Laboratory Medicine Curriculum

I Concepts in Laboratory Medicine .................................................. 1
II Diseases of Red Blood Cells .......................................................... 15
III White Blood Cells and Platelets ..................................................... 31
IV Transfusion Medicine ..................................................................... 38
V Bleeding and Thrombotic Disorders .............................................. 43
VI Cerebrospinal and Serous Body Fluids .......................................... 56
VII The Liver and Biliary Tract ............................................................ 72
VIII Pancreatic Disorders ....................................................................... 81
IX The Cardiovascular System ............................................................ 88
X Autoimmune Disorders .................................................................. 94
XI The Kidney ..................................................................................... 101
XII The Endocrine System .................................................................... 114

1
Advances in medical laboratory technology have driven major changes in the practice of laboratory medicine over
the past decades. The development of automated analyzers has sped up the processes of laboratory analysis, and
has also brought up newer tests and enhanced the accuracy, sensitivity and specificity of the tests already
available.
However, the heart of this process has seldom changed. The doctor and his understanding of his patient’s
condition remains key to a successful diagnosis. Doctors who wish to detect the ailment by demanding every
available test will stumble in the dark, cost the hospital and the patient a great deal of money, and may even end
with a wrong diagnosis.
Besides knowing how to interpret test results, the physician must also know:
 When to demand a test (indications)
 How to take the sample (and be able to teach the patient on this matter if necessary “e.g. urine sample”)
 The rational for ordering tests (which test to order first and why)
 The limitations of the test in hand,
 The conditions surrounding the findings. (e.g. a moderate yet rapid increase in serum potassium level is
riskier than a higher but slower increase)
ANALYTICAL AND STATISTICAL CONCEPTS IN DATA ANALYSIS
I-I The Reference Range
Comparison of a laboratory result versus a reference or “normal” range is often one of the most important aspects
of medical decision making.
To obtain a reference range, individuals without disease and on no medications donate samples for testing. A
distribution of these values, which should be numerous enough to be statistically reliable, is plotted. Statistical
methods are used to identify the central 95% of values. This range, representing
the middle 95% of results, is the reference range. As an indication that being
outside the reference range does not always reflect the disease, 5% of normal
healthy, non-medicated individuals who donated samples for the reference range
determination now fall outside of what has become the reference range for the
test. On the other hand, it is important to understand that individuals with values
inside the reference range may have subclinical disease, despite the presence of
an apparently normal value.
The reference range is dependent on the instrument and reagent used to perform the test. The reference ranges
are ideally established inside the laboratory where the test is being performed. Reference ranges supplied by
instrument and reagent manufacturers are not likely to correspond perfectly to ranges generated within an
individual laboratory. This is because the population used to establish the range by the manufacturer and/or the
instruments and reagents used by the manufacturer are likely to be different from those in an individual clinical
laboratory.
This means that if a normal,
healthy patient undergoes 20
unindicated laboratory tests
he may end up with 1 or more
“abnormal” results.

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Not all reference ranges follow the 95% rule. For some analysts, the reference range is defined as “less than” or
“greater than” a certain “threshold” value; for example, a prostate-specific antigen (PSA) level of 4 ng/mL is often
used to distinguish patients who require no further follow-up
(“normal”) from those who require a prostate biopsy
(“abnormal”). Also, some reference ranges have been defined by
professional organizations without adherence to the 95% rule. A
paradigm of this is the recommendation of American and
European cardiology associations that “an increased value for
cardiac troponin for the diagnosis of MI should be defined as the
measurement exceeding the 99th percentile of a reference
control group”. For other analysts (e.g. Cholesterol/lipids),
laboratories frequently provide therapeutic target ranges (called
Desirable Ranges) that represent recommendations based on
clinical trials and/or epidemiologic studies. For example, when apparently healthy, non-medicated individuals
provide samples for reference range determinations and the central 95% of values from this population provided
an inappropriately high reference range (e.g. cholesterol testing in a population with high-fat diet). In this
situation, desirable or prognosis-related ranges are established by groups of experts associating laboratory test
results with clinical outcome.
Example: Hemoglobin level of 13 may be abnormal for a person who lives 5000ft above sea level.
However, in Gaza, hemoglobin level of 11 is needed to consider the diagnosis of anemia at UNRWA
clinics.
Figure 1: Hemoglobin distribution at 5,000 ft
Medical decision limit
A cutoff value that is associated with specific diseases. For example, a fasting plasma glucose of 126 mg/dL is used to classify
diabetes.
Critical Values
Critical values, otherwise referred to as panic or alert values, are medical
decision level concentrations that would indicate a potentially or imminently
life-threatening situation. It is the concentration of analyte, or body fluid
sample being analyzed, at which some medical action is indicated for proper
patient care. There may be several medical decision levels for a given analyte.
‘‘Normal’’ and ‘‘Normal Ranges’’
How can we be sure that everyone in the
population that is surveyed to assess
these ‘‘normal’’ ranges is truly healthy?
How do we know that there is not an
overlap with truly diseased values?
These and other logical problems have
caused the word ‘‘normal’’ to be replaced
by ‘‘reference.’’
When a critical value is obtained, it
is necessary to quickly notify the
clinical team for immediate patient
evaluation and treatment.

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Example. Mild hypercalcemia is an indication to keep the patient adequately hydrated. However, sever hypercalcemia (>14
mg/dL) is an indication for more aggressive therapy that includes immediate volume expansion, administration of salmon
calcitonin and the concurrent administration of zoledronic acid.
I-II Measures of Test Performance
Sensitivity and Specificity
The 2 x 2 “Truth” table
The 2 × 2 table is the standard form for displaying test results in relation to disease status. Disease status
categories (diseased and well) are diagrammed in the vertical columns. Test results (positive, negative) are
diagrammed in the horizontal dimension.
Table 1: The 2X2 table
Cells in the 2 × 2 Table
• Positive (P) and Negative (N) refer to the actual results of the test in study.
• True (T) and False (F) refer to the agreement of test results with the “gold standard.”
• True Positives (TP): Diseased people who are correctly classified as positive.
• True Negatives (TN): Well people who are correctly classified as negative.
• False Positives (FP): Well people who are misclassified as positive.
• False Negatives (FN): Diseased people who are misclassified as negative.
Sensitivity: The proportion of people with disease who are correctly classified by the test as positive.
• Sensitivity = TP/All people with disease
𝒔𝒆𝒏𝒔𝒊𝒕𝒊𝒗𝒊𝒕𝒚 = (
𝒕𝒓𝒖𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆𝒔
𝒕𝒓𝒖𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆𝒔 + 𝒇𝒂𝒍𝒔𝒆 𝒏𝒆𝒈𝒂𝒕𝒊𝒗𝒆𝒔) 𝑿 𝟏𝟎𝟎
• Location on 2 × 2 table: left column
• Highly sensitive tests identify most, if not all, possible cases.
• Important to consider when there is a consequence associated with missing the detection of disease.
The population of individuals who have disease is the focus of sensitivity. The sensitivity of a laboratory test is its
capacity to identify all individuals with disease. In Figure 2, a threshold was chosen which maximizes sensitivity by
placing all those with disease above the line. This placement of the diagnostic threshold would decrease the
number of false-negatives (those with disease who fall below the line), because everybody with the disease would
have a positive test result. However, there is a significant misclassification of individuals without disease. As the
diagnostic threshold is lowered, an increasing number of patients without disease would be told they have a
positive test result, and by implication, the disease in question.

4
Figure 2: A clinical situation in which a diagnostic threshold is selected to maximize sensitivity.
Specificity: The proportion of well people who are correctly classified by the test as negative.
• Specificity = TN/All well people
𝑺𝒑𝒆𝒄𝒊𝒇𝒊𝒄𝒊𝒕𝒚 = (
𝒕𝒓𝒖𝒆 𝒏𝒆𝒈𝒂𝒕𝒊𝒗𝒆𝒔
𝒕𝒓𝒖𝒆 𝒏𝒆𝒈𝒂𝒕𝒊𝒗𝒆𝒔 + 𝒇𝒂𝒍𝒔𝒆 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆𝒔) 𝑿 𝟏𝟎𝟎
• Location on 2 × 2 table: right column
• Highly specific tests identify most, if not all, well people (i.e., not diseased), will give few FP results.
• Considered when FP results can harm the patient
The population of individuals without disease is the focus of specificity. Specificity is a statistical term that
indicates the effectiveness of a test to correctly identify those without disease. When used to describe a laboratory
test, it does not refer to its ability to diagnose a “specific” disease among a group of related disorders. One could
maximize specificity by raising the threshold shown in Figure 3 to place all those without disease below the line.
This would decrease the number of false-positives because everyone without disease would have a negative test
result. However, there would be a significant misclassification of the individuals with disease. As the diagnostic
threshold is raised, an increasing number of patients with disease would be told they have a negative test result
and, by implication, no disease.
Figure 3: A clinical situation in which a diagnostic threshold is selected to maximize specificity.
It is desirable to have a test that is both highly sensitive and highly specific. This is frequently not possible.
Typically, there is a trade-off. In many situations, clinical tests indicate people who are clearly normal, some
clearly abnormal, and some that fall into the gray area between the two.

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Effect of Altering the Test Cutoff
The trade-off illustrated further
When a test cutoff is altered, an inverse relationship between sensitivity and specificity is noted, and a trade-off
between the numbers of FP and FN results can be seen. Altering a cutoff changes a test’s sensitivity and specificity
because it relates to overlapping normal and abnormal patient distributions along the test value continuum (see
Fig. 4). For tests where high values indicate disease, lowering the cutoff (i.e., moving the cutoff line to the left) will
lead to more diseased patients being classified as abnormal. Thus, in Figure 4, changing the cutoff from C to B
increases sensitivity. If the cutoff is moved to A, then all diseased persons will have a positive test, and the
sensitivity will be 100%. However, increased sensitivity is associated with decreased specificity, and the number
of non-diseased persons with a positive test (FPs) increases as the cutoff is moved from C to B to A. If the cutoff is
raised (i.e., the cutoff line is moved to the right), more non-diseased patients are classified correctly, and
specificity increases. If the cutoff is moved to E, then all non-diseased persons will have a negative test, and the
specificity will be 100%. However, this will be accompanied by concomitant decreased sensitivity and additional
FN results.
Figure 4: Effects of varying the test cutoff on overlapping populations of
The Identification of the Appropriate Value for the Diagnostic Threshold
For diseases that are serious and treatable, and for which a second confirmatory laboratory test exists, it is
important to maximize sensitivity as in Figure 2. For example, for diagnosis of AIDS, it is better to have a few false-
positives that can be subsequently correctly identified with a confirmatory test than to fail to identify individuals
with HIV infection who might unknowingly infect others. However, for diseases that are serious and not curable,
a false-positive result is catastrophic for the patient. For such diseases, such as pancreatic cancer, it is better to
use the threshold shown in Figure 3 for diagnosis because if individuals with disease are missed, it will have no
effect on the treatment or outcome.
Predictive Value of a Test
Predictive Values: A measure of the test which represents the percentage of test results that match the diagnosis
of the patient. These values are predicted by the disease prevalence in the given population.
Positive Predictive Value (PPV): The proportion of people with a positive test result who are diseased. (i.e., that
a person with a positive test is a true positive)

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• Positive Predictive Value = TP/All people with a positive test result
• Positive Predictive Value = TP/(TP + FP)
• Location on 2 × 2 table: top row
• ↑ specificity = ↑ PPV
The population of individuals with a positive test result is the focus of positive predictive value. The positive
predictive value for a laboratory test indicates the likelihood that a positive test result identifies someone with
disease. It should be noted that the predictive value of a positive test is greatly influenced by the prevalence of
the disease in the area where testing is performed. As an example, a screening test for HIV infection is more likely
to be confirmed as positive in an area where many individuals are infected with HIV, as opposed to a location
where there is only a rare case of HIV infection. In the latter situation, most of the positive HIV tests in the initial
evaluation of a patient are found to be false-positives by confirmatory tests.
Negative Predictive Value (NPV): The proportion of people with a negative test result who are well. (i.e., that a
person with a negative test is a true negative)
• Negative Predictive Value = TN/All people with a negative test result
• Negative Predictive Value = TN/(TN + FN)
• Location on 2 × 2 table: bottom row
• ↑ sensitivity = ↑ NPV
The population of individuals with a negative test result is the focus of the negative predictive value. The negative
predictive value for a laboratory test indicates the likelihood that a negative test result identifies someone without
disease. It is not greatly influenced by the prevalence of disease because false-positives are not included in the
formula for negative predictive value.
 Note: sensitivity and specificity are not affected by prevalence in the population.

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The Difference Between Prevalence and Incidence
The prevalence of a disease reflects the number of existing cases in a population. It is usually expressed as a
percentage of a certain population. Incidence refers to the number of new cases occurring within a period of time,
usually 1 year. For example, sore throat has a low prevalence because considering the size of the population there
is a low percentage of individuals at a given time afflicted with sore throat. However, it has a high incidence
because many new cases of sore throat appear each year.
Precision versus Accuracy
Figure 5: Precision versus accuracy
Precision (Reliability) refers to the ability to test 1 sample and repeatedly obtain results that are close to each
other. This does not infer that the mean of these very similar numbers is the correct number (see Figure 5). Some
analyses have great precision but are very inaccurate. The accuracy (Validity) reflects the relationship between
the number obtained and the true result. Thus, a sample could have high accuracy but low precision if it provides
the correct answer but has substantial variability as the sample is repeatedly tested.
• Precision is a necessary, but insufficient, condition for accuracy.
I-III Types of Laboratory Tests
Lab tests are classified into three categories:
 Screening tests (high sensitivity)
 Confirmatory tests (high specificity)
 Diagnostic tests (both highly sensitive and specific)
The primary purpose of screening tests is to detect early disease or risk factors for
disease in large numbers of apparently healthy individuals. The purpose of a
diagnostic test is to establish the presence (or absence) of disease as a basis for
treatment decisions in symptomatic or screen positive individuals (confirmatory
test).

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Table 3: Screening Vs. Diagnostic Tests
Screening tests Diagnostic tests
Purpose To detect potential disease indicators To establish presence/absence of disease
Target
population
Large numbers of asymptomatic, but
potentially at risk individuals
Symptomatic individuals to establish
diagnosis, or asymptomatic individuals
with a positive screening test
Test method Simple, acceptable to patients and staff maybe invasive, expensive but justifiable as
necessary to establish diagnosis
Positive result
threshold
generally chosen towards high sensitivity
not to miss potential disease
Chosen towards high specificity (true
negatives). More weight given to accuracy
and precision than to patient acceptability
Positive result Essentially indicates suspicion of disease
(often used in combination with other risk
factors) that warrants confirmation
Result provides a definite diagnosis
Cost Cheap, benefits should justify the costs
since large numbers of people will need to
be screened to identify a small number of
potential cases
Higher costs associated with diagnostic test
maybe justified to establish diagnosis.
In the real world you never have a test that is 100% Sensitive and 100% Specific. We are usually faced with a
decision to use a test with high Sen (and lower spec) or high Spec (and lower Sen). Usually a test with high
sensitivity is used as the Initial Screening Test. Those that receive a positive result on the first test will be given a
second test with high specificity that is used as the Confirmatory Test. In these situations, you need both tests to
be positive to get a definitive diagnosis. Getting a single positive reading is not enough for a diagnosis as the
individual tests have either a high chance of FP or a high chance of FN. For example, HIV is diagnosed using 2
tests. First an ELISA screening test is used and then a confirmatory Western Blot is used if the first test is positive.
There are also specific situations where having a high specificity or sensitivity is really important. Consider that
you are trying to screen donations to a blood bank for blood borne pathogens. In this situation you want a super
high sensitivity, because the drawbacks of a false negative (spreading disease to a recipient) are way higher than
the drawbacks of a false positive (throwing away 1 blood donation). Now consider you are testing a patient for
the presence of a disease. This particular disease is treatable, but the treatment has very serious side effects. In
this case you want a test that has high specificity, because there are major drawbacks to a false positive.
Gold standard refers to the best possible test to which all other tests can be compared. When a new diagnostic
test is introduced, we compare it to the "gold standard".
• Gold standard tests have both high sensitivities and high specificities.
Why not use the gold standard at all times? The "gold standard" test may have the potential for complications
or is expensive or time consuming.

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Example: The diagnosis of hemochromatosis
Figure 6: Algorithm for the diagnosis of hemochromatosis
Screening Programs
What screening means. Screening refers to the application of a medical procedure or test to people who as yet
have no symptoms of a particular disease, in order to permit early detection of risk factors, asymptomatic
infection, or early stages of clinical disease, thus allowing early diagnosis and early intervention or treatment.
The screening procedure itself does not diagnose the illness. Those who have a positive result from the screening
test will need further evaluation with subsequent diagnostic tests or procedures.
Why we do screening. The goal of screening is to reduce morbidity or mortality from the disease by detecting
diseases in their earliest stages, when treatment is usually more successful.
• Examples of Screening Tests: Pap smear, mammogram, clinical breast exam, blood pressure
determination, cholesterol level, eye examination/vision test, and urinalysis.
Now that we have multiple tests in hand for a specific disease, we should ask:
 Should we screen for the disease?
 In whom to screen?
 How to do the screening (which test)?
 When should screening start and how often?
The perfect screening test would be:
 Always correct,
 Repeatable,
 Safe, painless, quick, inexpensive,
 Makes a clinical difference.
However, all of these cannot be found in one test, so we humbled down a little bit.

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Criteria for an effective screening program
1. Life-threatening diseases, such as breast cancer, and those known to have serious and irreversible
consequences if not treated early, such as congenital hypothyroidism, are appropriate for screening.
2. Treatment of diseases at their earlier stages should be more effective than treatment begun after the
development of symptoms. For example, cancer of the uterine cervix develops slowly, taking more than a
decade for the cancer cells to progress to a phase of invasiveness. During this pre-invasive stage, the cancer
is usually asymptomatic but can be detected by screening using the Pap smear. Treatment is more effective
during this stage than when the cancer has become invasive. On the other hand, lung cancer has a poor
prognosis regardless of the stage at which treatment is initiated. Early diagnosis and treatment appear to
prolong life little more than therapy after symptoms have developed. Screening to detect early stage lung
cancer using currently available techniques would not be beneficial.
3. The prevalence of the detectable preclinical phase of disease has to be high among the population screened.
This relates to the relative costs of the screening program in relation to the number of cases detected and to
positive predictive value. The expenditure of resources on screening must be justifiable in terms of eliminating
or decreasing adverse health consequences.
A screening program that finds diseases that occur less often could only benefit few individuals. Such a
program might prevent some deaths. While preventing even one death is important, given limited
resources, a more cost-effective program for diseases that are more common should be given a higher
priority, because it will help more people.
In some cases, though, screening for low prevalence diseases is also cost effective, if the cost of screening
is less than the cost of care if the disease is not detected early. For example, phenylketonuria (PKU) is a
rare disease but has very serious long-term consequences if left untreated. PKU occurs in only 1 out of
every approximately 15,000 births, and if left untreated can result in severe mental retardation that can
be prevented with dietary intervention. The availability of a simple, accurate and inexpensive test has led
many states, including New York State, to require PKU screening for all newborns.
4. A suitable screening test must be available. Suitability criteria includes adequate sensitivity and specificity,
low cost, ease of administration, safe, imposes minimal discomfort upon administration, and is acceptable to
both patients and practitioners.
5. There must also be appropriate follow-up of those individuals with positive screening results to ensure
thorough diagnostic testing occurs.
I- IV Errors in Laboratory Performance
There are 3 phases of laboratory analysis. The first of these is the pre-analytical phase. This time frame is from
patient preparation for the laboratory test, through the time of sample collection, until the sample arrives in the
laboratory. Most of the errors in laboratory test performance occur in this phase. Examples of pre-analytical
errors are: inappropriate preparation of the patient, such as not fasting for a particular test in which fasting is
required; ingesting drugs that will interfere with the laboratory tests; collection of the specimen in the wrong
tube; delayed transport of the specimen to the laboratory; storage of the sample at an incorrect temperature;
and collection of an inadequate amount of blood in vacuum tubes containing a fixed amount of anticoagulants.

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All these errors occur before the sample arrives for analysis and make it impossible, no matter how great the
analytical precision within the laboratory, to provide a test result that truly reflects the patient’s condition.
The second phase is the analytical phase, which is the time that the sample is being analyzed in the laboratory.
Errors can occur during this process, but they are much less common now because of the high level of automation
of many laboratory instruments. Examples of analytical errors are: incorrect use of the instrumentation and the
use of expired reagents. The third phase of laboratory test performance is the postanalytical phase, which begins
when the result is generated and ends when the result is reported to the physician. Example of errors in this phase,
which are more common than analytical errors, but less common than pre-analytical errors are: delay in time to
enter a completed result into the laboratory information system and reporting results for the wrong patient.
Review Questions
A. A new screening test is applied to a representative sample of 1,000 people in the population.
Based on the data presented in the following table, calculate the requested screening test
measures.
1. What is the sensitivity of the screening test?
2. What is the specificity of the screening test?
3. What is the positive predictive value of the screening test?
4. What is the accuracy of the screening test?
5. What is the number of false positive test results?
6. What is the prevalence of disease, assuming screening of a representative sample?
Explanations
1. Sensitivity = TP/All diseased people = 90/100.
2. Specificity = TN/All well people = 840/900.
3. PPV = TP/All test positives = 90/150.
4. Accuracy = (TP + TN)/All screened people = 930/1,000.
5. False positives = Well people who are misclassified by the test = 60.
6. Prevalence = All diseased people/All screened people = 100/1,000.
B. The Centers for Disease Control and Prevention “CDC” is concerned about optimizing the detection
of a disease that poses a serious public health threat. CDC health officials are considering lowering
the usual screening test cutoff point from X to Y.

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1. Moving cutoff in the manner being considered by the CDC causes the number of false positives to
A. increase
B. decrease
C. remain unchanged
D. cannot be determined
2. Moving the cutoff in the manner being considered by the CDC causes the positive predictive value to
A. increase
B. decrease
C. remain unchanged
3. Moving the cutoff in the manner being considered by the CDC causes the accuracy to
A. increase
B. decrease
C. remain unchanged
4. 4. Moving the cutoff in the manner being considered by the CDC causes the sensitivity to
A. increase
B. decrease
C. remain unchanged
5. Assuming that everyone who receives a positive test result is referred for medical follow-up, moving
the cutoff in the manner being considered by the CDC will cause the numbers of screened people
who are referred for follow-up to
A. increase
B. decrease

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C. remain unchanged
D. Cannot be determined
6. At Cutoff Point X, sensitivity is
A. 100%
B. 85%
C. 50%
D. 25%
E. 0%
Explanations
1. Answer: A. At Y, FP will increase as more well people are misclassified.
2. Answer: B. Although there will be more TP at Cutoff Y, there will be a large increase in numbers of FP.
The ratio, TP/(TP + FP), will decrease. A positive test result will be less predictive of actual disease.
3. Answer: B. X is the point of overlap and the point of maximal accuracy. Moving to Y will decrease
accuracy.
4. Answer: A. At Y, more diseased people will receive a (correct) positive test result. They will be TP. TP,
the numerator for sensitivity, will increase while the denominator (total people with disease) will be
unchanged.
5. Answer: A. Larger numbers of people would be screened positive at Cutoff Y and referred for follow-up.
6. Answer: B. Notice that Cutoff Point X separates the curve of diseased people into two areas; above the
cutoff point, approximately 85% of diseased people receive a (correct) positive test result. They are true
positives. Sensitivity = TP /All people with disease.

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Erythropoietin regulates red cell production. Normal erythropoiesis involves the maturation of pluripotent stem
cells into proerythroblasts- erythroblasts- reticulocytes. Immature RBCs, which have lost their nucleus but
retained their RNA, can be identified on a standard Wright's stained peripheral blood smear because the
cytoplasmic RNA stains a gray-purple color (polychromasia). These same cells, also called reticulocytes, can be
quantified by special stains or flow cytometry, yielding a reticulocyte count. The mature red blood cells contain
no RNA and survive for approximately 120 days. Throughout their life span, RBCs pass repeatedly through the
spleen, where old or damaged cells are ingested by macrophages. The hemoglobin
is catabolized into its heme (protoporphyrin ring+ iron) and globin components.
The porphyrin ring is metabolized into unconjugated (indirect, water insoluble)
bilirubin, which, when bound to albumin (now water soluble), is then transported
to the liver, where it is conjugated. Iron released from heme (or absorbed in the
intestine from the diet) is transported by transferrin, the blood plasma protein, to
the bone marrow and to other tissues where it is stored as ferritin and hemosiderin.
Anemia is a reduction below normal limits of the total circulating red cell mass. Hb concentration is the parameter
most widely used to diagnose anemia, based on 1967 World Health Organization (WHO) recommendations (Table
1). This definition is not universally accepted, and numerous alternatives have been proposed over the years,
usually suggesting slightly higher values and race-specific values. It is important to remember also that the normal
ranges for Hb and Hct are different for infants, children, adult men, adult women, pregnant women, and the
elderly. Further, attention to age- and gender-appropriate normal ranges is important in the evaluation of anemia.
Patients with anemia have normal SaO2 and PaO2, but they have reduced oxygen content due to the low level of
hemoglobin.
Signs of anemia include palpitations, dizziness, angina, pallor of skin and nails, weakness, claudication, fatigue,
and lethargy.
Figure 7: Erythropoiesis
Table1: WHO Definition of Anemia

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The Complete (Full) Blood Count “CBC”
The CBC is a deceptively simple test to order and interpret. CBC is a bargain; its cost can be much less than modern
imaging studies, but its value is lost without appropriate analysis and interpretation.
Parameters
Hemoglobin concentration (Hb): Units: g/dL or g/L.
Defines anemia (Hb <lower limit of normal adjusted for age and
gender).
Hematocrit: (packed cell volume) It is ratio of the volume of red cell
to the volume of whole blood.
High PCV: Polycythemia (any cause). Low PCV: Anemia (any cause).
Red cell count (RCC): Unit: × 106
/µL or × 1012
/L.
Most clinicians pay little attention to the red cell count but this
parameter is useful in the diagnosis of polycythemia disorders and
thalassemias (the latter results in the increased production of red
cells that are smaller than usual and contain low quantities of
haemoglobin, i.e. are microcytic and hypochromic).
Important causes of a low red cell count include:
 Hypoproliferative anemias, e.g. iron, vitamin B12 and folate
deficiencies.
 Aplasias e.g. idiopathic, drug-induced (e.g. chemotherapy),
or parvovirus infection.
Important causes of high red cell count:
 Polycythemia (Rubra) Vera.
 Thalassemia.
Mean cell volume (MCV): Normal range: 80 - 100 femtoliter (fL),
10–15
L
This index provides a useful starting point for the evaluation of anemia. (See figure 7)
Mean corpuscular hemoglobin (MCH): The average mass of hemoglobin per red
blood cell in a sample of blood. MCH value is diminished in hypochromic anemias.
Normal value is 27 to 31 picograms/cell.
Mean corpuscular hemoglobin concentration (MCHC): is the average
concentration of hemoglobin per unit volume of red blood cells. Of value in
evaluation of microcytic anemias. Normal range: 32-36 g/dL.
Note: many instances measure MCHC in percentage (%), as if it were a mass
fraction.
Red cell distribution width (RDW): Measures the range of red cell size in a sample
of blood, providing information about the degree of red cell anisocytosis, i.e. how
much variation there is between the size of the red cells.
Of value in some anemias: e.g. MCV with normal RDW suggests thalassaemia trait.
MCV with high RDW suggests iron deficiency.
MCV:
small = microcytic
normal = normocytic
large = macrocytic
MCH/MCHC:
decreased = hypochromic
normal = normochromic
elevated = hyperchromic
MCHC is elevated in
spherocytosis and
sickle cell anemia.

17
Rule of 3’s
The Three Basic Measures:
A. RBC count
B. Hemoglobin
C. Hematocrit
In other words: HcT (± 3%) should be 3 times the Hb (i.e. Hgb of 10 gm/dl should make Hct 30%). Hb should be 3
times the RBC count.
Check whether this holds good in given results. If not, it indicates micro, macrocytosis or hypochromia.
Assessment of iron status
Iron plays a pivotal role in many metabolic processes and the average adult contains between 3 and 5g of iron of
which two-thirds are present in the O2-carrying molecule, hemoglobin. Somewhat surprisingly, there is no specific
excretion mechanism in humans. Iron balance is controlled at the level of gut absorption.
Iron is absorbed from the gut by means of ferroportin, a transmembrane protein that transports iron through the
cell walls of enterocytes and macrophages and subsequently releases this iron to transferrin in the hepatoportal
circulation. Ferroportin itself is controlled by hepcidin, the key regulator hormone for iron hemostasis. Hepcidin
levels are decreased in low iron states and increased in iron overload states. Hepcidin binds ferroportin, causing
a decrease in the release of iron into the bloodstream. So, high levels of hepcidin cause decreased iron absorption,
while low levels allow for increased iron absorption. Hepcidin is also an acute phase protein that is increased in
response to inflammatory cytokines (especially interleukin-6).
Iron Studies
Transferrin is a blood plasma protein that binds iron and transports it to the tissues. Synthesis of transferrin is
inversely proportional to the body iron stores, with increased transferrin concentration when iron stores are
reduced.
Serum iron is a measurement of circulating iron bound to transferrin. Note: Free iron is toxic to the tissues.
Total iron-binding capacity (TIBC) indirectly measures transferrin by determining the total amount of iron the
blood can bind. Generally, it is not necessary to order both a transferrin level and TIBC. TIBC is less expensive than
a direct measurement of transferrin.
The ratio of serum iron to TIBC, measured as a percentage, is called transferrin saturation. For example, a value
of 15% means that 15% of iron-binding sites of transferrin are being occupied by iron. The reference range of the
transferrin saturation varies by age. In adults it is 20%-50%. Subnormal saturation is a useful index of iron
deficiency, but low values are also obtained in chronic disorders, and consequently lack specificity. Fasting
transferrin saturation of 45-50% may indicate hemochromatosis.
Soluble transferrin receptor (sTfR) concentration is elevated in iron deficiency and normal in anemia of chronic
disease (ACD). It is mainly used for differentiating ACD from iron deficiency anemia as its level is not affected by
inflammation.
Within the cell, iron is stored in protein complexes as ferritin or hemosiderin. In equilibrium conditions, serum
ferritin level is a good indicator of total iron stores. It is low in iron deficiency anemia, high-normal to high in
anemia of chronic disease, and high in hemochromatosis. Ferritin is also an acute-phase reactant and can be
elevated with inflammation, malignancy or chronic disease. A low C-reactive protein (CRP) helps rule out
inflammation.
A x 3 = B
B x 3 = C

18
Assessment of B12 & folate status
Measurement of the serum B12 and red cell folate levels is necessary in the
investigation of macrocytic anemia.
Deficiency of either vitamin leads to megaloblastic anemia, where there is
disruption of cell division in all actively dividing cells (includes the bone
marrow and gut).
In the marrow there is nuclear: cytoplasmic asynchrony, where the
deficiencies result in a decrease in DNA synthesis, which slows and inhibits
DNA replication (nuclear division). Nuclear maturation is slowed, whereas
cytoplasmic maturation (largely dependent on RNA function and unaffected
by failure of thymidilate synthesis) is relatively unimpeded. In contrast to the
nucleus, the cytoplasm of megaloblastic cells is abundant with normal hemoglobinization. This disparity between
nucleus and cytoplasm is known as nuclear-cytoplasmic asynchrony. Although most noticeable in erythroid cells
failure of DNA synthesis also affects myeloid and megakaryocytes.
The impaired RBC production and destruction of defective RBCs in the marrow before
release into the peripheral blood (ineffective erythropoiesis) results in the anemia
(often with pancytopenia). A bone marrow biopsy and aspirate reveal erythroid
hyperplasia. Hypersegmented (>5 lobes) polymorphonuclear neutrophils, and even
megakaryocytes, are common.
Note: in the early stages, deficiency of either vitamin may present without anemia or
macrocytosis (these are late features of the disease). However, in most cases of
deficiency the marrow will show characteristic megaloblastic change.
Note: deficiency of B12 may cause neurological problems in the absence of anemia.
Interpretation of results:
Vitamin B12: Normal ranges are based on 2 standard deviations either side of
the mean, so there will be ‘normal’ people who have ‘abnormal’ B12 (or folate)
levels.
Diagnose B12 deficiency at level< 200 pg/mL. If the level is borderline low (200-
400 pg/mL), check methylmalonic acid (MMA) and homocysteine (HC). Both are
elevated in B12 deficiency. Only the HC is elevated in folate deficiency-and the serum folate level is decreased.
Once B12 deficiency is diagnosed, the etiology should be pursued.
Investigation of haemolytic anaemia
The normal red cell has a lifespan of ~120 days. Anemia resulting from decreased RBC lifespan is termed hemolytic.
May be inherited or acquired, and the basic underlying mechanisms may involve abnormalities of the RBC
membrane, RBC enzymes or hemoglobin.
Extravascular vs. intravascular
Extravascular hemolysis implies RBC breakdown by the RES (e.g. liver, spleen, and macrophages at other sites)
while intravascular hemolysis describes RBC breakdown in the circulation itself. There are many investigations
available which will help determine the predominant site of destruction, which in turn will help define the
underlying cause of hemolysis, which is why we do the tests in the first place.
Serum folate levels are an
unreliable measurement of
body stores of folate, use the
red cell folate level instead.
Folate is necessary for
efficient thymidilate synthesis
and production of DNA.
B12 is needed to successfully
incorporate circulating folic
acid into developing RBCs;
retaining the folate in the RBC.
Figure 1: Hypersegmented
Neutrophil

19
Detection of hemolysis itself
The main question is whether the patient’s anemia is due to hemolysis or some other underlying mechanism
such as blood loss, marrow infiltration, etc.
General tests of hemolysis:
Is hemolysis actually occurring? Suggestive features are
 Evidence of red cell destruction.
 Evidence of red cell production (to compensate for red cell loss).
 Evidence of autoantibody in the patient’s serum.
Evidence of RBC destruction
 serum bilirubin. Heme loses the iron and is converted to bilirubin and cleared in the urine or stool.
With excessive hemolysis of either type, more of the bilirubin is unconjugated (indirect).
 urinary urobilinogen.
 serum LDH (reflecting increased RBC turnover). LDH levels elevated = intra and extravascular
hemolysis.
 Plasma haptoglobins or absent. Haptoglobin low = hemolysis. In both intravascular and extravascular
hemolysis, released hemoglobin is quickly bound to haptoglobin and then engulfed by macrophages. The
resultant low level of haptoglobin can be used to diagnose hemolysis-but does not help distinguish the
type.
 Spherocytes or other abnormal RBCS, e.g. schistocytes on blood film.
Evidence of increased RBC production
 Reticulocytes (on film, manual or automated count). Not absolutely specific, will in brisk acute
bleed, e.g. GIT.
 MCV (reticulocytes are larger than mature RBCs, and don’t forget folate deficiency which occurs
in hemolytic disorders).
Is it mainly intravascular?
 Plasma free Hb.
 Methemalbuminemia. Proteolytic breakdown of hemoglobin to form both heme and met-heme.
Metheme combines with blood plasma albumin to form methemalbumin.
 Haemoglobinuria, which indicates severe intravascular hemolysis overwhelming the absorptive capacity
of the renal tubular cells.
 Haemosiderinuria. Urine hemosiderin high = intravascular hemolysis. Iron is more frequently lost in the
urine with intravascular hemolysis and can be detected by the urine hemosiderin test.

20
Detection of the cause
What is the cause? Directed by clinical findings, the main causes are either:
Intrinsic:
 Molecular defect inside the cell (G6PD deficiency, hemoglobinopathies)
 An abnormality in membrane structure or function (hereditary spherocytosis)
Tests:
 RBC morphology “blood smear” (e.g. spherocytes in hereditary spherocytosis).
 Hb analysis. (e.g. electrophoresis in SC anemia)
 RBC enzyme assays (e.g. in G6PD deficiency).
Extrinsic: an environmental factor outside the cell (DIC, autoantibodies, TTP/HUS, HELLP).
Tests:
 Immune—check for autoantibodies (Coombs test).
Coombs test positive = antibody- or complement-
mediated hemolysis. The direct antiglobulin test (DAT),
or direct Coombs test, can help identify antibody or
complement on the red cell surface, which may
mediate hemolysis.
 Non-immune: check RBC morphology (e.g.
schistocytes, TTP/HUS).
 Is there some other underlying disease? Consider PNH
(rare).
Figure 2: Red cell breakdown and its products

21
Figure 3: Direct and Indirect Coombs Tests

22
Hemoglobin Electrophoresis
Electrophoresis is the separation of proteins through the application of voltage.
Most proteins have a net charge, usually a net negative charge, and when placed
into a semisolid medium (a gel) will move in response to a voltage. The distance
that a protein moves depends on its size and the magnitude of its charge, so that
different proteins can be separated from one another. The positively charged
electrode attracts negatively charged proteins and is called the anode. Proteins
that end up closest to the anode are called fast-migrating or anodal. Proteins
that end up farthest from the anode are considered slow-migrating or cathodal.
If RBC are lysed, the predominant protein within the lysate is Hb. In the normal
adult, this Hb is largely HbA, with about 2% to 3% HbA2. When this lysate is
applied to a gel across which a voltage is applied, the result is a prominent band
(HbA) near the anode (fast-migrating) and a dim, slower band (HbA2) near the
cathode. Any deviation from this pattern is indicative of a hemoglobinopathy or
thalassemia.
Thalassemia, being a quantitative defect in production of entirely normal Hbs, does not produce abnormal bands
on the electrophoresis. Instead, β-thalassemia is diagnosed by the presence of “thalassemic indices” (low Hct,
increased RBC count, and low MCV) and a quantitatively increased HbA2. α-Thalassemia has “thalassemic indices”
and normal HbA2. True hemoglobinopathies are due to production of a structurally abnormal Hb molecule that
usually produces a distinct band on electrophoresis. The identity of most abnormal Hbs can often be determined
by routine electrophoresis, particularly when supplemented with some clinical information and CBC data.
Figure 4: 1, Normal adult; 2 and 3,
17-year-old with sickle cell anemia;
5 and 6, patient with sickle cell
anemia, recently transfused; 4 and
7, Hbs A/F/S/C standard
Figure 5: Patterns of hemoglobin electrophoresis.

23
Figure 6: Classification of anemia
Iron Deficiency Anemia
Iron deficiency is the most common cause of anemia. Worldwide, the most common cause of iron deficiency is a
dietary lack of iron.
Iron from the diet is absorbed principally in the duodenum. It is carried by transferrin to the marrow, where it is
internalized into erythroblasts and incorporated into protoporphyrin to yield heme. Iron not utilized in this way is
stored bound to ferritin. When there is inadequate iron intake or excessive iron loss (Table 2), the ferritin iron
stores of the reticuloendothelial system become progressively depleted. Red cells are produced that contain an
inadequate concentration of Hb, giving rise to the appearance of small, hypochromic red cells.
Table 2: Causes of Iron Deficiency

24
Note: The finding of iron deficiency produces an obligation to identify and treat the underlying cause.
Iron deficiency is not an ‘all-or-nothing’ phenomenon. In progressive deficiency there is a gradual loss of iron with
subtle alterations of iron-related parameters during which the red cells may look entirely normal. In the initial
stages of developing iron deficiency macrophages become depleted of iron and the serum ferritin to the lower
end of the normal range; during this ‘latency’ period the Hb is normal. As the deficiency progresses plasma iron
levels and TIBC . RBC protoporphyrin accumulates, and eventually hypochromic RBCs appear in the peripheral
blood. At this stage a full blood count will usually show Hb, MCV, MCH and MCHC, and the peripheral blood
film will show microcytic hypochromic red cells.
Diagnosis:
In many cases, the CBC and peripheral blood findings are highly characteristic: low RBC count, low MCV, low mean
corpuscular hemoglobin concentration (MCHC), and high red cell distribution width (RDW). The platelet count is
often elevated (reactive thrombocytosis). The peripheral blood shows hypochromic, microcytic red cells with
scattered elliptocytes. This is in contrast to the most commonly entertained other diagnostic consideration,
thalassemia, in which the RBC count is high, the RDW tends to be lower, elliptocytes are not seen, and target cells
and basophilic stippling are more frequent.
To confirm the diagnosis of iron deficiency, the best single test is the serum ferritin. A ferritin above 15 μg/L
essentially excludes iron deficiency, and the serum ferritin in iron deficiency is often below 10 μg/L. Lowered
ferritin is the earliest finding in iron deficiency and persists throughout the course of the illness. The diagnostic
difficulty with the use of ferritin is that it is an acute-phase reactant, an analyte that increases in response to
inflammation. It may also be spuriously elevated in hepatic insufficiency, due to impaired clearance. Thus, other
assays may occasionally be needed to make a diagnosis of iron deficiency anemia.
In established iron deficiency, the serum iron is typically low, the total iron binding capacity (TIBC) is elevated, and
the percent transferrin saturation is low. These findings are somewhat in contrast to those seen in ACD (see
below). Serum soluble transferrin receptor is elevated whenever there are
cells depleted of iron; thus, it is elevated in iron deficiency anemia and in
erythroid hyperplasia (hemolytic anemia, polycythemia).
As a last resort, marrow iron stores can be examined directly under the
microscope if an adequate bone marrow aspirate is obtained.
Anemia of Chronic Disease
Sustained systemic inflammation alters iron utilization in the marrow, suppresses hematopoiesis, and blunts the
response of EPO to anemia. This combination of factors leads to a mild, refractory, hyporegenerative anemia that
is usually normocytic and normochromic, but is microcytic in up to 1/3 of cases.
Table 3: Stages of Iron Deficiency
Microscopic examination of
bone marrow aspirate is the
“gold standard” for assessing
marrow iron store.

25
Although iron deficiency is the most common cause of anemia worldwide, ACD is the most common cause of
anemia in both hospitalized and ambulatory hospital patients in the United States. The vast majority of cases are
due to rheumatoid arthritis, collagen vascular disease, such as lupus, chronic infection, and malignancy.
Diagnosis:
The diagnosis of ACD is made difficult by the presence of numerous comorbid factors, in patients who, by
definition, are ill. In such patients, ACD may be coincident with iron deficiency, folate deficiency, renal
insufficiency, and/or frequent phlebotomy. Furthermore, in up to 30% of those with iron indices characteristic of
ACD, no chronic illness can be identified.
The laboratory diagnosis of ACD depends on demonstrating a hypoproliferative (low reticulocyte count)
normocytic or microcytic anemia in the presence of characteristic iron studies. The iron studies should document
increased iron stores (normal to high serum ferritin or increased stainable iron in a bone marrow biopsy) and a
low serum iron, low transferrin, and low TIBC.
A normal or elevated ferritin level is crucial for distinguishing
ACD from iron deficiency. However, interpretation of the
results for ferritin can be problematic because ferritin is an
acute phase reactant. Thus, while a low ferritin is essentially
diagnostic of iron deficiency, a normal ferritin does not
entirely exclude it. In confusing situations, use the soluble
serum transferrin receptor. This analyte is increased in iron
deficiency anemia and normal in ACD.
Thalassemia
Mutations in the genes that encode globin chains may result
in 2 broad categories of disease:
 Some mutations lead to the production of a structurally abnormal globin chain, resulting in a
hemoglobinopathy such as hemoglobin S (sickle cell disease and sickle cell trait).
 Other mutations lead to reduced production of a structurally normal globin chain, resulting in thalassemia.
A Hb molecule is composed of 4 polypeptide chains. The major adult Hb, hemoglobin A (HbA), is composed of 2
alpha chains and 2 beta chains. The minor adult hemoglobin (HbA2) is composed of 2 alpha chains and 2 delta
chains. The major fetal hemoglobin (HbF) is composed of 2 alpha chains and 2 gamma chains. The one constant
feature of all Hbs is the alpha chain. The alpha chain genes are located on chromosome 16. Each chromosome 16
contains 2 separate alpha chain genes, for a total of 4 genes per normal cell, each transcriptionally active. Thus,
to render an individual completely deficient of alpha chains, inheritance of 4 mutated genes is required.
The beta, gamma, and delta chain genes are located on chromosome 11. Each chromosome 11 contains 1 beta, 1
gamma, and 1 delta gene. Should a mutation occur in the beta chain, there can be a degree of compensation by
increasing the production of gamma, delta, or both. There is no such substitute for the alpha chain.
With decreased alpha chain production, α-thalassemia arises. Harm comes to the red cell, however, not from a
deficiency of alpha chain, but from an excess of non-alpha chains (e.g., beta). The excess chains form precipitates
in the cell, leading to ineffective erythropoiesis, microcytosis, and enhanced splenic red cell destruction. Likewise,
decreased beta chain production (β-thalassemia) leads to precipitation of excess alpha chains and subsequent red
cell destruction. Disease severity reflects the genotype.
Diagnosis
Since alpha chains are present in utero, α-thalassemia can be diagnosed at birth. The diagnosis of β-thalassemia
is somewhat delayed, since beta chains are not produced to adult levels until 3 to 6 months of age. The CBC is
Table 4: Fe Deficiency Vs. Anemia of Chronic Disease

26
notable for microcytosis, usually in the presence of a normal or high RBC count and normal RDW. The peripheral
smear often displays target cells and may display basophilic stippling. When there are microcytosis, “thalassemic”
indices, and normal iron studies, the diagnosis of thalassemia is essentially assured.
In the case of β-thalassemia, an Hb electrophoresis displays increased HbA2 and sometimes HbF. In α-thalassemia
(recall that the alpha chain is needed for all Hb types), the proportion of Hbs appears normal. These findings are
usually sufficient for the diagnosis. If further definition is required, molecular genetic testing is available.
Folate Deficiency
Diagnosis: The blood smear shows the classic features of megaloblastic anemia: marked oval macrocytosis,
hypersegmented neutrophils, and large platelets. The diagnosis can be confirmed by measuring the serum or RBC
folate. However, there are several confounding factors in the use of these tests. Several balanced meals can
quickly normalize the serum folate, but the RBC folate reflects folate status better over time.
Vitamin B12 Deficiency
Diagnosis: Patients present with a macrocytic anemia, pancytopenia, and slight indirect hyperbilirubinemia (from
the continuous low-level intramedullary hemolysis). The blood smear shows the classic features of megaloblastic
anemia: marked oval macrocytosis, hypersegmented neutrophils, and large platelets. The diagnosis can be
confirmed by measuring serum B12 levels.
Like iron-deficiency anemia, once B12 deficiency is diagnosed, the etiology should be pursued. For pernicious
anemia (PA), the presence of anti-intrinsic factor (IF) antibodies supports the diagnosis.
Sickle Cell Anemia
A hemoglobinopathy is a structural defect in Hb, usually resulting from a germline single-nucleotide point
mutation in 1 of the Hb genes. Worldwide, hemoglobin S remains most common hemoglobinopathy.
Homozygous sickle cell anemia (genotype SS, sickle cell disease) is associated with abnormal polymerization of
Hb in red cells, leading to a cell with an altered shape that is rapidly cleared from the circulation. Polymerization
Table 5: Causes of Folate Deficiency
Table 6: Causes of Vitamin B12 Deficiency

27
of hemoglobin S is enhanced in hypoxic conditions. The red cells in SS have an average lifespan less than 30 days.
The clinical course in hemoglobin SS patients is one of chronic hemolysis punctuated by a wide range of
complicating events (crises).
Chronic hemolysis leads to a chronic anemia with growth retardation, delayed puberty, impaired exercise
tolerance, jaundice, and cholelithiasis (due to the formation of pigmented gallstones). The patients are usually in
need of intermittent transfusions.
Episodic complications include vaso-occlusive events (e.g., stroke, avascular necrosis of bone, and splenic
autoinfarction), splenic sequestration crises, aplastic crises (due most often to marrow infection with parvovirus
B19), bacterial sepsis, and hyperhemolytic crises. The risk of bacterial infection is related to an underlying
functional asplenia that affects most sickle cell patients by late childhood. This confers a particular susceptibility
to infection by encapsulated bacterial organisms such as Haemophilus influenzae and Streptococcus pneumoniae.
The most common cause of death in sickle cell disease is infection, followed by stroke and other thromboembolic
events.
Hb electrophoresis shows that the red cells contain mostly
hemoglobin S, with small quantities of hemoglobin F and
hemoglobin A2.
Heterozygotes (genotype SA, sickle cell trait) are essentially
asymptomatic and have normal red cell indices. The presence
of sickle Hb can be detected by Hb electrophoresis, where it is
found to represent about 35% to 45% of total Hb.
Diagnosis: The identification of variant Hbs is usually
performed with Hb electrophoresis. There are a number of
screening tests for sickle Hb. These are based on the tendency
of hemoglobin S to polymerize. A positive sickle screen is not
specific for sickle cell disease. Furthermore, a negative screening test does not entirely exclude hemoglobin S,
particularly in infants who may still have significant quantities of hemoglobin F.
Hereditary Spherocytosis
Cardinal features of HS are chronic hemolysis, jaundice, and splenomegaly. It is a fairly common condition, usually
transmitted as an autosomal dominant trait (25% autosomal recessive).
Diagnosis:
The peripheral blood film shows numerous spherocytes. These appear as red cells that lack central pallor. Larger
polychromatophilic cells are often numerous, reflecting an increased reticulocyte count. While spherocytes are
typically smaller than normal red cells, the MCV may be low, normal, or high, owing to reticulocytosis. The MCHC
is characteristically increased.
When numerous spherocytes are observed on a peripheral blood film, the 2 primary considerations are immune
hemolysis and HS. Immune hemolysis can usually be excluded with a negative direct antiglobulin test (DAT,
Coombs test).
The osmotic fragility test can be useful in supporting the diagnosis of HS (The reduced surface: volume
ratio makes spherocytes more susceptible to osmotic stress). However, spherocytes from any cause will result in
a positive test.
Glucose-6-phosphate Dehydrogenase (G6PD) Deficiency
This is the most common red cell enzyme defect. Over 400 variants of the X-linked G6PD gene exist, affecting over
200 million people. Since red cells lack a nucleus, they lack the capacity to make new enzymes. Even normal red
Figure 7: Electrophoresis Patterns in Sickle Cell Anemia

28
cells have greater enzymatic capacity when young than when old. However, if the activity of a critical enzyme
significantly degrades before the average red cell lifespan (120 days), then the cell dies prematurely. Red cells rely
on G6PD to produce glutathione that absorbs oxidant stress to protect Hb from oxidation. Oxidized Hb forms
precipitate within the red cell, known as Heinz bodies, whose excision by splenic macrophages results in bite cells.
There are numerous defective forms (alleles) of G6PD.
Most abnormal alleles result in a functionally normal enzyme but have a shortened lifespan within the red cell.
Uncommon alleles result in decreased G6PD production, and even young cells have low activity in these cases. In
most forms of the disease, young red cells, especially reticulocytes, have normal G6PD activity, whereas, in other
forms, enzyme activity is universally decreased. As such, most G6PD-deficient persons are clinically well until
exposed to excess oxidant. Such exposures arise in the form of ingestion (e.g., fava beans), medication use (e.g.,
nitrofurantoin, antimalarials, and sulfa drugs), or infection. In most individuals, there is preferential destruction
of older red cells.
Diagnosis
The peripheral smear shows a combination of bite cells and Heinz bodies. The latter require special staining in
order to be visualized. Laboratory assays are available for measuring G6PD activity. G6PD activity may appear
normal (false-negative) during an acute episode, because only nonhemolyzed, younger cells are available to be
assayed. Measure G6PD levels 2-3 months after the hemolytic event to avoid a false-negative result.
Autoimmune Hemolytic Anemia
When an antibody attaches to a red cell, the consequences depend largely on the nature of the antibody. Some
antibodies are capable of activating complement and producing brisk intravascular hemolysis. Others behave as
opsonins, promoting red cell destruction in the spleen. Some antibodies react only in a cold environment, some
only in warmth. Some coat the red cell and do nothing more. Most cases of immune hemolytic anemia are due to
antibodies of either the IgG or IgM type.
These disorders present with the typical manifestations
of anemia, with variable rates of onset. Mild
splenomegaly is common when hemolysis is
extravascular. Dark urine, abdominal or back pain, and
fever may accompany intravascular hemolysis. In severe
IgM-induced cold autoimmune hemolytic anemia, the
skin may have a livedo reticularis pattern, and there may
be acrocyanosis on exposure to cold.
Warm autoimmune hemolytic anemia is mediated by IgG
autoantibodies that optimally bind RBC at body
temperature (37°C). The red cell antigens most
commonly the target are the Rh antigens. IgG molecules
must form cross-links to activate complement, and the target red cell antigens in warm autoimmune hemolytic
anemia are usually insufficiently dense on the red cell surface to permit this. Thus, IgG antibodies opsonize the
red cell, leading to membrane damage mediated by splenic macrophages with the formation of small, spherocytic
cells (microspherocytes). In some cases, there is concomitant immune thrombocytopenia, and this association is
known as Evans syndrome.
Cold autoimmune hemolytic anemia is mediated by IgM antibodies that bind RBC at lower temperature ranges.
The target antigens are usually the red cell antigens i. Those binding over a limited thermal amplitude (e.g., 0 to
22°C) will obviously not produce clinical consequences. However, these antibodies may cause difficulty in the
laboratory, where studies are routinely carried out at room temperature, which could be within this thermal
Figure 8: Livedo Reticularis

29
amplitude. Antibodies with broader thermal amplitude may bind to red cells in the extremities, where
temperature falls a bit below core body temperature, resulting in acrocyanosis.
Both Warm and cold autoimmune hemolytic anemias are often idiopathic conditions.
However, a significant number are secondary to another underlying condition,
including lymphoid neoplasms (e.g., chronic lymphocytic leukemia), medication use,
systemic autoimmune diseases (e.g., systemic lupus erythematosus),
immunodeficiency, and infection (infectious mononucleosis, HIV, and Mycoplasma
pneumoniae).
The DAT, also known as the direct Coombs test, is pivotal for the diagnosis of immune hemolysis. This test is
capable of demonstrating the presence of antibodies or complement on the surface of RBC.
Additional laboratory findings include anemia, reticulocytosis, indirect hyperbilirubinemia, decreased
haptoglobin, and an increased LDH. The peripheral blood smear often demonstrates spherocytes, polychromasia,
and, in severe cases, nucleated red cells. In cold agglutinin disease, red cell clumping is seen.
Review Questions
1. Which of the following is NOT a cause of
microcytic-hypochromic anemia?
A. Disorders of iron metabolism
B. Disorders of porphyrin synthesis
C. Disorders of globin synthesis
D. Disorders of vitamin B12 absorption
2. Which form of anemia is caused by a deviation in
mitochondrial metabolism?
A. Iron deficiency
B. Microcytic-hypochromic
C. Sideroblastic
D. Megaloblastic
3. Which of the following is TRUE regarding post-
hemorrhagic anemia?
A. A healthy person can tolerate loss of 2000 ml of
blood without showing signs or symptoms.
B. results in normocytic and normochromic anemia.
C. Hypertension may be present.
D. There is increased venous return.
4. Which of the following is TRUE regarding fetal and
adult hemoglobin?
A. Fetal hemoglobin is composed of two α- and two
γ-chains.
B. Adult hemoglobin is composed of two α- and one
γ-chain.
C. Adult hemoglobin is HbF and HbA.
D. Fetal hemoglobin is not found in blood after age 6
months.
5. Which of the following terms describes a person
with Hb(s) and another normal Hgb allele?
A. Sickle cell anemia
B. Sickle cell thalassemia
C. Sickle cell trait
D. Hereditary spherocytosis
6. Which of the following is TRUE regarding G6PD
deficiency?
A. Most commonly found in females
B. Most common manifestation is polycythemia
C. Patients are dependent on blood transfusion
D. Associated with fava bean ingestion
7. Which of the following is a TRUE statement
regarding sickle cell disease?
A. Sickle cell trait is the homozygous form of the
disease.
B. Sickle cell disease means a person has HbA and
HbS genes.
8. Which of the following is a common trigger for
sickling?
A. Hypoxemia
B. Decreased hydrogen ions
C. Decreased plasma osmolality
D. High temperature
Red cell autoantibodies
tend to interfere with
pre-transfusion testing.

30
C. It is most common in European descent.
D. It is an autosomal recessive disease
9. A 16-year-old boy has had a low energy level for
as long as he can remember. On physical
examination he is pale and has a palpable spleen
tip. A CBC shows Hgb of 8.8 g/dL, Hct 24.1%, MCV 65
fL, RDW 12, platelet count 187,000/microliter, and
WBC count 7400/microliter. His serum ferritin is
normal. Which of the following is the most likely
diagnosis?
A. G6PD deficiency
B. Beta-thalassemia
C. Sickle cell anemia
E. Malaria
10. A clinical study is conducted involving adults from
18 to 80 years of age who underwent splenectomy
for blunt force abdominal trauma. An age-matched
control group of patients consists of patients who
have congestive splenomegaly. The laboratory
findings from these subjects are analyzed. Which of
the following laboratory test findings is most likely to
be observed only in the study group following
splenectomy?
A. Thrombocytopenia
B. RBC Howell-Jolly bodies
C. Decreased RBC distribution width
D. Leukopenia
E. Nucleated RBCs
11. A 25-year-old African-American man is given
anti-malarial prophylaxis for a trip to West Africa.
Over the next week he develops increasing fatigue.
On physical examination there are no abnormal
findings. Laboratory studies show a hematocrit of
30%. Examination of his peripheral blood smear
shows red blood cells with numerous Heinz bodies.
There is a family history of this disorder, with males,
but not females, affected. Which of the following is
the most likely diagnosis?
A. Beta-thalassemia
B. Sickle cell anemia
C. Alpha-thalassemia
E. G6PD deficiency
12. A clinical study is performed with subjects who
are adults found to have anemia. Their clinical
histories and laboratory findings are reviewed. It is
observed that ingestion of a drug preceded
development of the anemia in some of the subjects,
but not in others. Which of the following conditions
is most likely to be found in persons without a
history of drug ingestion?
A. G6PD deficiency
B. Autoimmune hemolytic anemia
C. Macrocytic anemia
D. Aplastic anemia
E. Microcytic anemia
13. Transient cessation in red blood cell production
resulting in acute anemia is termed?
A. Vaso-occlusive crisis
B. Aplastic crisis
C. Sequestration crisis
D. Hyperhemolytic crisis
14. What is diagnostic value of reticulocyte count in
the evaluation of anemia?
A. Determines response and potential of the bone
marrow
B. Determines compensation mechanisms for anemia
C. Determines the corrected RBC count after the
calculation
D. Determines the potential sampling error for RBC
count

31
The automated differential white cell count is provided as part of the CBC. A typical CBC will show the total white
cell count and the 5-part differential white cell count, broken down into the 5 main white cell subtypes in
peripheral blood which include:
 Neutrophils.
 Lymphocytes.
 Monocytes.
 Eosinophils.
 Basophils.
The printed CBC will show either the % of each type of white cell or the absolute count. Unless the absolute WBC
(as × 109
/L or 103
/µl) is known or calculated, the % count is of little value.
 As a general rule ignore the % count—you cannot detect abnormalities such as neutropenia unless you have
the absolute values.
A decrease in one or more of the components of the white cell count. The following patterns can occur:
1. Leukopenia. Here all the white cell types are reduced.
2. Neutropenia. This is by far the commonest. The term Granulocytopenia means a decrease in all the
granulocytes, sometimes including the monocytes, but with normal lymphocyte counts. However, many
use the term granulocytopenia interchangeably with neutropenia.
3. Lymphocytopenia.
Leukopenia
A reduction in all the white cell series. It is relatively uncommon,
and it occurs almost always as part of severe bone marrow
destruction or suppression, in which case the red cells and the
platelets are also usually affected (pancytopenia).
Neutropenia
Normal values for the total WBC and absolute neutrophil count
(ANC) change from childhood into adolescence. Values of the ANC
from 1 year of age slowly increase throughout childhood until the
adult value is achieved during adolescence.
Figure 8: Causes of pancytopenia

32
Neutropenia is defined as the reduction in the absolute number of
neutrophils in the blood circulation. The absolute neutrophil count
(ANC) is either derived by multiplying the total leukocyte count by
the percentage of band neutrophils and segmented neutrophils or
obtained directly from an electronic cell counter. The neutropenic
patient is increasingly vulnerable to infection by gram-positive and
gram-negative bacteria and by fungi. The risk of infection is related
to the severity of neutropenia.
Neutropenia is classified as mild, moderate or severe according
to number of cells/µl:
 Mild neutropenia (1000 ≤ ANC < 1500): minimal risk of infection
 Moderate neutropenia (500 ≤ ANC < 1000): moderate risk of infection
 Severe neutropenia (ANC < 500): severe risk of infection.
ANC < 100 is termed profound neutropenia.
Neutropenia may also be classified as congenital or acquired.
Congenital neutropenia:
Patients with “chronic benign neutropenia” are free of infection despite very
low stable neutrophil levels; they seem to respond adequately to infections
and inflammatory stimuli with an appropriate neutrophil release from the
bone marrow.
In contrast, the neutrophil count of patients with cyclic neutropenia
periodically oscillate (usually in 21-day cycles) between normal and low, with
infections occurring during the nadirs.
Both cyclic neutropenia and congenital neutropenia represent problems in
mutations in the neutrophil elastase genes ELA-2 or ELANE.
Acquired neutropenias: are more common than congenital neutropenias.
They can be seen after infections or after exposures to certain drugs, in the setting of autoimmunity, nutritional
deficiency, or hypersplenism, or as a consequence of a hematologic malignancy. In addition, a significant number
of patients with neutropenia have chronic idiopathic neutropenia.
The new onset of an isolated neutropenia is most often due to an
idiosyncratic reaction to a drug, and agranulocytosis (complete
absence of neutrophils in the peripheral blood) is almost always
due to a drug reaction. In these cases, examination of the bone
marrow shows an almost complete absence of granulocyte
precursors with other cell lines undisturbed. This marrow finding is
also seen in pure white blood cell aplasia, an autoimmune attack
on marrow granulocyte precursors.
The definition of neutropenia in infants is
different from that in adults. In infants
aged 2 weeks to 1 year, the lower limit of
the normal neutrophil count is 1000/µL.
After the first year of life, the lower limit
is 1500/µL, as in adults.
The ANC in benign chronic
neutropenia ranges usually
between 500/µL and 1500/µL,
and the clinical course is
asymptomatic.
Neutropenia is a relatively
frequent finding, whereas
congenital neutropenias are
quite rare.
Hypersplenism is a condition in which
the spleen becomes increasingly active
and then rapidly removes the blood cells.
It can result from any splenomegaly. It is
most common with splenomegaly
secondary to portal hypertension and
hematological disorders.

33
Neutropenia in the presence of a normal bone marrow may be due to immunologic peripheral destruction
(autoimmune neutropenia), sepsis, or hypersplenism. The presence in the serum of antineutrophil antibodies
supports the diagnosis of autoimmune neutropenia. Felty syndrome is an immune neutropenia associated with
seropositive rheumatoid arthritis and splenomegaly.
In the clinical setting, where either drug- or infection-associated neutropenia
is suspected, appropriate immediate measures include discontinuation of the
presumed offending agent, close monitoring of daily CBC, and consideration
of treatment with a myeloid growth factor in patients with uncontrolled
bacterial or fungal infection.
Lymphocytopenia
Lymphocytopenia is diagnosed from the results of a complete blood count. A lymphocyte level below 1,000
cells/µl in adults or below 2500 cells/µl in children is diagnostic.
Like neutropenia, it may also be congenital (rare) or acquired.
The most common cause of temporary lymphocytopenia is a recent infection,
such as the common cold or influenza.
The possibility of recent therapy with immunosuppressive drugs, including
corticosteroids, chemotherapy and anti-lymphocyte monoclonal antibodies,
must be considered in treating the patient with lymphopenia.
Other causes include critical illness including sepsis, autoimmune and
connective tissue diseases including lupus and rheumatoid arthritis,
sarcoidosis, chronic renal failure, excess alcohol use, older age, thymoma,
and tuberculosis and other bacterial infections.
Leukocytosis is defined as a total WBC more than two standard deviations above the mean, or a value of greater
than 11,000/µl in adults. Leukocytosis to values in excess of 50,000 cells/µl, when due to causes other than
leukemia, is termed a leukemoid reaction.
The first step in evaluating an increased WBC count (leukocytosis) is to examine the WBC differential to determine
which WBC type is in excess. The increase in WBCs may be secondary to either immature precursors or blasts
(acute leukemia), or expansion of mature leukocytes (granulocytes, lymphocytes, monocytes). Peripheral blood
smear (PBS) can aid in excluding the possibility of acute leukemia.
Non-neoplastic elevations in peripheral WBC count are commonly found in patients with infections and other
inflammatory states, such as those associated with autoimmune disorders.
Neutrophilia
Neutrophilia is ANC above 7700/µl.
A mild (physiologic) increase in circulating neutrophils can occur without
disease after strenuous exercise, during menstruation, and in the course
of pregnancy.
Fevers during neutropenia is
considered as infectious until
proven otherwise.
Febrile neutropenia is a life-
threatening circumstance.
In viral infections the virus first
causes lymphopenia, followed
by lymphocytosis in a few
days.
Corticosteroid therapy causes
a temporary lymphopenia on
commencement of therapy
due to temporary retention of
lymphocytes by the lymphoid
tissues, but normalizes within
2 days.
The definitions of leukocytosis
(e.g. neutrophilia) are defined as
total counts more than two
standard deviations above the
mean; thus it is logical to expect
2.5% of suspected patients to be
normal.

34
Non-physiologic neutrophilia represents either a reactive phenomenon (leukemoid reaction) or a myeloid
malignancy.
As is true for the approach to any medical problem, there is no substitute for an accurate history and physical
examination. However, before this process is started, the clinician must make sure that there is no laboratory
error involved. Specifically, blood counts that do not make sense within the context of the clinical findings should
be repeated before extensive evaluation is undertaken.
A leukemoid reaction often is associated with infection, inflammation, malignancy, or use of drugs including
glucocorticoids, psychiatric medications, and myeloid growth factors. Patient history and findings on physical
examination dictate whether further laboratory investigation is necessary to determine the cause of the increased
WBC count. Further evaluation, if indicated, starts with a PBS which may show circulating blasts (suggesting acute
leukemia) or simply left-shifted neutrophilia. Left-shifted neutrophilia suggests either CML or a leukemoid
reaction; those are distinguished using leukocyte alkaline phosphatase (LAP) score and peripheral blood FISH for
bcr/abl.
Eosinophilia
The degree of eosinophilia can be categorized into mild (500 to 1500 cells/µl), moderate (1500 to 5000 cells/µl)
or severe (>5000 cells/µl).
An increase in circulating eosinophils is most commonly found in patients with allergic disorders and those with
asthma. An increase in circulating eosinophils is also found in patients with certain parasitic infections and in
patients with dermatologic disorders such as eczema. Increases in eosinophils can also be caused by some drugs
and some autoimmune disorders. Finally, increases in eosinophils can be seen in certain neoplastic conditions such
as Hodgkin lymphoma and T-cell lymphomas.
The first step in treating a patient with blood eosinophilia is to exclude the possibility of “secondary” eosinophilia
caused by parasite infestation, drugs, comorbid conditions such as asthma and other allergic conditions,
vasculitides, lymphoma, and metastatic cancer. Therefore, the initial approach should include obtaining a good
patient history and ordering a stool test for ova and parasites.
Basophilia
Seen when absolute basophil count exceeds 200/ul.
Basophils are elevated with Allergic or inflammatory reactions (e.g. hypersensitivity reactions, ulcerative colitis),
some endocrinopathies (e.g. myxedema), and infections, including viral infections, tuberculosis, helminth
infections.
Peripheral blood basophilia is an extremely rare condition that suggests chronic basophilic leukemia. Such a
finding requires a bone marrow examination and a prompt hematology consultation.
Monocytosis
Monocytosis is present when the absolute monocyte count exceeds 800/ul.
The peripheral monocyte count is increased in a number of situations where the lymphocyte count is also
increased, such as tuberculosis. Rheumatoid arthritis, systemic lupus erythematosus, and other connective tissue
diseases also may be associated with a monocytosis.

35
Absolute monocytosis that is persistent should be considered a marker of a myeloproliferative disorder (eg,
chronic myelomonocytic leukemia) until proved otherwise by bone marrow examination and cytogenetic studies.
Lymphocytosis
In individuals older than 12 years, lymphocytosis is defined as an ALC (absolute lymphocyte count) >4000 cells/µl
(also expressed as >4000/µl or >4.0 x 109
/L). Levels of blood lymphocytes are higher in neonates and young
children, who may have normal blood absolute lymphocyte counts as high as 8000/µl.
Patients can develop a lymphocytosis in a variety of different conditions such as acute viral infections (e.g.,
hepatitis, chicken pox), certain bacterial infections (e.g., pertussis, tuberculosis), lymphoma and lymphocytic
leukemia.
The first step in the evaluation of lymphocytosis is a complete history and physical examination, together with a
complete blood count and examination of the peripheral blood smear (PBS) to review the morphology of the
excess lymphocytes.
Further tests should not be ordered if the clinical scenario is consistent with viral infection; after the patient
recovers, the CBC and PBS should be repeated to see whether the abnormality has resolved.
Non-resolving lymphocytosis with normal-appearing small-lymphocyte morphology suggests B-cell chronic
lymphocytic leukemia (CLL). A spectrum of other morphologic abnormalities characterizes other lymphoid
neoplasms.
Thrombocytopenia is defined as a platelet count less than 150,000/µl (150 x 109
/L), keeping in mind that 2.5% of
the normal population will have a platelet count lower than this.
Note: A recent fall in the platelet count by 50%, while still in the normal range, may herald severe clinical problems,
and requires active follow-up.
The first step in treating thrombocytopenia is to exclude the possibility of spurious thrombocytopenia caused by
EDTA-induced platelet clumping. The situation is clarified by either examining the PBS or repeating the CBC using
sodium citrate as an anticoagulant. Another important point to consider before starting a costly search for disease
is the fact that healthy women may experience mild to moderate thrombocytopenia (platelets, 75,000-150, 000)
during pregnancy, and such incidental thrombocytopenia of pregnancy requires no further investigation.
The second step in treating patients with thrombocytopenia is to exclude the life-threatening possibilities of
thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) or disseminated intravascular
coagulation (DIC) because of the urgency for specific therapy for these diagnoses.
 PBS (to look for schistocytes); serum levels of haptoglobin and LDH (to assess for concomitant hemolysis) and
creatinine; and coagulation tests including plasma levels of D-dimer, are recommended in most instances of
thrombocytopenia. Both TTP/HUS and DIC are characterized by microangiopathic hemolytic anemia and thus
display schistocytes on PBS, an increased LDH level, and a decreased haptoglobin level. Coagulation studies
are usually normal in TTP/HUS, whereas clotting times are prolonged in disseminated intravascular
coagulation.
The third step is consideration of both drug-related thrombocytopenia and hypersplenism in all instances.
Thrombocytopenia is more likely to occur in the presence of hypersplenism associated with cirrhosis. The most
frequently implicated drugs in thrombocytopenia are antibiotics including TMP/SMX, cardiac medications (eg,
quinidine, procainamide), thiazide diuretics, antirheumatics including gold salts, and heparin.

36
Next, after microangiopathic hemolytic anemia, drug-induced thrombocytopenia, and hypersplenism have been
ruled out, idiopathic thrombocytopenic purpura (ITP) becomes the major contender in the differential diagnosis
of isolated thrombocytopenia. ITP is a diagnosis of exclusion. Secondary ITP is associated with connective tissue
disease, lymphoproliferative disorders, and certain infections (e.g. HIV, Hepatitis C). Hence, laboratory tests for
HIV, antinuclear antibodies, and monoclonal protein are recommended. Primary ITP is a diagnosis of exclusion,
so rule out other causes before issuing the diagnosis.
Do not order an antiplatelet antibody test. The result is too nonspecific to be helpful! Bone marrow biopsy is
also not indicated in the work-up of most patients with isolated thrombocytopenia that is consistent with ITP.
Finally, Rare causes of isolated thrombocytopenia include hereditary thrombocytopenias (e.g. Bernard-Soulier
syndrome, and X-linked Wiskott-Aldrich syndrome), Myelodysplastic syndrome (MDS) (rarely presents with
isolated thrombocytopenia), amegakaryocytic thrombocytopenia and posttransfusion purpura (a rare
complication of blood transfusion). In all the aforementioned situations, a hematology consultation is advised.
Figure 2: Diagnostic approach to thrombocytopenia

37
Platelet count >500,000/µl.
Thrombocytosis may represent either a myeloid malignancy (primary
thrombocytosis [PT]) or a secondary process related to various clinical
conditions including iron deficiency anemia (IDA), surgical asplenia, infection,
chronic inflammation, hemolysis, tissue damage, and nonmyeloid malignancy
(reactive thrombocytosis [RT]).
The distinction between PT and RT is clinically relevant because the former but not the latter is associated with
increased risk of thrombohemorrhagic complications.
Once thrombocytosis has been reported, it should be confirmed by repeat testing and examination of the
peripheral blood smear, in order to exclude errors or cases of spurious thrombocytosis (e.g. Cytoplasmic
fragments).
In general, the degree of thrombocytosis is a poor discriminator of PT and RT. Patient history and physical findings
are most helpful in making this distinction, and are complemented by other findings on CBC and PBS: increased
Hgb level, MCV, or WBC count favors a diagnosis of PT associated with polycythemia vera or CML, whereas
microcytic anemia suggests RT associated with IDA. Howell-Jolly bodies are seen in splenectomy.
Platelet counts are elevated
in several acute conditions;
thus, it is sometimes called a
“poor man’s ESR”.
Figure 3: Diagnostic approach to thrombocytosis.

38
Blood transfusion is the replacement of lost (or deficient) blood by blood or its products donated by another
person. Early transfusions used whole blood, but modern medical practice commonly uses components of the
blood.
Blood products include:
• Packed red blood cells (pRBC)
• Platelets
• Frozen Plasma
• Cryoprecipitate
• Plasma Derivatives:
– Albumin
– Intravenous immune globulin (IVIG)
– Factor concentrates
Blood Collection
Blood donors are screened for behaviors or medical conditions that might make blood donation unsafe for them
(e.g., anemia, coronary artery insufficiency) or the donated blood hazardous for the transfusion recipient (e.g.,
exposure to viral hepatitis, use of a teratogenic medication).
To qualify for blood donation, the prospective donor must also pass a basic physical screening that includes
temperature, blood pressure, pulse, and examination of the arms for signs of intravenous drug use, and have a
hemoglobin level of at least 12.5 g/dL from a fingerstick or venous blood sample.
Figure 1: An overview of blood collection, processing, and transfusion

39
Component Preparation
Almost all of the whole blood collected is separated into its components—RBCs, platelets, and plasma—in order
to be able to store each under optimal conditions.
Packed RBCs remaining in the primary collection bag may be stored for up to 35 days at 1°C to 6°C. Platelets are
stored at 20°C to 24°C for up to 5 days, whereas the various plasma-derived components are stored frozen (≤−18°C
for 1 year; ≤−65°C for 7 years).
FFP can be used to prepare another useful component, called cryoprecipitated anti-hemophilic factor (or
“cryoprecipitate”). Cryoprecipitate contains factor VIII, von Willebrand factor, factor XIII, and fi brinogen that are
present in a small volume of plasma.
Blood components also may be donated by a procedure known as apheresis, in which whole blood is removed
from the donor, the component of interest (plasma or platelets most commonly, but RBCs as well) is removed,
and the remaining blood elements are returned to the donor.
Testing of Donated Blood
Donated blood is held in quarantine following collection while a variety of laboratory tests are performed using
blood specimens obtained from the donor. The ABO and Rh types are determined on an RBC sample obtained at
each donation, and the donor serum or plasma is screened for the presence of unexpected RBC alloantibodies.
The concern is that such alloantibodies could cause destruction of a transfusion recipient’s RBCs if they express
the target antigen. Plasma or platelets from a donor with an alloantibody are not used for transfusion, although
RBCs are generally safe, particularly if they have been saline washed.
Infectious Disease Testing
To minimize infectious disease transmission, blood donors are screened for evidence of infection and for
participation in activities that may have exposed them to infectious agents. In addition, each blood donation is
subjected to several tests for infectious agents before it is made available for transfusion.
The specific tests are different between countries. In Palestine, testing for hepatitis B, hepatitis C and HIV is
performed on all donors. Syphilis testing is performed in certain situations.
Compatibility testing
Prior to transfusion, the compatibility of donor RBCs with the intended transfusion recipient must be established.
Part of this process involves various serologic tests. But an equally important part of this process is the proper
identification of the patient when the blood bank specimen is obtained, and again when the transfusion is
initiated. Misidentification of patients and mislabeling of specimens are the most common serious errors
encountered in transfusion. ABO mistransfusion as a result of this kind of error is far more frequent than the
transmission of HIV and all of the hepatitis viruses, combined.
Compatibility testing includes:
 The identification of patient and proper labeling of the specimen for compatibility testing. The blood bank
specimen (tube of blood) must be labeled at the bedside. The label must include 2 patient identifiers (typically
name and medical record number) and the date.
 The determination of the ABO and Rh type of the donor.

40
 The determination of the ABO and Rh type of the patient on a current specimen, and a comparison to previous
records, if any.
 A screen of the recipient’s serum/plasma for unexpected RBC alloantibodies. If unexpected antibodies (i.e.,
not anti-A or anti-B) are found, the antigen specificity of these antibodies must be identified to establish the
risk of a hemolytic transfusion reaction (HTR) and to help identify potentially compatible donor RBCs that lack
the target antigen. A record check for previously identified alloantibodies must also be made.
 The performance of a crossmatch.
 The identification of the patient when the transfusion is initiated.
To determine if the patient has an alloantibody to a RBC antigen, an antibody screen (indirect antiglobulin test) is
performed. In this test, the patient’s serum or plasma is combined with 2 or 3 reagent RBCs that are specifically
chosen because they bear a number of the antigens to which clinically significant RBC alloantibodies are made.
These cells are group O so that they will not be agglutinated by the anti-A or anti-B isoagglutinins that may be
present. If the patient serum does not produce agglutination of the reagent screening cells, then no unexpected
RBC alloantibodies are present.
The crossmatch procedure is very similar to the antibody screen and is based on the indirect antiglobulin
technique, except in this case the patient’s serum is combined with RBCs from the donor unit. If the patient has
an alloantibody to the donor RBCs, the antibody will become bound to the donor RBCs during the incubation step
and the cells will be agglutinated by the antiglobulin reagent added in the final step. If agglutination occurs, the
crossmatch is incompatible and the unit of RBCs should not be transfused to that patient. If the RBCs from this
donor were mistakenly transfused, they would be destroyed prematurely. If there is no agglutination, the patient
does not have alloantibodies to the antigens present on this donor’s RBCs and the crossmatch is compatible.
INDICATIONS FOR TRANSFUSION
Red Blood Cells A quarter of a century ago, optimal treatment of
surgical and critically ill patients targeted hemoglobin levels greater than
or equal to 10 g/dL and hematocrit values greater than or equal to 30%.
Subsequent understanding of the risks inherent in transfusion prompted
investigations designed to reestablish a minimum baseline for acceptable
hemoglobin concentrations.
Active hemorrhage resulting in shock is one of the few evidence-based established indications for transfusion.
In anemia, randomized trials comparing the clinical outcomes of liberal and stringent RBC transfusion triggers that
have consistently failed to demonstrate any benefit of transfusing patients for hematocrits of 30% (10 g/dL)
compared to triggers as low as 21% (7 g/dL).
 Indications for transfusion of different blood components are illustrated in table 1 below.
Massive Transfusion
Massive transfusion is defined, in adults, as replacement of >1 blood volume in 24
hours or >50% of blood volume in 4 hours (adult blood volume is approximately 70
mL/kg).
No single measure can replace
good clinical judgment as the basis
for decisions regarding transfusion.
In absence of ongoing
losses, 1 unit of packed
RBCs should increase the
hemoglobin by 1 g/dL.

41
The definition of massive transfusion has evolved over time to reflect modern transfusion practice.Although one
patient blood volume in 24 hours remains the “classic” definition, recent authors expand this definition to reflect
up to 50 units of blood in 24-48 hours.
Massive transfusions carry increased risks of complications like hypothermia and coagulopathy and
thrombocytopenia.
COMPLICATIONS OF BLOOD TRANSFUSION
Acute hemolytic transfusion reaction is a medical emergency
caused by rapid, intravascular hemolysis, commonly due to an ABO
incompatibility-and most often a result of a clerical error. The initial
signs may be only fever and chills. So, if a patient receiving a
transfusion develops fever and chills, stop the transfusion
immediately-prognosis worsens as ore blood is given. Provide
supportive care, including normal saline infusion.
Diagnostic tests include Coombs testing, serum-free hemoglobin, hemolysis labs (indirect bilirubin, haptoglobin,
LDH), urine for hemoglobin testing, and repeat type and cross on transfused RBCs, as well as any blood left in the
Table 1: Indications for Transfusion
complications of transfusion can be
classified as immunologic, infectious,
or due to the chemical or physical
characteristics of blood components.

42
transfusion bag. Plasma is pink and peripheral smear shows schistocytes. Alert the blood bank immediately
because another patient may also be receiving the wrong blood.
Delayed hemolytic transfusion reaction is caused by extravascular hemolysis associated with Rh incompatibility
or minor antigen mismatches. Patients present approximately 7 days after transfusion with anemia, mild fever,
and mild unconjugated bilirubin elevation. No treatment is necessary in the absence of brisk hemolysis. Future
transfusions should be matched appropriately.
Febrile transfusion reactions. This is the most common transfusion rreaction. Fever and chills after a transfusion
are common and represent nonhemolytic reactions to leukocytes in the blood product. A normal peripheral smear
differentiates this mild, benign reaction from the more dangerous acute hemolysis. Stop the transfusion and
assess for hemolysis by sending off the same labs in the "acute hemolytic" category above. If the Coombs is
negative, the symptoms are probably due to anti-HLA antibodies against the WBCs, which are transfused along
with the component blood product. Give antipyretics. Filters are used to remove WBCs in the transfused product
(leukocyte-depletion) to minimize this reaction.
Transfusional hemosiderosis is iron overload from chronic repeat transfusions, usually in patients with sickle cell
disease, thalassemia, or transfusion-dependent myeloproliferative or myelodysplastic disorders. Each 250 cc of
packed RBCs contains approximately 250 mg of iron. Patients can become symptomatic after as few as 20 units.
After 100 units (20-25 grams of iron), patients almost always show some symptoms of iron overload, which
include:
 Glucose intolerance
 Cirrhosis
 Cardiomyopathy
 Hypogonadism
Diagnosis of transfusional hemosiderosis is established by an elevated ferritin and an iron-laden liver biopsy. Start
iron chelation treatment (deferoxamine) before symptoms appear, because symptoms are typically not reversible.
Consider chelation after 20-25 units of packed red cells (approximately 5 grams of iron) if transfusions are ongoing.
Transfusion-related acute lung injury (TRALI) is a severe pulmonary reaction caused by antibodies present in
transfused FFP. The timing of TRALI is typically during or shortly after transfusion. Clinically, there is sudden onset
of respiratory distress. This may include alveolitis, noncardiogenic pulmonary edema, and acute respiratory
distress syndrome (ARDS). Treatment is supportive and may include mechanical ventilation. Stop the transfusion
and never use blood products from that donor again!
Allergic reactions to transfused blood: Simple urticaria and anaphylaxis can occur. Recipient lgA deficiency leads
to anti-lgA antibody formation, and donor blood with normal lgA levels can provoke anaphylaxis. lgA-deficient
donors should be used for such recipients.
Infectious complications are most likely to occur with platelet products because they are stored at room
temperature. Skin flora and gram-negative bacteria (E. coli, Yersinia, and Pseudomonas) are the usual organisms.
Graft vs. host (GVH) reaction. Immunocompromised patients may develop GVH from lymphocytes in transfused
blood. Also in immunocompetent patients, 1st
degree relative donations carry some risk because they may be
HLA-haploidentical, and lymphocytes may engraft. Order irradiated blood in both circumstances in order to ensure
that the potentially harmful lymphocytes have been destroyed.

43
Normal hemostasis is the controlled activation of coagulation factors and platelets leading to clot
formation, with subsequent clot lysis, in a process that stops hemorrhage without excess clotting
(thrombosis). Effective hemostasis is a rapid and localized response to an interruption in vascular
integrity (vessel wall injury), such that clots are formed only when and where they are needed.
Clot formation involves platelet activation and the subsequent generation of fibrin via the coagulation
cascade.
Platelet plug formation is initiated in vivo by exposure of platelets to vascular subendothelium when a
vessel is injured. The platelets adhere to the subendothelium, spread out along the surface, and release
substances that promote the aggregation of other platelets at that site. The platelets also accelerate
fibrin clot formation by providing a reactive surface for several steps in the coagulation cascade. The
coagulation factor pathway is an enzymatic cascade with sequential conversion of proenzymes
(zymogens) to fully activated enzymes, which then convert other zymogens to their activated forms.
There are 2 major regulatory pathways that determine the rate at which the cascade is amplified. One
of these is the protein C–protein S anticoagulant pathway, which degrades Factors Va and VIIIa, reducing
the flux through the coagulation sequence by removing these 2 activated cofactors. The other
mechanism involves the inhibitory action of antithrombin (formerly known as antithrombin III), which
inhibits the activity of thrombin and other serine proteases, namely factors IXa, Xa, XIa, and XIIa.
Antithrombin has a limited anticoagulant effect of its own, but is activated in the presence of heparin or
selected other negatively charged heparin-like molecules.
Figure 9: Clotting Cascade

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