HEMATOLOGY I
INTRODUCTION TO HISTORY
HEMATOLOGY The first scientists, such as
Athanasius Kircher in 1657,
The average human possesses 5
described "worms" in the blood, and
LITERS of blood.
Functions of Blood: Anton van Leeuwenhoek in 1674
1. Transports oxygen from lungs gave an account of RBCs,
to tissues; but it was not until the late 1800s
2. Clears tissues of carbon that Giulio Bizzozero described
dioxide; platelets as "PETITES PLAQUES."
3. Transports glucose, proteins,
The development of the Wright
and lipids;
4. Moves wastes to the liver and stain by James Homer Wright in
kidneys. 1902 opened a new world of visual
The liquid portion is plasma, which, blood film examination through
among many components, provides the microscope.
coagulation enzymes that Although automated analyzers now
protect vessels from trauma and differentiate and enumerate blood
maintain the circulation. Plasma
cells, Wright's Romanowsky-type
transports and nourishes blood
cells. stain (polychromatic, a mixture of
There are Three categories of acidic and basic dyes), and
blood cells: refinements thereof, remains the
1. Red blood cells (RBCs), or foundation of blood cell
erythrocytes; identification.
2. White blood cells (WBCs), or
In the present-day hematology
leukocytes; and
3. Platelets (PLTs), or laboratory, RBC, WBC, and platelet
thrombocytes. appearance is analyzed through
HEMATOLOGY is the study of these automation or visually using
blood cells. By expertly staining, 500X to 1000X light microscopy
counting, analyzing, and recording examination of cells fixed to a glass
the appearance, phenotype, and microscope slide and stained with
genotype of all three types of cells,
Wright or Wright-Giemsa stain
the medical laboratory professional is
able to predict, detect, and diagnose (Chapters 3, 12, and 13).
blood diseases and many systemic The scientific term for cell
diseases that affect blood cells. appearance is morphology, which
Physicians rely on hematology encompasses cell color, size,
laboratory test results to select shape, cytoplasmic inclusions,
and monitor therapy for these and nuclear condensation.
disorders; consequently, a complete
blood count (CBC) is ordered on
RED BLOOD CELLS
nearly everyone who visits a
physician or is admitted to a
RBCs are ANUCLEATE,
hospital.
BICONCAVE, DISCOID CELLS
filled with a reddish protein,
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HEMATOLOGY I
Hemoglobin, which transports Thoma pipette, was used routinely
oxygen and carbon dioxide (Chapters until the advent of automation. The
7 and 8). RBCs appear salmon pink diluted blood was transferred to a
and measure 7 to 8 um in glass counting chamber called a
diameter with a zone of pallor that hemacytometer (Figure 11.1). The
occupies one third (1/3) of their microscopist observed and counted
center, reflecting their biconcavity. RBCs in selected areas of the
Note: “Zone of central pallor” hemacytometer, applied a
should be approximately 1/3 the mathematical formula based on the
diameter of the entire red cell (cells dilution and on the area of the
like this are called normochromic). hemacytometer counted (Chapter
If it's much larger, that means that 11), and reported the RBC count in
the cell does not have enough cells per microliter (μL, mcL, also
hemoglobin (cells like this are called called cubic millimeter, mm³),
hypochromic) and the patient is milliliter (mL, also called cubic
anemic. centimeter, or cc), or liter (L).
Since before 1900, physicians and Visual RBC counting was
medical laboratory professionals developed before 1900 and,
counted RBCs in measured volumes although inaccurate, was the only
to detect anemia or polycythemia. way to count RBCs until 1958, when
Anemia means loss of oxygen- automated particle counters
carrying capacity and is often became available in the clinical
reflected in a reduced RBC count laboratory.
or decreased RBC hemoglobin The first electronic counter, patented
concentration (Chapters 16 to 25). in 1953 by Joseph and Wallace
Polycythemia means an increased Coulter of Chicago, Illinois, was
RBC count reflecting increased used so widely that today automated
circulating RBC mass, a condition cell counters are often called
that leads to hyperviscosity Coulter counters, although many
(Chapter 32). high-quality competitors exist
Historically, microscopists counted (Chapter 12).5 The Coulter
RBCs by carefully pipetting a tiny principle of direct current
aliquot of whole blood and electrical impedance is still used to
mixing it with 0.85% (normal) count RBCs in many automated
saline. blood cell analyzers.
Normal saline matches the
osmolality of blood; consequently,
the suspended RBCs retained their
intrinsic morphology, neither swelling
nor shrinking.
A 1:200 dilution was typical for
RBC counts, and a glass pipette
designed to provide this dilution, the
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Fortunately, the widespread reagent, hemoglobin is converted to
availability of automated cell stable cyanmethemoglobin
counters has replaced visual RBC (hemiglobincyanide), and the
counting, although visual counting absorbance or color intensity of the
skills remain useful where automated solution is measured in a
counters are unavailable. spectrophotometer at 540 nm
wavelength. The color intensity is
compared with that of a known
standard and is mathematically
converted to hemoglobin
concentration. Modifications of the
cyanmethemoglobin method are
used in most automated
applications, although some
automated blood cell analyzers
replace it with a formulation of the
ionic surfactant (detergent)
sodium lauryl sulfate to reduce
environmental cyanide.
HEMATOCRIT is the ratio of the
Figure 1.1 Composite of Cells Found in Peripheral volume of packed RBCs to the
Blood of Healthy Individuals. (A), Erythrocyte (red volume of whole blood and is
blood cell, RBC); (B), neutrophil (segmented manually determined by transferring
neutrophil, NEUT, SEG, polymorphonuclear blood to a plastic tube with a uniform
neutrophil, PMN); (C), band (band neutrophil, bore, centrifuging, measuring the
BAND); (D), eosinophil (EO); (E), basophil (BASO); column of RBCs, and dividing by the
(F), lymphocyte (LYMPH); (G), monocyte (MONO); total length of the column of RBCs
(H), platelet (PLT). (Wright-Giemsa stain, 1000. plus plasma. The normal ratio
approaches 50%.
HEMOGLOBIN, HEMATOCRIT, AND Hematocrit is also called PACKED
RED BLOOD CELL INDICES CELL VOLUME (PCV), the packed
cells referring to RBCs. Often one
RBCs also are assayed for
can see a light-colored layer between
1. Hemoglobin (HGB)
the RBCs and plasma. This is the
concentration
buffy coat and contains WBCs and
2. Hematocrit (HCT)
platelets, and it is excluded from the
Hemoglobin measurement relies on a
hematocrit determination.
weak solution of potassium
The medical laboratory professional
cyanide (KCN) and potassium
may use the three numerical
ferricyanide (K3[Fe(CN)6]),
results
called Drabkin reagent.
a. RBC count
An aliquot of whole blood is mixed
b. HGB
with a measured volume of Drabkin
c. HCT,
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HEMATOLOGY I
to compute the RBC indices mean cell visually review RBC morphology,
volume (MCV), mean cell hemoglobin commenting on RBC diameter, color
(MCH), and mean cell hemoglobin or hemoglobinization, and shape and
concentration (MCHC). the presence of cytoplasmic
inclusions.
The MCV All these parameters, RBC count,
- femtoliters (fL), HGB, HCT, indices, and RBC
- reflects RBC diameter on a morphology, are employed to detect,
Wright-stained blood film. diagnose, assess the severity of, and
The MCHC monitor the treatment of anemia,
- grams per deciliter (g/dL), polycythemia, and the numerous
- reflects RBC staining intensity systemic conditions that affect RBCs.
and the amount of central Automated blood cell analyzers are
pallor. used in nearly all clinical laboratories
The MCH to generate these data, although
- picograms (pg) visual examination of the Wright-
- expresses the mass of stained blood film is still essential to
hemoglobin per cell and verify abnormal results.
parallels the MCHC.
A fourth RBC index, RBC
RETICULOCYTES
distribution width (RDW),
- expresses the degree of variation
In the Wright-stained blood film,
in RBC volume.
0.5% to 2.5% of RBCs exceed the
- Extreme RBC volume variability is
7-to 8-um average diameter and
visible on the Wright-stained blood
stain slightly blue-gray. These are
film as variation in diameter or
polychromatic
anisocytosis.
(polychromatophilic)
The RDW is based on the standard
erythrocytes, newly released from
deviation of RBC volume and is
the RBC production site: the bone
routinely reported by automated
marrow (Chapters 4 and 5).
blood cell analyzers.
Polychromatic erythrocytes are
In addition to aiding in diagnosis of
closely observed because they
anemia, RBC indices provide stable
indicate the ability of the bone
measurements for internal quality
marrow to increase RBC production
control of automated blood cell
in anemia caused by blood loss or
analyzers.
excessive RBC destruction.
Sample reference intervals for RBC
Methylene blue dyes, called
parameters are included on the
nucleic acid stains or vital stains,
inside front cover; these vary by age
are used to differentiate and count
and gender.
these young RBCs.
Medical laboratory professionals
Vital (or "supravital") stains are
routinely use light microscopy at
dyes absorbed by live cells.
500X or 1000x magnification to
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Young RBCs contain ribonucleic acid The acid causes RBCs to lyse or
(RNA) and are called reticulocytes rupture; without it, RBCs, which are
when the RNA is visualized using 500 to 1000 times more numerous
vital stains (Chapter 11). than WBCs, would obscure the
Counting reticulocytes visually by WBCs.
microscopy was (and remains) a The WBC count reference intervals
tedious and imprecise procedure are included on the inside front
until the development of automated cover.
reticulocyte counting by the TOA Visual WBC counting has been
Corporation (presently Sysmex) in largely replaced by automated
1990. Now all fully automated blood blood cell analyzers, but it is
cell analyzers provide a relative accurate and useful in situations in
reticulocyte percentage, an absolute which no automation is available.
reticulocyte count, and, in addition, Medical laboratory professionals who
an especially sensitive measure of analyze body fluids such as
RBC production, the immature cerebrospinal fluid or pleural fluid
reticulocyte fraction (Chapter 12). may employ visual WBC counting.
However, it is still necessary to A decreased WBC count is called
confirm automated analyzer counts leukopenia,
visually from time to time, so and an increased WBC count is called
medical laboratory professionals leukocytosis, but the WBC count
must retain this skill. alone has modest clinical value.
The microscopist must differentiate
WHITE BLOOD CELLS the categories of WBCs in the blood
WBCs, or leukocytes, are a loosely by using a Wright-stained blood
related category of cell types film and light microscopy.
dedicated to protecting their host The types of WBCs found in
from infection and injury. peripheral blood in healthy
WBCs are transported in the blood individuals are as follows:
from their source, usually bone 1. Neutrophils (NEUTs, segmented
marrow or lymphoid tissue, to neutrophils [SEGs],
their tissue or body cavity polymorphonuclear neutrophils
destination. [PMNs]; Figure 1.1B).
WBCs are so named because they - are phagocytic cells whose
are nearly colorless in an unstained major purpose is to engulf and
cell suspension. WBCs may be destroy microorganisms and
counted visually using a microscope foreign material, either
and hemacytometer. directly or after they have
The technique is the same as RBC been labeled for destruction
counting, but the typical dilution is by the immune system.
1:20, and the diluent is a dilute acid - The term segmented refers
solution. to their multilobed nuclei.
The cytoplasm of neutrophils
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contains pink- or lavender- - *The distribution of basophils
staining granules filled with and eosinophils in blood is so
bactericidal substances. small compared with that of
- An increase in neutrophils is neutrophils that the terms
called neutrophilia and often eosinopenia and basopenia
signals bacterial infection. are theoretical and not
- A decrease is called used.
neutropenia and has many Neutrophils, bands, eosinophils, and
causes, but it is often caused basophils are collectively called
by certain medications or granulocytes because of their
viral infections. prominent cytoplasmic granules,
- Bands (band neutrophils, although their functions differ.
BANDs; Figure 1.1C). Bands 4. Lymphocytes (LYMPHS; Figure
are slightly less mature 1.1F). Lymphocytes comprise a
neutrophils with a complex system of cells that
nonsegmented nucleus in a U provide for host immunity.
or S shape. An increase in - Lymphocytes recognize
bands also signals bacterial foreign antigens and mount
infection and is customarily humoral (antibodies) and
called a left shift. cell-mediated antagonistic
2. Eosinophils (EOs; Figure 1.1D). responses.
Eosinophils are cells with round, - On a Wright-stained blood
bright orange-red cytoplasmic film, most lymphocytes are
granules filled with proteins nearly round, are slightly
involved in immune system larger than RBCs, and have
regulation. round featureless nuclei and a
- An elevated eosinophil count thin rim of nongranular
is called eosinophilia and cytoplasm.
often signals a response to - An increase in the lymphocyte
allergy or parasitic count is called lymphocytosis
infection. and often is associated with
3. Basophils (BASOs; Figure 1.1E). viral infections.
Basophils are cells with dark - Accompanying lymphocytosis
purple, irregular cytoplasmic are often reactive lymphocytes
granules that obscure the with characteristic
nucleus. morphology.
- The basophil granules contain - An abnormally low lymphocyte
histamines and various count is called lymphopenia
other proteins. or lymphocytopenia and is
- An elevated basophil count is often associated with drug
called basophilia. Basophilia therapy or
is rare and often signals a immunodeficiency.
hematologic disease.
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5. Monocytes (MONOS; Figure leukemia or chronic lymphocytic
1.1G). The monocyte is an leukemia, or acute, for example,
immature macrophage passing acute myeloid leukemia or acute
through the blood from its point lymphoblastic leukemia.
of origin, usually the bone Leukemias may involve any of the
marrow, to a targeted tissue cell lines and are categorized by their
location. respective immunophenotypes and
- Macrophages are the most genetic aberrations.
abundant cell type in the body Some leukemias are more common
although monocytes comprise a in a specific age group; chronic
minor component of peripheral blood lymphocytic leukemia is more
WBCs. prevalent in people older than 65
- Macrophages occupy every body years, whereas acute
cavity; some are motile and some lymphoblastic leukemia is the
are immobilized. Their tasks are to most common form of childhood
identify and phagocytize (engulf leukemia.
and consume) foreign particles Medical laboratory scientists
and assist the lymphocytes in participate in characterization of
mounting an immune response leukemias using Wright-stained
through the assembly and bone marrow smears, flow
presentation of antigen epitopes. cytometric immunophenotyping,
- On a Wright-stained blood film, molecular diagnostic technology,
monocytes have a slightly larger cytogenetics, and, occasionally,
diameter than other WBCs, blue-gray cytochemical staining.
cytoplasm with fine azure granules,
and a nucleus that is usually PLATELETS
indented or folded.
Platelets, or thrombocytes, are true
- An increase in the number of
blood cells that maintain blood
monocytes is called monocytosis.
vessel integrity by initiating
- Benign monocytosis may be found
vessel wall repairs
in certain infections or in
Platelets
inflammation.
a. rapidly adhere to the surfaces of
- Medical laboratory professionals
damaged blood vessels,
seldom document a decreased
b. form aggregates with
monocyte count, so the theoretical
neighboring platelets to plug the
term monocytopenia is seldom
vessels, and
used.
c. secrete proteins and small
molecules that trigger
Leukemia is an uncontrolled
thrombosis (clot formation).
proliferation of a clone of malignant
Platelets are the major cells that
WBCs.
control hemostasis (physiological
Leukemia may be chronic, for
process by which bleeding ceases) ,
example, chronic myeloid
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a series of cellular and plasma-based Mean platelet volume (MPV)
mechanisms that… - The presence of predominantly
a. seal wounds, larger platelets generates an
b. repair vessel walls, and elevated MPV value, which
c. maintain vascular patency sometimes signals a
(unimpeded/unobstructed blood regenerative bone marrow
flow). response to platelet
Platelets are only 2 to 4 mm in consumption.
diameter, round or oval, anucleate Elevated platelet counts, called
(for this reason some hematologists THROMBOCYTOSIS, signal
prefer to call platelets “cell inflammation or trauma.
fragments”), and slightly granular ESSENTIAL THROMBOCYTHEMIA
(Figure 1.1H). is a rare malignant condition
Their small size makes them appear characterized by extremely high
insignificant, but they are essential platelet counts and uncontrolled
to life and are extensively studied for platelet production. – is a life-
their complex physiology. threatening hematologic disorder
Uncontrolled platelet and A low platelet count, called
hemostatic activation is THROMBOCYTOPENIA
responsible for deep vein - is a common consequence of
thrombosis, pulmonary emboli, acute drug treatment and may be
myocardial infarctions (heart life threatening.
attacks), cerebrovascular accidents - is usually accompanied by
(strokes), peripheral artery disease, easy bruising and uncontrolled
and repeated spontaneous abortions hemorrhage.
(miscarriages). - Thrombocytopenia accounts
The microscopist counts platelets for many hemorrhage-related
using the same technique used in emergency department visits.
counting WBCs on a Accurate platelet counting
hemacytometer, although a contributes to patient safety because
different counting area, diluent, and it provides for the diagnosis of
dilution is usually used. thrombocytopenia in many disorders
Owing to their small volume, or therapeutic regimens.
platelets are hard to distinguish
visually in a hemacytometer, and
phase microscopy provides for
easier identification.
Automated blood cell analyzers COMPLETE BLOOD COUNT
have largely replaced visual platelet
counting and provide greater A COMPLETE BLOOD COUNT
accuracy. (CBC)
The reference interval for the platelet
count is on the inside front cover.
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- is performed on automated platelet counts using dilution
blood cell analyzers and pipettes, hemacytometers, and
- includes the RBC, WBC, and microscopes, most laboratories
platelet measurements. employ automated blood cell
The medical laboratory analyzers to generate the CBC.
professional may collect a blood Many blood cell analyzers also
specimen for the CBC, but often a provide comments on RBC, WBC,
phlebotomist, nurse, physician and platelet morphology.
assistant, physician, or patient In case of abnormal result, the
care technician may also collect instrument provides an indication
the specimen. of this, sometimes called a flag.
No matter who collects, the In this case a “reflex” blood film
medical laboratory professional is examination is performed.
responsible for the integrity of the The BLOOD FILM
specimen and ensures that it is EXAMINATION is a specialized,
submitted in the appropriate demanding, and fundamental CBC
anticoagulant and tube and is activity.
free of clots and hemolysis Nevertheless, if all blood cell
(red-tinted plasma indicating RBC analyzer results are within
damage). reference intervals, the blood
The specimen must be of film examination is usually
sufficient volume, because “short omitted from the CBC.
draws” result in incorrect However, physicians may request
anticoagulant-to-blood ratios. a blood film examination on the
The specimen must be tested or basis of clinical suspicion even
prepared for storage within the when the analyzer results fall
appropriate time frame to within their respective reference
ensure accurate analysis intervals.
accurately registered in the work
list, a process known as
specimen accession.
Accession may be automated,
relying on bar code or
radiofrequency identification
technology, thus reducing
instances of identification error.
Although all laboratory scientists
and technicians are equipped to
perform visual RBC, WBC, and BLOOD FILM EXAMINATION
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the microscopist prepares a ENDOTHELIAL CELLS
“wedge-prep” blood film on a
glass microscope slide, allows it seldom studied in the
to dry, and fixes and stains it hematology laboratory
using Wright or Wright-Giemsa
stain. COAGULATION
The microscopist visually
performs an estimate of the WBC Most hematology laboratories
count (with the 40 or 50 objective include a blood coagulation
at 400 or 500 magnification) and testing department
platelet count (with the 100 oil Platelets are a key
immersion objective at 1000 component of hemostasis, as
magnification) for comparison previously described; plasma
with their respective analyzer coagulation is the second
counts, and investigates component
discrepancies. The coagulation system
Next, the microscopist employs a complex sequence
systematically reviews, identifies, of plasma proteins, some
and tabulates 100 (or more) enzymes, and some enzyme
WBCs to determine their percent cofactors to produce clot
distribution. This process is formation after blood vessel
referred to as determining the injury.
WBC differential (“diff ”). The a third system of enzymes and
WBC differential relies on the cofactors digests clots to
microscopist’s skill, visual acuity, restore vessel patency, a
and integrity, and it provides process called fibrinolysis.
extensive diagnostic information.
The medical laboratory
Finally, the microscopist
professional focuses especially
examines the morphology of
on blood specimen integrity
WBCs, RBCs, and platelets by
for the coagulation laboratory,
light microscopy for abnormalities
because minor blood specimen
of shape, diameter, color, or
defects, including clots,
inclusions using 1000
hemolysis, lipemia, plasma
magnification.
bilirubin, and short draws,
Results of the CBC, including all
render the specimen useless
automated blood cell analysis and
blood film examination NOTES:
parameters and interpretive
comments, are provided in paper Cells of the erythroid series are
or digital formats for physician precursors to RBCs
review with abnormal results
highlighted.
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myeloid series cells mature to form
bands and neutrophils, eosinophils,
and basophils
megakaryocytes produce platelets
The glucose-6-phosphate
dehydrogenase assay
phenotypically detects an inherited
RBC enzyme deficiency causing
episodic hemolytic anemia
erythrocyte sedimentation rate,
detects inflammation and roughly
estimates its intensity
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