Laboratory Hematology
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LABORATORY HEMATOLOGY
Laboratory plays an indispensable role in the diagnosis and management of almost all
hematologic disorders. The purpose here is to introduce you with the common laboratory
tests and explain their significance. Some of the common tests in a hematology laboratory
are the following but we will discuss only the first 4 tests. Other tests are discussed
elsewhere
 Complete blood count with differential
 Reticulocyte count
 Peripheral blood smear evaluation
 Detection of fetal hemoglobin
 Serum and urine protein electrophoresis (SPEP & UPEP)
 Hemoglobin electrophoresis and/or HPLC for hemoglobin
 Cytochemistry
 Leukocyte immunophenotyping
 Coagulation tests
COMPLETE BLOOD COUNT:
This is the most common of all hematology tests. A standard CBC is referred to the
following tests. Some parameters are actual measurements whereas others are just
calculated by the analyzer based on other measured values
1. White blood cell (Leukocyte) count (WBC) with – measured
 5-part differential count
 5-part absolute count
2. Red blood cell (Erythrocyte) count – measured
3. Hemoglobin concentration – measured
4. Hematocrit percentage – measured or calculated
5. Mean cell volume (MCV) – calculated
6. Mean cell hemoglobin (MCH) – calculated
7. Mean cell hemoglobin concentration (MCHC) – calculated
8. Red cell distribution width (RDW) – calculated
9. Platelet count – measured
10. Mean platelet volume (MPV) – calculated
An EDTA-anticoagulated blood is usually used for CBC determination. Although a 10 cc
or a 5 cc blood collection tube is usually used, the amount of blood utilized for CBC is a
very small fraction (<< 1 cc).
Any abnormality “flagged” by automated counter triggers a manual review of the
peripheral smear and cell counting.
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.
Laboratory Hematology
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AUTOMATED BLOOD ANALYSIS:
Several automated hematology analyzers are available for this purpose. One of the most
hematology analyzer is the one from Coulter called Coulter counter. Coulter method of
cell counting and sizing is based on the detection and measurement of changes in the
electrical resistance produced by cells suspended in a conductive liquid and traversing a
small aperture. Suspended blood cells are “focused” into a single cell stream and passed
through a small aperture with electrodes on either side. The cells act as discrete “nonconductors” or insulators of current as they pass from the electrode and through the
aperture. The momentary drop or interruption of current produced by each cell is
measured as a pulse.
 Cell count is proportional to the number of detected signals (or pulses)
 Cell size is proportional to the magnitude (amplitude) of each signal
[This picture is adapted from BLOOD: Principles & Practice of Hematology by Handin et al. Lippincott Press]
There are 2 channels of coulter counter.
(a) No lytic agent / No shrinkage agent channel – Used for RBC and platelet
evaluations (gives accurate RBC and platelet volumes)
(b) RBC lytic agent / WBC shrinkage agent – Used for WBC evaluation and Hb
measurement
Each channel is analyzed by 3 distinct apertures for 4 seconds each. The total time for
measurements and calculations is <1 minute.
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.
Laboratory Hematology
A typical CBC report from a normal count is shown below:
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.
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Laboratory Hematology
Some of the CBC reports from actual patients are shown below:
NEUTROPHILIA, absolute and relative:
EOSINOPHILIA, absolute and relative:
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.
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Laboratory Hematology
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LYMPHOCYTOSIS, absolute and relative:
PANCYTOPENIA:
White Blood Cell (WBC) evaluation:
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WBC count measured as number of cells >35 fl remaining in the “lysed” channel.
Reported as cells x 109/L
The 5-part differential and absolute count is made possible by two modalities
(i)
Conductivity – distinguishes among cells based on characteristics of the
nucleus, granules, and chemical composition of the cells
(ii)
Light scattering – distinguishes among cells based on characteristics of the
cell surface
Data generated by all three interrogation modalities are used to generate a 3-D
scatter plot to separate different WBCs.
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.
Laboratory Hematology
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Red Blood cell (RBC) evaluation:
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RBC count [M 4.5 – 5.5 x 1012/L, F 4 – 5 x 1012/L] is measured as number of
cells with sizes >36 fl in the “unlysed” channel. WBC are also counted but these
represent <0.1% of the cells counted. Reported as cells x 1012/L. RBC count is
increased in polycythemia & decreased in decreased production by the marrow.
Hemoglobin concentration [Males 13 – 18 g/dl, Females 12 – 16] is measured
spectrophotometrically in the “lysed” channel. Reported as g/dl. Increased in
polycythemia and decreased in anemia.
Mean Cellular Volume (MCV) [80-100 fl] is determined from measured RBC
size distribution (histogram) and is reflective of true RBC size. Reported in fl
(Femto liter). Increased in macrocytosis such as seen in megaloblastic anemia and
decreased in microcytic anemia such as iron deficiency anemia and thalassemia.
Reticulocytes are also larger than mature RBCs and thus increased reticulocytes
may “artifactually” increase the MCV. In patients with combined macrocytosis
and microcytosis as seen in combined nutritional deficiencies of vitamin
B12/folate and iron may in the end result in a falsely normal MCV. That is why
examination of a peripheral blood smear is so important!
Hematocrit [M 42% - 52%, F 37% - 48%] is calculated by coulter counter by a
formula = RBC x MCV/10). Reported in %. It is increased in polycythemia and
decreased in anemia. It is, however, directly affected by the plasma volume. In
dehydrated states, Hct is increased whereas in volume overload is low.
Mean cell hemoglobin (MCH) [28 – 33 pg/cell] is calculated by Hb/RBC x 10
and reported in pg (pico grams). It represents the amount of hemoglobin present
in one red blood cell! It is decreased in hypochromia.
Mean cell hemoglobin concentration (MCHC) [32 – 36 g/dl] is calculated by
Hb/Hct x 100 and reported in g/dl. It represents the amount of Hb present in a
“population” of red cells and takes into account any variation in the cell size and
shape. A low MCHC reflects hypochromia.
Red cell distribution width (RDW) [12% – 15%] is the coefficient of RBC size
variation, which is calculated from the measured RBC size in histogram. It
provides a quantitative expression of the size spread of the RBC populations
(anisocytosis). Reported as %. It allows the presence of more than one cell size
population to be appreciated quantitatively. The RDW is often used to
provisionally distinguish microcytic anemia caused by iron deficiency (in which
RDW is increased) and thalassemia (in which RDW is normal). In macrocytic
anemia it helps in distinguishing megaloblastic anemia (in which RDW is
increased) and other macocytic anemias (in which RDW is often normal).
Platelet evaluation:
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Platelet count [150 – 450 x 109/L] is determined from measurements of number
of cells with sizes 2-20 fl in the “unlysed channel” Reported as cells x 109/L
Mean platelet volume [6-8 fl] is measured from a histogram and is reflective of
the true platelet volume! It is rarely clinically used. Reported in fl
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.
Laboratory Hematology
[This ptable is adapted from BLOOD: Principles & Practice of Hematology by Handin et al. Lippincott Press]
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.
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Laboratory Hematology
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RETICULOCYTE COUNT:
Reticulocytes are newly produced RBCs that have extruded their nuclei but still show
ribosomal RNA material that imparts a reticulated appearance to RBCs. They constitute
about 1-2% of peripheral blood population and can be recognized as slightly larger than
normal RBCs and with some blue/purple appearing fine dots (ribosomal RNA) causing
polychromasia. The most common method of highlighting reticulocytes is a supravital
stain to precipitate intracellular RNA. After staining, a thin peripheral smear is made and
reticulocytes are counted by manual method. These cells can also be counted by flow
cytometry. Reticulocytes are increased in any condition in which there is increased output
of new RBC in the peripheral blood such as a response to bleeding and hemolysis and
they are decreased in conditions of decreased production of RBC by the marrow.
PERIPHERAL BLOOD EVALUATION:
This is an important part of evaluation of many hematologic disorders. A peripheral
blood smear is made under four main conditions:
(i)
The automated Coulter counter “flagged” an abnormality that triggers
further evaluation
(ii)
A physician requests evaluation of peripheral blood smear by a pathologist
based on clinical history, and
(iii) At a pathologist’s request to further evaluate any hematologic disease
process even when the CBC is normal such as suspected presence of
parasites in the blood and within cells such as Ehrlichia
(iv)
As part of routine quality control mechanism by laboratories
[The detailed description of a peripheral blood smear will be provided during the session
for peripheral blood evaluation toward the end of the course]
DETECTION OF FETAL HEMOGLOBIN:
(By Kleihauer-Betke test)
Detection of fetal hemoglobin (hemoglobin F) in individual RBCs is useful to establish
the diagnosis of feto-maternal transfusion and also in the evaluation of Hb-F in patients
with hemoglobinopathies. This test is based on the principle that Hb-F is resistant to
elution by an acid whereas Hb-A washes off leaving ghosts of cells. A manual count
yields the % of F cells present.
The contents & pictures in this handout are derived from various sources including books, journal articles and patient
material for teaching purposes only. No commercial incentives are sought or intended.