Hemolytic Anemias - Strom 03.16.2015

Question 1

Hemolytic anemia

Answer 1

1) Anemia due to intravascular rupture
2) Anemia due to increased uptake of red cells by phagocytes in spleen and liver (called the “reticuloendothelial system”)

Question 2

If you rule in anemia due to only one cause, are you finished?

Answer 2

Most likely not; many anemic patients are anemic because they have 2+ concurrent issues.

Question 3

Free hemoglobin

Answer 3

• Looking for hemolytic anemia
• Can see free hemoglobin just by holding tube up to the light (ensure its presence is not due to traumatic blood draw)
• Free plasma hemoglobin is usually filtered out by the kidneys, but it will be present in urine in severe hemolysis and/or renal disease (ensure you’re not just seeing RBC in urine by using microscopy)

Question 4

LDH

Answer 4

• Looking for hemolytic anemia
• Large amount of LDH in RBC, so if they’re lysing, a large amount of LDH will be present in serum
• Make sure LDH in serum is not due to lysing of other cells, like lymphoblasts (would be indicative of acute leukemia)

Question 5

Unconjugated bilirubin (end product of heme catabolism)

Answer 5

• Looking for hemolytic anemia
• Serum will turn yellow as it fills with free hemoglobin
• Serum increase is due to inability of liver to keep with the large amount of free hemoglobin being dumped into the blood (ensure your patient doesn’t just have a benign liver disease)

Question 6

Haptoglobin

Answer 6

• An acute phase reactant increased in inflammatory states
• Also the hemoglobin recycling bin
• If lots of free hemoglobin is being cleared, then haptoglobin is reduced; however, if patient is in inflammatory condition, then levels will actually remain increased.

Question 7

Reticulocyte count

Answer 7

Count reticulocytes on a peripheral blood smear to look for evidence of hemolysis

Question 8

How does the workup of any hematologic disease begin?

Answer 8

With a review of the peripheral blood smear

Question 9

Plasma - what is it?

Answer 9

The liquid portion of the blood

Question 10

Serum - what is it?

Answer 10

It’s what is leftover if you let a tube of blood clot

Question 11

Hereditary spherocytosis

AKA: hereditary eliptocytosis, pyropoikilocytosis, or stomatocytosis

Answer 11

• Genetic defects of the red cell membrane in “cables”, “anchor points”, and associated proteins. Result: loss of membrane renders cells round instead of disc-shaped.
• Genetic
• Peripheral Destruction –> Extravascular Hemolysis –> RBC destruction by reticuloendothelial system because the rounded cells are less able to maneuver through splenic sinusoids and therefore consumed by splenic macrophages. Anemia results.
• Diagnosis: Perform an osmotic fragility test: see what fraction of red cells survive after they are exposed to a hypotonic environment
• On a smear, see spherocytes and Howell-Jolly bodies (fragments of nuclear material in RBCs)

Question 12

Describe how we should think of the red cell membrane

Answer 12

A pressurized balloon with a number of anchor points on its surface; cables run between the anchor points on the interior of the membrane to stabilize the structure

Question 13

Genetic cause of hemoglobin variants: unstable hemoglobin variants

Answer 13

• Recall that hemoglobin is packed into a red cell to the point just below where it will precipitate. Minor changes in the globin amino acid sequence can push the system over that limit, resulting in crystallization of the hemoglobin and resultant instability of the red cell
• Hemoglobulinopathies include Hgb S, Hgb C (or SC), Hgb E, etc.
• Diagnosis: Hemoglobin electrophoresis

Question 14

Genetic cause of hemolysis: ATP generating system (glycolysis)

Pyruvate Kinase Deficiency

Answer 14

• Recall that ATP is needed for the Na+/K+ pump that maintains cell osmolarity, that after it matures out of the bone marrow it loses its mitochondria, and that it sustains itself through glycolysis
• Defects in glycolytic enzymes like pyruvate kinase are lethal to the cell bc it loses its ability to make the ATP that then helps its pump work. Cell swelling and bursting results
• Systemic effect: The bone marrow tries to keep up with RBC loss and therefore pumps out immature red blood cells with POLYCHROMASIA (bluish discoloration.
• Diagnosis: use an enzyme activity test
• On a smear, see increased reticulocytes and nucleated RBCs, which are immature and more immature red cells, respectively

Question 15

Genetic cause of hemolysis: Anti-oxidant system

G6PD deficiency

Answer 15

• Recall that disulfide bonds can form between hemoglobin molecules and also that peroxide is generated in red cells. The anti-oxidant system of red cells is needed for cell survival.
• G6PD is an enzyme in the pentose phosphate pathway that ultimately serves to regenerate NADPH. NADPH helps reduce glutathione, which can then dispose of peroxide.
• Peripheral destruction –> intravascular hemolysis
• X-linked; common genetic defects in malaria-endemic areas; methemoglobinemia (oxidation of heme iron to Fe3+ state) can also result
• Diagnosis: enzyme activity test
• On a smear: Heinz bodies (oxidized hemoglobin) in RBC, visualized with methylene blue; bite cells, caused by splenic macrophages taking a bite out of the RBC in an attempt to rid of Heinz body/repair cell; blister cells
• If you have G6PD deficiency, do not eat lava beans or take anti-malarial drugs and bactrim

Question 16

Methemoglobinemia

Answer 16

Extensive oxidation of heme iron

RBC carry O2 next to ferrous (2+) iron; if ferrous is oxidized to ferric form (3+), it no longer works

Can occur in conjunction with G6PD deficiency or cytochrome B5 redcutase deficiency (the latter is an enzyme that converts 3+ back to 2+)

Question 17

If your patient has a G6PD deficiency, what drugs should be avoided?

Answer 17

Generally, any drug that causes red cells to lyse.

Specifically, dapsone and primaquine, which are used to treat malaria. But recall that malaria is endemic is areas where the G6PD deficiency is highest, so definitely test your patient for the deficiency before prescribing.

There are other drugs listed, but he mentioned those specifically.

Question 18

Neoplastic/acquired genetic hemolysis: Unimpaired complement fixation

Paroxysmal nocturnal hemoglobinuria

Answer 18

• Recall that plasma proteins that initiate complement fixation bind red cells at a low rate all of the time via the alternative pathway. If process goes to completion, MAC complex forms, and red cell is lysed. To prevent lysing, red cells use DAF to accelerate the decay of surface-bound complement proteins before said proteins can put together the MAC.
• This is not an inherited condition; rather, it is an acquired clonal non-malignant mutation. The mutation is in the enzyme PIG-A, which is involved with anchoring the DAF to the cell surface.
• Peripheral destruction –> Intravascular hemolysis –> Anemia
• Diagnosis: flow cytometry to check for expression of relevant surface proteins
• Treatment: bone marrow transplant or a really expensive monoclonal antibody called Ecluzimab

Question 19

Infectious hemolysis: Malaria

Answer 19

• Check for merozoites and gametocytes on a peripheral smear
• Check for which species is present, as one is more significantly fatal than the others: Plasmodium falciparum.
• Peripheral Destruction –> Intra-vascular hemolysis

Question 20

Infectious hemolysis: Babesia

Answer 20

• Transmitted by ticks
• Endemic in MA, RI, and NY
• Causes hemolysis

Question 21

Infectious hemolysis: Bartonella bacilliformis

Answer 21

• Transmitted by sand flies
• Endemic in Peru, Ecuador, and Columbia
• Causes severe anemia in acute phase and a cutaneous rash in its chronic
• Presents with fever, hemolytic anemia, and splenomegaly
• Look at blood smear under scope!

Question 22

Infectious hemolysis: C. perfringens

Answer 22

• A normal skin flora that secretes a toxin (alpha toxin) that causes hemolysis and gas gangrene
• Seen in trauma, septic abortions, and cancer
• Very severe hemolysis

Question 23

Autoimmune hemolytic anemias: List two outcomes of a red cell being tagged by an antibody

Answer 23

1) Simplest case: Caused by Ab specific for red cell surface antigens; when macrophages come to clean up the tagged red cells, they either phagocytose them or completely, or they sometimes decide to just take a bite out of them instead, leaving behind a red cell that has a reduced ratio of membrane to cell volume. The red cell subsequently takes on a spherical, instead of bi-concave, disc. They come to be called “microspherocytes”, which can be seen on a peripheral blood smear.
2) A RBC can be tagged by an antibody, and instead of being rapidly cleared as in the above scenario, complement fixation can be induced. Multi-step process: C1 and C3b can bind to the RBC. Macrophages have a C3b receptor, allowing them to find and clear RBCs in a process called extravascular hemolysis. If complement fixation goes to completion, the MAC complex will form, and the RBC will lyse (called “intravascular hemolysis”).

Question 24

Autoimmune hemolysis: Warm autoimmune hemolytic anemia

Answer 24

• On a smear: polychromasia; basophilic stippling; microspherocytes; nucleated red cells due to greatly accelerated bone marrow red cell production; NO BLASTS OR MYELOCYTES
• Lab Testing: 1) Direct Coomb’s Test, now called the DAT, or direct anti globulin test. Must test positive for antibodies and complement at 37 degrees. The DAT is not specific and must be supported with clinical findings and lab findings of hemolysis. A possible downfall of this method is that tagged red blood cells may be cleared too quickly to be detected in a blood test; 2) Can also incubate the patient’s plasma with a set of 3 exogenous, commercial, different “reagent” red cell preparations; patient with warm autoimmune hemolytic anemia will test positive for all three.

Question 25

Is there a single, definitive lab test for autoimmune hemolytic anemia?

Answer 25

No.

Question 26

Cold reactive antibodies (AKA: cold agglutinins)

Answer 26

• A group of anti-red cell antibodies that are difficult or impossible to detect at body temperature but can be detected at lower temperatures.
• Usually IgM
• PATHOGENIC cold agglutinins either have a high titer or a broad thermal amplitude (reactive at room temperature or higher).
• They usually bind only in the periphery (fingers, toes) and dissociate in the body core, where the RES is. This is the best case scenario. Seen sometimes as Raynaud’s phenomenon.
• OR they’ll cause significant hemolysis (not so good) by initiating complement fixation
• Agglutination is easily seen in a blood smear
• Get the lab to warm up your specimen before attempting to count red cells and measure hematocrit, as an analyzer will count a clump of agglutinated red cells as one cell.

Question 27

Cold autoimmune hemolytic anemia

Answer 27

• Cause by antibodies that bind RBCs at temperatures of 4-30 degrees and fix complement, leading to formation of MAC and subsequent lysis of red cell
• Diagnostic clues: Raynaud’s phenomenon; hemolysis by serum testing (looking for LDH, unconjugated bilirubin); you’ll see a positive result for antibody screen when tested at 30 degrees, but negative result at 37 (EVERYONE has agglutinins at 4 degrees, so that wouldn’t be specific for people who have the disease); DAT positive for complement
• On the smear: polychromasia; basophilic stippling; microspherocytes; nucleated red cells; NO BLASTS OR MYELOCYTES
• Ultimately, the diagnosis depends on concomitant clinical and lab findings

Question 28

Warm autoimmune hemolytic anemia: Associations, Treatments, Prognosis

Answer 28

Associated with: lymphoma, other malignancies, autoimmune conditions like SLE and RA, drug-associated, or idiopathic

Treatment: steroids, then splenectomy

Prognosis: Poor

Question 29

Cold autoimmune hemolytic anemia

Answer 29

Associated with: Viral syndromes, mycoplasma pneumonia, lymphoma, autoimmune conditions like SLE and RA, or idiopathic

Treatment: Steroids, then splenectomy

Prognosis: Chronic, seasonal

Question 30

Schistocytes

Answer 30

The term for red cells that have been sliced into fragments due to excess fibrin deposition. Excess fibrin deposition damages red cells, slicing them into pieces called schistocytes. This type of hemolysis is called microangiopathic.

Question 31

AdamTS13

Answer 31

A protease responsible for slicing the vWF cables to their proper size. Want cables to be the proper size bc if they’re too long, too much platelet adherence occurs, and thromboses in small-medium sized vessels results.

If the protease does not work due to antibodies that target them, the cables become too long, and the patient develops thrombotic thrombocytopenia purpura.

Treatment: plasmapheresis (pretty much a plasma exchange)

Question 32

Autoimmune hemolysis: Thrombotic Thrombocytopenic purpura (TTP)

Answer 32

vWF cables are too long due to a mutation in AdamTS13 and are attracting too many platelets

Causes thromboses, hemorrhage, and microangiopathic hemolytic anemia

On a smear: See schistocytes

Absence of sepsis, Clinical triad of fever, renal failure, and fluctuating CNS symptoms

Treatment: plasmapheresis

Question 33

Autoimmune hemolysis: Thrombotic Thrombocytopenic purpura (TTP)

Answer 33

vWF cables are too long and are attracting too many platelets

Thromboses, hemorrhage, microangioplastic hemolytic anemia, and schistocytes

Absence of sepsis, Clinical triad of fever, renal failure, and fluctuating CNS symptoms

Treatment: plasmapheresis

Question 34

Iatrogenic/toxic hemolysis: Hemolytic transfusion reaction

Answer 34

Occurs with production of antibodies to antigens on transfused red cells (bc those antigens are not on recipient’s cells)

Question 35

Major antigens and blood transfusions

Answer 35

If you’ve got a major antigen wrong, then you’ll know immediately or within a few hours, as the patient with have an immune response

Question 36

Minor antigens and blood transfusions

Answer 36

Example: Jk^a and Jk^b. If receive Jk^a when you’re Jk(b/b), you can develop antibodies to them and have an immune response

Response will be: delayed hemolytic reaction, 1-7 days after transfusion.

Question 37

Typical lab findings of hemolytic anemias

Answer 37

1) Free hemoglobin
2) Increased LDH (non-specific)
3) Increased un-conjugated bilirubin
4) Reduced haptoglobin as it tries to recycle increased hemoglobin
5) Look to the peripheral smear to count reticulocytes. Determine if they are elevated or low in order to decide if you’re looking at an extra-vascular or intra-vascular hemolysis situation.
6) Hemoglobinuria

Question 38

Hemoglobin C

Answer 38

• Caused by a change in globin amino acid sequence that causes Hb to precipitate (glutamic acid replaced by lysine)
• Genetic
• Peripheral Destruction –> Extravascular hemolysis –> Mild anemia
• A positive note: heterozygotes have resistance to malaria
• Diagnosis: hemoglobin electrophoresis
• On a smear, see target cells and hemoglobin clumping

Question 39

Enzyme responsible for anchoring DAF to the red cell membrane

Answer 39

PIG-A

Mutation in this enzyme leads to paroxysmal nocturnal hemoglobinuria.

Question 40

Reticuloendothelial system

Answer 40

Antibodies bind to red cell surface antigens in hemolytic autoimmune diseases. Red cells with said antibodies bound get cleared by tissue phagocytes, usually in the spleen or liver. These macrophages are collectively called the RES.

Question 41

Microspherocytes

Answer 41

In autoimmune hemolytic diseases, the RES dudes sometimes just take a bite out of an antibody-bound RBC in an attempt to leave behind as much hemoglobin as possible What’s left after the bite is called a microspherocyte.

Question 42

Microangiopathic hemolysis

Answer 42

A type of hemolysis that cuts red blood cells into little slices called schistocytes