Pathophysiology of Anemia I

Question 1

What is Mean corpuscular volume (MCV)?

Answer 1

average volume of a RBC

Question 2

What is a good range for MCV?

Answer 2

80-100 fl

Question 3

If a patient is anemic what two underlying causes should you think of?

Answer 3

the patient is not making enough RBC or is losing them in the bloodstream

Question 4

If MCV is low (ie. the RBCs are smaller than usual), what basic cause of the anemia should you think of?

Answer 4

the patient is not making enough RBC

Question 5

What is the most common cause of anemias in which the red cells are small?

Answer 5

Iron deficiency

i.e. it’s the first element in your differential diagnosis of microcytic anemias

Question 6

General rule: After the H&P and CBC, review of the peripheral blood smear is the best place to start in evaluating any hematologic disease

Answer 6

General rule: After the H&P and CBC, review of the peripheral blood smear is the best place to start in evaluating any hematologic disease

Question 7

What do iron deficient RBCs look like?

Answer 7

red cells without much hemoglobin.

In addition to being small, the area of central pallor in these biconcave discs is enlarged. As a rule of thumb, if that area’s diameter is greater than a third of the red cell’s diameter, the cell is hypochromic (lacking color - i.e. lacking hemoglobin). aka microcytosis

Question 8

What does Anisocytosis mean?

Answer 8

Anisocytosis means there is variation in their size

Question 9

What does Poikilocytosis mean?

Answer 9

poikilocytosis means variation in shape.

Both are characteristic of (but not specific for) severe iron deficiency

Question 10

Will anisocytosis and poikilocytosis appear before or after hypochromia occurs with iron deficient anemia?

Answer 10

After, they are indicative of severe anemia while hypochromia is more indicative of moderate anemia

Question 11

Will terms like anisocytosis and poikilocytosis be generated on an automated CBC?

Answer 11

No, just a manual differential. But the hematology analyzer will tell you if the red cells size distribution is increased (the “red cell distribution width”, or RDW), which correlates well with morphologic anisocytosis.

Question 12

Increased RDW is consistent with what?

Answer 12

morphologic anisocytosis.

Question 13

Patients with iron deficiency, either due to poor intake or chronic blood loss or both, have depleted their storage pool iron. Total serum iron is also reduced, since whatever is available is rapidly transported to the red cell production facility (the bone marrow). What happens when serum iron is low?

Answer 13

When serum iron is low, storage pool macrophages increase the amount of transferrin receptors on their surfaces – and we can detect that as an increased “soluble transferrin receptor” measurement.

This is not commonly used, but is available as a backup lab method should the more common tests yield ambiguous findings

Question 14

T or F. We can also detect the reduced storage pool iron directly as a reduced serum ferritin.

Answer 14

T. That’s the most reliable and important test for iron deficiency.

Question 15

What happens to transferrin production in iron deficiency anemia?

Answer 15

In an effort to move any remaining storage pool (or dietary) iron to the production facility (the bone marrow), transferrin production is ramped up.

Question 16

How is increased transferrin production measured clinically?

Answer 16

increased total iron binding capacity (TIBC).

If you see both reduced serum ferritin and increased TIBC, iron deficiency is confirmed. Then you have to find out how it happened.

Question 17

What is the the most common cause of iron deficiency anemia?

Answer 17

Red cell loss- could be chronic blood loss

So the next step in the clinical evaluation is to find out whether and where red cell loss could be occurring (covered in separate lecture)

Question 18

What is your second thought in DD of microcytic anemia?

Answer 18

beta thalassemia

Question 19

What does ‘thalassemia’ mean?

Answer 19

Reduced globin production can result from any of dozens of genetic defects grouped together under the term thalassemia

Question 20

Globin synthesis requires what?

Answer 20

normal alpha and beta globin genes (and amino acids)

Question 21

How does thalassemic red cells look?

Answer 21

Defects that result in reduced beta globin production yield the expected red cell appearance: small cells lacking hemoglobin

Question 22

How do thalassemic red cells compare to red cells in iron deficiency?

Answer 22

They are usually smaller (a mean cell volume, or MCV, of under 70 pL, with normal being between 90 and 100 pL) and showing target forms on the peripheral blood smear.

Question 23

Target forms are nonspecific for beta thalassemia. Where else are they seen?

Answer 23

Target forms are not specific; they are commonly also seen in association with liver disease.

Question 24

A reliable clue that your patient with a microcytic anemia has a thalassemia rather than iron deficiency is the ____ of red cells.

Answer 24

NUMBER. It’s reduced in iron deficiency, and is usually normal or increased in thalassemias.

Normal RBC count for male: 4.7-6.1; female: 4.2-5.4 x10^6/ul

Question 25

If you see those morphologic features, you need to confirm that a thalassemia is present by doing what?

Answer 25

ordering a hemoglobin electrophoresis test.

Question 26

What does a hemoglobin electrophoresis test do?

Answer 26

This will detect abnormal hemoglobins, such as those with variant amino acid sequences (the condition called hemoglobinopathy; sickle cell is the one most people are familiar with, but there are many others). It works via separation/quantification of the intact hemoglobin tetramer vs. any variant forms that might be present.

It is actually most commonly done now by high performance liquid chromatography, but the term electrophoresis is usually used when one orders this type of study.

Question 27

But most beta thalassemias involve reduced or absent expression of one (or both) beta globin genes. The residual beta globin is structurally normal. So how is it that hemoglobin electrophoresis can tell you that your patient has a beta thalassemia?

Answer 27

Two copies of the delta protein that replaces the beta protein bind two copies of the alpha subunit to form a quite functional hemoglobin molecule (hemoglobin A2) which migrates during electrophoresis a bit differently than the predominant adult form (hemoglobin A).

Question 28

Describe the structure of the beta globin locus (chromosome 11).

Answer 28

there are SIX variant copies (homologues) of the gene on each copy (allele) of the chromosome. Three are expressed in utero (epsilon, g-gamma, and a-gamma); a fourth, psi-beta, is a pseudogene awaiting an evolutionary assignment; the fifth (delta) is primarily expressed in the fetus, with a low level of expression after birth; and the last (beta) is the form expressed in adults.

Question 29

What happens if expression of a beta globin allele is impaired by a mutation?

Answer 29

The relative amount of delta globin is increased (sometimes to high levels)

Question 30

What does the increased delta global do?

Answer 30

Two copies of this protein bind two copies of the alpha subunit to form a quite functional hemoglobin molecule (hemoglobin A2) which migrates during electrophoresis a bit differently than the predominant adult form (hemoglobin A).

Question 31

What electrophoresis result will you see most reliably if your patient has a beta thalassemia?

Answer 31

Increased Hemoglobin A2

Question 32

How else can beta thalassemia appear on electrophoresis in extreme cases?

Answer 32

In severe cases, a fetal hemoglobin (alpha2- gamma2, called hemogobin F) will also be detected. Hemoglobin F is the predominant circulating form in the human fetus; it normally declines to undetectable levels a few months after birth.

Question 33

The severity of different forms of beta thalassemia is a highly variable function of the mutation type, and of whether it is present in one (heterozygous) or two (homozygous) copies.

Answer 33

The severity of different forms of beta thalassemia is a highly variable function of the mutation type, and of whether it is present in one (heterozygous) or two (homozygous) copies.

Question 34

What is ‘beta thalassemia minor’?

Answer 34

In general, individuals who are heterozygous for a beta thalassemia allele have only a mild anemia that does not require transfusion or other types of therapy; it is usually only discovered on a routine CBC.

The clinical term used for these patients is beta thalassemia minor.

Question 35

What is ‘beta thalassemia major’?

Answer 35

In general, homozygotes have severe anemia, often require transfusion, and have other, sometimes crippling clinical manifestations. aka beta thalassemia major

Question 36

What is ‘beta thalassemia intermedia’?

Answer 36

This term is defined by clinical presentation, NOT by the genotype

Specifically, the term applies to patients with moderate anemia that does not require transfusion but is complicated by other clinical manifestations. It turns out that this clinical picture can be due to either to heterozygosity for an allele that severely impairs globin production, OR by homozygosity for an allele that only mildly impairs it, OR by compound heterozygosity for two alleles that mildly impair it.

Question 37

T or F. there is no system for regulating the production of alpha monomers when beta monomer production is impaired

Answer 37

T. So when beta production is impaired, alpha globin accumulates. But alpha globin does NOT form functional tetramers.

Question 38

What does alpha global do when it accumulates in the absence of beta globin?

Answer 38

Instead it precipitates and causes membrane damage so severe that the damaged red cell precursors usually lyse before they can even get out of the bone marrow. The term for this is ineffective erythropoiesis, and it’s responsible for most of the severe clinical manifestations of thal intermedia and thal major.

Question 39

How does ineffective erythropoiesis work?

Answer 39

The system’s built-in means of correcting low oxygen delivery to the tissues is to make more EPO and generate more RBC precursors. That regulatory scheme is dysfunctional in the context of beta thalassemia; it just generates a large mass of RBC precursors that expands the bone marrow but fails to correct the poor oxygen transport.

Question 40

What are the main consequences of ineffective erythropoiesis?

Answer 40

enlarged marrow spaces that are evident as skeletal abnormalities, and

increased iron uptake via impaired hepcidin function

Question 41

Iron overload in beta thalassemia can lead to organ damage of which organs?

Answer 41

liver, myocardium, and endocrine organs

It can be made worse by frequent blood transfusions, since they dump still more iron into the system,

Question 42

Hemoglobin alpha globin loci are located on which chromosome?

Answer 42

16

Question 43

Describe the structure of the alpha globin gene locus.

Answer 43

Like beta globin, this gene has been replicated - but only four times. Two of these homologues are primarily expressed in utero (zeta 2 and 1), two in adults (alpha 2 and 1).

Question 44

How many functional haplotypes of alpha globin genes are there?

Answer 44

10. Four basic haplotypes for each allele (4x4= 16- 6 duplicates=10)

Question 45

What would a defect in one alpha allele manifest as?

Answer 45

alpha thalassemia 1 trait; basically a silent carrier state.

almost no clinical/lab findings

Question 46

What would a defect in two alpha allele manifest as?

Answer 46

Alpha thalassemia 2 trait.

Clinical significance: Clinicians often rule out the top three or four causes of a microcytic anemia, then simply say “probably alpha thalassemia” and leave it at that. It’s up to you to decide, given the potential genetic counseling issues, whether that’s acceptable.

Question 47

How would alpha thalassemia 2 trait appear in labs?

Answer 47

excess Hgb Bart’s (gamma)4 at birth and normal Hgb electrophoresis in adults

Question 48

Alpha thalassemia 2 trait is common in which ethnic group?

Answer 48

3% African American

Question 49

What would a defect in three alpha allele manifest as?

Answer 49

Hemoglobin H disease. There is an excess of the hemoglobin beta protein in the red cells of individuals with this type of genotype. It turns out that beta globin monomers can form a functional tetramer - and they do, in this circumstance. This contrasts sharply with the situation in beta-thalassemia, where excess alpha monomers do NOT form a functional globin tetramer. The beta tetramer (hemoglobin H) can be detected by hemoglobin electrophoresis

Question 50

What would a defect in all four alpha allele manifest as?

Answer 50

Hgb Bart’s

lethal in utero or shortly after birth - apparently before normal developmental expression of hemoglobin beta can produce significant amounts of Hgb H. Common in southeast Asia

Question 51

What happens if DNA synthesis is impaired for RBCs?

Answer 51

Effect on the bone marrow: If DNA synthesis is impaired, the red cell production line gets held up after one or two cell divisions - at a stage where the nucleus is large, and the chromatin is not condensed down into the darker material called heterochromatin. But the cytoplasm continues to mature: RNA, which yields a blue color with the stains we use, begins to get degraded, and hemoglobin’s reddish color begins to predominate. The appearance is called megaloblastic, and it can be seen in B12 deficiency OR folate deficiency OR other conditions.

Effect on peripheral blood: In addition to anemia, impaired bone marrow DNA synthesis is associated with big red cells (macrocytosis). This is evident as an increased mean cell volume (MCV). They also look bigger on the peripheral blood smear (in comparison to those small, round, mature lymphocytes). Hypersegmented neutrophils are another characteristic finding. These are neutrophils with 5 or more nuclear lobes, in comparison to the 3-4 one usually sees.

Question 52

We use the term macrocytic anemia in reference to any anemia showing large red cells. We use the term megaloblastic anemia to describe the subset of macrocytic anemias in which the characteristic bone marrow findings have been documented.

Answer 52

Most macrocytic anemias turn out to be megaloblastic

Question 53

What are some exceptions to the rule that most microcytic anemias turn out to be megaloblastic?

Answer 53

Exceptions include:

acute alcohol toxicity, in which a very high MCV may resolve over 24-48 hours;

recovery from acute anemia, in which the reticulocyte count is increased (recall that they are bigger than mature red cells);

and artifactually increased MCVs due to antibody-induced red cell clumping.

Question 54

What are the top 4 for DD of megaloblastic anemia?

Answer 54

-Impaired B12 uptake
(Pernicious anemia)

-Impaired folate uptake
malabsorption
malnutrition

-Drug effect
     nucleoside analogues
        (HAART)
     Ribonucleotide reductase
        inhibitors (Hydroxyurea) 

-Intrinsic Bone marrow dysfunction
     Myelodysplastic syndrome(s)

Question 55

Can humans make B12?

Answer 55

No, but we are very efficient at using what we get in diet.

Question 56

How is B12 bound upon entering the body?

Answer 56

HC (haptocorrin) is the first binding agent (a protein in saliva), followed by binding of Cbl by intrinsic factor (IF). IF is made by parietal cells in the stomach – it binds B12 after the HC is digested away in the jejunum, then shepherds it down to the distal ileum, where the complex gets taken up and transported into the bloodstream.

Question 57

What things can cause B12 deficiency/uptake in the stomach?

Answer 57

• Pernicious anemia (lack of IF), or defect in IF
• gastric bypass, gastrectomy
• acid/blocking drugs
• atrophic gastritis

Question 58

What things can cause B12 deficiency/uptake in the pancreas?

Answer 58

chronic pancreatitis

Question 59

What things can cause B12 deficiency/uptake in the jejunum?

Answer 59

bacterial overgrowth
parasites
sprue

Question 60

What things can cause B12 deficiency/uptake in the ileum?

Answer 60

ileum resection

Crohn’s disease

Question 61

What causes folate deficiency?

Answer 61

Although it can take several years of impaired uptake to functionally deplete the body’s stores of B12, it can take only a matter of months to have the same effect on folate stores. We see it most commonly in the context of alcohol abuse.

Question 62

Megaloblastic anemia is a side effect of which drugs?

Answer 62

Methotrexate (inhibits dihydrofolate reductase) and Hydroxyurea (inhibits ribonucelotide reductase)

Question 63

How is erythropoietin production in the kidney regulated?

Answer 63

by oxygen sensors.

Question 64

Where are the oxygen sensors in the kidney located?

Answer 64

They are located in peritubular cells in the renal cortex, and when they sense reduced availability of oxygen they secrete erythropoietin.

(Note that the urge to breath is regulated differently, by CO2 concentration in the bloodstream – NOT by oxygen concentration).

Question 65

When is hepcidin up-regulated?

Answer 65

when iron levels are high and in chronic and acute inflammatory conditions

This can complicate the care of patients in a large number of clinical settings. Just about any chronically ill patient can get it, including those with many types of cancer, autoimmune conditions, chronic hepatitis, or any hard-to-cure infection.

This is called anemia of chronic inflammation

tons of iron in storage but transport system is clamped down on

Question 66

Why would hepcidin be unregulated in inflammatory states?

Answer 66

Bacteria really need iron. That is probably why several bacterial pathogens secrete proteins that lyse red cells. So when you’re fighting off a bacterial infection, it is probably a good idea to clamp down on the availability of iron in the bloodstream.

Question 67

Is transferrin saturation increased or decreased in anemia of chronic inflammation?

Answer 67

The “transferrin saturation” (serum iron divided by TIBC) is actually increased (pathogenesis of that is a bit unclear), but the net flux of iron through the system is reduced – and red cell precursors feel the effects of that, even while macrophages sitting right next to them in the bone marrow might be stuffed with ferritin.

Question 68

Pathologists use cyanide to stain iron compounds in tissue sections – it yields the blue pigment in the bone marrow macrophages.

In general, when you suspect this you should at least think about getting a bone marrow biopsy to confirm it. That’s because in most of the contexts shown at left, the CBC and iron studies do not rule out other bad things happening in the bone marrow, such as an acute leukemia, a lymphoma, metastases, or a large number of granulomas.

Answer 68

Pathologists use cyanide to stain iron compounds in tissue sections – it yields the blue pigment in the bone marrow macrophages.

In general, when you suspect this you should at least think about getting a bone marrow biopsy to confirm it. That’s because in most of the contexts shown at left, the CBC and iron studies do not rule out other bad things happening in the bone marrow, such as an acute leukemia, a lymphoma, metastases, or a large number of granulomas.

Question 69

A patient with reduced serum ferritin and increased serum transferring has?

Answer 69

iron deficiency

Question 70

Would MCV be high or low in iron deficiency?

Answer 70

Low- microcytic