Hemolytic Anemias Flashcards
Hemolysis:
means premature destruction of circulating red cells
– Normal red cell survival in circulation is 120 days (3 months)
hemolytic anemia:
is when there is hemolysis and bone marrow cannot compensate
compensated hemolytic anemia:
– Can increase red cell production by 7 times
(reticulocytosis)
– Results in increased erythropoiesis in bone marrow (bone marrow erythroid hyperplasia)
What are the 5 categories of hemolytic diseases?
– Membrane defects (congenital or acquired)
– Metabolic defects (congenital or acquired)
– Hemoglobin Defects (congenital; RARELY acquired)
– Mechanical destruction(congenital or acquired) – Immune Destruction (congenital or acquired)
intravascular hemolysis:
lysis of red cells in the VASCULAR TREE
extravascular hemolysis:
– Phagocytic destruction of red cells by reticuloendothelial (RE) system, i.e. macrophages
– Occurs in spleen, liver, bone marrow
what is released in intravascular hemolysis?
cells destroyed in circulation release LDH and Hgb
the FREE hgb binds to HAPTOGLOBIN
the hemoglobin-haptoglobin complex is CLEARED by hepatocytes
What happens when there is poorly compensated intravascular hemolysis?
serum haptoglobin level goes DOWN (more bound to free hgb)
LDH level goes up (more cells destroyed)
Products of Hgb breakdown go UP
what happens to lab markers in SEVERE intravascular hemolysis?
haptoglobin?
LDH?
- Haptoglobin becomes saturated (very low haptoglobin) – bound to Hgb
- LDH level gets very high
What is hemoglobinemia?
Free Hgb presen in teh blood
(and Hgbx3 – HCT can be > +2)
What is hemoglobinuria?
alpha-beta dimers can be filtered by glomerulus and pass in urine
what is hemosiderinuria:
renal tubular cells take up and metabolize the alpha-beta dimers to form hemosiderin granules
what percent of RBCs are destroyed every day extravascularly in normal adults?
11%!
– Senescent RBCs, abnormally shaped RBCs
– Accelerated in pathologic hemolysis
• intrinsic or extrinsic RBC abnormalities
Where does extravascular hemolysis occur?
Red cells degraded by macrophages in RE system
– spleen, liver, bone marrow
What happens in pathologic extravascular hemolysis?
some free hgb spills into circulation
– Decreased haptoglogin
– Free hemoglobin in severe cases
How is Hgb broken down?
Hgb is broken down to Fe, heme and globin
Fe is recycled/stored in…
RE cells as ferritin or hemosiderin
Heme is broken down to…
bilibrubin
• Transported to liver by albumin
• Conjugated in liver, excreted as bile
• Converted to urobilinogen, urobilin and stercobilinogen by bacterial flora; eliminated in feces
• Some urobilinogen is reabsorbed and excreted in urine
Globin is broken down into…
AA
What are consequences of chronic hemolytic anemia?
- increased indirect (unconj) bilirubin
- erythroid hyperplasia of bone marrow
- splenomegaly
- Fe overload
What hematologic changes are associated with hemolytic anemias?
• Usually normochromic normocytic
– May be macrocytic due to reticulocytosis and/or folate
deficiency
• RBC morphology
– Poikilocytosis (red cell shaped changes) can give clues both to the presence of hemolysis and the cause of hemolysis (if someone notices it on the blood smear)
• Reticulocytosis
– Proportional to severity of anemia and bone marrow ability to
respond
• Erythroblastemia (circulating nucleated red cells)
• Bone marrow changes
– Hypercellular: erythroid hyperplasia – Iron overload
• Chronic hemolytic anemia- aplastic crisis
– Erythroid aplasia with severe exacerbation of anemia – Parvovirus B19 erythroid maturational arrest
reticulocyte ?
polychromatophili red cell that contains RNA
reticulocyte count?
Percentage or absolute number of reticulocytes can be used to assess bone marrow response to blood loss
caveats re reticulocyte count?
– Can be increased with hemorrhage or hemolysis, or erythropoietin therapy, or bone marrow recovery after toxic, nutritional or other insult!
– Don t forget to correct a % reticulocyte count for degree of anemia!
– A reticulocyte response may not occur for up to 72 hours after hemolysis becomes severe enough to cause anemia!
If the bone marrow is fully functional, an increase in erythropoietin caused by acute blood loss or hemolysis will result in increased numbers of circulating reticulocytes within approximately ____ hours
72
Why should we correct %reticulocytes?
If the box on the left represents the normal number and the normal percentage of reticulocytes in a measured volume of blood, the box on the right has a “normal” number of reticulocytes
for volume but the percentage is very high
RPI <2
failed erythropiesis
RPI >3
marrow hyperproliferration or APPROPRIATE response
when counted by an automated machine, how are reticulocytes reported?
as an absolute number in a measured volume of total red cells
what is a leukoerythroblastic reaction and what does it indicate?
• Nucleated red cells and immature granulocytes in blood reported on the leukocyte differential count • Indicates severe marrow stress – Hemolysis – Hemorrhage – Sepsis – Marrow infiltration • Carcinoma • Leukemia/lymphoma • Infection, e.g. granulomas – Early newborn period (physiologic)
what is haptoglobin?
• Produced by liver
• Binds to free hemoglobin so is decreased or absent
in hemolysis
low haptoglbobin is seen in?
– advanced liver disease
– recent massive transfusion
– genetic variant
– Megaloblastic anemias (including B12 or folate deficiency!)
how is haptoglobin an acute phased reactant?
– increased 3-4 x in inflammation, infection, tissue necrosis
(e.g. pneumonia, myocardial infarction)
– Inflammation or infection may mask the expected decrease in haptoglobin caused by hemolysis!
is haptoglobin sensitive or specific for hemolysis?
NOOO!
what is LDH?
Increases due to red cell destruction with leakage of enzymes, including LDH
why is LDH1 more useful than LDH?
LDH is a Nonspecific test
– Increased due to any type of tissue damage
– LDH isoenzymes more useful; LDH1 found predominantly in red cells and myocardium
is LDH sensitive or specific for hemolysi?
SENSITIVE but not specific
an increase in ______ supports a diagnosis of hemolysi?
indirect (unconjugated) bilirubin
– Increased bili load presented to liver faster than conjugation can occur
** In well-compensated hemolytic anemia, total bili may be <3 mg/dL; no clinical jaundice
What are other causes of an elevated indirect bili?
– Hereditary disorders of conjugation (Crigler-Najjar
syndrome, Gilbert s disease) – Breast milk jaundice
Increased free Hgb indicates ….
acute intravascular hemolysis
macroscopically appears as pink or cherry red plasma
When should you suspect there is an increase in free plasma Hgb? How do you confirm this?
Suspect when (Hemoglobin x 3 - Hematocrit) >+2
• Can be confirmed with a plasma free hemoglobin level
What urine tests can be done to look for hemolysis?
• Urine hemoglobin
– Indicates hemoglobinemia
– Cloudy, smoky, dark-red or coke-colored urine
• Urine hemosiderin
– Hemosiderin granules must be intracellular to be
significant
– Plasma free hemoglobin, positive urine hemosiderin
– Low haptoglobin (can occur with liver disease and marked extravascular hemolysis)
are typical of…
INTRAVASCUlAR hemolysis
– Elevated unconjugated (indirect) bilirubin
extravascular hemolysis
Your 22 year old female patient has a low haptoglobin level, an elevated LDH and elevated total bilirubin level which, when fractionated, is almost entirely indirect bilirubin. What further test(s) might you order first that could help you to distinguish between hemolysis and a megaloblastic anemia (such as vitamin B12 or folate deficiency)?
A. A bone marrow biopsy
B. A reticulocyte count and a blood smear for morphology C. A urine pregnancy test
D. A fractionated LDH
E. An ethics consult.
B?
67 year old African-American male admitted UMMC with chest pain RBC L Hgb L HCT L MCV 78 RDW H MCHC H Direct bili H
???
what RBC membrane abnormalities can lead to hemolysis?
– Lipid bilayer
– Structural skeleton laminates inner surface of bilayer
What is the MC congenital hemolytic anemia?
• Hereditary Spherocytosis is the most common congenital hemolytic anemia
– Congenital defect in Band 3 of spectrin molecule
– 1/2000 families
– All ethnic groups, but more common in Northern Europeans
– VARIABLE from nearly asymptomatic, to death in utero
– Clinical clues are anemia, jaundice, splenomegaly
– Other complications include biliary obstruction (gallstones), aplastic crisis due to parvovirus B19
what are lab clues for hereditary spheocytosis?
• Elevated reticulocyte count, indirect bilirubin and LDH
• Spherocytes on Blood Smear
– May be scarce
– Morphologically can look identical to other causes of spherocytes (transfused red cells, immune hemolysis, etc)
how do you diagnose herediatry spherocytosis?
• Increased osmotic fragility
– Graph % of cells hemolyzed for sequential
dilutions of saline
– Does not distinguish HS from other causes of increased spherocytes
• NEW TEST BY FLOW CYTOMETRY!!
– Since we ve figured out the membrane defect is in the Band 3 of spectrin, we can look for the altered spectrin by flow cytometry!
what is LCAT deficiency?
– Ridiculously rare
– You need LCAT to esterify cholesterol
– High levels of unesterified cholesterol cause cholesterol to be taken up by red cells membranes first making them BIG (big target shaped red cells) then STIFF (“acanthocytes” or spur shaped red cells) and fragile
– Much more common with ACQUIRED liver disease
What are the two mechanisms for metabolism in RBCs?
- Anaerobic embden meyerhof pathway to replenish ATP ( metabolizes 90% of glucose)
- remainder of glucose is metabolized by the hexose monophosphate shunt –> supplies NADPH to regenerate reduced glutathione (GSH) that protects Hgb and RBCs from oxidative damage
What is the most prevalent inborn metabolism disorder affecting RBCs?
G6PD deficiency
what is G6PD def?
G6PD deficient red cells cannot maintain sufficient levels of reduced glutathione (GSH)
• Impairs ability to deal with toxic insults or oxidative stress
– If you don t have enough G6PD to make enough GSH then Superoxide binds to hemoglobin causing it to crystalize.
– Crystalized oxidized hemoglobin is called Heinz bodies
– Heinz bodies cause lysis of red cells in spleen, i.e.
extravascular hemolysis
G6PD is characterized by….
Episodic anemia induced by infection or drugs that cause oxidative stress
– Minority have chronic hemolytic anemia
G6PD def is what type of inheritance?
sex linked (on x chromosome)
What are the two isotypes of G6pD def?
– B isoform is most common; found in all populations
– A isoform is present in about 20% of black men in US ( Has similar enzymatic activity to B isoform)
- G6PD Mediterranean found in Sicilians, Greeks, Sephardic Jews and Arabs
what drugs are associated wtih hemolysis in G6PD def?
ANTIMALARIALS
SULFONAMIDES
• Other antibacterials, e.g. nitrofurantoin, nalidixic acid
• Sulfones
• Miscellaneous
– Dimercaprol, naphthalene (mothballs), methylene blue, TNT, fava beans
what are the clinical findings in G6PD def?
• Acute hemolysis in previously healthy person following ingestion of oxidant drug
– 1-3 days following drug administration
– Hemolysis is acute in infection
• Hemoglobinemia, hemoglobinuria, jaundice
• Usually self-limited in G6PDA – Young RBC have enough enzyme
• G6PD Mediterranean may require transfusion
How do you dx G6PD?
– Start with blood smear morphology (can be characteristic)
• Bite cells, helmet cells, or veil cells due to removal of precipitated material by spleen
– HEINZ BODY PREPARATION
• Heinz bodies, particles of denatured hemoglobin and
membrane proteins adhere to membrane
• Need specific Heinz body stain (HEINZ BODY PREP)
• Limitations: Not interpretable in splenectomized patients
mechanical hemolysis occurs w/in the vasuclar system so you see ….
increased LDH, low haptoglobin, possibly serum free hemoglobin, hemoglobinuria, hemosiderinuria
how do you dx mechanical hemolysis?
blood smear morphology (fragmented red cells)
immune hemolysis?
circulating antibodies directed against red cell membrane antigen
what is congenital immune hemolysis?
Congenital immune hemolysis occurs when maternal antibodies directed against fetal red cells are transmitted across the placenta
• The placenta transfers IgG but not IgM antibodies
• IgG antibodies survive approximately 3 months
• The laboratory evaluation of IgG mediated immune hemolysis will be presented with the acquired hemolytic anemias
what are types of acquired membrane defects leading to hemolytic anemia and how do you diagnose them?
- PNH - lack membrane protein PIG
> blood smear, flow cytometry for abnormal surface protein (loss of PIG and DAF antigen) - denaturation of membrane spectrin protein (burns, toxins, venom)
> blood smear, increased LDH and unconj bili
When do acquired metabolic defects cause hemolytic anemia?
• Metabolic defects (acquired)
– Occurs when there is sufficient oxidative stress to overcome normal ability to compensate with G6P-D producing NADH and NADPH to recharge glutathione (GSH) and protect hemoglobin
What are common causes of G6PD def?
– Examples are Dapsone or chronic use of Pyridium or when sepsis is combined with Septra and Pyridium
– Findings are identical to congenital Heinz body hemolysis (except that G6P-D level is normal)
What causes large vessel vs small vessel hemolysis? Both result in ____ bon blood smear.
Large vessel hemolysis
• Malignant HTN
• Prosthetic heart valves
Small vessel hemolysis • DIC • TTP/HUS • Autoimmune vasculitis • March hemoglobinuria
Results in schistocytes (a fancy name for a red cell fragment)
what causes this?
– Thrombi form in small vessels
– Results in excessive shearing forces due to high- pressure gradient in circulation –> Direct external impact destroys red cells
microangiopathic thrombotic hemolysis
DIC
TTP/HUS
What is DIC?
• Disturbed balance between procoagulant factors and coagulation inhibitors
– Uncontrolled generation of thrombin and fibrin in microcirculation
• RBCs fragmented by strands of fibrin
– Results in RBC fragments, i.e. schistocytes
• If associated with significant hemolysis: Microangiopathic hemolytic anemia
• BUT, TRUST ME ON THIS ONE, YOU ALMOST NEVER SEE FRAGMENTS ON THE BLOOD SMEAR IN DIC SO BLOOD MORPHOLOGY IS NOT A GOOD DIAGNOSTIC TEST FOR DIC!
what is a common CAUSE of DIC?
tissue factor expression by….
– Tumor tissue
• adenocarcinomas, e.g. breast, GI, prostate • some leukemias
– Damaged normal tissue
• Placental abruption
• Extensive burns
– Vascular endothelium • Gram-negative sepsis
What do you see clinically in DIC?
bleeding and thrombosis
What is seen lab wise and on smear for DIC?
• Schistocytes (fragments on blood smear) rare
– Normal red cell morphology does not exclude DIC!
– Following coagulation tests is more useful
• Falling platelet count • Decreased fibrinogen
– Fibrinogen is an acute phase reactant so need serial determinations
• Increased fibrin degradation products
– Due to fibrinolysis
– D-dimer is most helpful
• Detects cross-linked domains of fibrin-degradation products
What is cardiac hemolysis and how do you test for it?
• Intracardiac lesions that alter hemodynamics, generating excessive shear force.
• May be due to cardiac valve replacement
– Risk increased by synthetic material, small valvular
area, thrombotic valve or perivalvular leaks – Also aortofemoral bypass
• Native valvular lesions
– Severe aortic stenosis, coarctation of aorta, ruptured
aneurysm of the sinus of Valsalva.
DIAGNOSTIC TESTING: Starts with blood smear for morphology
what is march hemoglobinuria?
Uncommon form of intravascular hemolysis occurs after strenuous marching or running on hard surface in thin shoes
->Red cells are prone to external impact when flowing through small vessels over bony prominences
What are the two subtypes of autoimmune hemolytic anemia?
warm (37)
cold (4)
Warm vs. Cold AIHA
WARM 70%, usally IgG: ABRUPT, jaundice and splenomegaly, severe anemia
- drugs
- lymphoma
COLD, IgM: INSIDIOUS, NO jaundice/splen, usually idiopathic
- lymphoma, infection (EBV, CMV, mycoplasma)
How does warm AIHA lead to extravascular hemolysis?
– Antibody coated red cell recognized by splenic
macrophages
– Fc receptor-mediated immune adherence and phagocytosis of the antibody bound red cells
– If complement binds to the red cell-antibody complex it can lyse in the circulation but it also enhances macrophage binding in the spleen and liver
– When RE cells in the spleen or liver remove antibody from the red cell it produces spherocytes that can be seen on the blood smear
• Shortened red cell survival leads to anemia, reticulocytosis, elevated indirect bilirubin and LDH
What is the CLASSIC lab clue for warm AIHA?
– Positive screen on the Type and screen – Blood bank struggling to cross match
What is the pathophys for cold AIHA?
• Primarily IgM autoantibodies ( cold agglutinins )
– Because 10 antigen binding sites are present on each IgM, red cell agglutination occurs at low temps
• Occurs in peripheral areas of circulation
• Red cells susceptible to trauma and mechanical
hemolysis
– If Ab active at temps approaching 37o C
• classical complement pathway is activated – intravascular complement-mediated lysis or
– extravascular hemolysis in spleen
What is the classic lab clue for cold AIHA?
The lab is having trouble doing a CBC because the red cells are sticking together in the machine ( Cold Agglutinin )
What is a good way to differentiate WARM vs COLD hemolysis?
Warm IgG spherocytes present
COLD IgM RBC agglutination
What is a direct antiglobulin test?
incubate with ANTIBODIES –> pos oombs test
• Detects immunoglobulin adsorbed to red cells
• Rabbit antihuman globulin reagent agglutinates human red cells that are coated with Ig or Complement
• Agglutination is graded from 0-+4
• Positive DAT not associated with hemolysis:
– Multiple myeloma, SLE, AIDS, cephalosporin therapy
• Negative DAT does not exclude hemolysis if red cell destruction has been massive and complete
What is an INDIRECT antiglobulin test?
intubate with RBCs —> agglutination with abs already there give pos coombs
• Detects serum antibody that coats red cells under test conditions
– Identifies its blood group specificity
– Characterizes temp of reactivity (i.e. cold vs warm
reactivity
– This is the screen in the Type and Screen !!
• Incubate test cells with patient serum
• Followed by washing and addition of DAT reagents to detect cell-bound complement and/ or immunoglobulin
If the DAT is positive how do you know what the antibody on the surface of the patient s red cells is directed against?
You ELUTE the antibody from the surface of the red cells and test it against laboratory red cells that have know antigens
Wash the Abs from the RBC surface and react the Abs in the wash water (eluate) with donor RBCs
What is alloimmune hemolytic anemia?
Immune system is sensitized and antibodies form in response to red cell alloantigens
– Blood transfusion
– During or after pregnancy
• May cause hemolytic disease of the newborn
• Caused by transplacental transfer of maternal IgG
– Following Hematopoietic Stem Cell transplantation
What are the three major types of drug-induced immune mediated hemolytic anemia?
- True autoantibody production similar to warm autoimmune hemolysis
• α-methyldopa - Hapten model
• drug binds red cell membrane and stimulates production of
antibodies that are directed at drug plus membrane
– Penicillin, cephalosporins - Innocent bystander
• Drug binds plasma proteins, then antibody attaches. Entire complex then binds to the innocent red cell that just happens to be there. The red cell-drug-plasma protein-antibody complex either activates hemolytic complement or activates RE cells in the spleen and liver
– quinine, quinidine, sulfonamides, thiazides.
What are nonspecific clues that a hemolytic anemia is present?
– Elevated LDH
– Unexplained drop in hemoglobin
– Dark urine
– Elevated unconjugated (indirect) bilirubin
– Unexplained elevated reticulocyte count
– Positive antibody screen on routine type and screen** new crossmatch may not pick up autoantibodies so order a DAT
how do you confirm the presence of a hemolytic anemia?
– BLOOD SMEAR MORPHOLOGY
– The BLOOD SMEAR MORPHOLOGY can point you in the direction of specific confirmatory tests
What are specific confirmatory tests that can be done in hemolytic anemia?
• Haptoglobin if intravascular hemolysis is suspected
• DAT if immune hemolysis is suspected
• Heinz body prep if Heinz body hemolysis is
suspected
• Membrane tests (flow cytometry for Hereditary Spherocytosis) if membrane defect is suspected
• Hemoglobin electrophoresis if hemoglobinopathy/thalassemia is suspected
• Flow cytometry for PNH if PNH is suspected
• ADAMTS 13 testing if TTP is suspected
