Week 1 - Monday

Question 1

Can you name some diseases associated with target cells on peripheral smear? (Try to name 6)

Answer 1

Target cells are associated with:

• Thalassemia
• Iron deficiency anemia
• Structurally aberrant hemoglobin disease: HbS, HbC – which also characteristically have the HbC crystal
• Splenectomy
• Liver disease
• Hereditary LCAT (lecithin-cholesterol acyl transferase) deficiency

HbC patients have mild hemolytic anemia

Question 2

What is the rate limiting step for the HMP shunt (Pentose Phosphate Pathway)? What does the enzyme generate?

Answer 2

The key enzyme is G6P dehydrogenase (G6PD), the rate-limiting enzyme; all steps of the oxidative phase are irreversible and are used to generate NADPH for reductive biosynthetic pathways

NADPH is used to reduce glutathione, a coenzyme for glutathione peroxidase which prevents oxidative damage by converting H2O2 → H2O. This is especially important in RBCs

Increased in tissues that consume NADPH in reductive pathways like adipose tissue for fatty acid synthesis, gonads and adrenal cortex for steroid synthesis, liver for fatty acid and cholesterol synthesis, and glutathione reduction inside RBCs

Question 3

Where does erythropoiesis take place?

Answer 3

Takes place in myeloid tissue (bone marrow).

Clinical Correlate: The presence of precursor cells, which normally reside in the bone marrow, in the peripheral blood is a useful marker for disease states.

Reticulocyte count can be used to gauge the ability of the marrow to respond to anemic states. It is particularly useful for differentiating anemia caused by increased destruction of RBCs from anemia caused by decreased production of RBCs.

For example, in cases of hemolytic anemia, the bone marrow responds by increasingRBC production, as such reticulocytes are released at a higher rate into the peripheral blood. Thus, reticulocyte count is increased in states of RBC destruction.

In contrast, in cases of decreased production, such as iron or B12 deficiency or aplastic anemia, the marrow is not able to adequately respond by releasing reticulocytes into the blood, thus reticulocyte count will be decreased.

Question 4

What enzyme acts on CO2 in the bloodstream? What is the final product?

Answer 4

CO2 transport: CO2 is converted to H2CO3 by carbonic anhydrase

H2CO3 (carbonic acid) dissociates to bicarbonate and a proton; the H+ binds to hemoglobin and thus has no effect on serum pH

Allosteric inhibition: CO2 also binds at the hemoglobin chain N terminus, favoring the deoxy Hb form

Question 5

Which nucleic acids require folate for synthesis?

Answer 5

Folate: needed for thymine and purine synthesis, especially in states of rapid cell division (pregnancy, erythropoiesis)

Deficiency → neural tube defects & megaloblastic anemia

Some conditions/actions result in an increased need for folate:

• pregnancy and lactation
• smoking
• malabsorption, e.g., celiac disease, alcoholism

Numerous medications can interfere with folate utilization, including the following:

Metformin (sometimes prescribed to control blood sugar in type 2 diabetes)

Sulfasalazine (used to control inflammation associated with Crohn’s disease, ulcerative colitis and rheumatoid arthritis)

Triamterene (a diuretic)

Anti-cancer drugs: 5-fluorouracil, methotrexate

Question 6

What is a schistocyte and when is it seen?

Answer 6

Schistocyte (“helmet cell”): RBC fragment, seen in any hemolytic anemia, formed in cases of mechanical trauma, can be an intrinsic abnormality of erythrocytes

Question 7

Describe the process behind the formation of RBC bite cells.

Answer 7

Bite cells are formed when the spleen removes a Heinz body from an RBC

Heinz bodies form in G6PD deficiency

Question 8

Why can the indirect Coombs test be used to cross-match blood?

Answer 8

Indirect Coombs Test: detects free-floating antibodies in serum

A serum sample is incubated with RBCs with known antigens

RBC agglutination = positive Indirect Coombs Test

Used to cross-match blood for transfusion and in antenatal antibody screening (anti-Rh antibodies against a RH+ fetus)

Question 9

Why does a deficiency of G6PD cause hemolytic anemia?

Answer 9

G6PD deficiency: hemolytic anemia when RBCs are exposed to oxidative stress because of inadequate NADPH production leading to less anti-oxidant activity of glutathione

Causes of oxidizing stress: infections, fava beans, drugs (e.g. sulfonamides, dapsone, primaquine)

Transmitted in X-linked recessive fashion with a predominance in Asia, the Mediterranean, and Africa (disease provides protection against Plasmodium falciparummalaria)

On a peripheral smear look for Heinz bodies (inclusions in RBCs composed of denatured Hemoglobin) and degmacytes (bite cells) (result of splenic macrophages removing Heinz bodies)

Question 10

Define the MCV cutoffs of macrocytosis and microcytosis.

Answer 10

MCV (mean corpuscular volume): One of the best lab values used for categorizing anemias.

<80 = microcytic, >100 = macrocytic. 80-100 = normocytic

Question 11

In heme degradation, which molecule is intermediate between heme and unconjugated bilirubin?

Answer 11

Heme Degradation: Heme → biliverdin → unconjugated bilirubin, transported to liver

Question 12

Which hormone controls the production of red blood cells?

Answer 12

Controlled by erythropoietin (EPO), a glycoprotein hormone produced in kidneys and liver.

Question 13

How does carbon monoxide compete with oxygen for hemoglobin?

Answer 13

Carbon monoxide: CO is a competitive inhibitor with 200x affinity for heme compared to O2

Carboxyhemoglobin is bright red and poisoned patients are commonly described as having a cherry-red appearance to their skin

Question 14

What is a dacrocyte? What are the 2 major conditions associated with these cells?

Answer 14

Teardrop cells (Dacrocytes): found in myelofibrosis (replacement of bone marrow by fibrous tissue aka myelophthistic anemia) and thalassemia major

Question 15

What happens to serum ferritin levels in iron deficiency anemia?

Answer 15

Labs: ↓ serum iron, ↓ ferritin, and ↑ TIBC

Serum ferritin is correlated to intracellular iron stores → serves as an excellent way to differentiate iron deficiency anemia (↓ ferritin) from anemia of chronic disease (↑ ferritin)

The increase of TIBC is due to a decrease in the total number of binding sites occupied by iron. Remember that TIBC and Transferrin always change in the same direction!

The change in transferrin is due to a negative feedback process. When the body senses a decrease in total iron → upregulation of Tf synthesis

↓ MCV (microcytic)

Question 16

The day after a physically intensive match, a 17-year-old soccer player develops an abscess. Cultures grow S. aureus but are also notable for extensive neutrophils. From which precursor are these cells differentiated?

Answer 16

Neutrophils, eosinophils, and basophils differentiate from myeloblasts. Monoblasts differentiate into monocytes. Lymphoblasts differentiate into B-cells/plasma cells, T-cells and NK cells.

Question 17

Disruption of heme synthesis results in which clinical diseases?

Answer 17

Disruption at 4 points in the pathway leads to accumulation of intermediates → porphyrias

Question 18

Explain how a direct coombs test works.

Answer 18

Direct Coombs Test

The direct Coombs test (aka direct antiglobulin test) is used to detect antibodies that are bound directly to the surface of RBCs.

RBCs are exposed to anti-human globulin. If anti-RBC antibodies are present on the surface of RBCs, the anti-human globulin will cause agglutination, thus constituting a positive test.

A positive Direct Coombs Test suggests autoimmune hemolysis.

Question 19

A 38-year-old female with gastroparesis that is treated with metoclopramide is prescribed sulfamethoxazole-trimethoprim for a urinary tract infection. After taking the first dose, she develops dyspnea, fatigue, and confusion. You note that her lips are blue.

Pulse oximetry shows an oxygen saturation of 91%. An arterial blood gas on room air returns the following:

pH 7.48
pCO2 30 mm Hg
pO2 100 mm Hg

What is the likely diagnosis?

Answer 19

Methemoglobinemia: oxidation to the Fe3+ (ferric) state leads to decreased affinity of O2 at these heme sites; however, at other non-oxidized heme sites, there is a compensatory increase in affinity → leading to a left shift of the oxygen-dissociation curve

Normally, oxidation is prevented via a reductive enzyme pathway (HMP shunt) in RBCs

Drugs that cause methemoglobinemia: Metoclopramide, Procaine, Nitrites, Antimalarials, Sulfonamides, Dapsone.

Can be easily remembered with mnemonic: A Methemoglobinemic Patient is Not Always Something Deadly.

Question 20

In a centrifuged blood sample, what components of blood will be in each fraction?

Answer 20

In a centrifuged blood sample, plasma is the top layer, leukocytes and platelets are in the middle layer, and RBCs are in the bottom layer.

Question 21

What is the main treatment for anemia of chronic disease?

Answer 21

Treatment of underlying inflammatory condition is paramount. Do NOT give iron!

Question 22

Aside from iron deficiency, name 3 diseases that cause microcytic anemia.

Answer 22

Microcytic Anemias: Thalassemias, Anemia of chronic disease, Iron deficiency, Lead poisoning.

Can be remembered with mnemonic TAIL

Thalassemias will also show target cells, basophilic stippling

Sideroblastic anemia is associated with myelodysplastic syndrome (“pre-leukemia”) as well as chronic alcoholism and lead poisoning.

Anemia of Chronic disease commonly presents as microcytic anemia

The best way to differentiate microcytic anemias is by serum ferritin levels.
Thalassemia: Normal iron studies. (There is no deficiency of iron.)
Anemia of Chronic disease: ↑ Ferritin, ↓ % saturation of transferrin, ↓ serum iron.

Why?

• Inflammatory state (from chronic infection, immune disorders or neoplasms) → IL-6 production → ↓ release of iron from iron stores.
• Because iron stores are normal or elevated, ferritin is increased.
• However, circulating iron in the blood is low → ↓ % saturation of transferrin and serum iron.

Iron Deficiency Anemia: ↓ Ferritin because of deficient iron stores.

TIBC can also be used to differentiate iron deficiency anemia from anemia of chronic disease.
Recall that TIBC is inversely proportional to the body’s iron stores —
- When iron stores are low (as in iron deficiency) → TIBC is increased
- When iron stores are adequate (as in anemia of chronic disease) → TIBC is decreased.

Question 23

What is plasma?

Answer 23

Plasma: Fluid portion of blood, contains proteins.

Albumin: maintains oncotic pressure of the blood.

Clinical Correlate: Burn victims and patients with nephrotic syndrome lose albumin (through their skin and urine, respectively), resulting in edema due to loss of oncotic pressure.

Gamma and beta globulins: transport hormones, metal ions, and lipids.

Blood coagulation proteins, including fibrinogen → fibrin is the reaction that forms the fibrin clot.

Plasma without fibrinogen is serum.

Question 24

List 4 factors that encourage oxygen dissociation from hemoglobin.

Answer 24

1) ↓ pH: relative acidic environment, like peripheral tissues
2) CO2: produced by cellular metabolism
3) 2,3-DPG (diphosphoglycerate, the same as bisphosphoglycerate): stabilizes the *taut *conformation, produced by glycolysis
4) ↑ temperature

These factors shift the O2 dissociation curve to the right – a higher O2 pressure is needed to maintain the same level of hemoglobin saturation

Question 25

What is the most common cause of iron deficiency anemia?

Answer 25

Iron deficiency anemia is most commonly the result of chronic blood loss: menorrhagia, gastrointestinal bleeding (e.g. colonic polyp or cancer, Meckel’s diverticulum in children).

Can also occur in breast fed infants; human milk has low iron.

Also, note that iron is absorbed in the duodenum, so any disease process of the small bowel (i.e. Crohn’s, celiac) that affects the duodenum may compromise iron absorption.

Question 26

What is the difference between poikilocytosis and anisocytosis?

Answer 26

Poikilocytosis = abnormally shaped RBCs

Occurs when RBCs are traumatized (DIC, TTP/HUS) or have membrane abnormalities (spherocytes, elliptocytes, Burr cells)

Anisocytosis = normal-shaped RBCs but with variation in size

Question 27

A disheveled 43-year-old male is brought into the emergency department after being found unconscious on a park bench. A review of his medical records indicates a history of homelessness, alcohol abuse, schizophrenia and seizure disorder. A CBC is sent:

Leukocyte count: 12,100/mm3
Hemoglobin: 8.7 g/dL
Hematocrit: 26%
Platelet count: 121,000/mm3
MCH: 26.2 pg/cell
MCHC: 32.3% Hb/cell
MCV: 112 μm3

What is the likely cause of this man’s anemia?

Answer 27

Macrocytic (megaloblastic) Anemias: B12 and folate deficiencies

His EtOH abuse puts him at risk for B12 and folate deficiencies

B12 (cobalamin) deficiency: associated neurologic defects

Folate deficiency: no neurologic defects

Question 28

What does blood contain?

Answer 28

Blood contains plasma, platelets, leukocytes, and red blood cells.

Question 29

How frequently (in days) is the entire RBC population replaced?

Answer 29

Average survival: 120 days ∴ every 120 days, the entire RBC population is replaced

Question 30

In cyanide poisoning, which form of hemoglobin can be used to bind cyanide?

Answer 30

Cyanide poisoning: CN- preferentially binds to Fe3+ and inactivates cytochrome c oxidase in the electron transport chain → stops cellular respiration

Nitrites can be used to convert Hb to methemoglobin → methemoglobin then binds the CN- → use sodium thiosulfate to chelate this CN- and yield thiocyanate → renally excreted

Question 31

Which enzyme is defective in acute intermittent porphyria?

Answer 31

Acute intermittent porphyria: autosomal dominant defect of Porphobiligen-deaminase

Characterized by attacks of peripheral/autonomic neuropathies with intermittent, symptom free periods. It is unclear how porphyrin intermediates cause these symptoms.

Symptoms can include abdominal pain (severe and several days’ duration), seizures, tachycardia, hypertension, coma, dark or reddish-brown urine, and psychiatric manifestations (e.g. depression).

Question 32

What is the triad of Plummer-Vinson syndrome?

Answer 32

Plummer-Vinson Syndrome: Triad of anemia, glossitis and esophageal webs. Be suspicious if a patient presents with symptoms of dysphagia and has concurrent anemia.

Patients with Plummer Vinson syndrome may have increased risk of developing esophageal squamous cell cancer.

Question 33

Which enzyme is defective in porphyria cutanea tarda?

Answer 33

Porphyria cutanea tarda: autosomal dominant defect of UROD (uroporphyrinogen decarboxylase)

80% of cases are acquired, 20% genetic (autosomal dominant).

Episodes are precipitated by hepatotoxic agents (e.g. EtOH, hepatitis)

Initial symptoms: fragile skin that blisters with minimal sun exposure. Patients may also present with pink or dark colored urine, but this is more commonly seen with Acute Intermittent Porphyria.

Pharmacology Correlate:
Barbiturates (and other inducers) induce the P450 system → consumption of heme → loss of heme’s negative feedback on ALA synthase (the rate limiting step of heme biosynthesis) → accumulation of heme intermediates.

Accumulation of these intermediates causes acute exacerbations of porphyrias.

Question 34

Elevated levels of lead inhibit which 2 key enzymes?

Answer 34

Lead poisoning: symptoms primarily via inhibition of ALA dehydrogenase (also called ALAdehydratase or PBG synthase) but also inhibits ferrochelatase

Causes microcytic anemia

Inhibition of ALA dehydratase → accumulation of ALA; if ferrochelatase is inhibited, protoporphyrin IX accumulates.

Symptoms can closely mimic those of acute intermittent porphyria, or can be more subtle (loss of developmental milestones, temperamental lability). Lead poisoning in children → encephalopathy, seizures, mental deterioration.

Question 35

Which substrates can erythrocytes use as metabolic fuel?

Answer 35

Glucose is the sole fuel (glycolysis only)

Also uses glucose for NADPH via HMP shunt to prevent oxidative damage

Question 36

What activates the enzyme ALA synthase? What inhibits it? What is the required co-factor?

Answer 36

ALA synthase has important regulation:

• Activated by P450 inducers
• Inhibited by hemin, glucose
• Requires vitamin B6 as a cofactor

Question 37

What is the first step in the synthesis of heme?

Answer 37

Heme synthesis starts in mitochondria with succinyl-CoA from the citric acid cycle. All the steps in heme synthesis are as follows (Steps critical to know for Step 1 are bolded):

δ-aminolevulinate acid(ALA) is synthesize from glycine and succinyl-CoA via the enzyme ALA synthase in the mitochondrial matrix.

ALA → porphobilinogen via the enzyme ALA dehydratase in the cytosol

Porphobilinogen → hydroxymethylbilane via the enzyme porphobilinogen deaminase (aka uroporphyrinogen I synthase)

Hydroxymethylbilane → uroporphyrinogen III via the enzyme uroporphyrinogen IIIsynthase

Uroporphyrinogen III → coproporphyrinogen III via the enzyme uroporphyrinogen decarboxylase

Coproporphyrinogen III → protoporphyrinogen III via the enzyme coproporphyrinogen oxidase

Protoporphyrinogen III → protoporphyrin IX via the enzyme protoporphyrinogen oxidase

Protoporphyrin IX combines with Fe 2+ → heme via the enzyme ferrochelatase in the mitochondrial matrix

The first and last 3 steps of heme synthesis occur in mitochondria, the rest occur in the cytosol.