MLS advanced heme exam 1

Describe the structure of the red cell membrane

Lipids bilayer composed of phospholipids and cholesterol. Peripheral proteins in the cytoplasm like spectrin and ankyrin. Integral proteins are incorporated into phospholipid bilayer. Over 300 antigen structures

State the functions of spectrin

Provides structural support, long, flexible protein forming a mesh-like network

State the functions of ankyrin

Anchors spectrin to the membrane, maintains membrane stability

Describe difference in metabolism between a reticulocyte and a mature red cell

Reticulocytes are metabolically active, have aerobic and anaerobic metabolism, protein synthesis and more efficient energy production while mature RBC are minimalist survival cells with only anaerobic metabolism and no protein synthesis.

State the major energy source of mature RBCs

Glucose

List the erythrocyte metabolic processes that require energy

State the major metabolic pathway by which the mature red cell generates its energy

Embden Meyerhoff anaerobic pathway produces 90% of ATP in RBCs

Three ancillary pathways associated with the major metabolic pathway and purpose of each

1. Hexose monophosphate (HMP) shunt → produce glutathione 2. Methemoglobin reductase pathway → maintain Fe2+ form of iron in heme 3. Rapoport-Luebering Pathway→ produce 2,3-BPG

Explain the importance of G6PD in the formation of Heinz bodies

G6PD is a key enzyme in HMP shunt so when deficient, hemoglobin becomes oxidized and denatured hemoglobin precipitates leading to Heinz bodies. These will damage the membrane and lead to hemolysis

Hemolysis definition

Destruction of red blood cells leading to release of hemoglobin into plasma or macrophages

The major site of normal red cell breakdown

Primarily occurs in the spleen

Describe the pathway of heme degredation

Hemoglobin split into globin chains and heme→ iron (reused/stored) and protoporphyrin → bilirubin

Site of intra and extravascular hemolysis

Intra: inside blood vessels, extra: macrophages in the spleen, liver or bone marrow

Describe the processes of intravascular and extravascular hemolysis, including site of hemolysis, catabolic products, and relative timeframe for the appearance of those products after hemolysis.

Describe the fate of the different components of hemoglobin as red cells are broken down by macrophages (extravascular hemolysis).

Globin is broken into amino acids and reused, iron is stored a ferritin/hemosiderin and transported via transferrin and heme (protoporphyrin)→biliverdin→ unconjugated bilirubin→travels to liver

Describe the fate of hemoglobin following hemolysis

Binds haptoglobin

Match the plasma proteins to the RBC degradation product that binds to the proteins

Haptoglobin binds free hemoglobin, hemopexin binds free heme, albumin binds unconjugated bilirubin and methemoglobin, transferring binds iron (Fe3+→2+)

Increased rate of RBCs destruction which leads to a shortened RBC lifespan

Definition of hemolysis

Definition of hemolytic anemia

Rate of RBC destruction exceeds RBC production

Two general categories of hemolytic anemia

Intra and extravascular hemolysis

State the site of hemolysis in intravascular hemolysis forms and describe the fate of hemoglobin

Within blood vessels, free hemoglobin is released into circulation→ binds to haptoglobin forming hb-hp complex→ removed by macrophages in liver and spleen. When haptoglobin gets used up in severe hemolysis → hemoglobinemia

State the site of hemolysis in extravascular hemolysis forms and describe the fate of hemoglobin in each instance

In spleen or liver, macrophage engulfs RBC and breaks down hemoglobin→ iron and protoporphyrin, →iron stored and protoporhyrin →biliverdin→ unconjugated bilirubin→ urobilinogen excreted in stool

General PBSM features of hemolytic anemia

Reticulocytosis (increased immature RBCs), anisocytosis, poikilocytosis, possible nucleated RBCs

What hemolytic anemias will you see spherocytes in?

Hereditary spherocytosis, immune hemolytic anemia (IgG), thermal injury

What hemolytic anemias will you see elliptocytes/ovalocytes in?

Hereditary elliptocytosis aka southeast asian elliptocytosis

What hemolytic anemias will you see acanthocytes in?

Severe liver disease

What hemolytic anemias will you see Burr cells in?

Pyruvate kinase (PK) deficiency, uremia

What hemolytic anemias will you see schistocytes in?

Immune hemolytic anemia (IgM- larger and immediate fragmentation of RBC, binds complement), cardiac trauma, microangiopathic hemolytic anemia- fragmentation hemolysis

What hemolytic anemias will you see RBC Agglutination in?

Cold agglutinin diseases, immune hemolytic diseases

What hemolytic anemias will you see erythrophagocytosis in?

Damage to RBC surface by complement-fixing antibodies

Can you see erythrophagocytosis on PBSMs?

Not seen as morphology on PBSM, seen in severe case of complement…

Etiology and PBSM of hereditary spherocytosis

Ankyrin mutation (ANK1 most frequently) causing instability→ vesicles form and split off RBC→ membrane loss, decreased S:V ratio→ spherocytes. Extravascular hemolysis (get stuck in spleen). PBSM: spherocytes, HJ bodies in moderate-severe, increased osmotic fragility

Laboratory findings of hereditary spherocytosis

Deecreased HGB, increased MCHC(greater than 36g/dL, analyzer flag), retics, spherocytes and polychromasia. Negative DAT, decreased haptoglobin, increased LDH and indirect biliruben, increased osmotic fragility test

What to associate osmotic fragility test with?

Hereditary spherocytosis or thalassemia

Hereditary spherocytosis

Etiology and lab findings of hereditary elliptocytosis

Spectrin mutation (SPTA or SPTb), most patients are asymptomatic, 10% are moderate-severe, elliptical shaped RBC

Hereditary elliptocytosis

Etiology and laboratory feature of hereditary pyropoikilocytosis

Marked thermal sensitivity- morphology changes after 1 hr incubation, now considered a severe form of HE (spectrin mutation), severe anemia with poikilocytosis, bizarre shapes and forms, decreased MCV 50-65, PBSM: fragments, microspherocytes, elliptocytes

Hereditary pyropoikilocytosis

Describe the mechanisms of spherocyte formation in immune hemolytic anemia

IgG binds to RBC, targeting for removal. Splenic macrophages partially phagocytose the membrane causing a loss of membrane surface area, while volume stays the same→ spherocyte

Whats a lab procedure used to differentiate immune spherocytosis from hereditary spherocytosis

DAT (Direct Antiglobulin test) aka Coombs test. Immune: positive, hereditary: negative

State the mechanism of hemolysis in oxidant hemoytic states

Ex. G6PD enzyme deficiency→ oxidative damage→ Heinz bodies→ splenic macrophages remove these damaged portions leading to extravascular hemolysis

Describe the typical blood smear morphology in the G6PD

Heinz bodies (on supravital stain), positive auto hemolysis

List and explain factors which lead to acquired extrinsic types of hemolysis

Immune mediated destruction- antibodies bind to RBC leading to hemolysis, infections- pathogens can invade RBC causing destruction, mechanical (traumatic) damage- physical force shears RBC in circulation, chemical or drug induced, hypersplenism- enlarged spleen, thermal injury

Explain the principle and significance of the osmotic fragility test

Read on a spectophotometer, starts out at with a titration of sodium chloride and an isotonic amount of water, then it increases in hypotonic solution. It tells apart thalassemia and hereditary spherocytosis.

Explain the red cell abnormality and mechanism of cell destruction in paroxysmal nocturnal hemoglobinuria (PNH)

Caused by HSC mutations that result in lack of GPI-anchored proteins, such as CD55 and CD59(prevent compliment activation), causes chronic intravascular hemolytic anemia

Explain the principles of laboratory tests used to identify PNH

Flow cytometry- absence of CD55 and CD59(both needed to have complement and lysis so w/o causes chronic hemolysis, also hemoglobinemia, hemoglobinuria, decreased serum haptoglobin, increased unconjuated bilirubin and LDH, hemosiderinuria, elevated retics

Compare and contrast cold and warm types of immune hemolytic anemias

Both autoimmune. Warm: autoantibodies react at body temperature (37 C), primarily IgG, DAT positive, symptoms of anemia and can be life threatening. Cold: IgM antibodies cold reacting (4C) and activates complement. Can be secondary to viral infections, walking pneumonia and mono. Mild anemia

Describe the typical blood smear morphology seen in the warm hemolytic anemia

Mainly Spherocytes, polychromasia but also anisocytosis and poikilocytosis

Describe the typical blood smear morphology seen in the cold types of hemolytic anemia

List the parasites that can be found in blood

Malaria- p. Falciparum, vivax, ovale, malariae, knowlesi. Babeiosis- babesia microti tickborne disease

Morphology of Plasmodium spp.

Morphology of babesia spp.

Aplastic anemia causes

80-85% are acquired, 15-20% inherited: Fanconi anemia, Dyskeratosis congenital, Schwachman-Bodian-Diamond syndrome

Aplastic anemia overview

Rare but potentially fatal bone marrow failure syndrome

Aplastic anemia characteristics and lab findings

Pancytopenia, bone marrow hypocellularity, depletion of HSC, ANC decreased, Hgb <10g/dL, MCV increased to normal, retics decreased, bone marrrow sh