Reticulocyte Count
To be useful the reticulocyte count must be adjusted for the patient's
hematocrit. Also when the hematocrit is lower reticulocytes are released earlier
from the marrow so one must adjust for this phenomenon. Thus:
Absolute retic= Patients retic x (Patients Hct/45)
Corrected retic= Absolute retic count/Maturation time
(Maturation time= 1 for Hct=45%, 1.5 for 35%, 2 for 25%, and 2.5 for 15%.)
OR
Total number of reticulocytes=Absolute retic x RBC number
Increased reticulocytes (greater than 2-3% or 100,000/mm3 total) are seen
in blood loss and hemolytic processes, although up to 25% of hemolytic anemias
will present with a normal reticulocyte count due to immune destruction of red
cell precursors. Retic counts are most helpful if extremely low (<0.1%) or
greater than 3% (100,000/mm3 total).
Anemia: Etiologies
1. Production defects:
- Nutritional deficiencies-Vitamin B12, folate or iron deficiency.
- Inflammation/chronic disease.
- Primary marrow disorders-pure red cell aplasia, myelodysplasia.
2. Sequestration (hypersplenism)-usually associated with mild pancytopenia.
3. Dilutional-common in hospitalized patients. Patient's plasma volume increase
with
laying down and also when they quit smoking. May be responsible for as
much as a 3-6% drop in the hematocrit in the first two days of
hospitalization.
4. Blood loss
.
5. Blood destruction.
Iron Deficiency: Diagnosis
1. RBC indices are of little diagnostic value unless the MCV is below 70fl which
is only seen in iron deficiency and thalassemia.
2. Serum iron can be decreased in a variety of states including iron deficiency,
inflammation and stress. The serum iron level varies tremendously from morning
to evening and from day to day. The minuscule amount of iron in a multi-vitamin
can falsely elevate the serum iron for up to 24 hours.
3. The total iron binding capacity is very specific for iron deficiency (near
100%) but has poor sensitivity (less than 30%).
4. The iron saturation (Fe/TIBC x 100) can be decreased below sixteen percents
in both anemia of chronic disease and iron deficiency and is of little help in
distinguishing between the two.
5. In the normal patient the serum ferritin is directly correlated with iron
stores. This relationship holds true even in inflammatory states although the
curve is "shifted to the left". That is, for a given level of storage iron in
a patient with an inflammatory state the serum ferritin is higher. A ferritin
level of greater than 100ng/ml rules out iron deficiency anemia in any patient.
The only exception are in acute hepatitis or liver necrosis (but not chronic
liver disease) when the serum ferritin will be massively elevated due to release
of liver stores of iron. Ferritin may be falsely elevated also in disseminated
TB and Hodgkin's disease. Despite these minor exceptions, the measurement of the
serum ferritin is the most useful and cost effective test of iron stores.
Microcytic Anemia: Differential Diagnosis
1. Iron Deficiency. The lack of iron results in decreased hemoglobin
available to the developing red cell. Thus the erythrocytes that are produced
are underhemoglobinized which result in smaller cells. The earliest sign of iron
deficiency is decreased iron stores. This stage has a normal CBC and indices,
although one can see microcytic/hypochromic cells on the smear. The anemia
gradually evolves into the classic microcytic- hypochromic anemia. Diagnosis is
made by showing decreased iron stores on bone marrow examination. Biochemically
the diagnosis is establish by a high TIBC or a low ferritin. The major
diagnostic difficulty is distinguishing iron deficiency from anemia of chronic
disease.
2. Anemia of Chronic Disease. (anemia of defective iron utilization). In
patients with inflammatory states iron is sequestered in the RE system and is
unavailable for use by the developing red cell (defective iron utilization).
Thus at the erythrocyte level the defect is identical to iron deficiency and
therefore results in production of underhemoglobinized red cells. This can
result in a microcytic/hypochromic anemia. Additional factors including shorten
red cell survival and decrease responsiveness to erythropoietin add to the
hypoproliferative state. The inflammatory state also leads to a decreased serum
iron and decreased TIBC. Recently the spectrum of diseases associated with
anemia of chronic disease has expanded. Besides the classic association of
temporal arteritis (may be a presenting sign), rheumatoid arthritis, cancer etc.,
anemia of chronic disease has been found in patients with non-inflammatory
medical conditions such as congestive heart failure, COPD and diabetes. Patients
with anemia of chronic disease can have hemoglobins decreased into the lower 20%
range and many (20-30%)will have red cell indices in the microcytic range.
Diagnosis is made by demonstrating ample bone marrow iron stores with
decrease sideroblasts (iron containing red cell precursors). Biochemically anemia
of chronic disease is a diagnosis of exclusion. The key test is to rule out iron
deficiency. The RDW is of ABSOLUTELY no value in differentiating anemia due to
iron deficiency from those associated with chronic disease. The serum iron is
decreased in both conditions and the TIBC is low in states where iron deficiency
and chronic disease co-exists thus rendering these tests useless. The finding
of an elevated ferritin over 100ng/ml is an adequate demonstration of good iron
stores. In the older patient or one with back pain, one should also rule-out the
presence of multiple myeloma by perform a serum protein electrophoresis. In dif-
ficult cases one can resort to assessing bone marrow stores of iron.
3. Thalassemia. In this disorder it is the defective production of
hemoglobin that leads to microcytosis. The main types are the beta-thalassemia,
alpha-thalassemia and Hemoglobin E.
Patients who are heterozygotes for beta-thalassemia have microcytic indices
with mild (30ish) anemias. Homozygotes have very severe anemia. Peripheral
smear in heterozygotes reveals microcytes and target cells. Diagnosis is
established in by hemoglobin electrophoresis which shows an increased HbA2. One
should check iron stores since an elevated HbA2 will not be present in patients
with both thalassemia and iron deficiency. Beta-thalassemia occurs in a belt
ranging from Mediterranean countries, the Middle East, India, Pakistan to
Southeast Asia. Patients with beta-thalassemia trait who are of child bearing
age need to have their spouse screened for beta-thalassemia and Hemoglobin E.
Alpha-thalassemia also presents with microcytosis. Patients with alpha-thalassemia will have normal hemoglobin electrophoresis. The diagnosis of alpha-thalassemia is made by excluding other causes of microcytosis, a positive family
history of microcytic anemia, and a life-long history of a microcytic anemia.
Exact diagnosis requires DNA analysis. Alpha-thalassemia is distributed is a
similar pattern to beta-thalassemia except for it very high frequency in Africa
(up to 40%).
Hemoglobin E is actually an unstable beta-hemoglobin chain that presents
in a similar fashion to the thalassemia. It is believed to be the most common
hemoglobinopathy in the world. Hemoglobin E occurs in Southeast Asia, especially
in Cambodia, Laos and Thailand. Patients who are heterozygotes are not anemic
but are microcytic. Patients who are homozygotes are mildly anemic with
microcytosis and target cells. The importance of Hemoglobin E lies in the fact
that patients with both genes for Hemoglobin E and beta-thalassemia have severe
anemia and behave in a similar fashion to patients with homozygote beta-thalasse-
mia.
4. Sideroblastic Anemia. Defective production of the heme molecule is the
basis of this disorder. The deficit of heme leads to the underhemoglobinazation
of the erythroid precursors and microcytosis. Sideroblastic anemia can be
congenital, can be due to toxins such as alcohol, lead, INH, or can be an
acquired bone marrow disorder. The peripheral smear may show basophilic
stippling in lead poisoned patients, a dimorphic (macrocytic and intensely
microcytic red cells) in patient with acquired sideroblastic anemia, or stigmata
of a myelodysplastic syndrome. Diagnosis is made by the finding of ringed
sideroblasts on the bone marrow iron stain. Iron studies in patients with
sideroblastic anemia usually show sign of iron-overload.
Hemolytic Anemias
Hemolytic anemias are either acquired or congenital. The laboratory signs
of hemolytic anemias include:
1. Increased LDH (LDH1)-sensitive but not specific.
2. Increased indirect bilirubin-sensitive but not specific.
3. Increased reticulocyte count-specific but not sensitive
4. Decreased haptoglobin-specific but not sensitive.
5. Urine hemosiderin-specific but not sensitive.
The indirect bilirubin is proportional to the hematocrit, so with a
hematocrit of 45% the upper limit of normal is 1.00 mg/dl and with a hematocrit
of 22.5% the upper limit of normal for the indirect bilirubin is 0.5mg/dl. Since
tests for hemolysis suffer from a lack of sensitivity and specificity, one needs
a high index of suspicion for this type of anemia.
In autoimmune hemolytic anemias (AIHA) one usually see microspherocytes on
the peripheral smear and splenomegaly may be present on exam. The diagnosis is
established by the finding of a positive direct antibody test (direct Coombs).
AIHA may be idiopathic or associated with malignancies, drugs or other autoimmune
disorders. Not all patients with a positive direct antibody test will have AIHA.
Microangiopathic hemolytic anemias are hemolytic anemias in which intravas-
cular destruction of red cells is present. One sees schistocytes in the peri-
pheral smear and an elevated LDH. The most common associated diseases are
disseminated intravascular coagulation, thrombotic thrombocytopenic purpura,
hemolytic-uremic syndrome, aortic valvular disease or the presence of an
artificial heart valve.
Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia that
is due to a clonal proliferation of erythrocytes abnormally sensitive to the
action of compliment. Hemolysis may be more conspicuous at night leading to the
characteristic hemoglobinuria. The routine lab abnormalities of hemolysis are
present. The diagnosis is made by performing a Ham's test (acid-serum lysis)
which is based on the abnormal cells unique sensitivity to complement.
The most common congenital cause of hemolysis is hereditary spherocytosis.
In this disease the red cell membrane is abnormal leading to increased splenic
destruction. Spherocytes are present on the peripheral smear and splenomegaly
is present on exam. Patients often have a family history of gallstones. The
laboratory values are consistent with hemolysis and the MCHC is elevated. The
diagnosis is established by the finding of increased osmatic fragility. Other
rare causes of hereditary hemolysis includes hereditary elliptocytosis.
Enzyme deficiencies such as glucose-6-phosphate dehydrogenase deficiency
are also important causes of hereditary hemolytic syndromes. The same population
at risk for thalassemia are also at risk for G-6-PD deficiency. It is sex linked
and thus only affects males. This defect is in the hexose monophosphate shunt
and renders the RBC to be unable to withstand oxidative stress. Most people with
this disease have hemolysis only with such stressors as infections and intake of
oxidative drugs. There are two main subtypes-African (A-) and Mediterranean
which tends to be more severe. Such drugs as dapsone and sulfamethoxazole may
provoke severe hemolysis in these patients. Diagnosis is establish by measuring
enzyme activity. Since the reticulocyte has increased G-6-PD activity one may
get a false negative normal level during times of hemolysis.
Macrocytosis
Round macrocytosis-due to abnormal lipid composition of the erythrocyte
membrane. Common etiologies include:
1. Alcoholism.
2. Liver Disease.
3. Renal Disease.
4. Hypothyroidism ("myxedema of the red cell").
Oval macrocytosis (macroovalocytes) is a sign of problem with cell DNA
replication. The developing red cell has difficulty in undergoing cell division
but RNA continues to be translated and transcribed leading to growth of the
cytoplasm while the nucleus lags behind. Often one or more cell division are
skipped leading to a larger than normal cell. Common causes are:
1. Drug effect including cytotoxic chemotherapy.
2. Folate Deficiency
3. Myelodysplasia
4. Vitamin B12 deficiency
Folate/Vitamin B12 deficiency
Although the classic sign of these two nutritional deficiencies is a
megaloblastic anemia, many patient who are folate or vitamin B12 deficient will
have a normal MCV. This is due to co-existing microcytic anemia or can be seen
in early stages of the anemia. All patients who are folate and vitamin B12
deficient will have macroovalocytes and hypersegmented neutrophils (six or
greater lobes in one neutrophil or greater than 5% with five lobes) present on
careful review of the smear.
It is now being realized that a presenting sign of vitamin B12 depletion
is neuropsychological problems. This can occur in patients with low-normal
vitamin B12 levels. These include paresthesia, ataxia, weakness, reflex changes
and orthostatic hypotension. In fact non-anemic patients may have the worst
neurologic defects. Although some of these patients are not anemic,
macroovalocytes and hypersegmented neutrophils are present on the smear. Low
normal vitamin B12 levels in patients with neuropsychological problems or in the
elderly may represent a true deficient state and should not be ignored. The role
of the Schilling test remains controversial in the management of vitamin B12
deficiency. One use is in the young patients to better define the "lesion" in
vitamin B12 metabolism. For best results patients should be replaced with
vitamin B12 2 months before the test to allow for the intestinal mucosa to heal
and prevent misdiagnosis of a malabsorption syndrome.
Since vitamin B12 absorption is complex, many patient are at risk for
deficiencies. These would include the elderly (achlorhydria); patients with
pancreatic insufficiency, small bowel disease, and autoimmune disorders
(pernicious anemia).
Folate deficiency is due to malabsorption or nutritional deficiency.
Patients at risk include alcoholics, patients with hemolysis, pregnant patients,
goat's milk imbibers, and patients on dilantin. Prophylactic administration of
folate may be indicated in these at risk patients. Red cell folate is the most
accurate diagnostic test. One must be sure of adequate vitamin B12 store before
initiating folate replacement to prevent exacerbation of any neuropathy induced
by vitamin B12 deficiency.
Myelodysplasia
The myelodysplastic syndromes are a group of bone marrow diseases marked
by various cytopenias, morphologically abnormal blood cells, dysplastic bone
marrow changes and a propensity to evolve into acute leukemia. The changes on
the peripheral smear can range from very abnormal looking blood smears to subtle
changes. Hallmarks on the peripheral smear are pseudo-Pelger Huet cells (two-lobed neutrophils), macroovalocytes and hyposegmented neutrophils. This is
commonly a disease of older patients and manifests itself as an anemia with
normal iron, vitamin B12, and folate studies. Often these patients are
misdiagnosed and treated ineffectually with iron or vitamin shots. Another group
of patients in whom the myelodysplastic syndromes are common is patients who have
undergone therapy for malignancy. Diagnosis is by bone marrow examination.
Often cytogenetic abnormalities will be present and aid in diagnosis and
assessing prognosis.
Aplastic Anemia/Pure Red Cell Aplasia
Destruction of the hemopoietic cells of the marrow by whatever means leads
to the clinical condition of aplastic anemia. The patient with this condition
presents with pancytopenia. A thorough history should be taken from the patient
to try to identify any possible drug or toxin exposure, although in most patients
the cause is unknown. Splenomegaly is absent. The peripheral smear shows a
decreased number of normal red cells. Diagnosis is established by bone marrow
biopsy which reveals a hypocellular marrow. Since paroxysmal nocturnal
hemoglobinuria can initially present as aplastic anemia, when marrow functions
recovers, a Ham's test should be preformed. In young patients with aplastic
anemia, bone marrow transplantation is the treatment of choice. Since prognosis
significantly worsen in these patients if they had receive transfusion, one
should not transfused these patients unless absolutely necessary.
Pure red cell aplasia is the condition where the red cell precursor are
selective destroyed. Since this condition can be associated with thymomas (and
responsive to its removal), this should be sought with radiographic studies.
Parvovirus B19, which is selectively toxic to the developing red cell, can cause
a chronic infection which leads to clinical picture resembling pure red cell
aplasia in susceptible patients. Parvovirus infection is also hazardous in
patients dependent on increased RBC production such as those with congenital
hemolytic anemia or sickle cell anemia. In those patients parvovirus infection
may lead to a dramatic "aplastic crisis".
Anemia and Alcoholism
Anemia of complex etiologies is often present in the alcoholic patient.
Alcohol has a direct toxic effect on the bone marrow which leads to decreased red
cell production. Folate metabolism is also interfered with leading to functional
folate deficiency. This is aggravated by the poor dietary intake of folate by
alcoholics and impaired absorption of folate in these patients. The alcohol
abusing patient may also have increased blood losses due to gastrointestinal
bleeding and trauma. Hypersplenism due to liver disease can be a contributing
factor to the anemia. This group of patients will often have co-existent inflam-
matory states which will lead to defective iron utilization. Heavy alcohol use
can even lead to a sideroblastic anemia.
Diagnosis of specific defects in the alcoholic can be difficult due to the
myriad of problems. The serum ferritin is a dependable gauge of marrow iron
stores. Although the MCV may be normal, most alcoholics with folate deficiency
will have either macroovalocytes or hypersegmented neutrophils present on the
peripheral smears. Sources for blood lost should be aggressively sought. Often
a bone marrow examination is required to fully elucidate the etiology(s) of the
anemia.
Anemia and HIV Disease
Patients infected with HIV can also be anemic for multiple reasons. HIV
infection itself leads to decreased hematopoiesis through multiple direct effects
on the bone marrow. Opportunistic infections such a MAI, histoplasmosis and
other pathogens can infect the marrow. Chronic infection with parvovirus B19 can
lead to a profound anemia. Marrow invasion by malignancies such as non-Hodgkin's
lymphoma and Kaposi's sarcoma may be a cause of anemia. AZT is toxic to
hematopoietic cells, especially red cell precursors and its use leads to a
megaloblastic anemia. Other drugs such as DHPG and trimethoprim-sulfa can have
marrow suppressive effects. Since the gastrointestinal tract is often a target
of many infections, this can be a major portal of blood loss. Malabsorptive
syndromes have been reported in HIV disease and when combined a with poor
nutritional status can lead to deficiency of folate and vitamin B12.
The anemic patient with HIV disease should receive aggressive evaluation
to rule out reversible causes of anemia. Nutritional deficiency should be sought
out by appropriate testing. The direct antibody test (direct Coombs) is often
positive in this group of patients without signs of clinically apparent hemolysis
and should be disregarded unless there is evidence of on going hemolysis such as
microspherocytes. In patients with profound anemia and a very low reticulocyte
count a bone marrow should be done to look for parvovirus B19 infection. Pure
red cell aplasia secondary to AZT has been reported. Bone marrow biopsy can also
show marrow invasion by tumor or pathogens. Erythropoietin levels should be
measured because patient with levels of less than 500u/ml are responsive to this
hormone. Since blood transfusion are immunosuprressive, it may be preferable to
treat anemic AIDS patietns with erythropoietin.
Indications for a Bone Marrow Aspiration and Biopsy
1. Pancytopenia.
2. Leukoerythroblastic blood smear (presence of immature white cells and
nucleated red cells).
3. Staging of the lymphomas and of small cell lung cancer (not in the diagnosis
of these diseases!).
4. Unexplained anemia.
5. Blood smear suggestive of myelodysplasia or of leukemia.
6. Monoclonal gammopathy.
7. Anemia with a very low (less than 0.1%) reticulocyte count.
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