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MEDICATION-INDUCED
BLOOD DYSCRASIAS:
Diagnosis, Treatment And
Prevention
Jassin M. Jouria, MD
Dr. Jassin M. Jouria is a medical doctor, professor of
academic medicine, and medical author. He graduated from Ross University School
of Medicine and has completed his clinical clerkship training in various teaching
hospitals throughout New York, including King’s County Hospital Center and
Brookdale Medical Center, among others. Dr. Jouria has passed all USMLE medical
board exams, and has served as a test prep tutor and instructor for Kaplan. He has
developed several medical courses and curricula for a variety of educational
institutions. Dr. Jouria has also served on multiple levels in the academic field
including faculty member and Department Chair. Dr. Jouria continues to serves as a
Subject Matter Expert for several continuing education organizations covering
multiple basic medical sciences. He has also developed several continuing medical
education courses covering various topics in clinical medicine. Recently, Dr. Jouria
has been contracted by the University of Miami/Jackson Memorial Hospital’s
Department of Surgery to develop an e-module training series for trauma patient
management. Dr. Jouria is currently authoring an academic textbook on Human
Anatomy & Physiology.
ABSTRACT
Although drug-induced hematologic disorders are less common than other
types of adverse reactions, they are associated with significant morbidity
and mortality. Some agents, such as hemolytics, cause predictable
hematologic disease, but others induce idiosyncratic reactions not directly
related to the drug’s pharmacology. The most important part of managing
hematologic disorders is the prompt recognition that a problem exists. The
main mechanisms to manage hematologic disorders include vigilance to
observe signs and symptoms indicating a blood disorder and patient
education of the warning symptoms to alert them of the need to report a
condition to their primary care provider or an emergency health team.
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Continuing Nursing Education Course Director & Planners
William A. Cook, PhD, Director, Douglas Lawrence, MA, Webmaster,
Susan DePasquale, MSN, FPMHNP-BC, Lead Nurse Planner
Policy Statement
This activity has been planned and implemented in accordance with the
policies of NurseCe4Less.com and the continuing nursing education
requirements of the American Nurses Credentialing Center's Commission on
Accreditation for registered nurses. It is the policy of NurseCe4Less.com to
ensure objectivity, transparency, and best practice in clinical education for
all continuing nursing education (CNE) activities.
Continuing Education Credit Designation
This educational activity is credited for 4 hours. Nurses may only claim credit
commensurate with the credit awarded for completion of this course activity.
Pharmacology Content is 0.5 hour (30 minutes).
Statement of Learning Need
Clinicians need to know how to manage the risk of hematologic disorders
induced by medication. Understanding the risk, recognizing the signs and
symptoms that may indicate a blood disorder, and being skilled in how to
educate the patient are essential knowledge needs of clinicians to ensure
patients, caregivers and health teams are able to recognize the warning
symptoms of hematologic disorders.
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Course Purpose
To provide nurses and health team associates with knowledge about
medication-induced dyscrasias to better recognize, treat, and educate
patients, caregivers and other health team members on acute and long-term
management.
Target Audience
Advanced Practice Registered Nurses and Registered Nurses
(Interdisciplinary Health Team Members, including Vocational Nurses and
Medical Assistants may obtain a Certificate of Completion)
Course Author & Director Disclosures
Jassin M. Jouria, MD, William S. Cook, PhD, Douglas Lawrence, MA
Susan DePasquale, MSN, FPMHNP-BC – all have no disclosures
Acknowledgement of Commercial Support
There is no commercial support for this course.
Activity Review Information
Reviewed by Susan DePasquale, MSN, FPMHNP-BC
Release Date: 5/21/2016
Termination Date: 5/21/2019
Please take time to complete a self-assessment of knowledge, on
page 4, sample questions before reading the article.
Opportunity to complete a self-assessment of knowledge learned will
be provided at the end of the course.
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1. The complete blood count (CBC) is sometimes referred to as
a.
b.
c.
d.
the
the
the
the
cellular components test (CCT).
peripheral blood count (PBC).
EDTA test.
three-phase test.
2. Microscopic evaluation of a blood smear is best when the slide
is prepared
a.
b.
c.
d.
indefinitely if properly stored.
within 8 hours of collection.
within 3 hours of collection.
up to 24 hours after collection.
3. True or False: Freezing of blood samples is essential to
preserving the samples for a valid, complete blood count (CBC)
test.
a. True
b. False
4. The complete blood count (CBC) analyzes
a.
b.
c.
d.
concentration of leukocytes (white blood cells).
volume of RBCs (red blood cells).
weight of RBCs (red blood cells).
All of the above
5. ___________________ provides the best morphologic
preservation of blood cells and prevents coagulation of the
blood specimen.
a.
b.
c.
d.
Cold agglutination
Dipotassium (K2) EDTA
IgM antibodies
Romanowsky stains
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Introduction
Certain medication, such as hemolytics, cause predictable hematologic
disease, but others can induce idiosyncratic reactions not directly related to
the drug’s pharmacology. In the first course of this two part series, it was
emphasized that the most important part of management of a hematologic
disorder is the prompt recognition when a problem exists. This is done by
two mechanisms: firstly, vigilance for signs and symptoms that may indicate
a blood disorder; and, secondly, patient education about the warning
symptoms that should alert them to the need to urgently contact their
medical provider or emergency services if a prompt medical appointment is
not possible. This second course focuses more specifically on the
identification and management of drug-induced blood dyscrasias, including
prevention through vigilant monitoring, hygiene and vitamin intake.
Diagnosis Of Blood Dyscrasia:
The Complete Blood Count (CBC)
One of the most common tests to identify and monitor a condition of blood
dyscrasia is the complete blood count (CBC) with differential. It is one of the
most common laboratory tests performed, which informs clinicians about
blood cell production and the ability of the red blood cells (RBC) to carry
oxygen and of the white blood cells (WBC) to fight infection. Hematology
medicine (concerned with the diagnosis and treatment of blood health and
disease) laboratory testing helps to identify disease states that may be
associated side effects of certain drugs that cause blood dyscrasias.
The performance of a complete blood count (CBC) has three phases: preexamination (before testing), examination (testing), and post-examination
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(reporting). The pre-examination phase includes the proper identification of
the patient and proper collection and handling of the specimen. The
specimen is analyzed in the examination phase.
The CBC (sometimes referred to as the peripheral blood count (PBC)) is a
primary screening test that provides information regarding the cellular
components of the blood as they circulate in the peripheral blood. The
concentration of leukocytes (white blood cell (WBC)), erythrocytes (red
blood cell (RBC)), and platelets as well as a categorization of the different
WBC subsets is included. Additional information regarding RBCs is also
integrated into the CBC and includes, at minimum, the concentration of
hemoglobin and the packed cell volume of RBCs, called the hematocrit.
Finally, a CBC can also provide what are known as the RBC indices that are
used to depict the volume and the total weight of each RBC and
concentration of hemoglobin in it. The CBC can be determined by automated
and/or manual methods. The post-examination phase includes reporting and
interpreting the data.
Based on the information collected from the CBC, the laboratory professional
can provide diagnostic criteria or meaningful recommendations for any
follow-up testing (reflex testing) to the medical provider to support quality
patient care. The CBC report can often be submitted for medical review in an
emergency situation within minutes; however, manual differentials by a
trained laboratory professional will take longer. This section highlights the
varied phases of the CBC test and final report for medical review,
interpretation and diagnosis of a blood disorder.3,49,62-68
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Phases of the CBC
Pre-examination
Patient identification
Specimen collection and handling
Examination
Automated results
Evaluation and analysis of peripheral blood smear
Post-examination
Interpretation of data
Reporting results
Pre-Examination Phase of the CBC
The pre-examination phase of the CBC encompasses patient identification,
blood collection, and specimen handling. Briefly, patient identification
includes the patient’s name and a second identifier that can be a hospital
number but more commonly is the patient’s date of birth. This
documentation must be available from the patient at the time of the blood
collection. Once the patient has been properly identified, the laboratory
professional performing the phlebotomy must be well acquainted with the
various collection devices, their safety features and requirements, and the
anticoagulants or additives within the sample collection tubes. This individual
should have a thorough knowledge of the phlebotomy procedure safety
issues including methods to prevent exposure to blood-borne pathogens.
Although other blood collection tube additives can be used for hematologic
analysis, almost all specimens for a routine CBC are collected in a purplelavender-top sample collection tube that contains ethylenediaminetetraacetic
acid (EDTA). Dipotassium (K2) EDTA provides the best morphologic
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preservation of blood cells and prevents coagulation of the blood specimen.
The anticoagulated specimen allows a laboratory professional to generate
multiple blood smears from one tube of blood, a technique that must be
performed within 3 hours of blood collection.
Proper transport is of great concern within a clinical facility and is even more
important when samples are brought in from locations outside the facility.
Samples from outside facilities must be delivered in a manner that complies
with limited temperature variations and time restraints. Freezing or
excessive heat will damage the blood cells and render analysis invalid.
Although microscopic evaluation of a blood smear is best when the slide is
prepared within 3 hours of collection, the instrument analysis can be delayed
for 6–8 hours without deterioration of the data. Some parameters are valid
for up to 24 hours after sample collection if properly stored. Often, however,
the hematology instrument’s manufacturer provides the recommended time
points for performing the automated CBC to ensure that the analysis
produces data for a patient of the highest quality. The information generated
from manual and instrument analysis is only as good as the specimen that is
tested. Therefore, the pre-examination stage of testing is of primary
importance.
Examination Phase of the CBC
Laboratory professionals use automated instruments to determine the CBC
information for the majority of patient samples. Proper instrument
preparation includes quality control and assessment to confirm the normal
function of the instrument. Commercial controls, patient controls, or moving
averages are used to determine the analytical reliability of the automated
instrument. Once the analytical reliability of the instrument has been
confirmed, examination of patient samples can begin.
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Automated Results
Throughout the examination phase, laboratory professionals must follow
safety protocols designed to minimize risk of exposure to biohazards,
chemical hazards, and physical hazards and dispose of biological waste
appropriately. Each hematology instrument utilizes different but overlapping
technologies (i.e., impedance, optical light scattering) to determine the
reporting parameters of the CBC. A typical panel of CBC parameters includes
information regarding the cells produced by the hematopoietic system, the
RBCs, WBCs, and platelets. Because other organs and organ systems also
affect hematopoiesis, a myriad of disease states can be evaluated from CBC
data to determine diagnosis, treatment, and prognosis for a patient.
In addition to the typical CBC parameters, hematology instruments generate
scatterplots and histograms that laboratory professionals interpret. Each
scatterplot and histogram contains information about the cell populations,
interfering substances, and instrument function and therefore serves as
forms of quality control for instrument and specimen integrity. For example,
the laboratory professional consults the scatterplots and histograms to
assess the volume of granulocytes (increased volume indicates immaturity
or nuclear hypersegmentation whereas decreased volume of lymphocytes
can indicate chronic lymphocytic leukemia). The scatterplots and histograms
are also helpful in assessing RBC parameters that can be affected by
conditions such as cold agglutination (RBC clumping at temperatures below
body temperature) and a severely elevated WBC count. The parameters and
reference intervals of atypical mature (adult) CBC are listed in the following
table.
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Parameters and Reference Intervals of a Typical Adult CBC
White blood cell count
4.5−11.0×103/mcL (4.5−11.0×109/L)
Red blood cell count
4.0−5.5×106/mcL (4.0−5.5×1012/L)
Hemoglobin
12.0–17.4 g/dL (120–174 g/L)
Hematocrit
36–52% (0.36–0.52 L/L)
Mean cell volume
80–100 fL
Mean cell hemoglobin
28–34 pg
Mean cell hemoglobin
concentration
32–36 g/dL
Red cell distribution width
11.5–14.5%
Reticulocyte count Relative (%)
Absolute
I.
II.
Platelet count
150−400×109/L
Mean platelet volume
6.8–10.2 fL
Automated WBC Differential
Relative
(%)
Absolute (×109/L)
Neutrophils
40–80
1.8–7.0
Lymphocytes
25–35
1.0–4.8
Monocytes
2–10
0.1–0.8
Eosinophils
0–5
0–0.4
Basophils
0–1
0–0.2
0.5–2.0%
(×109/L)
Male and female reference intervals are combined. For age- and sex-specific reference
intervals, see the front cover of this textbook. Additional parameters are dependent
upon instrumentation. Data are shown as conventional units (SI units).
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Leukocyte, Erythrocyte, Hematocrit and Hemoglobin Report
The leukocyte count (WBC count), erythrocyte count (RBC count),
hematocrit (Hct), and hemoglobin (Hb) are determined using automated
instrumentation. The WBC and RBC counts are reported as the number of
cells per liter. The WBCs are reported as billions of cells per liter (×109/L),
and the RBCs are reported as trillions of cells per liter (×1012/L). The
hematocrit measures the volume that the RBCs occupy within whole blood
and is reported as a percentage (%) or as the volume of RBCs in liters
divided by the volume of whole blood in liters [L/L]. In automated analyzers,
the hematocrit is usually calculated from the measured MCV and RBC count
using the following formula:
The hemoglobin is measured spectrophotometrically after it has been
released from lysed erythrocytes. It is reported in grams per deciliter or
grams per liter. The laboratory professional interprets the accuracy of the
RBC count, hematocrit, and hemoglobin values using a quick mathematical
check called the rule of three. Simply, the RBC count x3 = hemoglobin x3
= hematocrit (%). If the calculated values do not agree within ±3% of the
measured values, a measurement error or instrument malfunction could
have occurred, or the patient could have a pathology that requires
investigation.
A diurnal variation in blood cell concentration occurs in which the value for
the WBC count is lowest in the morning and highest in the afternoon,
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whereas the RBC count, Hct, and Hgb are just the opposite; the higher
values are observed in the morning.
Erythrocyte Indices
The erythrocyte indices help classify the erythrocytes by their size and
hemoglobin content. Hemoglobin, hematocrit, and erythrocyte count values
are used to calculate the three indices: mean cell volume (MCV), mean cell
hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC).
When calculating the indices, it is important to note that the conversion
factors used in the formulas vary depending on the use of conventional units
or Systeme International (SI) Units for hemoglobin and hematocrit. These
indices suggest how the RBCs will appear microscopically and provide
significant diagnostic information (most commonly for the diagnosis of
anemias). Laboratory professionals correlate the indices with the Hct, Hgb,
and RBC count to ensure that technical problems are identified when they
occur.
Mean Cell Volume
The MCV denotes the average volume of individual erythrocytes and is
expressed in femtoliters (fL or 10-15 L). It is measured by automated
instrumentation and can be calculated from the Hct and RBC count.
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Classification of Erythrocytes Based on MCV
Terminology
Description
Normocytic
80.0–100.0 fL
Microcytic
Red cells with a reduced volume (<80 fL)
Macrocytic
Red cells with an increased volume (>100 fL)
Anisocytosis
Increased variation in the range of red cell sizes
The MCV is used to classify cells as normocytic, microcytic, or macrocytic
and usually correlates with the appearance of cells on stained blood smears
(i.e., cells with an increased MCV appear larger (macrocytic), and cells with
a decreased MCV appear smaller (microcytic)). However, it must be
remembered that the MCV is a measurement of volume, whereas estimation
of the size of flattened cells on a blood smear is a measurement of cell
diameter. Cell diameter and cell volume are not the same.
Example:
A patient has an Hct of 0.45 L/L and an RBC count of 5.0×1012/L;
90.0 fL = 0.45×10005. The value, 90.0 fL, indicates that the cell has a
volume that falls within the reference interval (80–100 fL) and is
therefore classified as normocytic.
Spherocytes usually have a normal or only slightly decreased volume (MCV),
but on a stained smear, they cannot flatten as much as normal erythrocytes
because of a decreased surface area and increased rigidity. Spherocytes,
therefore, often appear to have a smaller diameter than normal cells. On the
other hand, codocytes can appear larger due to an increased diameter, but
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the MCV is often normal. Generally, abnormalities in the MCV are clues to
disease processes of the hematopoietic system.
Mean Cell Hemoglobin
The MCH is a measurement of the average weight (in picograms, 10−12 g)
of hemoglobin in individual erythrocytes. The MCH is calculated from the
hemoglobin and erythrocyte count.
Example:
A patient has an Hgb concentration of 15 g/dL and an RBC count
of 5.0×1012/L. 30 pg = 15×105. The value, 30.0 pg, indicates that
the RBCs contain an average weight of hemoglobin that is within the
reference interval (28.0–34.0 pg).
The MCH does not take into account the size of a cell; it should not be
interpreted without taking into consideration the MCV because the MCH
varies in a direct linear relationship with the MCV. Cells with less volume
typically contain less hemoglobin while cells with larger volume typically
contain more hemoglobin.
Mean Cell Hemoglobin Concentration
The MCHC is the ratio of hemoglobin mass to volume in which it is contained
(i.e., average concentration of hemoglobin in a deciliter of erythrocytes,
expressed in g/dL). The MCHC is calculated from the Hgb and Hct.
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MCHC (g/dL) =
Example:
A patient has an Hgb concentration of 15 g/dL and an Hct of 0.45 L/L.
33.3 g/dL = 150.45. The value, 33.3 g/dL, reveals that the cells
contain a normal concentration of hemoglobin (32.0–36.0 g/dL) and
are therefore normochromic.
The MCHC indicates the concentration of hemoglobin in the general cell
population and is described by the suffix -chromia, meaning color. Cells can
be classified morphologically as hypochromic if the area of central pallor
is >1/3 of the cell size. The term hyperchromic should be used sparingly (if
ever).
The only erythrocyte that is hyperchromic with an MCHC >36.0 g/dL is the
spherocyte. Spherocytes have a decreased surface-to-volume ratio due to a
loss of membrane but have not lost an appreciable amount of their
hemoglobin. In certain conditions, the indices MCV, MCH, and MCHC can be
falsely elevated.
Classification of Erythrocytes Based on MCHC
Normochromic
32.0–36.0 g/dL
Hypochromic
<32.0 g/dL
Hyperchromic
>36.0 g/dL
Red Cell Distribution Width
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Because the MCV represents an average of erythrocyte volume, it is less
reliable in describing the erythrocyte population when considerable variation
in erythrocyte volume/size (anisocytosis) occurs. The red cell distribution
width (RDW) is the coefficient of variation of the MCV and may be referred
to as the RDW-coefficient of variation (RDW-CV). The formula for the RDWCV, a calculated index from hematology instruments to help identify
anisocytosis, follows:
Abnormal increased RDW values (714.5%) indicate an increase in the
heterogeneity of erythrocyte size. No known abnormalities are represented
by a decreased RDW.
Caution must be used in interpreting the RDW-CV because it reflects the
ratio of the standard deviation of cell volume and the MCV. An increased
standard deviation (heterogeneous cell population) with a high MCV can give
a normal RDW-CV. Conversely, a normal standard deviation (homogenous
cell population) with a low MCV can give an increased RDW-CV. Examination
of the erythrocyte histogram and stained blood smear gives clues as to the
accuracy of the RDW-CV in these cases. When the standard deviation is
increased, indicating a true variability in cell size, the base of the erythrocyte
histogram is broader than usual. Because of this interpretation issue,
automated instruments often report the RDW-CV and RDW-standard
deviation (RDW-SD). The RDW-SD is directly measured and not affected by
the MCV.
Reticulocyte Count
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Immature, anuclear erythrocytes containing organelles and residual
ribosomes for hemoglobin synthesis are known as reticulocytes, which
usually spend 2–3 days in the bone marrow and an additional day in the
peripheral blood before their RNA is degraded and they become mature
erythrocytes. The peripheral blood reticulocyte count indicates the degree of
effective bone marrow activity and is one of the most useful and costeffective laboratory tests in monitoring response to therapy and
pathophysiology of anemia. Reticulocytes can sometimes be identified
as polychromatophilic erythrocytes (erythrocytes with a bluish tinge) on
Romanowsky-stained smears. The polychromatophilia is due to the presence
of basophilic RNA within ribosomes mixed with acidophilic hemoglobin.
A supravital stain such as new methylene blue or brilliant cresyl blue must
be used to definitively identify the presence of reticulocytes. Although
automated methods for reticulocyte enumeration are available on some
hematology instruments, many laboratories use a manual method. Test
results are expressed as a percentage of reticulocytes in relation to the total
RBC count (relative count) or as the absolute number (see the following
section). In the automated method, >30,000 RBCs are assessed, so the
method is more precise than the manual method (which assesses only 1000
RBCs) and is more accurate when the reticulocyte count is very low.
Absolute Reticulocyte Count
The absolute reticulocyte count is a more informative index of erythropoietic
activity than the relative reticulocyte count. When reported as a percentage,
the reticulocyte count does not indicate the relationship between the
peripheral blood erythrocyte mass and the number of reticulocytes being
produced. The reticulocyte count reported as a percentage can appear
increased because of either an increase in the number of reticulocytes in the
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circulation or a decrease in the number of total RBCs. Therefore, it is
recommended that in addition to the percentage of reticulocytes, laboratory
professionals report the absolute reticulocyte count to provide a more useful
estimate of reticulocyte production.
Automated analyzers can provide the absolute count and can be calculated
when using manual methods for reticulocytes:
Absolute reticulocyte (×109/L) =
RBC count (×1012/L)×Reticulocyte count (%)
Example:
A patient has an RBC count of 3.5×1012/L and a 10% reticulocyte
count; 350 ×109/L = 3.5×10. The value 350×109/L represents an
increase in reticulocyte production since the mean normal value
is 90×109/L.
Platelet Count and Mean Platelet Volume
Automated hematology instruments generate the platelet count, which is
reported as billions of platelets per liter (number of platelets×109/L). The
mean platelet volume (MPV) is similar to the MCV for erythrocytes because it
represents the average volume of individual platelets. The laboratory
professional utilizes both the platelet count and the MPV to assess
thrombopoiesis and pathologic conditions related to platelets. A decreased
platelet count generally represents decreased thrombopoiesis, increased
platelet destruction, or consumption. Reactive or malignant conditions can
cause an increase in the platelet count.
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WBC Differential
The WBC differential is an analysis and enumeration of the various subtypes
of WBCs. An altered concentration of one specific type of leukocyte most
commonly causes an increase or decrease in the total WBC count. For this
reason, an abnormal total WBC count should be followed by a WBC
differential, also known as a diff.
The WBC differential can be performed by automated instruments or
manually. To perform a manual WBC differential, a blood smear stained with
a Romanowsky-type stain (usually Wright’s stain) is viewed microscopically.
A total of 100 leukocytes are viewed and each leukocyte subtype is
classified. The differential results are reported as the percentage of each cell
type counted. To accurately interpret whether an increase or decrease in cell
types exists, the absolute concentration of each cell type is calculated using
the results of the WBC count and the differential.
The Peripheral Blood Smear
Each testing location and institution sets the parameters that trigger the
necessity of a manual morphologic examination of the peripheral smear, but
generally a peripheral blood smear is prepared for microscopic examination
when CBC values obtained from an automated instrument differ from what is
considered normal. A laboratory professional reviews the blood smear for
overall quality, the morphology of white blood cells, red blood cells, and
platelets, and performs a WBC differential. The peripheral smear is
correlated with the parameters reported by the instrument as the
culminating interpretation of the CBC.
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Briefly, a wedge smear is made by placing a small drop of blood at one end
of a microscope slide and spreading that drop to create a thin smear (or
film) of blood. After the blood has dried, the slide is stained using a
Romanowsky-type stain. A well-made and properly stained blood smear is
required for accurate interpretation of the CBC. The slide is examined
macroscopically and microscopically to ensure that the blood was spread and
stained properly. The optimal blood smear is pinkish purple in color and
transitions to a feathered edge.
Information such as RBC agglutination (appears as a grainy blood smear),
lipidemia (represented as holes within the smear), and multiple myeloma
(bluish-colored smear) can be suspected during this important macroscopic
evaluation of the blood smear and should be noted by the laboratory
professional before moving on to the microscopic evaluation. This
macroscopic assessment determines whether the blood smear is acceptable
for microscopic analysis.
Low-Power Magnification
The laboratory professional first assesses the general appearance and
distribution of WBCs, RBCs, and platelets using low power magnification 10x
objective (100x magnification). A high concentration of WBCs at the furthest
edge of the smear (the feathered edge) indicates poor cell distribution and is
sufficient evidence that a new smear should be made. In addition, very large
or abnormal WBCs are often pushed to the outer edges of the smear. Cells
that have ruptured are called smudge cells. These are often B lymphocytes
and their presence is characteristic of pathological conditions such as chronic
lymphocytic leukemia. The laboratory professional also performs an estimate
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of the number of WBCs (i.e., WBC estimate) under low power (either 100x
or 400x) and correlates the WBC estimate to the WBC count.
On a well-made blood smear, the erythrocytes are evenly distributed and
well separated on the feathered edge of the smear. Stacking or aggregating
of cells is associated with certain pathologic states. The following table
summarizes specifics of the peripheral blood smear examination.
Summary of the Microscopic Peripheral Blood Smear Examination
10×Objective
(100×magnification)
WBCs
Scan for abnormal or
large cells
40×Objective
(400×
magnification)
Perform WBC
estimate
Smudge cells
RBCs
Scan for rouleaux and
agglutination
Platelets
Scan for clumps and
satellitism
100×Objective
(1000×
magnification)
Evaluate leukocyte
morphology
Perform 100-cell
differential
Determine the critical
area
Evaluate erythrocyte
morphology:
Assess size, shape,
color, presence of
inclusions
Perform platelet
estimate
Evaluate platelet
morphology:
Assess size and
granularity
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In the presence of IgM antibodies (cold agglutinins) directed against
erythrocyte antigens, erythrocytes can agglutinate forming irregular clusters
of varying sizes. This agglutination forms irregular, grapelike clusters that
are readily differentiated from rouleaux.
On automated hematology analyzers, a CBC with an elevated MCV and low
RBC count but a normal hemoglobin suggests the presence of cold-reacting
erythrocyte agglutinins. In addition, the calculated hematocrit will be falsely
decreased and the MCH and MCHC will be falsely increased. The effect of
cold agglutinins is overcome by keeping the blood at 37 °C. When
performing blood counts, the diluting fluid also must be kept at 37 °C. The
following table provides helpful information regarding abnormalities that may
be seen in erythrocyte arrangement during laboratory testing.
Abnormalities in Erythrocyte Arrangement
Terminology
Description
Associated Physiologic State
Agglutination
Irregular clumps
of red blood cells
Due to antigen–antibody interaction
Rouleaux
Red blood cells
arranged in rolls
or stacks
Usually associated with abnormal or increased
plasma proteins (red blood cells can be dispersed
by mixing cells with saline)
Rouleaux is an alignment of erythrocytes one on top of another resembling a
stack of coins. This phenomenon occurs normally when blood is collected
and allowed to stand in tubes. It can also be seen in the thick portion of
blood smears. In certain pathologic states that are accompanied by an
increase in fibrinogen or globulins, rouleaux becomes marked and is readily
seen in the feathered edge of blood smears. When the erythrocyte assumes
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abnormal shapes, such as sickled forms, rouleaux formation is inhibited.
Rouleaux is also inhibited when erythrocytes are suspended in saline. The
presence of rouleaux or agglutination is a possible indication that a new
smear should be made.
The platelets must also be evaluated using low power magnification. The
manner in which the platelets have spread on the slide is checked because
platelet clumps can be pushed to the outer edge of the smear, and in some
cases, platelets can adhere to neutrophils (a phenomenon called satellitism).
This can result in falsely decreased estimation of the platelet count. Platelet
clumps and satellitism can be eliminated using sodium citrate as an
anticoagulant. Finally, fibrin strands can be observed in this scan of the
blood smear, indicating that the blood sample was coagulated (likely due to
improper mixing following the venipuncture). In either case, a new sample
should be obtained from the patient and a new smear should be made.
The final task at low power magnification is to determine the critical area of
the smear that will be used to perform the morphologic examination of cells.
This critical area is usually identified using the 40x objective (400x
magnification) and is characterized by the proximity of RBCs to each other
(the area of the smear in which very few RBCs overlap or touch and are
generally distributed in a uniform manner). At high power magnification, the
critical area is used to evaluate RBC morphology and perform the WBC
differential and platelet estimate.
High-Power Magnification
Following the quick, yet important, scan of the blood smear on low
magnification, the laboratory professional evaluates the smear on high
power, often at 1000× magnification. Ultimately, interpretation of the
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microscopic findings in the peripheral blood smear and correlation, or lack
thereof, with the automated instrument report is made for all parameters of
the CBC. A WBC differential is performed in which 100 cells are observed
and classified to determine the relative number of leukocytes as a
percentage and to identify the presence of morphologic abnormalities.
A platelet estimate is performed and compared with the instrumentgenerated platelet count; the morphology of the platelets is noted. Finally,
the RBC morphology is assessed for size, shape, color, and inclusions using
either the 40× or 50× objective and compared with the instrument report
for the RBC indices. To evaluate abnormalities including inclusions, the
laboratory professional should review the slide with the 100× objective
(1000× magnification).
Erythrocyte Morphology
The erythrocyte is sometimes called a discocyte because of its biconcave
shape. On a Romanowsky-stained blood smear, the erythrocyte appears as a
disc with a central area of pallor surrounded by a rim of pink-staining
hemoglobin (the center stains lighter in color compared with the rim). The
area of pallor is caused by the closeness occurring between the two concave
portions of the membrane when the cell becomes flattened on a glass slide.
Normally the area of pallor occupies about one-third the diameter of the cell.
Anisocytosis denotes a nonspecific variation in the size of the cells. Some
variation in size is normal because of the variation in age of the erythrocytes
with younger cells being larger and older cells smaller. Poikilocytosis is the
general term used to describe a nonspecific variation in the shape of
erythrocytes. It is important to note that some abnormal morphology can be
artifactual because of poorly made or improperly stained smears.
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Anisocytosis
Anisocytosis can be detected by examining the blood smear and/or by
reviewing the MCV and RDW (discussed earlier in this chapter). Normal
erythrocytes have a diameter of about 7−8 mcM (μm) and an MCV of 80–
100 fL. If the majority of cells are larger than normal, they are macrocytic; if
smaller than normal, they are microcytic. If there is a significant variation in
size with microcytic, normocytic, and macrocytic cells present, the MCV can
fall within the reference interval because it is an average of cell volume. In
this case, the RDW is helpful.
An RDW of >14.5% suggests that the erythrocytes are heterogeneous in
size, which makes the MCV less reliable. Microscopic examination of the cells
is especially helpful when the RDW is elevated. To evaluate erythrocyte size
microscopically, the cells are compared with the nucleus of a normal small
lymphocyte. Normocytic erythrocytes are about the same size as the
lymphocyte nucleus.
Poikilocytosis
Most laboratories report only significant poikilocytosis. The stained smear
should be reviewed while keeping in mind the overall context of the
laboratory results and the significance of the reported findings. To determine
the significance of and to decide whether to report poikilocytes, the following
should be considered:
1. Will it assist in differential diagnosis of the disease (likely anemia)?
2. Will it make a difference in the management of the patient?
3. Is the dominant poikilocyte significant in this setting?
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4. Do the specific constellation of findings indicate a particular pathologic
state?
Poikilocytes are further subclassified according to the specific cell shape.
As much as possible, the CBC report should specify the red blood cell
shape of diagnostic significance existing within the umbrella term of
poikilocytosis or poikilocytes. Only when there are too many red blood cell
shapes or when some cell shapes cannot be described would the term
poikilocytosis suffice to identify erythrocyte morphology; otherwise the
individual red blood cell shapes should be identified.
Acanthocytes also called spur cells, are small spherical cells with irregular
thorn like projections. Often the projections have small bulb-like tips.
Acanthocytes do not have a central area of pallor. These cells have
membranes with free cholesterol accumulating preferentially in the outer
bilayer of the membrane leading to decreased fluidity. Remodeling by the
spleen results in spheroidal cells with irregular surface projections. These
cells are readily trapped in the spleen.
Codocytes, also called Target cells, are thin, bell-shaped cells with an
increased surface-to-volume ratio. On stained blood smears, the cells have
the appearance of a target with a bull’s-eye in the center. An achromic zone
and a thin outer ring of pink-staining hemoglobin surround the bull’s eye.
The typical appearance of these cells is discernible in the area of the slide
only where the cells are well separated but not in the extreme outerfeathered edge where all cells are flattened. Target cells can appear as
artifacts when smears are made in a high-humidity environment or when a
wet smear is blown dry rather than fan dried. Target cells have an increased
surface-to-volume ratio of the cell.
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Dacryocytes, also called teardrops, are erythrocytes that are elongated at
one end to form a teardrop or pear-shaped cell. The teardrop morphology
can form after erythrocytes containing cellular inclusions have traversed the
spleen. Erythrocytes with inclusions are more rigid in the area of the
inclusion, and this portion of the cell has more difficulty passing through the
splenic filter than the rest of the cell. As splenic macrophages attempt to
remove this rigid inclusion, the cell is stretched into an abnormal shape. The
teardrop cannot return to its original shape because the cell either has been
stretched beyond the limits of deformability of the membrane or has
remained in the abnormal shape for too long.
Sickle cells, also called drepanocytes, are elongated, crescent-shaped
erythrocytes with pointed ends. Some forms have more rounded ends with a
flat rather than concave side. These modified forms of sickle shape can be
capable of reversing to the normal discocyte. Sickle cell formation can be
observed in stained blood smears from patients with sickle cell anemia. The
hemoglobin within the cell is abnormal and polymerizes into rods at
decreased oxygen tension or decreased pH. The cell first transforms into a
holly leaf shape and as the hemoglobin polymerization continues, it
transforms into a sickle-shaped cell with increased mechanical fragility.
Some holly-leaf forms can be observed on stained blood smears in addition
to the typical sickle shape.
Echinocytes, also called burr cells, are usually smaller than normal
erythrocytes with regular, spine-like projections on their surface. Their
presence is most often artifact in stained blood smears because of the glass
effect of the slide. The glass releases basic substances that raise the pH of
the medium surrounding the cell and induce echinocyte formation. Plasma
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provides a buffering effect on the cells, and for this reason, blood films made
from whole blood may show only certain areas of echinocyte transformation.
To determine the in vivo or in vitro nature of echinocytes, a wet preparation
can be made in which a drop of blood is enclosed between two plastic cover
slips and the unstained individual erythrocytes are observed. If no
echinocytes are present in the wet preparation but were noted on the
stained blood smears, the cell abnormality occurred as an in vitro artifact.
Echinocytes can appear in blood that has been stored at 4°C for several
days. Consequently, blood specimens from patients receiving transfusions
can have echinocytes if blood is taken from the patient immediately after
transfusion; however, after a few minutes, the buffering action of patient’s
plasma causes the transfused echinocyte to resume a normal discoid shape.
For true in vivo echinocytes, the characteristic appearance is not related to
tonicity of the medium in which the cells are suspended. The shape change
is instead thought to result from an increase in the area of the outer leaflet
of the lipid bilayer as compared with the inner layer. Echinocyte formation is
reversible (i.e., the cell can revert to a discocyte). However, an echinocyte
can eventually assume the shape of a spherocyte, presumably because the
spleen grooms (removes) the membrane spines; in this circumstance, the
cell cannot revert to a normal shape.
Elliptocytes, also called pencil cells and cigar cells, vary from elongated oval
shapes (ovalocytes) to elongated rodlike cells. Some laboratory professionals
may use the terms elliptocytes and ovalocytes interchangeably, whereas
others may use distinct guidelines to delineate the two morphologies. True
elliptocytes have parallel sides and a central area of biconcavity with
hemoglobin concentrated at both ends. Elliptocytes are formed after the
erythrocyte matures and leaves the bone marrow because reticulocytes and
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young erythrocytes in patients with elliptocytosis are normal in shape. The
mechanism of formation is not known but is assumed to involve alterations
of the erythrocyte membrane skeleton. Elliptocytes are the predominant
shape of erythrocytes in hereditary elliptocytosis. On the other hand,
ovalocytes are fatter on one end than the other and appear to have an egg
shape. Ovalocytes are formed in a manner similar to elliptocytes.
Keratocytes, also called helmet cells, have a concavity on one side and two
hornlike protrusions on either end. Keratocytes are produced when a fibrin
strand impales an erythrocyte. The two halves of the erythrocyte hang over
the strand as saddlebags; the membranes of the touching sides fuse,
producing a vacuole-like inclusion on one side. This cell with an eccentric
vacuole is called a blister cell. The vacuole bursts, leaving a notch with two
spicules on the ends.
Knizocytes are cells with more than two concavities. This cell’s appearance
on stained blood smears can vary depending upon how the cell comes to
rest on the flat surface; however, most knizocytes have a dark-staining band
across the center with a pale area on either side surrounded by a rim of
pink-staining hemoglobin. The mechanism of formation is unknown.
Leptocytes are thin, flat cells with normal or larger than normal diameter.
Although the cell’s diameter is normal or increased, its volume is usually
decreased. The cells have an increased surface-to-volume ratio either as a
result of decreased hemoglobin content or increased surface area. The
leptocyte is usually cup-shaped like stomatocytes, but the cup has little
depth. Target cells can be formed from leptocytes on dried blood smears
when the depth of the cup increases.
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Schistocytes are erythrocyte fragments caused by mechanical damage to the
cell. They appear in a variety of shapes such as triangular, comma, and
helmet-shaped. Because schistocytes are fragments of erythrocytes, they
are usually microcytic. They maintain normal deformability, but their survival
in the peripheral blood is reduced. The fragments can assume a spherical
shape and hemolyze or can be removed in the spleen.
Spherocytes are erythrocytes that have lost their biconcavity because of a
decreased surface-to-volume ratio. On stained blood smears, the spherocyte
appears as a densely stained sphere lacking a central area of pallor.
Although the cell often appears microcytic on stained blood smears, the
volume (MCV) is usually normal. The spherocyte is the only erythrocyte that
can be called hyperchromic because of an increased MCHC.
Stomatocytes, or mouth cells, appear as small cup-shaped uniconcave discs
(in wet preparations). Upon staining, these cells exhibit a slitlike (mouthlike)
area of pallor. Normal discocytes can be transformed under certain
conditions to stomatocytes and, eventually, to spherostomatocytes. The
stomatocyte shape is reversible, but the spherostomatocyte is not. Cationic
drugs and low pH cause a gradual loss of biconcavity leading to the
stomatocyte and eventually the formation of a sphere.
Stomatocytosis is the opposite of echinocytosis; the shape change in
stomatocytosis is thought to be the result of an increase in the lipid content
or area of the inner leaflet of the membrane lipid bilayer. Stomatocytes also
can appear as an artifact on stained blood smears; thus, care should be used
in identifying them.
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The following is a helpful table that lists the varied erythrocyte (red cell)
morphologies, cell descriptions (shapes) and disease states associated with
the cell description.
Erythrocyte Morphologies
Terminology
Synonyms
Description
Associated Disease
States
Poikilocytosis
—
Increased variation in the
shape of red cells.
See disease states
associated with specific
poikilocytes on this table.
Acanthocyte
(spike)
Spur cell
Red cells with spicules of
varying length irregularly
distributed over the
surface; no area of
pallor.
Abetalipoproteinemia;
alcoholic liver disease;
disorders of lipid
metabolism; post
splenectomy; fat
malabsorption; retinitis
pigmentosa.
Codocyte
(bell)
Target cell
Thin, bell-shaped, with
increased surface-tovolume ratio; on stained
blood smears, appears as
a target with a central
bull’s-eye, surrounded by
achromic zone and outer
ring of hemoglobin.
Hemoglobinopathies;
thalassemias; obstructive
liver disease; iron
deficiency anemia;
splenectomy; renal
disease; LCAT deficiency.
Dacryocyte
(tear)
Teardrop
Round cell with a single
elongated or pointed
extremity; may be
microcytic and/or
hypochromic.
Myelophthisic anemias;
primary myelofibrosis
(PMF); thalassemias.
Drepanocyte
(sickle)
Sickle cell
Contain polymerized
hemoglobin showing
various shapes: sickle,
crescent, or boat shaped.
Sickle cell disorders.
Echinocyte
(sea urchin)
Burr cell;
crenated
cell
Spiculated red cells with
short equally spaced
projections over the
entire surface.
Liver disease; uremia;
pyruvate kinase
deficiency; peptic ulcers;
cancer of stomach;
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Terminology
Synonyms
Description
Associated Disease
States
heparin therapy.
Elliptocyte
(oval)
Ovalocyte;
pencil cell;
cigar cell
Oval to elongated
ellipsoid cell with central
area of pallor and
hemoglobin at both ends.
Hereditary elliptocytosis;
iron deficiency anemia;
thalassemia; anemia
associated with leukemia.
Keratocyte
(horn)
Helmet
cell; hornshaped cell
Red cells with one or
several notches with
projections that look like
horns on either end
Microangiopathic
hemolytic anemias; heartvalve hemolysis; Heinzbody hemolytic anemia;
glomerulonephritis;
cavernous hemangiomas
Knizocyte
—
RBC with more than two
concavities; on stained
blood smears has a dark
band of hemoglobin
across the center with a
pale area on either side.
Conditions in which
spherocytes are found.
Leptocyte
(thin)
Thin cell
Thin, flat cell with
hemoglobin at periphery;
usually cup-shaped, MCV
is decreased but cell
diameter is normal.
Thalassemia; iron
deficiency anemia;
hemoglobinopathies; liver
disease.
Schistocyte
(cut)
Schizocyte;
fragmented
cell
Fragments of red cells;
variety of shapes
including triangles,
commas; microcytic.
Microangiopathic
hemolytic anemias; heartvalve hemolysis;
disseminated intravascular
coagulation; severe
burns; uremia.
Spherocyte
—
Spherocytic red cells with
dense hemoglobin
content
(hyperchromatic); lack
an area of central pallor.
Hereditary spherocytosis;
immune hemolytic
anemias; severe burns;
transfusion with ABO
incompatibility; Heinzbody hemolytic anemias.
Stomatocyte
(mouth)
Mouth cell;
cup form;
mushroom
Uniconcave red cells with
the shape of a very thick
cup; on stained blood
Hereditary
stomatocytosis;
spherocytosis; alcoholic
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Terminology
Synonyms
Description
Associated Disease
States
cap
smears cells have an oval
or slit like area of central
pallor.
cirrhosis; anemia
associated with Rh null
disease; lead intoxication;
neoplasms.
Variation in Hemoglobin (Color)
Normal erythrocytes have an MCH of approximately 30 pg. However, the
MCHC is a better indicator of chromia or color of erythrocytes on
Romanowsky-stained smears. Normally, on stained smears, the erythrocyte
has a central area of pallor approximately one-third the diameter of the cell.
In certain conditions, RBCs contain less hemoglobin than normal and appear
to have a larger than normal central pallor (hypochromia). On the other
hand, the only erythrocyte that contains more hemoglobin than normal in
relation to its volume is the spherocyte.
Hypochromic cells are poorly hemoglobinized erythrocytes with an
exaggerated area of central pallor (>1/3 the diameter of the cell) on
Romanowsky-stained blood smears. Although occasionally normocytic,
hypochromic cells are usually microcytic. Hypochromic cells are the result of
decreased or impaired hemoglobin synthesis. When visualizing a blood
smear, correlating the automated findings from hematology analyzers to the
appearance of cells is important. In the case of hypochromia, the MCHC
value will be decreased.
Polychromatophilic erythrocytes (reticulocytes) are usually larger than
normal cells with a bluish tinge on Romanowsky-stained blood smears. The
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bluish tinge is caused by the presence of residual RNA in the cytoplasm.
Large numbers of these cells are associated with decreased erythrocyte
survival or hemorrhage and an erythroid hyperplastic marrow.
Erythrocyte Inclusions
Erythrocytes do not normally contain any particulate inclusions. When
present, inclusions can help direct further investigation because they are
associated with certain disease states.
Basophilic Stippling
Erythrocytes with basophilic stippling are cells with bluish-black granular
inclusions distributed across their entire cell area. The granules can vary in
size and distribution from small diffuse to coarse and punctate. The
granules, which are composed of aggregated ribosomes, are sometimes
associated with mitochondria and siderosomes. Basophilic stippling is not
believed to be present in living cells; instead, stippling probably is produced
during preparation of the blood smear or during the staining process.
Electron microscopy has not shown an intracellular structure similar to
basophilic stippling. Cells dried slowly or stained rapidly can demonstrate
fine, diffuse stippling as an artifact. Pathologic basophilic stippling is more
coarse and punctate.
Cabot Rings
Cabot rings are reddish-violet erythrocytic inclusions usually occurring in the
formation of a figure eight or oval ring. Cabot rings are thought to be
remnants of spindle fibers, which form during mitosis. They occur in severe
anemias and in dyserythropoiesis.
Howell-Jolly Bodies
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Howell-Jolly bodies are dark purple or violet spherical granules in the
erythrocyte. These inclusions are nuclear (DNA) fragments usually occurring
singly in cells, rarely more than two per cell. Howell-Jolly bodies are
associated with nuclear maturation abnormalities. They are thought to occur
as a result of an individual chromosome failing to attach to the spindle
apparatus during mitosis, and, thus, it is not included in the reformed
nucleus. When the nucleus is extruded, the Howell-Jolly body is left behind
(until removed by splenic macrophages).
Variations in Erythrocyte Color
Terminology
Description
Associated Physiological or
Disease States
Hypochromia
Decreased concentration of
hemoglobin in the red cell.
Red cells have an increased area of
central pallor (>1/3 diameter of
cell).
May be present in iron
deficiency anemia, thalassemia,
and other anemias associated
with a defect in hemoglobin
production.
Polychromasia
Young red cells containing residual
RNA.
Stain a pinkish-gray to pinkishblue color on Wright’s stained
blood smears.
Usually appear slightly larger than
mature red cells.
Found in increased numbers in
hemolytic anemias, newborns,
recovery from acute
hemorrhage.
Heinz Bodies do not stain with Romanowsky stains but can be visualized with
supravital stains or with phase microscopy of the living cell. They appear as
2–3 mcM round masses lying just under or attached to the cell membrane.
Heinz bodies are composed of aggregated denatured hemoglobin.
Iron Inclusions refer to particulate iron molecules that can be detected in
erythrocytic cells in both normal and abnormal conditions. Intracellular
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siderotic granules represent iron that has not been incorporated into
hemoglobin.
Sideroblasts are erythroblasts that contain stainable iron granules
whereas siderocytes are non-nucleated, mature erythrocytes that contain
stainable iron granules. Sideroblasts and siderocytes can be identified with
Perl’s Prussian blue iron stain, which stains iron aggregates blue. The
granules do not stain with Romanowsky stains. About 20–60% of all
erythroblasts in the marrow contain iron that can be visualized with Perl’s
Prussian blue stain. This number decreases in some pathologic states and
can be markedly increased in others. Reticulocytes and erythrocytes in the
peripheral blood do not normally contain stainable iron aggregates unless
the patient has been splenectomized.
Abnormal Erythrocyte Inclusions
Terminology
Description
Associated Disease
States
Basophilic
stippling
Round or irregularly shaped granules of
variable number and size, distributed
throughout the RBC.
Composed of aggregates of ribosomes (RNA).
Stain bluish black with Wright’s stain.
Lead poisoning;
anemias associated
with abnormal
hemoglobin
synthesis;
thalassemia.
Cabot rings
Appear as a figure-8, ring, or incomplete ring.
Thought to be composed of the microtubules
of the mitotic spindle.
Stain reddish violet with Wright’s stain.
Severe anemias;
dyserythropoiesis.
Howell-Jolly
bodies
Small, round bodies composed of DNA usually
located eccentrically in the red cell.
Usually occurs singly, rarely more than two
per cell.
Stains dark purple with Wright’s stain.
Post splenectomy;
megaloblastic
anemias; some
hemolytic anemias;
functional asplenia;
severe anemia.
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Terminology
Description
Associated Disease
States
Pappenheimer
bodies
Clusters of granules containing iron that are
usually found at the periphery of the cell.
Visible with Prussian blue stain and Wright’s
stain.
Sideroblastic
anemia;
thalassemia; other
severe anemias.
Heinz bodies
Bodies composed of denatured or precipitated
hemoglobin.
Not visible on Wright’s stained blood smears.
With supravital stain appear as purple, roundshaped bodies of varying size, usually close to
the cell membrane.
Can also be observed with phase microscopy
on wet preparations.
G6PD deficiency;
unstable hemoglobin
disorders; oxidizing
drugs or toxins; post
splenectomy.
Pappenheimer bodies are damaged secondary lysosomes and mitochondria
variable in their composition of iron and protein. This type of inclusion
appears as clusters of small granules in erythrocytes and erythroblasts and
stains with both Romanowsky and Perl’s Prussian blue stains. Romanowsky
stains reveal Pappenheimer bodies by staining the protein matrix of the
granules whereas Perl’s Prussian blue is responsible for staining the iron
portion of the granules. Pappenheimer bodies occur only in pathologic
states.
Normal Erythrocytic Cell Inclusions
Terminology
Description
Reticulofilamentous
substance
(reticulocyte)
 Artifactual aggregation of ribosomes.
 Not visible on Wright’s stained smears; supravital stain (i.e.,
new methylene blue) must be used.
 Appears as deep blue reticular network.
Sideroblast
 Iron granules found in erythroblasts.
 Stains blue with Perl’s Prussian blue stain but not with
Romanowsky stains.
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Terminology
Description
Siderocyte
 Iron granules found in erythrocytes.
 Stains blue with Perl’s Prussian blue stain not stain with
Romanowsky stains.
Professional Review of the CBC
The role of the laboratory professional is to analyze and interpret the CBC
data generated by the automated hematology analyzer and the manual,
peripheral blood smear review. The interpretation is essentially a correlation
of the various components of the CBC in order to identify the likelihood of
abnormal results, pathology, and discrepancies in the generated data. The
hematology instrument or laboratory information system produce alerts that
include delta checks or that indicate the presence of interfering substances,
both of which the laboratory professional must resolve.
Delta checks compare a patient’s current clinical values for a test with
previous values. This type of quality control can detect sudden changes in a
patient’s physiology or can be useful in identifying instrument error. Delta
checks are particularly important in diagnosis and in monitoring therapy.
Abnormal results and the presence of interfering substances (i.e., lipemia,
hemolysis) must also be noted and corrected. In the event that the data can
be correlated for diagnosis, the laboratory professional should be able to
recommend subsequent testing to the patient’s medical provider.
Bone Marrow Disease And Examination11,43,63,69-86
The hematopoietic system consists of the bone marrow, liver, spleen, lymph
nodes, and thymus. The bone marrow is one of the body's largest organs,
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representing 3.4 to 6 percent of total body weight and averaging about 1500
grams in adults. This section discusses bone marrow disease and indications
to proceed with examination, including the procedure of bone marrow
aspiration and biopsy.11,43,63,69-86
Hematopoiesis (blood cell production and maturation) can be seen at
different anatomic locations (yolk sac, liver, spleen, axial and radial bones),
depending on the gestational and postnatal period. In normal adults,
hematopoiesis is seen mainly in the bone marrow. The bone marrow–derived
pluripotential hematopoietic stem cells, under the influence of various
cytokines or growth factors, or both, differentiate into myeloid
(granulocytes, monocytes, megakaryocytes, erythrocytes) and lymphoid cell
lineages. Benign conditions and malignant diseases related to these cells are
called hematolymphoid disorders.
Nonhematolymphoid diseases may also involve the bone marrow; therefore,
examination of the bone marrow has a wide application in clinical practice.
Because hematologic diseases involving the bone marrow can result in
morphological abnormalities of the peripheral blood cells, the bone marrow
examination should be interpreted in conjunction with a peripheral smear
examination. Severe thrombocytopenia is generally not a contraindication to
the procedure. In experienced hands and with the needles currently
available, the bone marrow aspiration and biopsy carry minimal risk.
The hematopoietic marrow is organized around the bone vasculature. An
artery entering the bone branches out toward the periphery to specialized
vascular spaces called sinuses. Several sinuses combine in a collecting sinus,
forming a central vein that returns into the systemic circulation.
Hematopoietic cords, in which hematopoiesis takes place, lie just outside of
the sinuses. After maturation in the cords, the hematopoietic cells cross the
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walls of the sinuses and enter the blood. Hematopoietic cell colonies are
compartmentalized in the cords.
Structurally, bone marrow consists of hematopoietic cells (erythroid,
myeloid, lymphoid, and megakaryocyte), adipose tissue, bone and its cells
(osteoblasts and osteoclasts), and stroma. The main function of the marrow
is to supply mature hematopoietic cells into the peripheral blood in a steadystate condition as well as to respond to increased demands.
A semidormant pool of pluripotential stem cells maintains a self-renewal
property. Granulocytic, monocytic, eosinophilic, erythroid, and
megakaryocyte progenitor cells are influenced in their differentiation by
colony-stimulating factors (CSFs). CSFs are produced by T lymphocytes, as
well as stromal cells, fibroblasts, endothelial cells, and macrophages, when
stimulated by monocyte interleukin-1 (IL-1) and tumor necrosis factor
(TNF). Some CSFs, such as IL-3 and granulocyte-monocyte CSF, have a
broad influence and are required throughout proliferation and differentiation
of progenitor cells. Others, which include granulocyte, monocyte, and
eosinophil CSFs, are lineage specific, and regulate division and differentiation
only of corresponding, committed progenitor cells. In addition,
erythropoiesis is influenced by erythropoietin produced in the kidney.
In the process of cell egression from the cords to the circulation, a number
of releasing factors are identified. The best characterized of these are
granulocyte colony–stimulating factor (G-CSF) and granulocyte–macrophage
colony–stimulating factor (GM-CSF), but other factors, such as components
of a complement system, androgenic steroids, and endotoxins, may play a
role. The endothelial lining of the sinusoids forms a continuous, veil-like wall
through which the mature cells migrate from extravascular sites into the
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circulation. This is accomplished by close contact between mature
hematopoietic cells and endothelial cells. A transient migration pore is
formed during such contact through which the mature cells pass into the
circulation without loss of plasma to the extravascular pool. It is evident that
the bone marrow is subjected to a complex regulation by many cellular and
humoral systems of the body, and any disease that affects these systems is
likely to affect hematopoiesis.
Bone marrow studies are frequently used in the diagnosis of hematopoietic
disorders. Once a formidable task, obtaining bone marrow tissue has
become, with current improved techniques, a standard procedure. Several
techniques have been devised, each having its own merits and limitations.
Bone marrow aspiration and bone marrow biopsy are usually performed
concurrently. Although obtaining the bone marrow for examination carries
little procedural risk for the patient, the procedure is costly and can be quite
painful. For this reason, bone marrow studies should be performed only
when clearly indicated or whenever the medical provider expects a beneficial
diagnostic result for the patient.
Hematologic diseases affecting primarily the bone marrow and causing a
decrease or increase of any cellular blood elements are among the most
common indications. It is not unusual for more than one blood element to be
increased or decreased, as occurs in leukemias and some refractory
anemias. In these situations, bone marrow study affords specific
information, and it usually precedes any other diagnostic procedure.
Systemic diseases may affect the bone marrow secondarily and require bone
marrow studies for diagnosis or monitoring patients' conditions. Patients
having any of the solid malignant tumors may undergo bone marrow studies
when the initial diagnosis is established for evaluation of the degree of tumor
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spread and staging of the disease. On occasion, a bone marrow study may
result in a diagnosis of unsuspected metastatic malignant tumor. During the
course of malignant disease, additional marrow studies may be performed
periodically to monitor the status of tumor burden and its therapeutic
response.
Infections manifesting clinically as fever of unknown origin may exhibit
granulomas, focal necrosis, or histiocytic proliferations. Intracytoplasmic
organisms may be seen in the marrow. Material for morphologic studies and
bacterial cultures may be collected simultaneously during a single procedure.
The suspected diagnoses of disseminated tuberculosis, fungal infections
(particularly histoplasmosis and cryptococcosis), and some protozoan
infections are frequently confirmed through such studies. Hereditary and
acquired conditions occasionally involve the bone marrow histiocytes (i.e.,
Gaucher's disease, sea blue histiocytosis, hemophagocytic syndrome, and
others). A simple procedure such as bone marrow aspiration or biopsy may
establish the diagnosis.
Indications for Bone Marrow Evaluation
A primary objective of a bone marrow examination is to assess the quantity
and development of hematopoietic cells. Bone marrow evaluation is
necessary for diagnosing, managing, making prognoses, and following up a
variety of hematologic and nonhematologic disorders. Because the cells in
the peripheral blood often reflect changes in the bone marrow, the bone
marrow should always be evaluated in conjunction with the results of the
peripheral blood count and smear review. Thrombocytopenia, coagulation
factor deficiency, or anticoagulant therapy is not a contraindication for the
bone marrow procedure. However, a bone marrow biopsy should not be
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performed when a bone marrow procedure will not be useful in diagnosing or
evaluating the patient’s condition.
Conditions for Which a Bone Marrow Evaluation Is Indicated
Purpose
Condition
Primary diagnosis of hematopoietic
and lymphoid malignancies






Staging of lymphoid malignancies
and solid tumors
 Lymphomas
Post-treatment follow-up
 After chemotherapy and radiation therapy
for neoplasms
 After stem cell transplant
Detection of infection and/or source of
fever of unknown origin
 Mycobacterium and fungal infections
 Granulomas
 Unknown infectious agents using cultures
and special stains
 Hemophagocytic syndrome
Primary diagnosis of systemic diseases
 Metabolic disorders (i.e., Gaucher’s
disease)
 Systemic mastocytosis
Miscellaneous
 Evaluation of storage iron
 Evaluation of unexplained cytopenias
Acute leukemias
Chronic myeloproliferative disorders
Chronic lymphoproliferative disorders
Myelodysplastic syndromes
Hodgkin’s and non-Hodgkin’s lymphomas
Plasma cell neoplasms
Obtaining and Preparing Bone Marrow for Hematologic Studies
The sites for bone marrow studies in adults are most commonly the posterior
superior iliac crest, occasionally the sternum, and very rarely the anterior
superior iliac crest and spinal processes or vertebral bodies. Sternal
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aspiration should be avoided in children, as well as in patients with multiple
myeloma and metastatic carcinoma, because these diseases can cause
thinning and erosion of bone that may increase the chance of perforation
and can cause potentially fatal cardiac complications.
Occasionally, when a localized bone lesion is visualized on roentgenogram or
computed tomographic (CT) scan, a directed or open bone marrow biopsy of
the lesion may be performed by a radiologist or surgeon in an operating
room while the patient is under anesthesia. In newborns and infants, a bone
marrow sample can be obtained from the upper end of the tibial bone.
Before performing the procedure, the physician should inform the adult
patient or the parent or guardian of a child of the procedure, its risks, and its
expected benefits for the diagnostic process. The bone marrow procedure
cannot be performed until a consent form is signed and witnessed by a
second person, commonly the patient's nurse. The actual procedure is often
performed with the assistance of a clinical laboratory scientist. While the
physician performs the procedure and the nurse attends to the patient, the
clinical laboratory scientist gives full attention to the processing of the
specimens.
It is the clinical laboratory scientist's responsibility to ensure that the
samples are adequate. If they are not, the physician is informed immediately
so that the procedure can be repeated before the patient is discharged.
Samples are preserved appropriately for histologic, flow cytometric,
cytogenetic, microbiologic, electron microscopic, molecular, and other
studies as indicated in a particular case.
In experienced hands, complications of bone marrow biopsy and aspirate are
very rare (0.1%). These rare complications include bleeding or infection at
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the biopsy site, transient neuropathy, and osteomyelitis. After the
procedure, the patient is advised to lie on the biopsy site, which should be
re-evaluated in 15 to 30 minutes for any bleeding or oozing. Bone marrow
aspiration and biopsy can be performed on patients with severe
thrombocytopenia, coagulation factor deficiencies (more than 50% activity is
required), and in those receiving anticoagulant therapy. The only valid
contraindication is failure to meet the indication criteria.
Equipment
The instrument tray used to perform a bone marrow procedure should
contain enough equipment to complete the procedure and to prepare the
tissues obtained for the appropriate studies. Complete bone marrow trays
are sold as disposable equipment, which is convenient, and also avoids the
risk of transmitting infectious diseases. Because of potential disease
transmission, nondisposable bone marrow trays are rarely used today.
The required materials to perform a bone marrow procedure include:

30-mL, 20-mL, 10-mL and 5-mL syringes

2% Lidocaine

Iodine prep

Alcohol (70%) prep

23-Gauge and 21-Gauge needles

Bone marrow biopsy/aspiration needle 11-gauge × 4 in.

Filter papers

Buffered formalin 10% with a pH of about 6.8 or other fixative for
histologic processing of bone biopsy and marrow particles

Tube containing liquid EDTA anticoagulant

One box of slides

One slide folder
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
One rubber bulb

Pasteur pipet

Petri dish

Sterile blades

Gloves (several pairs of different sizes)

Sterile gauze and cotton balls

Applicator sticks

Bandage

Culture bottles for bacterial culture. (Note: Some bone marrow
specimen should be saved in a syringe for tuberculosis and fungal
cultures, when indicated).

Pencil to label slides

No. 11 Bard Parker blades
Several different styles of aspiration and trephine bone biopsy needles or
instruments are commonly used. Most instruments used today are patterned
on the needle introduced by Jamshidi. These instruments are produced in
several sizes for both adult and pediatric patients. Modifications of the
original aspiration and trephine needles have been developed by different
companies and are manufactured as disposable equipment.
Aspiration of Bone Marrow
A bone marrow aspiration may be performed as an independent procedure
or in conjunction with a bone marrow biopsy. The procedure can be
performed in the outpatient setting in clinics or in the physician's office. As a
rule, children and very apprehensive patients receive a mild sedative before
the procedure. The site selected is shaved, if needed, and washed with soap.
Then an antiseptic is applied, and the area is draped with sterile towels. A
local anesthetic such as 1% to 2% lidocaine (Xylocaine) is infiltrated into the
skin, in the intervening tissues between the skin and bone, and in the
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periosteum of the bone from which the marrow is to be obtained. A cut of
about 3 mm is made through the skin with a Bard-Parker blade to facilitate
piercing skin and subcutaneous tissue.
The physician penetrates the bone cavity with an aspiration needle,
assembled with guard and stylet locked in place. When the marrow cavity is
penetrated, the stylet is removed, a syringe is attached to the free end of
the needle, and the plunger is quickly pulled, drawing 1.0 to 1.5 mL of
marrow particles and sinusoidal blood into the syringe. Because the vacuum
created in the syringe is important for rapid and efficient suctioning of the
cells and particles, the syringe should be 10 mL or larger with a well-fitting
plunger.
Despite the use of local anesthesia, the patient normally experiences
discomfort during the aspiration process (aspiration pain). Accomplishing the
aspiration with a quick and continuous pull on the plunger diminishes the
patient's discomfort and decreases the chance of clotting the specimen. A
clotted specimen is useless for smear preparation because the fibrin threads
strip the cytoplasm off of the cells and hamper their spreading. Keeping the
volume of the initial aspirate small also prevents dilution of the sample with
large amounts of sinusoidal blood, thus improving the quality of the aspirate.
The first-aspirated material is used immediately for preparing smears.
Additional aspirate may be obtained in separate syringes if needed for flow
cytometry, chromosome studies, bacterial cultures, and other tests. Once an
adequate aspirate is obtained, the quality of the smear depends entirely on
the clinical laboratory scientist's skill and speed in preparing the smears and
preserving the morphology of the marrow cells. Part of the first aspirate is
used for the preparation of direct and marrow particle smears. Another
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portion is placed in an ethylene diaminetetraacetic acid (EDTA)
anticoagulant-containing tube for use as a particle preparation. If some
aspirate still remains, it can be left to clot. The clot may be fixed in 10%
buffered formalin or another chosen fixative and processed for histologic
examination.
The preferred anticoagulants for ancillary studies performed on the bone
marrow aspirate are EDTA for flow cytometry and sodium heparin for
cytogenetics. If the aspiration attempt is unsuccessful (a dry tap), an
additional core biopsy may be obtained (placed in saline or RPMI) for flow
cytometric studies. In these situations, touch preparations of the core biopsy
are useful for Wright-Giemsa stained morphologic evaluation, as well as
cytochemical and fluorescence in situ hybridization (FISH) studies, if needed.
Preparation of Bone Marrow Aspirate
All necessary materials, preservatives, and slides should be meticulously
clean and in readiness to avoid any delay. The aspirate in the first syringe
contains mostly blood admixed with fat, marrow cells, and particles of
marrow tissue, which should be used for smears. Several direct smears can
be prepared immediately, using the technique for blood film preparation. A
small drop is placed on a glass slide, and the blood and the particles are
dragged behind a spreading slide with a technique similar to that for
preparing blood films. Although this method of preparation preserves the cell
morphology well, it is inadequate for the evaluation of the cells in
relationship to each other and for the estimation of marrow cellularity.
Smears of marrow particles are prepared by pouring a small amount of the
aspirate on a glass slide. The marrow tissue is seen as gray particles floating
in blood and fat droplets. The particles are aspirated selectively with a
plastic dropper or Oxford pipette and transferred to a clean glass slide,
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which is covered gently with another slide. The two slides are pulled in
opposite and parallel directions to smear the particles without crushing the
cells. Some people recommend an alternate technique using two cover
glasses. In this process, the marrow particles are squashed between two
cover slips, which are then gently pulled apart.
Techniques for preparing particle smears vary from person to person and
from laboratory to laboratory. The aspirate may be transferred into a watch
glass and the particles collected with a capillary pipette or the broken end of
a wooden stick applicator. With experience, one usually adapts a technique
that facilitates production of high-quality slides. The clinical laboratory
scientist should prepare an adequate number of slides of smeared marrow
particles. In cases of newly diagnosed acute leukemia, no fewer than 10
slides should be prepared. These are needed for histochemical stains such as
myeloperoxidase, Sudan black B, naphthyl AS-D chloroacetate esterase,
alpha-naphthyl butyrate esterase, and others.
Marrow particle smears are used in the evaluation of cellularity (usually
marrow biopsy is ideal) and the relationship of the cells to each other. Wellprepared smears have the added advantage of excellent cell morphology,
allowing subtle changes in cell maturation and cytoplasmic inclusions to be
recognized easily. All direct and particle smears should be labeled at the
bedside with the patient's name, identification number, and the date and
then air-dried.
Histologic Marrow Particle Preparation
The leftover marrow particles obtained during the aspiration procedure can
be processed for histologic examination. The tissue particles, admixed with
blood, may be left to clot, then fixed in 10% buffered formalin and
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processed for histologic sectioning. However, better results are obtained if
the blood and particles are transferred to an EDTA anticoagulant–containing
tube before clotting sets in. The blood and particles are then filtered through
histo-wrap filter paper, and the concentrated particles enfolded in the paper
are fixed in 10% buffered formalin. In the histology laboratory, these
particles are collected through scraping the paper, and then embedding the
particles in paraffin for further processing.
Bone Marrow Core Biopsy
A bone marrow core biopsy is especially indicated when the marrow cannot
be aspirated or is a dry tap, owing to pathologic alterations encountered in
acute leukemias, myelofibrosis, hairy cell leukemia, and other disorders. A
trephine bone marrow core biopsy is also performed for the diagnosis of
neoplastic and granulomatous diseases. In multiple myeloma and for staging
of lymphomas or solid tumors, bilateral posterior superior iliac crest biopsies
are recommended, as increased sampling size enhances the likelihood of
capturing a focal process. An adequate biopsy sample is at least 15 mm in
length.
When a bone marrow biopsy is performed in conjunction with a marrow
aspiration, customarily the biopsy sample is obtained after the aspirate. This
sequence is usually achieved by changing the direction of the needle to
avoid the aspiration artifact. However, this technique may result in an
aspiration artifact with hemorrhage into the area of the biopsy site, leading
to difficulties in evaluating cellularity and morphology. Therefore, a core
biopsy sample should be obtained before the aspiration or the marrow
biopsy procedure is to be performed through a new puncture site in the
anesthetized area.
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In some cases when flow cytometry is indicated, and the aspirate is difficult
to obtain (i.e., in patients with hypercellular marrow, hairy cell leukemia, or
marrow fibrosis) an additional core marrow biopsy may be obtained in
normal saline or RPMI (Roswell Park Memorial Institute) solution. Adequate
cell suspensions can then be made from this biopsy sample and processed
for flow cytometric studies.
Preparation of Trephine Biopsy
Touch Preparation:
The bone marrow core biopsy sample is supported lightly without pressure
between the blades of forceps and touched several times on two or three
clean slide surfaces. The biopsy core sample should not be rubbed on the
slide, because rubbing destroys the cells. The slides are air-dried. The touch
preparations are fixed in absolute methanol and stained with Wright-Giemsa
stain.
In the absence of a good aspirate smear, the touch preparations may be the
only source for studying cellular details and the maturation sequence of the
bone marrow biopsy sample. For example, in a case of hairy cell leukemia,
dry taps are common and cellular morphology by Wright-Giemsa stained
touch preparation may be a useful clue to the diagnosis and allow
cytochemical staining procedures, such as tartrate-resistant acid
phosphatase (TRAP). Sometimes the touch preparations contain enough cells
to obtain differential counts and blast evaluation, and to perform
histochemical studies.
Histologic Bone Marrow Biopsy Preparation:
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The biopsy specimen is immersed without delay in B-5 or 10% buffered
formalin fixative. Histology laboratories may have a choice of other preferred
fixatives such as Zenker's solution, Carnoy's solution, and others. After
fixation, the biopsy specimen undergoes standard histologic processing of
decalcification, dehydration, embedding in paraffin blocks, sectioning of 2- to
3-µm thick sections, and histologic staining. The advantage of the bone
marrow biopsy is that it represents a large sample of marrow and bone
structures in their natural relationships.
A variety of different stains can be used to demonstrate marrow iron,
reticulum, and collagen. However, because of decalcification, the core biopsy
may not be a good method for studying marrow iron stores, as the
processing leaches iron from the tissue, which may be underrepresented in
the iron stain. Acid-fast organisms and fungi in granulomatous diseases may
be detected quickly with specific stains, offering great advantages in
diagnosing these infections. For instance, mycobacterial cultures may
require weeks of incubation to show growth of organisms, whereas on tissue
sections, the histologic and etiologic diagnosis may be made within 10 to 12
hours. When metastatic tumors and lymphomas are found in the bone
marrow, immunohistochemical stains can be used on histologic sections to
demonstrate specific tumor markers. Thus, a very precise diagnosis of the
origin of a tumor can be made without elaborate, expensive, and invasive
techniques.
A disadvantage of the bone marrow biopsy is that fine cellular details are
lost in the processing; therefore, it is of little value in the diagnosis of
myelodysplastic syndromes and subtyping of acute leukemias. In these
situations, the Wright-Giemsa stained aspirates or core biopsy touch
preparations may supply the missing morphologic details. Multiple touch
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preparations also offer an opportunity for histochemical stains
(myeloperoxidase, Sudan black B, naphthyl AS-D chloroacetate esterase, αnaphthyl butyrate esterase, etc.), which are essential in the classification of
leukemias. Polymerase chain reaction (PCR) and FISH can be performed on
paraffin-embedded, formalin-fixed marrow biopsy specimens. PCR
technology may be used to evaluate B-cell or T-cell lymphomas, various
leukemias, and minimal residual disease.
Trephine bone marrow biopsy specimens may be embedded in methyl
methacrylate, a synthetic plastic medium, and sectioned into 1 to 2 µm thin
sections without decalcification. The morphological quality of the cells is
extremely well preserved, and a differential count can be done on
hematoxylin–eosin (H&E) or Giemsa-stained slides. However, this technique
requires specially trained personnel, equipment, and separate handling in
the histology laboratory, which increases the cost of the procedure. The
processing time of the tissue also increases, which may not be acceptable if
rapid diagnoses are required. In addition, tissue embedded in plastic media,
instead of paraffin, may not be suitable for immunohistochemical studies of
bone marrow.
Bone Marrow Examination
The examination of the bone marrow aspirate smears should start at low
magnification with a dry objective of 10×. Scanning the slide permits
selection of a suitable area for examination and the differential count. Bare
nuclei should be avoided; such nuclei result from destruction of the marrow
cells by squashing or stripping of their cytoplasm by fibrin threads. An area
is selected in which the cells are well spread, intact, and not diluted by
sinusoidal blood.
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When marrow particles are examined, such areas are found at the periphery
of the particles. At this low magnification, marrow cellularity is also
evaluated. The megakaryocytes are usually noted adjacent to a spicule,
about five to ten per low-power field. Nonhematopoietic tumor cells
infiltrating the bone marrow may also be seen at this magnification. These
are usually larger than the granulocytic or erythropoietic precursors and are
scattered in small groups and crowded clusters. Some show a glandular
configuration.
After the initial scan, immersion oil is applied to the slide and the
examination continues on high magnification (oil immersion objective 50× or
100×). The high magnification provides details of the nuclear and
cytoplasmic maturation process. The iron in histiocytes is visualized as
brown-blue granules. Cytoplasmic inclusions of a diagnostic nature can be
seen in histiocytes and granulocytes. Differential counts of bone marrow are
performed with the oil immersion objective.
Estimation of Bone Marrow Cellularity
Cellularity is reflected in the ratio of nucleated hematopoietic cells to fat
cells. Bone marrow cellularity normally varies with age, and the estimated
cellularity must be compared to age-related normal ranges. At birth the
normal marrow cellularity is 100%. Thereafter, the cellularity gradually
decreases. Overall marrow cellularity in adults is about 50% (± 10%). The
general rule to estimate age-related normal ranges is 100 minus age ± 10.
For example, the estimated normal marrow cellularity of a 40-year-old
person would be 100–40 ± 10 (i.e., a range from 50% to 70%).
Cellularity may vary from area to area, and, therefore, estimated cellularity
should represent the average percentage. If hypercellular (90%) and
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hypocellular (10%) areas are seen, this finding should be mentioned
descriptively in the report because in such cases an average cellularity may
be difficult to estimate. The immediate subcortical region of adult bone
marrow is usually hypocellular as compared with the deeper medullary area.
Therefore, sections that contain predominantly subcortical bone are
frequently suboptimal for assessing true marrow cellularity.
The bone marrow biopsy specimen is most reliable for assessment of
cellularity, because it offers a large amount of tissue for evaluation.
However, the evaluation of cellularity can also be done on well-prepared
aspirate smears or marrow particles. The best area for examination of
cellularity in smears is the area between two uncrushed particles. The ratio
of cells to fat is evaluated at low magnification (objective 10×), so that
larger areas are included in the field of observation. The empty spaces that
result from the spreading of the cells but are not occupied by fat cells are
disregarded and treated as an artifact.
The terms decreased or increased cellularity is used when fewer or more
than the expected normal number of cells are found. Precise evaluation can
be achieved with experience, and good reproducibility can be attained
among several observers. The marrow cellularity can be expressed in
percentages, but this is best done on histologic sections of biopsy
specimens. Marrow cellularity has diagnostic value when it is related to the
M:E ratio, which is calculated after a differential count is performed.
It is always important to look for any abnormal changes in the bony
trabeculae. Various conditions can alter the morphological appearance of
these trabeculae. Marked thickening of trabeculae can be seen in
myeloproliferative disorders (myelofibrosis with myeloid metaplasia),
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whereas thinning of trabeculae can be seen in older adults, in patients with
acquired immunodeficiency syndrome (AIDS) or other cachectic conditions,
and after chronic steroid administration. Various metabolic disorders can
also alter the morphology of bony trabeculae. Examples include a mosaic
pattern, seen in patients with Paget's disease, and resorption and cyst
formation, in persons with hyperparathyroidism and chronic renal failure.
When lymphoid aggregates are seen in the bone marrow biopsy specimen,
the differential diagnosis includes benign lymphoid aggregates and
malignant lymphoma. Usually benign aggregates are small and well
demarcated, nonparatrabecular, and composed predominantly of small,
round lymphocytes with plasma cells at the periphery and blood vessels
present within the aggregate. Conversely, malignant follicles are usually
large with ill-defined borders, paratrabecular, composed of atypical
lymphocytes, and lack plasma cells at the periphery. However, a neoplastic
lymphoid infiltrate can also be interstitial, diffuse and patchy. In some cases,
immunohistochemical stains can be performed on the marrow core biopsy to
differentiate between benign lymphoid aggregates and malignant lymphoma.
Bone marrow fibrosis may be found in patients with hairy cell leukemia, in
myeloproliferative and myelodysplastic syndromes, sometimes in acute
leukemia, after radiation, and after toxic injury to the marrow. On routine
H&E sections, streaming of marrow stroma and dilated sinusoids suggests
marrow fibrosis; this finding can be confirmed and graded by performing
reticulin and trichrome stains. Normally, occasional reticulin-positive fibers
may be seen around the blood vessels. The fibrosis can be graded as mild,
moderate, or severe. In addition, a description of the fibrosis as fine or
coarse, and focal or diffuse, may be helpful, especially when following the
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improvement of a patient with myelofibrosis after treatment or bone marrow
transplantation.
Trichrome stain is usually performed to detect any collagenous fibrosis,
which if present may indicate irreversible fibrosis. Fibrosis and other bone
marrow conditions such as marked hypercellularity in acute leukemias may
result in a dry tap when attempting the aspiration procedure. However, if
flow cytometry is needed, it is a good practice to obtain two bone marrow
biopsy samples. One biopsy sample can be put in formalin and processed for
morphological examination. The other core biopsy sample can be put in
saline or RPMI medium; a cell suspension is then made for flow cytometric
studies.
Bone Marrow Differential Count
A bone marrow differential count is an excellent tool for training a novice in
bone marrow morphology and is widely used in diagnosing and following up
patients with leukemias, refractory anemias, and myelodysplastic and
myeloproliferative syndromes. Because of the compartmentalization of the
hematopoietic cells and high cellularity of marrow, at least 500 to 1000
nucleated cells need to be classified for a representative differential count.
In infants during the first month after birth, dramatic alterations occur in the
distribution of the different marrow compartments. At birth there is a
predominance of granulocyte precursors, which switches within a month to a
predominance of lymphoid elements. In early infancy many lymphocytes
have fine chromatin and a high nuclear-to-cytoplasmic ratio, and lack
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distinct nucleoli. They are called hematogones and represent normal
lymphoid progenitor cells. Hematogones may be misinterpreted as blasts if
the observer is unfamiliar with these characteristics. In children up to 3
years old, one-third or more of the marrow cellularity is made up of
lymphocytes. The lymphocyte number gradually declines to the normal adult
level thereafter.
In adult marrow the lymphocytes are distributed randomly among the
hematopoietic cells and within lymphoid follicles. This can introduce
significant variation in the differential count from sample to sample in the
same patient. The majority of adult marrow is composed of granulopoietic
and erythropoietic precursors. For the purpose of the differential count,
these are enumerated into different categories according to their stage of
maturation. When adequate numbers of cells are tabulated, the percentage
of each category is calculated. The ratio between all granulocytes and their
precursors and all nucleated red cell precursors represents the M:E ratio.
Some hematologists prefer to exclude the segmented neutrophils from the
differential count as being part of the neutrophil storage pool of the marrow.
The normal M:E ratio in this case is between 1.5 and 3. However,
pathologists and hematologists who interpret the bone marrow histologic
sections of particle clot and biopsies in conjunction with marrow smears
include the segmented neutrophils in the differential counts, because these
cannot be excluded in the evaluation of histologic specimens and are part of
the marrow cellularity. The normal M:E ratio then is slightly higher and
ranges between 2 and 4. The granulopoietic tissue occupies two to four
times greater marrow space than the erythropoietic precursors, owing to the
shorter survival of the granulocytes in the circulation (i.e., neutrophils, 6 to
10 hours, versus erythrocytes, 120 days). Changes in the survival of
granulocytes and erythrocytes are reflected in changes in the M:E ratio.
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Megakaryocytes (large bone marrow cells necessary for normal blood
clotting) are not included in the differential count. Megakaryocytes are
unevenly distributed, and a differential count is a poor means for their
evaluation. Usually five to ten megakaryocytes are seen per microscopic field
at low magnification (objective 10×). When clusters of megakaryocytes and
promegakaryocytes are seen in every field, it is an indication of
megakaryocytic hyperplasia. In a normocellular marrow, finding fewer than
two megakaryocytes per field on screening may indicate megakaryocytic
hypoplasia. A marked increase or decrease in the number of
megakaryocytes is easy to evaluate, whereas slight to moderate changes are
difficult to judge and are better estimated on histologic sections of biopsy
and particle specimens.
Bone Marrow and Peripheral Blood Interpretation Based on Cellularity and
M:E Ratio Changes
A bone marrow aspirate or biopsy sample represents a minute part of a very
large and dynamic organ. Its activity and responses are reflected in blood
changes; therefore, evaluation of the bone marrow should always be done in
conjunction with evaluation of the peripheral blood. In adults with 50%
marrow cellularity, about 30% to 40% represents granulopoiesis and 10% to
15% erythropoiesis, with an average M:E ratio of 4:1. An increase or a
decrease in marrow cellularity with the normal M:E ratio usually indicates a
balanced granulocytic and erythrocytic hyperplasia or hypoplasia,
respectively. However, if cellularity changes occur simultaneously with the
M:E ratio change, the interpretation requires a broader understanding of
hematopoietic tissue physiology and its reactions during disease.
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Cell morphology and the M:E ratio are well represented in random bone
marrow specimens. The variations are not significant even when samples are
compared from sternal and iliac crest aspirates. However, marrow cellularity
is poorly represented in random smears; thus, this interpretation should be
considered with some degree of reservation. Even large biopsy specimens
may have a great degree of variation in cellularity. For these reasons, in
diseases in which marrow cellularity is crucial for the diagnosis (aplastic
anemia, marrow hypoplasia), more than one bone core biopsy may be
required.
Bone Marrow Iron Stores
The storage iron of the bone marrow is in the form of hemosiderin. The iron
content of hemosiderin is higher than that of ferritin. Other components of
hemosiderin are protein, ferritin aggregates, some lipids, and membranes of
cellular organelles. Hemosiderin can be seen on unstained smears as goldenyellow granules. On Wright–Giemsa-stained smears it appears as brownishblue granules. However, for more precise evaluation, Prussian blue reaction
is used to demonstrate the intracytoplasmic iron of histiocytes and red cell
precursors.
The evaluation of marrow iron stores is essential in the diagnosis of anemias
and especially in refractory and dyserythropoietic anemias. When the
morphological characteristic of the iron particles in the storage nutrient
histiocyte and erythroblastic precursors is an important diagnostic
consideration (i.e., in sideroblastic anemias), an iron stain is performed on a
particle smear. If the overall distribution of the amount of iron is of clinical
importance (i.e., iron-deficiency anemia, anemia of chronic diseases,
hemochromatosis, and others), then histologic sections of bone marrow
biopsy sample, and/or marrow aspirate, and marrow clotted particles are
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stained for iron. The biopsy sample and the particles are a more reliable
source of information, because they represent a large sample of
hematopoietic tissue. The EDTA chelating method, which does not affect the
storage iron, should decalcify bone marrow biopsy samples for iron studies.
Rapid-acid decalcifying solutions extract iron and must not be used in these
cases.
Bone Marrow Report
The bone marrow report must contain all relevant information and optimally
is composed of two components: clinical information and morphologic
interpretation. The clinician should provide the patient’s biographic data,
clinical differential diagnosis, and relevant therapeutic information. The
morphology and interpretation by the pathologist and laboratory professional
must include site of sampling (i.e., sternum), types of sample obtained,
differential counts from both peripheral blood and bone marrow, and
morphologic abnormalities in any cell lineages in the patient’s peripheral
blood or bone marrow.
The results must be interpreted in conjunction with any additional studies
(special stains, flow cytometry, cytogenetics, molecular studies). If the
additional studies are significant in establishing the diagnosis but are
unavailable when the bone marrow report is written, an addendum report
should mention the significance of these studies. The comparison of the
current marrow specimen to the previous tissue samples (marrow or other
nonmarrow biopsies) is essential in some situations.
Finally, the pathologist should render a diagnostic interpretation within a
reasonable period of time. For example, if 50% blasts were present in the
blood or the bone marrow, it is reasonable to call the clinician about the
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preliminary diagnosis of acute leukemia. However, the final diagnosis can be
made only after the special stains and flow cytometry studies have been
completed. Comments, if needed, should be concise and relevant to the case
and can include a recommendation for additional tests and a possible
differential diagnosis.
The bone marrow report usually encompasses the following:
1. The name of the laboratory or physician's office from which the report
originates.
2. The patient's data, including age and relevant clinical summary or
clinical diagnosis.
3. A description of material received for studies, such as smears of
aspirate, marrow particles, and bone biopsy (or biopsies).
4. Data from the complete blood count (CBC) and WBC differential count,
and a description of the blood smear, preferably from the day on which
the bone marrow specimen is obtained. A platelet count should be
included, as well as a reticulocyte count, if available.
5. The bone marrow differential count.
6. A description of cellularity, M:E ratio, granulopoiesis, erythropoiesis,
and megakaryocytopoiesis. Any change in the nonhematopoietic
elements of marrow, such as hemophagocytosis, granulomas,
microorganisms, metastatic tumor cells, histiocytic hyperplasia, or the
appearance of bony trabeculae, is included in this section of the report.
The status of iron stores and special staining procedures performed
are reported.
7. A description of histologic sections of marrow particles or bone marrow
biopsy.
8. The diagnostic conclusion. This should encompass separate diagnoses
of blood and bone marrow even where the same diagnosis is
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applicable to both. For example, 1) blood — pancytopenia, and bone
marrow, left posterior iliac spine aspirate and biopsy —
myelodysplastic syndrome, refractory anemia with ringed sideroblasts;
or 2) blood — acute myeloid leukemia minimally differentiated (WHO
classification), and bone marrow, left posterior iliac crest aspirate and
biopsy — acute myeloid leukemia minimally differentiated (WHO
classification).
Bone marrow can provide a representative picture of disease processes and
has wide application in clinical medicine. Marrow examination has a
significant role in the evaluation of leukemias, lymphomas, plasma cell
disorders, myeloproliferative disorders, myelodysplastic disorders,
myelofibrosis, metastatic tumors, various anemias, granulomatous diseases,
infectious diseases, metabolic diseases, in evaluating the status of
engraftment after bone marrow transplantation, and in assessing
chemotherapy effects. The clinical laboratory scientist's contribution in this
phase consists of preparing the optimum blood and bone marrow slides and
performing the differential count. Examination of the blood and bone
marrow, correlation with the clinical presentation, and diagnostic conclusions
on each specimen are the responsibility of a physician who has adequate
training and experience to integrate all of the available clinical and
laboratory information in reaching the correct diagnosis.
Treatment and Management
The most important part of management is the prompt recognition that a
problem exists. This is done by two mechanisms: firstly, vigilance for signs
and symptoms that may indicate a blood disorder and, secondly, patient
education about the warning symptoms that should alert them to the need
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to urgently contact their medical provider or emergency services if a prompt
provider appointment is not possible.
Treatment of blood dyscrasias requires specialist expertise. Any drugs
suspected of being involved in the reaction should be discontinued
immediately and short-term supportive treatments given to aid a potential
spontaneous recovery. Such support can include blood and platelet products,
antibiotic or antifungal agents and recombinant human hemopoietic growth
factors. Aplastic anemia may require immunosuppressive therapy and bone
marrow or stem cell transplant. Modern treatments for blood dyscrasias,
such as granulocyte colony-stimulating factor (G-CSF), have significantly
reduced mortality rates. Hemolytic anemia normally recovers within two to
three weeks of drug withdrawal, although corticosteroid therapy may be
beneficial.
The treatment for medication induced blood dyscrasias typically falls into one
(or more) of three categories as reviewed below. This section covers the
varied methods of treatment required depending on the type and severity of
patient response to medication and disease outcomes, as well as the
important of clinicians’ vigilance to be aware of and to educate their patients
of the potential for drug-induced dyscrasia, methods of prevention and
treatment.81,87
Removal of Drug
If a patient presents with one of the conditions discussed above, it is
imperative that the drug causing the reaction be discontinued immediately.
In many instances, this will require the provider to identify and initiate
alternate treatments for the patient. In some rare instances, the drug may
cause withdrawal symptoms in the patient. The provider will have to manage
these effects.
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Symptomatic Support
In addition to eliminating the drug, it is often necessary for the provider to
provide symptomatic support to the patient. All of the conditions above
produce a number of symptoms, many of which are significant. Patients will
require some level of treatment to reduce and/or eliminate the symptoms
caused by the drugs and the associated conditions.
Immunosuppressive Therapy
In some instances, patients may require immunosuppressive therapy to
mitigate the effects of the condition. Immunosuppressive therapy is used in
instances when there is a risk that the patient’s immune system may
interfere with treatment. This is most common when patients are receiving
transfusions or donations of blood, marrow, or organs. Since the patient’s
immune system may attack these foreign bodies, it is important to suppress
the immune system, thereby allowing the treatment to take effect. The
following table provides treatment information for each condition.
Aplastic Anemia
Because of the high mortality rate associated with severe and
very severe aplastic anemia, it is imperative that drug-induced
aplastic anemia be diagnosed quickly and therapy initiated
immediately. Treatment should be based on the severity of
disease, with the goal of therapy being to improve peripheral
blood counts, limit the requirement for transfusions, and minimize
the risk for infections.
As with all cases of drug-induced hematologic disorders, the first
step is to remove the suspected offending agent. Early withdrawal
of the drug can allow for reversal of the aplastic anemia.
Appropriate supportive care is also essential because the major
causes of mortality in patients with aplastic anemia are infections
(bacterial and fungal) and bleeding. Patients must receive
transfusion support with erythrocytes and platelets, as well as
appropriate antimicrobial prophylaxis or treatment during
neutropenic periods.
Routine use of growth factors such as recombinant human
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erythropoietin and granulocyte colony-stimulating factor has not
been shown to improve outcome and are not recommended for
the management of aplastic anemia. Current treatment guidelines
for aplastic anemia recommend the use of prophylactic antibiotic
and antifungal agents when neutrophil counts are below 500
cells/mm3 (0.5 × 109/L). If patients experience febrile
neutropenia, broad-spectrum IV antibiotics should be started
immediately.
Current guidelines do not recommend the use of prophylaxis for
viruses or Pneumocystis jiroveci. For patients who have been
heavily transfused, iron chelation therapy with agents such as
deferoxamine or deferasirox may be necessary to avoid the
serious consequences of iron overload.
The clinical course of aplastic anemia is variable. The condition
can progress to severe or very severe disease in some patients,
although it can remain relatively stable or even resolve in others.
The treatment of moderate disease ranges from no clinical
intervention to immunosuppressive regimens, and treatment
should be based on the degree of cytopenias. For patients with
disease requiring treatment, the two major treatment options for
patients with drug-induced aplastic anemia are allogeneic HSCT
and immunosuppressive therapy.
Factors that determine which therapy would be preferred include
age, disease severity, and availability of a human leukocyte
antigen– (HLA-) matched sibling donor. For patients younger than
the age of 40 years, the treatment of choice is allogeneic HSCT
from an HLA-matched sibling donor. This treatment modality is
associated with potential cure and results in a 5-year survival rate
of 77% in adults and up to 90% in children. Unfortunately, most
patients do not have a matched sibling donor. For young patients
who do not have an available HLA-matched sibling, allogeneic
HSCT from an unrelated donor may be considered but is usually
reserved for those who fail to respond to upfront
immunosuppressive therapy. When used in this setting, the 5year overall survival rate in these patients has improved to over
50%, primarily because of improvements in HLA typing and
unrelated donor selection.
For patients older than the age of 40 years and for those who are
not candidates for allogeneic HSCT, the preferred first-line
therapy is immunosuppressive therapy. Allogeneic HSCT in older
patients is associated with significantly higher transplant-related
morbidity and mortality. The highest mortality rate was seen in
older patients and those with poorer clinical status at the time of
transplantation. Complications of allogeneic HSCT, such as graftversus-host disease and graft rejection, require all patients to be
closely monitored for an extended period of time.
The current standard immunosuppressive regimen for the
treatment of acquired aplastic anemia is combination therapy with
antithymocyte globulin (ATG) and cyclosporine. This combination
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has been reported to achieve 5-year survival rates between 75%
and 85%, but the response rates in older patients are lower. ATG
is composed of polyclonal immunoglobulin G (IgG) against human
T lymphocytes derived from either horses or rabbits, and it has
been a standard component of immunosuppressive therapy for
aplastic anemia for many years. In a study comparing the horse
versus rabbit product, both given in combination with
cyclosporine, treatment with the horse-derived
Antithymocyte globulin product resulted in significantly higher
response rates (68% vs. 37%) and 3-year overall survival rates
(96% vs. 76%). Although the mechanism for this difference is not
completely understood, the greater depletion of CD4+ cells
associated with the rabbit ATG as compared with horse ATG may
be associated with adverse outcomes. Based on these results,
treatment with the horse-derived ATG product is preferred for
treatment.
Because response to immunosuppressive therapy is often delayed
(3–4 months), patients require continued supportive care until
recovery. Patients should be monitored for adverse effects,
including serum sickness, which can occur about 1 week after
ATG begins.
Cyclosporine plays an important role in immunosuppressive
therapy for aplastic anemia. Although cyclosporine monotherapy
has been used to treat moderate aplastic anemia, it is more often
used in combination with ATG. The addition of cyclosporine to
ATG therapy has been shown to increase response rate, improve
failure-free survival, and reduce the number of
immunosuppressive courses needed. Cyclosporine inhibits
interleukin-2 production and release and subsequent activation of
resting T cells.
Cyclosporine dosing has varied from 4 to 6 mg/kg per day to 10
to 12 mg/kg per day, with the most frequently reported initial
dose of 5 mg/kg per day in two divided doses. Cyclosporine doses
are titrated to a target blood concentration that can be patient
and institution specific but are usually in the range of 150 to 250
mcg/L (125–208 nmol/L) for adult patients.
Increased relapse rates have been observed when tapering
cyclosporine rapidly, and it is recommended that cyclosporine be
continued for at least 12 months after response and then tapered
slowly. Corticosteroids are added to ATG-based
immunosuppression because of their ability to reduce adverse
reactions associated with ATG administration. In an effort to
improve outcomes, several other agents have also been
investigated for treatment of aplastic anemia. The additive
benefits of other immunosuppressive agents such as
mycophenolate, cyclophosphamide, and sirolimus have been
evaluated. However, they have not been shown to be superior to
the combination of ATG and cyclosporine, and their place in
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therapy is not defined.
Allogeneic HSCT has long been the established treatment of druginduced aplastic anemia. Although current practices in allogeneic
stem cell procurement generally favors the use of peripheral
blood stem cell harvesting, recent experiences have suggested
that for patients with aplastic anemia, stem cells sourced from
bone marrow may be associated with better outcomes, because of
the relative lack of T cells in a bone marrow product, which is
thought to confer a decreased risk of graft-versus-host disease.
Data to support this theory are largely from single-center
experiences, and the benefit of one source has not been proven in
well-designed trials. Until it is clearer whether one stem cell
source is better than another, the choice of stem cell source
should be largely based on donor availability and preference.
Agranulocytosis
The primary treatment of drug-induced agranulocytosis is the
removal of the offending drug. After discontinuation of the drug,
most cases of neutropenia resolve over time, and only
symptomatic treatment (i.e., antimicrobials for infection
treatment and prophylaxis) and appropriate vigilant hygiene
practices are necessary.
Sargramostim (granulocyte-macrophage colony-stimulating factor
[GM-CSF]) and filgrastim (granulocyte colony-stimulating factor
[G-CSF]) have been shown to shorten the duration of
neutropenia, length of antibiotic therapy, and hospital length of
stay. Although the use of both agents has been reported in the
literature, a commonly reported regimen is G-CSF 300 mcg/day
via subcutaneous injection.
The only prospective, randomized trial to date did not confirm the
benefit of these growth factors. However, some experts have
questioned the validity of these results based on the small sample
size (n = 24) and the lower than standard dose of filgrastim used
(i.e., 100–200 mcg/day). One systematic review found that
patients with a neutrophil nadir less than 100 cells/mm3 (0.1 ×
109/L) had a higher rate of infections and fatal complications than
those with a higher nadir. Therefore, most clinicians recommend
the use of growth factors in patients with a neutrophil nadir less
than 100 cells/mm3 (0.1 × 109/L), regardless of the presence of
infection.
Hemolytic Anemia
Drug-Induced Immune Hemolytic Anemia
The severity of drug-induced immune hemolytic anemia depends
on the rate of hemolysis. Hemolytic anemia caused by drugs
through the hapten or adsorption and autoimmune mechanisms
tends to be slower in onset and mild to moderate in severity.
Conversely, hemolysis prompted through the immune complex
mechanism (innocent bystander) phenomenon can have a sudden
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onset, lead to severe hemolysis, and result in renal failure.
The treatment of drug-induced immune hemolytic anemia
includes the immediate removal of the offending agent and
supportive care. In severe cases, glucocorticoids can be helpful,
but their use outside of autoimmune hemolytic anemia is not
supported by strong evidence. Other agents such as the chimeric
anti-CD20 monoclonal antibody rituximab and IgG treatments
have been used, but their role is yet to be clearly defined.
Drug-Induced Oxidative Hemolytic Anemia
Removal of the offending drug is the primary treatment for druginduced oxidative hemolytic anemia. No other therapy is usually
necessary because most cases of drug-induced oxidative
hemolytic anemia are mild in severity. Patients with these enzyme
deficiencies should be advised to avoid medications capable of
inducing the hemolysis.
Megaloblastic
Anemia
When drug-induced megaloblastic anemia is related to
chemotherapy, no real therapeutic option is available, and the
anemia becomes an accepted side effect of therapy. If druginduced megaloblastic anemia results from cotrimoxazole, a trial
course of folinic acid, 5 to 10 mg up to four times a day, can
correct the anemia.
Folic acid supplementation of 1 mg every day often corrects the
drug-induced megaloblastic anemia produced by either phenytoin
or phenobarbital, but some clinicians suggest that folic acid
supplementation can decrease the effectiveness of the
antiepileptic medications.
Thrombocytopenia
The primary treatment of drug-induced thrombocytopenia is
removal of the offending drug and symptomatic treatment of the
patient. The use of corticosteroid therapy in the treatment of
drug-induced thrombocytopenia is controversial, although some
authors recommend it in severe symptomatic cases.
Corticosteroids are sometimes helpful when clinicians are initially
trying to distinguish between drug-induced thrombocytopenia and
idiopathic thrombocytopenic purpura (ITP).
In the case of HIT, the main goal of management is to reduce the
risk of thrombosis or thrombosis-associated complications in
patients who have already developed a clot. All forms of heparin
must be discontinued, including heparin flushes, and alternative
anticoagulation must begin immediately. The direct thrombin
inhibitors are the alternative anticoagulants most commonly used
in current practice. Three direct thrombin inhibitors are currently
available: lepirudin, argatroban, and bivalirudin.
Lepirudin, the first drug that was approved for the treatment of
HIT, is a recombinant analogue of hirudin, a natural anticoagulant
found in leeches. Lepirudin is renally eliminated and requires
dosage adjustment in those patients with kidney dysfunction.
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It is also important to note that antibodies to lepirudin develop in
about 30% of patients who receive this agent for the first time,
and it is therefore recommended that patients receive only one
course of lepirudin.
Argatroban is another IV thrombin inhibitor indicated for the
management of HIT. But unlike lepirudin, argatroban is
metabolized in the liver and can be used in patients with endstage renal disease. However, dosage adjustment is needed for
patients with significant hepatic impairment. The most recently
approved direct thrombin inhibitor is bivalirudin. It is similar to
lepirudin in that is a parenteral bivalent analogue of hirudin. It
requires dosage adjustment only in severe renal failure.
Fondaparinux, an anticoagulant pentasaccharide that inhibits
factor Xa, has been proposed by some as a potential treatment
for HIT because it does not appear to cause in vitro crossreactivity with HIT antibodies.
Clinical data, however, to support the use of fondaparinux in the
treatment of HIT-induced thrombosis are lacking. The most recent
guidelines by the American College of Chest Physicians suggest
that fondaparinux is most appropriately used in patients at
relatively low risk of having HIT but for whom the use of either
UFH or LMWH is not desired.
These agents should also be considered for the treatment of
patients who have acute HIT without thrombosis because of the
increased risk of thrombosis occurring in these patients. Because
of the increased risk of venous limb gangrene, warfarin should not
be used alone to treat acute HIT complicated by deep vein
thrombosis.
Drug induced thrombocytopenia, in most cases, resolves quickly
after removal of the offending agent. In some cases, however,
thrombocytopenia can persist for weeks or months, especially in
the case of chemotherapy-induced thrombocytopenia or
thrombocytopenia caused by immune mechanisms. In this
setting, limited options are available to maintain platelets in a
safe range while awaiting count recovery.
Historically, transfusions were used to maintain platelet counts
until bone marrow recovery. The emergence of thrombopoietin
analogs such as eltrombopag and romiplostim has raised the
question of using drug therapy to treat drug-induced
thrombocytopenia.
Current indications for these agents are limited to ITP, but
preliminary data suggest a potential benefit in patients with
prolonged drug-induced thrombocytopenia. Currently, this
treatment cannot be recommended routinely, but future studies
can help to elucidate if there is a role for these agents in the
management if drug-induced thrombocytopenia.
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Reporting A Drug-Induced Blood Dyscrasia
Because of the seriousness of drug-induced hematologic disorders, it is
necessary to track the development of these disorders to predict their
occurrence and to estimate their incidence. Reporting during post-marketing
surveillance of a drug is the most common method of establishing the
incidence of adverse drug reactions. This section briefly reviews aspects of
reporting an adverse drug reaction, which is an extensive are of health
research and patient care protocols guiding medical treatment and intended
to improve patient safety, which learners are encouraged to further
investigate and pursue with their health agency of employment should an
adverse medication event occur.1,7,36,85-87
The MedWatch program supported by the Food and Drug Administration is
one such program. Many facilities have similar drug-reporting programs to
follow adverse drug reaction trends and to determine whether an association
between a drug and an adverse drug reaction is causal or coincidental. In
the case of drug-induced hematologic disorders, these programs can enable
practitioners to confirm that an adverse event is indeed the result of drug
therapy rather than one of many other potential causes; general guidelines
are readily available.
Because drug-induced blood disorders are potentially dangerous,
rechallenging a patient with a suspected agent in an attempt to confirm a
diagnosis is not recommended. In vitro studies with the offending agent and
cells or plasma from the patient’s blood can be performed to determine
causality. These methods are often expensive, however, and require
facilities and expertise that are not generally available. Laboratory
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confirmation of drug causation is not always necessary to warrant
interruption or discontinuation of therapy. Therefore, it is extremely
important that practitioners be able to clinically evaluate suspect drugs
quickly and to interrupt therapy when necessary.
Throughout the past decades, lists of drugs that have been associated with
adverse events have been developed to help clinicians identify possible
causes. Unfortunately, these lists are extremely extensive, including a large
number of very commonly used drugs, making it difficult to determine the
cause of any abnormality. Furthermore, the absence of a drug from such a
list should not discourage the investigation and reporting of an agent
associated with an adverse event. It is imperative that clinicians use a
rational approach to determine causality and identify the agents associated
with a reaction.
The clinician should focus on the issue, perform a rigorous investigation,
develop appropriate criteria, use objective criteria to grade the response,
and complete a quantitative summary. A complete, thorough, and detailed
drug and exposure history must be obtained from the patient in order to
best determine any potential for drug causation. A systematic approach to
evaluate the information available in the literature also helps the clinician
focus and to intervene to treat the cause of the disorder.
Adverse Drug Reaction Probability Scale
A common tool used by clinicians to rate the likelihood of causality in
adverse drug reaction (ADR) investigations is an ADR probability scale
(algorithm). One such scale was developed and tested by Naranjo and
colleagues. This tool provides a series of scored questions that lead an
investigator to the likelihood that an ADR was caused by the suspected
medication. Depending on the aggregate score, the causality is rated as
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doubtful, possible, probable, or definite. The scale gives the most weight to
the temporal relationship of the reaction with relation to administration of
the drug, observations after a rechallenge of the suspected medication, and
alternate explanations for the ADR. As mentioned earlier, it is often unethical
to rechallenge patients who experience severe hematologic toxicities. Thus,
without a rechallenge, it is difficult to achieve a definite causality rating with
such an algorithm.
In determining the likelihood that an observed reaction is caused by a
particular medication, clinicians should review the medical literature for past
reports supporting the observation. Using an evidence-based approach, the
investigator assigns greater weight to prospective study designs such as
clinical trials or cohort studies than to case reports or expert opinion. This
provides a framework for the investigator’s confidence in published literature
describing adverse drug reactions.
Summary
Drug-induced thrombocytopenia is the most common drug-induced
hematologic disorders. Although drug-induced hematologic disorders are less
common than other types of adverse reactions, they are associated with
significant morbidity and mortality. Aplastic anemia has been identified as a
leading cause of death followed by thrombocytopenia, agranulocytosis, and
hemolytic anemia. Similar to most other adverse drug reactions, druginduced hematologic disorders are more common in elderly adults than in
the young; the risk of death also appears to be greater with increasing age.
Although many are idiosyncratic effects, some drugs with a well-known risk
of blood dyscrasias are widely used due to a lack of alternative agents with
similar effects. In the case of these, following specific monitoring advice may
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help avert adverse effects, but specific advice to patients on how to spot
symptoms indicative of blood dyscrasias is important. In the case of new
drugs, any suspected case of blood dyscrasias should be reported to
regulatory bodies. It is imperative that medical professionals know and
understand the risks that medications pose in order to recognize the
symptoms of a medication-induced blood dyscrasia at early onset and take
the necessary steps to rectify it. Rechallenge with a drug suspected of
causing toxicity is usually not advisable.
For some drugs, such as heparin, quinidine, and vancomyin, in vitro testing
has been performed and mechanisms for cytopenias elucidated. However,
such testing is not always possible given that for most there are no
standardized, commercially available assays and that reactions may be
related to metabolites as opposed to more easily tested parent compounds.
Because of the seriousness of drug-induced hematologic disorders, it is
necessary to track the development of these disorders to predict their
occurrence and to estimate their incidence. Reporting during post-marketing
surveillance of a drug is the most common method of establishing the
incidence of adverse drug reactions. Drug-induced blood disorders are a rare
adverse effect, whose deleterious effects can be mitigated by the vigilance of
health professionals to promote patient education and prevention of an
adverse drug reaction through close monitoring of potential risk factors,
early recognition and intervention, and proper reporting.
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Please take time to help NurseCe4Less.com course planners evaluate
the nursing knowledge needs met by completing the self-assessment
of Knowledge Questions after reading the article, and providing
feedback in the online course evaluation.
Completing the study questions is optional and is NOT a course
requirement.
1. The complete blood count (CBC) is sometimes referred to as
a.
b.
c.
d.
the
the
the
the
cellular components test (CCT).
peripheral blood count (PBC).
EDTA test.
three-phase test.
2. Microscopic evaluation of a blood smear is best when the slide
is prepared
a.
b.
c.
d.
indefinitely if properly stored.
within 8 hours of collection.
within 3 hours of collection.
up to 24 hours after collection.
3. True or False: Freezing of blood samples is essential to
preserving the samples for a valid, complete blood count (CBC)
test.
a. True
b. False
4. The complete blood count (CBC) analyzes
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a.
b.
c.
d.
concentration of leukocytes (white blood cells).
volume of RBCs (red blood cells).
weight of RBCs (red blood cells).
All of the above
5. ___________________ provides the best morphologic
preservation of blood cells and prevents coagulation of the
blood specimen.
a.
b.
c.
d.
Cold agglutination
Dipotassium (K2) EDTA
IgM antibodies
Romanowsky stains
6. True or False: Cells that have ruptured are called smudge cells.
a. True
b. False
7. The complete blood count (CBC) is a primary screening test
that provides information regarding the cellular components of
the blood as the components
a.
b.
c.
d.
circulate in the peripheral blood.
circulate in the lymphatic system.
form in the bone marrow.
circulate through the liver.
8. Ethylenediaminetetraacetic acid (EDTA) allows a laboratory
professional to generate multiple blood smears because
a.
b.
c.
d.
it is an anticoagulant.
it protects against blood-borne pathogens.
reflex testing is part of the initial CBC.
most tests are manual.
9. A complete blood count (CBC) can also provide what are known
as the RBC indices. which are used
a. to depict the volume of each red blood cell (RBC).
b. to depict the total weight of each RBC.
c. to measure the concentration of hemoglobin in RBCs.
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d. All of the above
10. True or False: The laboratory professional first assesses the
general appearance and distribution of WBCs, RBCs, and
platelets using high power magnification.
a. True
b. False
11. Because the MCV represents an average of erythrocyte
volume, it is _____ reliable in describing the erythrocyte
population when considerable variation in erythrocyte
volume/size (anisocytosis) occurs.
a.
b.
c.
d.
not
equally
less
more
12. The reticulocyte count reported as a percentage can appear
increased because of either an increase in the number of
reticulocytes in the circulation or a decrease in the number of
total
a.
b.
c.
d.
leukocytes.
RBCs.
anticoagulants.
stem cells.
13. The laboratory professional utilizes both the platelet count
and the MPV to assess _______________ and pathologic
conditions related to platelets.
a.
b.
c.
d.
hemostasis
agglutination
thrombopoiesis
avitaminosis
14. Information such as red blood count agglutination appears as
a __________ blood smear.
a. pinkish, purple
b. red
c. clear
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d. grainy
15. __________________, having a bluish-colored smear, may
be suspected during the macroscopic evaluation of a blood
smear.
a.
b.
c.
d.
Hepatic damage
Thrombopoiesis
Cytopenia
Multiple myeloma
16. The effect of cold agglutinins is overcome by keeping the
blood
a. frozen.
b. at room temperature.
c. at 37°C
d. at 2°C
17. The presence of IgM antibodies (cold agglutinins) directed
against erythrocyte antigens, erythrocytes can agglutinate
forming
a.
b.
c.
d.
irregular grapelike, clusters of varying sizes.
rows that look like stacked coins.
clusters similar to rouleaux.
even cell distribution.
18. Rouleaux is an alignment of erythrocytes that occurs normally
when blood is collected
a.
b.
c.
d.
and
and
and
and
smeared onto a slide evenly.
allowed to stand in tubes.
then frozen immediately.
then suspended in saline.
19. True or False: On a well-made blood smear, the erythrocytes
are evenly distributed and well separated on the feathered
edge of the smear.
a. True
b. False
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20. An abnormality in erythrocyte arrangement that appears as
rows that look like stacked coins is termed
a.
b.
c.
d.
irregular clusters.
agglutination.
rouleaux.
satellitism.
21. The final task at low power magnification is to determine the
_________________ the smear that will be used to perform
the morphologic examination of cells.
a.
b.
c.
d.
platelet estimate in
critical area of
RBC (red blood cell) size, shape and color in
RBC inclusions in
22. Low power magnification is usually identified using _______
magnification.
a.
b.
c.
d.
400x
100x
40x
20x
23. The platelets must be evaluated also using low power
magnification, and in some cases, platelets can adhere to
neutrophils, a phenomenon called
a.
b.
c.
d.
an irregular cluster.
a morphologic abnormality.
rouleaux.
satellitism.
24. A WBC (white blood cell) differential is performed in which
__________ cells are observed to determine the relative
number of leukocytes as a percentage.
a.
b.
c.
d.
random
100
10 to 20
10
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25. The _____________ is characterized by the proximity of
RBCs to each other (the area of the smear in which very few
RBCs overlap or touch and are generally distributed in a
uniform manner).
a.
b.
c.
d.
peripheral blood smear
area of pallor
the “glass effect”
critical area
26. To evaluate morphologic abnormalities including inclusions,
the laboratory professional should review the slide with the
a.
b.
c.
d.
40× objective.
50× objective (500x magnification).
100× objective (1000× magnification).
400x magnification.
27. On a Romanowsky-stained blood smear, the erythrocyte has a
central area of pallor caused by the closeness between
___________________ the membrane when the cell is
flattened on a glass slide.
a.
b.
c.
d.
the
the
the
the
rim and the area of pallor in
two concave portions of
biconcave shape and cell center within
rim and discocyte in
28. To evaluate erythrocyte size microscopically, the cells are
compared with the _________ of a normal small lymphocyte.
a.
b.
c.
d.
nucleus
rim
area of pallor
size
29. True or False: Normocytic erythrocytes are about the same
size as the lymphocyte nucleus.
a. True
b. False
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30. _____________ also called spur cells, are small spherical
cells with irregular thorn like projections.
a.
b.
c.
d.
Drepanocytes
Codocytes
Acanthocytes
Echinocyte
31. Which of the following denotes a nonspecific variation in the
size of erythrocyte cells?
a.
b.
c.
d.
Poikilocytosis
The “glass effect”
Elliptocytosis
Anisocytosis
32. Poikilocytosis is the general term used to describe a
nonspecific variation in the _________ of erythrocytes.
a. biconcavity
b. color
c. size
d. shape
33. Acanthocytes do not have
a.
b.
c.
d.
irregular surface projections.
a central area of pallor.
irregular thorn-like projections.
All of the above
34. Drepanocytes are elongated, crescent-shaped erythrocytes
with pointed ends that are also known as
a.
b.
c.
d.
elliptocytes.
ovalocytes.
sickle cells.
cigar cells.
35. In the case of hypochromia, the mean cell hemoglobin
concentration (MCHC) value will be
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a.
b.
c.
d.
increased.
decreased.
unchanged.
static.
36. Cabot Rings are reddish-violet erythrocytic inclusions usually
occurring in the formation of a figure eight or oval ring that
occur in
a.
b.
c.
d.
severe anemias.
dyserythropoiesis.
a., and b., above
None of the above
37. True or False: The bone marrow is one of the body's largest
organs, representing up to 6 percent of total body weight in
adults.
a. True
b. False
38. An artery entering the bone branches out toward the
periphery to specialized vascular spaces called
a.
b.
c.
d.
colonies.
cords.
nodes.
sinuses.
39. Bone marrow studies should be performed only when clearly
indicated or whenever a beneficial diagnostic result for the
patient is expected because
a.
b.
c.
d.
bone marrow examinations are risky.
bone marrow studies are not reliable.
bone marrow studies are painful to the patient.
of the danger of infection.
40. A primary objective of a bone marrow examination is to
assess the quantity and development of
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a.
b.
c.
d.
nonhematologic disorders.
hematopoietic cells.
leukocytes.
erythrocytes
Correct Answers:
1. b
11. c
21. b
31. d
2. c
12. b
22. a
32. d
3. b
13. c
23. d
33. b
4. d
14. d
24. b
34. c
5. b
15. d
25. d
35. b
6. a
16. c
26. c
36. c
7. a
17. a
27. b
37. a
8. a
18. b
28. a
38. d
9. d
19. a
29. a
39. c
10. b
20. c
30. c
40. b
References Section
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helpful material for further reading. Unpublished works and personal
communications are not included in this section, although may appear within
the study text.
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The information presented in this course is intended solely for the use of healthcare
professionals taking this course, for credit, from NurseCe4Less.com. The information is
designed to assist healthcare professionals, including nurses, in addressing issues
associated with healthcare.
The information provided in this course is general in nature, and is not designed to address
any specific situation. This publication in no way absolves facilities of their responsibility for
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