ability to
form and to remove a clot is truly a system dependent on many synergistic forces. Hemostasis depends on
a system of checks and balances between thrombosis and hemorrhage that includes both procoagulants
and anticoagulants.
This scale needs to be kept in balance. Thrombosis is an activation of the hemostatic system at an
inappropriate time in a vessel. Thrombi formed in this fashion are pathologic and beyond the normal
hemostatic mechanism. If physiological anticoagulants are decreased in the circulation there will be a
clot. If procoagulants or clotting factors are decreased, the scale will tip toward bleeding. Hemorrhage or
excessive bleeding may be due to blood vessel disease, rupture, platelet abnormalities, and acquired or
congenital abnormalities. Hemostasis is comprised of the vascular system, platelets, and a series of
enzymatic reactions of the coagulation factors. The role of hemostasis is to arrest bleeding from a vessel
wall defect, while at the same time maintaining fluidity within circulation.
Under physiological conditions, fluidity is maintained by the anticoagulant, profibrinolytic, and
antiplatelet properties of the normal endothelium.
Coagulation is divided into two major systems: the primary and secondary systems of hemostasis. The
primary system comprises platelet function and vasoconstriction. The secondary system involves
coagulation proteins and a series of enzymatic reactions. Once the coagulation proteins become involved,
fibrin is formed and this reinforces platelet plug formation until healing is complete. The product of the
coagulation cascade is the conversion of soluble fibrinogen into an insoluble fibrin clot. This is
accomplished by the action of a powerful coagulant, thrombin. Thrombin is formed by a precursor
circulating protein, prothrombin. Dissolution of the platelet plug is achieved by the fibrinolytic process.

VASCULAR SYSTEM

The vascular system prevents bleeding through vessel contraction, diversion of blood flow from damaged
vessels, initiation of contact activation of platelets with aggregation, and contact activation of the
coagulation system. The vessel wall contains varying amounts of fibrous tissue such as collagen and
elastin, as well as smooth muscle cells and fibroblasts. Arteries are the vessels that take blood away from
the heart and have the thickest walls of the vascular system. Veins return blood to the heart, and are larger
with a more irregular lumen than the arteries.

The Endothelium

The endothelium contains connective tissue such as collagen and elastin. This matrix regulates the
permeability of the inner vessel wall and provides the principal stimuli to thrombosis following injury to a
blood vessel. Circulating platelets recognize and bind to insoluble sub-endothelial connective tissue
molecules. This process is dependent on molecules that are in plasma and on platelets. Two factors, von
Willebrand (vWF) and fibrinogen, participate in the formation of the platelet plug and the insoluble
protein clot, resulting in the activation of the coagulation proteins. Receptor molecules not only adhere to
platelets and damaged vessel components but also allow platelets to use vWF and fibrinogen to bind
platelets and form a plug. Blood flows out through the wall and comes in contact with collagen. Collagen

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is an insoluble fibrous protein that accounts for much of the body’s connective tissue. Vessel injury leads
to the stimulation of platelets.

Events Following Vascular Injury

1. Thromboresistant properties of a blood vessel maintain blood in a fluid state.
2. After vascular injury, subendothelial components of collagen induce platelet aggregation, which
is mediated by vWF and platelet receptor glycoprotein Ib.
3. Further platelet recruitment occurs as a result from fibrinogen binding to its platelet receptor,
glycoprotein IIb/IIIa.
4. . Tissue factor generates thrombin, which results in cross-linked fibrin strands that reinforce the
platelet plug.
5. Platelet actomyosin mediates clot retraction to compact the platelet mass.

PRIMARY HEMOSTASIS

Platelets: An Introduction
Platelets were recognized in 1882 by Bizzozero as a cell structure different from red and white cells.
However, it was not until 1970 that platelets’ relationship to hemostasis and thrombosis became so
important. Every cubic millimeter of blood contains one-fourth of 1 billion platelets, resulting in
approximately a trillion platelets in the blood of an average woman. Each platelet makes 14,000 trips
through the bloodstream in its life span of 7 to 10 days.

Platelet Development

Platelets, or thrombocytes, are small discoid cells (0.5 to 3.0 μm) that are synthesized in the bone marrow
and stimulated by the hormone thrombopoietin. They are developed through a pluripotent stem cell that
has been influenced by colony-stimulating factors (CSF) produced by macrophages, fibroblasts, T
lymphocytes, and stimulated endothelial cells. The parent cells of platelets are called megakaryocytes.
These large cells (80 to 150 μm) are found in the bone marrow.
Megakaryocytes do not undergo complete cellular division but undergo a process called endomitosis or
endoreduplication creating a cell with a multilobed nucleus. Each megakaryocyte produces about 2000
platelets. Thrombopoietin is responsible for stimulating maturation and platelet release. This hormone is
generated primarily by the kidney and partly by the spleen and liver. There is no reserve of platelets in the
bone marrow: 80% are in circulation and 20% are in the red pulp of the spleen. Platelets have no nucleus
but do have granules: alpha granules, and dense granules.
These granules are secreted during the platelet release reaction and contain many biochemically active
components such as serotonin, ADP, and ATP. They are destroyed by the reticuloendothelial system
(RE). Platelet development occurs in the following sequence:
1. Megakaryoblasts are the most immature cell (10 to 15 μm) with a high nuclear to cytoplasmic
ratio and two to six nucleoli.
2. Promegakaryocyte is a large cell of 80 μm with dense alpha and lysosomal granules.
3. Basophilic megakaryocyte shows evidence of cytoplasmic fragments containing membranes,
cytotubules, and several glycoprotein receptors.
4. The megakaryocyte is composed of cytoplasmic fragments that are released by a process called
the budding of platelets.

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Platelet Structure and Biochemistry
Platelets have a complex structure comprised of four zones: the peripheral zone, the sol gel zone, the
organelle zone, and the membrane system.

Megakaryocyte, the platelet parent cell.

Platelet Function and Kinetics

Platelets play an important role in both the formation of a primary plug as well as the coagulation
cascade. The formation of a plug at the site of a cut vessel serves as the initial mechanical barrier. The
lumen of the vessel is lined with endothelial cells; a break in this will initiate a series of reactions.
There are four phases to platelet function:

1. REACTION 1 (ADHESION): Platelets adhere to collagen and undergo shape change from disc
to spiny spheres. Glycoprotein (GP) Ib and vWF aid in adhesion. This is primary aggregation and
is reversible. This reaction is mediated by the release of platelet granules.
2. REACTION 2 (AGGREGATION): In response to chemical changes, these events lead to
platelet aggregation in which platelets adhere to other platelets. Platelet shape change occurs.
3. REACTION 3 (RELEASE): Platelets release the contents of their dense granules. The release of
these granules constitutes a secondary aggregation that is irreversible. Thromboxane A2 is
released by platelets, which promotes vasoconstriction. ADP amplifies the process.
4. REACTION 4 (STABILIZATION OF THE CLOT): This reaction is responsible for thrombus
formation. The adherent and aggregated platelets release factor V and expose platelet factor 3 to
accelerate the coagulation cascade and promote activation of clotting factors and ultimately
stabilize the platelet plug with a fibrin clot. The platelet membrane contains important receptors
called GPs on the platelet surface. Further interactions are mediated by both plasma protein
receptors of vWF and fibrinogen. Other activators of platelets are thrombin, ADP, thromboxane
A2, serotonin, epinephrine, and arachidonic acid.
The receptor for vWF is GPIb-IX. GPIIb/IIIa are receptors for fibronectin, vWF, fibrinogen, and factors
V and VIII. This interaction recruits more platelets to interact with each other. Adhesion of platelets to
collagen and each other can occur without contraction or shape change. Contraction causes shape change
into a spiny sphere. Exposure of a negatively charged membrane leads to secretion of granular contents.
These activated platelets release ADP and synthesized thromboxane A2, which mediate activation of
additional platelets, resulting in the formation of a platelet plug.

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Platelet Aggregation Principle

Aggregation defines the ability of platelets to stick to one another. The formation of aggregates is
observed with a platelet aggregometer.
Primary aggregation is the first wave of aggregation and is preceded by a shape change except when
platelets are stimulated with epinephrine. Primary aggregation is a reversible process. The second phase
of platelet aggregation occurs when platelet granule contents are secreted. Secondary aggregation is
irreversible. Epinephrine, collagen, ADP, and arachidonic acid are the aggregating agents most frequently
used in clinical platelet aggregation.

Courtesy: Mbukam ED

39
 MLT 2002 Lecture 8 Notes 2013
SECONDARY HEMOSTASIS

Secondary hemostasis involves a series of blood protein reactions through a cascade-like process that
concludes with the formation of an insoluble fibrin clot. This system involves multiple enzymes and
several cofactors as well as inhibitors to keep the system in balance. Coagulation factors are produced in
the liver, except for factor VIII, which is believed to be produced in the endothelial cells. When the
factors are in a precursor form, the enzyme or zymogen is converted to an active enzyme or a protease.
The initiation of clotting begins with the activation of two enzymatic pathways that will ultimately lead to
fibrin formation: the intrinsic and extrinsic pathways. Both pathways are necessary for fibrin formation,
but their activating factors are different. Intrinsic activation occurs by trauma within the vascular system,
such as exposed endothelium. This system is slower and yet more important versus the extrinsic pathway,
which is initiated by an external trauma, such as a clot and occurs quickly.

Classification of Coagulation Factors

Coagulation factors may be categorized into substrates, cofactors, and enzymes. Substrates are the
substance upon which enzymes act. Fibrinogen is the main substrate. Cofactors accelerate the activities of
the enzymes that are involved in the cascade. Cofactors include tissue factor, factor V, factor VIII, and
Fitzgerald factor. All of the enzymes are serine proteases except factor XIII, which is a transaminase.
There are three groups in which coagulation factors can be classified:
1. The fibrinogen group consists of factors I, V, VIII, and XIII. They are consumed during coagulation.
Factors V and VIII are labile and will increase during pregnancy and inflammation.
2. The prothrombin group: Factors II, VII, IX, and X all are dependent on vitamin K during their
synthesis. This group is stable and remains preserved in stored plasma.
3. The contact group: Factor XI, factor XII, prekallikrein, and high-molecular-weight kininogen
(HMWK) are involved in the intrinsic pathway, moderately stable, and not consumed during coagulation.
Factor I, Fibrinogen
Substrate for thrombin and precursor of fibrin, it is a large globulin protein. Its function is to be converted
into an insoluble protein and then back to soluble components. When exposed to thrombin, two peptides
split from the fibrinogen molecule, leaving a fibrin monomer to form a polymerized clot.
Factor II, Prothrombin
Precursor to thrombin, in the presence of Ca2+, it is converted to thrombin (IIa), which in turn stimulates
platelet aggregation and activates cofactors protein C and factor XIII. This is a vitamin K–dependent
factor.
Factor III, Thromboplastin
Tissue factor activates factor VII when blood is exposed to tissue fluids.
Factor IV, Ionized Calcium
This active form of calcium is needed for the activation of thromboplastin and for conversion of
prothrombin to thrombin.
Factor V, Proaccelerin or Labile Factor
This is consumed during clotting and accelerates the transformation of prothrombin to thrombin. A
vitamin K–dependent factor, 20% of factor V is found on platelets.
Factor VI, Nonexistent

40
Factor VII, Proconvertin or Stable Factor
This is activated by tissue thromboplastin, which in turn activates factor X. It is a vitamin K–dependent
factor.
Factor VIII, Antihemophilic
This cofactor is used for the cleavage of factor X-Xa by IXa. Factor VIII is described as VIII/vWF:VIII:C
active portion, measured by clotting, VIII:Ag is the antigenic portion, vWF:Ag measures antigen that
binds to endothelium for platelet function; it is deficient in hemophilia A.
Factor IX, Plasma Thromboplastin Component
A component of the thromboplastin generating system, it influences amount as opposed to rate. It is
deficient in hemophilia B, also known as Christmas disease. It is sex linked and vitamin K–dependent.
Factor X, Stuart-Prower
Final common pathway merges to form conversion of prothrombin to thrombin, activity also related to
factors VII and IX. It is vitamin K–dependent and can be independently activated by Russell’s viper
venom.
Factor XI, Plasma Thromboplastin Antecedent
Essential to intrinsic thromboplastin generating of the cascade, it has increased frequency in the Jewish
population. Bleeding tendencies vary, but there is the risk of postoperative hemorrhage.
Factor XII, Hageman factor
This surface contact factor is activated by collagen. Patients do not bleed but have a tendency to
thrombosis.
Factor XIII, Fibrin Stabilizing Factor
In the presence of calcium, this transaminase stabilizes polymerized fibrin monomers in the initial clot.
This is the only factor that is not found in circulating plasma.
High-Molecular-Weight Kininogen
This surface contact factor is activated by kallikrein.
Prekallikrein, Fletcher Factor
This is a surface contact activator, in which 75% is bound to HMWK.

Physiological Coagulation (In Vivo)

The original theory of coagulation used a cascade or waterfall theory. This description depicted the
generation of thrombin by the soluble coagulation factors and the initiation of coagulation. This theory
identified two starting points for the generation of thrombin: the initiation of the Intrinsic pathway with
factor XII and surface contact, and the extrinsic pathway with factor VIIa and tissue factor. These two
pathways meet at the common pathway, where they both generate factor Xa from X, leading to a common
pathway of thrombin from prothrombin and the conversion of fibrinogen to fibrin. This process holds true
under laboratory conditions. The discovery of a naturally occurring inhibitor of hemostasis, tissue factor
pathway inhibitor (TFPI), is able to block the activity of the tissue factor VIIa complex, soon after it
becomes active.

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Laboratory Model of Coagulation

Hemostasis and coagulation tests are generally done for patients with bleeding disorders, vascular
injury or trauma, or coagulopathies. Laboratory testing looks at the in vitro effect of the coagulation
process which is measured by the prothrombin time (PT), activated partial thromboplastin time
(APTT), thrombin time (TT), fibrin degradation products (FDPs), and D-dimer. While the
coagulation cascade does not reflect what goes on in vivo, it provides a model in which the laboratory
relates to for testing. However, the coagulation cascade reflects the mechanisms that the laboratory uses
for results. The screening tests provide a tremendous amount of information to the physician. They can be
performed both quickly and accurately.
Courtesy: Mbukam ED

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