Diseases of red blood cells

Question 1

Hereditary spherocytosis

• Clinical presentation
• Incidence
• Inheritance
• Gene affected
• Laboratory findings

Answer 1

• Clinical presentation
  
  • neonatal jaundice
  • adult with gallstones and splenomegaly
  • anemia or compensating reticulocytosis
• Incidence in US is 1 in 5,000
  
  • common in Northern Europe with incidence of 1 in 1,000
  • most common red cell disorder in person of northern European descent
• Autosomal dominant usually
• ANK1 (ankyrin)
• Labs
  
  • increased MCHC
  • MCV and MCH are normal
  • elevated retic
  • extravascular hemolysis findings
    
    • increased LDH
    • increased bilirubin
  • osmotic fragility and autohemolysis tests abnormal
  • unlike autoimmune hemolytic anemia, which also shows spherocytes, the DAT is negative

Question 2

Hereditary elliptocytosis/hereditary ovalocytosis

• Inheritance
• Mutation
• Laboratory findings
• Incidence
• Types and clinical presentation

Answer 2

• Usually autosomal dominant
• Mutations in spectrin alpha chain
• Labs
  
  • elliptocytes are twice as long as they are wide, comprise >25% of red cells
  • mild extravascular hemolysis
• 1 in 2,500 in the US to 1 in 100 in parts of Africa
• Types
  
  1. common type
     
     • African Americans
     • hereditary pyropoikilocytosis
       
       • sensitivity of red cells to heat
       • this is a transient neonatal expression of common HE
  2. spherocytic type
     
     • double heterozygosity for HS and HE
  3. stomatocytic type, aka Southeast Asian Ovalocytosis
     
     • Malaysia
     • caused by band 3 protein defect
     • confers protection against P vivax

Question 3

G6PD deficiency

• G6PD is needed for
• pathophysiology
• Labs
• Geographic distribution
• Inheritance pattern
• Testing should be done when?

Answer 3

• G6PD is needed for production of NADPH and reduced glutathione for protection from oxidants
• G6PD deficient red cells are hypersensitive to oxidant stress, including
  
  • medications
    
    • methylene blue
    • sulfa drugs
    • nitrofurantoin
    • primaquine
  • fava beans
  • infection
• Hemolysis is episodic, precipitated by oxidant stress
• Labs
  
  • poikilocytosis
  • bite cells
  • blister cells
  • heinz bodies (with supravital stains)
  • findings of extravascular hemolysis (jaundice, increased LDH)
• Prevalent in Africa, southern Europe, Middle East, Southeast Asia, and Oceania
• X linked recessive inheritance
• testing for this shoud be done when patient is not hemolyzing to avoid false negative from reticulocytes with adequate G6PD

Question 4

Pyruvate kinase deficiency

• PK does what?
• Labs
• Inheritance pattern
• Geographic distribution

Answer 4

• PK catalyzes rate limiting step in the Embden-Meyerhof (glycolysis) pathway; main source of red cell ATP
• Labs
  
  • chronic hemolysis (extravascular) - jaundice, elevated LDH
  • echinocytes (dessicocytes)
• Autosomal recessive
• Worldwide

Question 5

Hemoglobin S

• amino acid change

Answer 5

• valine encoded instead of glutamate at position 6 of the beta chain (glu to val)

Question 6

Sickle cell trait

• % of hemoglobin types
• CBC and smear
• Screening test
• Clinical manifestations

Answer 6

• Genotype SA
• Hemoglobin types
  
  • 35-45% HbS
  • 50-65% HbA
  • <3% HbA2
• Normal CBC and smear
• Screening tests are positive
  
  • metabisulfite test
  • dithionate test
• Clinical presentation
  
  • usually asymptomatic
  • increased incidence of
    
    • hematuria
    • isothenuria
    • papillary necrosis
    • medullary carcinoma of the kidney
  • exercise induced rhabdomyolysis
  • splenic infarcts in hypoxic conditions, such as exposure to high altitude

Question 7

Sickle Cell Disease

• Genotype
• Length of time of RBC survival
• Hemoglobin types

Answer 7

• SS
• RBC survival is 17 days instead of 120 days
• Types of hemoglobin
  
  • >80% HbS
  • 1-2% HbF
    
    • inhibits HbS polymerization
    • at birth HbF prevents development of clinical symptoms
    • by 6 months HbS > 50%, when symptoms start
    • combined sickle cell disease hereditary persistence of fetal hemoglobin (SS-HPFH) is clinically mild
    • treatment with hydroxyurea is meant to increase HbF
  • 1-4% HbA2
  • 0% HbA

Question 8

Hb SC disease

Answer 8

• 50% HbS and 50% HbC
• clinical manifestations are intermediate in severity between SS and SA

Question 9

Hemoglobin SA/alpha thalassemia

percentages of hemoglobin types

Answer 9

• decreased percentage of HbS as compared to SA
• in single gene alpha deletion, there is 30%-35% HbS
• in 2 alpha gene deletions there is 25%-30% HbS

Question 10

SA/beta thalassemia

Answer 10

• increased proportion of HbS (>50%)
• 1-15% HbF
• disease manifestations can be severe

Question 11

Hemoglobin C

• amino acids
• trait (hemoglobin percentages, symptoms, smear)
• disease (hemoglobin percentages, symptoms, smear)

Answer 11

• glutamate becomes lysine at position 6 on beta chain
• Hemoglobin C trait (AC)
  
  • 40-50% hemoglobin C
  • generally asymptomatic
  • peripheral smear has target cells
• Hemoglobin C disease (CC)
  
  • Types
    
    • 90% HbC
    • 7% HbF
    • 3% HbA2
    • 0% HbA
  • Mild hemolytic anemia
  • Splenomegaly
  • Numerous target cells
  • Hexagonal or rod shaped crystals

Question 12

Hemoglobin E

• amino acids
• geographic distribution
• prevalence
• CBC
• smear
• symptoms

Answer 12

• Beta 26 glutamate to lysine
• Southeast Asia
• 2nd most common abnormal hemoglobin worldwide (after S)
• CBC shows thalassemic indices and the peripheral smear numerous target cells
• typically mild, but coinheritance with beta thalassemia may be severe

Question 13

Hemoglobin D and G

Answer 13

• Clinically normal
• HbD is beta chain defect
• HbG is an alpha chain defect
• Both run with HbS on alkaline gel
• absence of HbS can be determined by sickle screen study or by citrate gel

Question 14

Hemoglobin Lepore

• CBC
• geographic distribution
• electrophroesis
• genes

Answer 14

• Thalassemic red cell indices
• Mediterranean, especially Italy
• Runs with HbS on alkaline gel, but usually only ~15% of total Hb
  
  • Actual HbS is rarely present in this quantity, so Lepore should be suspected
• Sickle screening tests are negative
• Hb Lepore is result of fusion between delta and beta genes

Question 15

Hemoglobin Constant Spring

Answer 15

• thalassemic red cell indices
• Southeast Asia
• mutation in alpha gene stop codon, producing abnormally long transcript

Question 16

Unstable hemoglobins

• smear (also seen in what conditions?)
• types of Hb

Answer 16

• result in Heinz bodies and bite cells
• Hb Hammersmith, Ann Arbor, Koln
• Note: Hb Bart and HbH (severe alpha thal) are also associated with Heinz body anemia

Question 17

Methemoglobin

• what is it
• % of total Hb
• low level maintained by
• hereditary versus acquired
• appearance of blood
• ways to measure it
• treatment

Answer 17

• Hemoglobin with iron in the oxidized ferric (Fe+++) state
• Cannot combine with O2
• Normally, up to 1.5% of total Hb is Hi (methemoglobin)
  
  • low level maintained by the action of the NADH dependent methemoglobin reductase system
• hereditary methemoglobinemia
  
  • deficient methemoglobin reductase or abnormal HbM upon which this enzyme cannot act
  • cyanosis appears at 6 months of age
• acquired methemoglobinemia
  
  • exposure to drugs or chemical that increase formation of Hi
  • nitrites, quinones, phenacetin, and sulfonamides
• blood is grossly chocolate brown
• cooximeter can measure methemeglobin directly (pulse ox and ABG cannot detect Hi)
• treatment = methylene blue, which reduces Hi to Hb

Question 18

Sulfhemoglobin

• what is it
• smear
• % total hemoglobin normally and when cyanosis occurs
• precipitated by

Answer 18

• hemoglobin oxidized in presence of sulfur
• when further oxidized, SHb preciptates to form Heinz bodies
• SHb cannot transport oxygen
• normally, SHb <1% of total Hb
• cyanosis manifests at 3-4% or 0.5 G/dl
• SHb may increase after exposure to sulfonamides and in presence of C perfingens bacteremia (enterogenous cyanosis)

Question 19

Carboxyhemoglobin

Answer 19

• CO binds tightly to hemoglobin forming carboxyhemoglobin (HbCO)
• CO has even greater avidity for fetal hemoglobin
• CO directly toxic to intracellular oxidative mechanisms
• toxic effects vary with proportion of HbCO
• Normally HbCO is <=1%
• ABG and pulse ox cannot detect HbCO
• Smokers have up to 6%

Question 20

Thalassemic indices

Answer 20

• Microcytosis
• Low hematocrit
• Normal to elevated RBC count
• RDW normal to slightly increased (in contrast, markedly increased in IDA)
• MCV/RBC count ratio < 13 favors thalasemmia
• MCV/RBC count ratio > 15 favors IDA
• Smear shows microcytsosis, target cells, basophilic stippling

Question 21

Beta thalassemia

• genetics (chromosome, alleles, mutations)
• symptoms start when
• heterozygotes vs homozygotes (HbA2, HbF, HbA)
• intermedia vs major
• delta-beta thalassemia
• Hb Lepore - relative amounts of Hb
• tetramers in Beta thal

Answer 21

• one copy of the beta gene on each chromosome 11, which make two genes
• most beta thal results from point mutation
• pathogenic alleles B^o or B+
• Symptoms not be apparent until 6 months of age
• Heterozygotes (Beta thal trait) have
  
  • high HbA2 (>3.5%)
  • normal to increased HbF
  • may show normal A2 if patient is iron deficient
• Homozygotes have
  
  • increased HbF (50-95%)
  • normal to elevated HbA2
  • little to no HbA
• Beta thal intermedia and major are distinguished by dependence of the latter on transfusion
• in delta-beta thalassemia (deletion of delta and beta) there is a
  
  • normal HbA2
  • elevated HbF (5-20%)
• In Hb Lepore (fusion of delta and beta) there is
  
  • normal quantity of HbA2
  • slightly elevated HbF
  • band in S region comprising 6-15% (Hb Lepore)
• Form alpha tetramers

Question 22

Alpha thalassemia

• describe genes
• genetic alteration
• haplotypes
• symptoms and labs in single gene, 2 gene, 3 gene, and 4 gene deletions

Answer 22

• 2 copies of the alpha gene on each chromosome 16
• most thalassemia result from large structural deletion
• haplotypes characterized as
  
  • alpha thal 2 (alpha+ thalassemia)
    
    • chr. 16 has one normal and one deleted alpha gene
    • most common genotype in African Americans
  • alpha thal 1 (alpha0 thal)
    
    • chr 16 has 2 deleted alpha genes
    • Asians
• single gene deletions
  
  • asymptomatic
  • normal CBC
  • normal electrophoresis
• 2 gene deletions
  
  • alpha thal trait
  • thalassemic indices
  • normal percentage of A2 (normal electrophoresis)
  • in absence of iron deficiency, this can be interpreteted as c/w alpha thal trait
• 3 gene deletions
  
  • HbH disease (beta tetramers)
  • hemolytic anemia
  • Heinz bodies
  • fast moving HbH on electrophoresis
• 4 gene deletions
  
  • Hb Bart disease
  • fetal (gamma) hemoglobin tetramers
  • severe hemolysis - hydrops fetalis
  • hypochromic nRBCs
  • Hb Bart on electrophoresis

Question 23

S alpha thalassemia

Answer 23

30-35% HbS with one alpha gene and 25-30% with 2 alpha thal gene deletions

Question 24

Normal expression of HbF

Answer 24

• 30-40% of total hemoglobin at term
• < 10 % at 6 months
• <5% at 1 year
• <1% at 2 years

Question 25

Hereditary persistence of fetal hemoglobin

Answer 25

* Delayed switch from gamma to beta or delta * HbF in pancellular distribution * 1 in 100 of HbSS have HPFH * 25% HbF * do not have anemia or vasoocclusive episodes * Electrophoresis showing HbS, F, and A2 has two causes * Combined sickle cell HPFH (clinically mild) * Combined sickle cell Beta thal (clinically severe)

Question 26

Non hereditary causes of elevated HbF

Answer 26

* Megaloblastic anemia * Aplastic anemia * PNH * Fanconi anemia * Juvenile myelomonocytic leukemia * Acute eyrthroid leukemia * HbF heterocellular

Question 27

Warm autoimmune hemolytic anemia - describe antibody - lab tests - smear - secondary causes - how do cells react in a panel

Answer 27

* **IgG** against broad **Rh** antigens * DAT is positive usually * Hemolysis is **extravascular** * **spherocytes** * 5-10% have positive **DAT** without hemolysis * WAIHA may be primary or secondary * Secondary causes of WAIHA * **CLL/SLL** * **Inherited immunodeficiency** * **IgA deficiency** * **Bruton agammaglobulinemia** * **Collagen vascular disease** * **Thymoma** * All cells in panel react at **AHG phase**

Question 28

Cold autoagglutinin disease (CAD)/cold autoimmune hemolytic anemia (CAIHA) - list IgM antibodies - reactions occur at what phase in panels? - nonpathologic cold agglutinins - pathologic cold agglutinins - primary versus secondary CAIHA

Answer 28

* **IgM antibodies**, often against **I** * others include **i, H, Pr, and IH** * **Pr** is rare and destroyed by **enzymes**; present in cord and adult blood * **H and IH** are neutralized by **saliva** and are almost always benign * can activate **complement**, thus reactions seen at the antiglobulin phase using polyspecific sera (IS and AHG phases) * agglutinated by C3d but not IgG * Cold agglutinins may be pathological or nonpathologic * nonpathologic cold agglutinins * react at **4 degrees, up to 22** degrees * **titer \< 64** at 4 degrees * pathologic cold agglutinins * reactive over a broad thermal range and cause spontaneous autoagglutination at room temperature * **titer \> 1000 at 4 degrees** * CAIHA may be primary or secondary * **Idiopathic cold** autoimmune hemolytic anemia or cold agglutinin syndrome is chronic condition in older people * **monoclonal IgM** * **Secondary** CAIHA is a transient condition often associated with infection * **M pneumonia** associated with anti **I** * **EBV** associated with anti **i**

Question 29

Paroxysmal cold hemoglobinuria

Answer 29

* PCH is most often seen in children * Secondary to virus (e.g., measles, mumps, chicken pox, mono) * Originally described in syphilis * Presents with paroxysmal episodes of hemoglobinuria associated with cold exposure * Occasionally peripheral blood smear shows intraneutrophilic hemophagocytosis * Treatment: keep patient warm and transfusing prewarmed blood as needed * Caused by IgG biphasic hemolysin (Deonath-Landsteiner antibody) * Anti P * Biphasic because it produces hemolysis only when incubated at 4 degrees then 37 degrees

Question 30

Cryoglobulinemia - detection method - types - clinical findings - renal biopsy - EM findings - blood smears

Answer 30

* Cryoglobulins precipitate reversibly at low temperatures * **Detection** * Blood is drawn and kept at 37 degrees * Centrifuge at 37 degrees and the remaining serum is stored at 4 degrees for at least 3 days * Then centrifuged at 4 degrees; precipitate that forms is a cryoprecipitate * Precipitate can be subjected to electrophoresis * 3 types * Type I: monoclonal Ig found in multiple myeloma or Waldenstrom * Type II: mixture of monoclonal IgM and polyclonal IgG * IgM has RF (anti IgG) activity * This is the most common type of cryo * Types II and III affect people with **lymphoproliferative disorders, chronic infection, chronic liver disease, and autoimmune diseases** (especially SLE) * **HCV** is most common cause * Type III * polyclonal IgG and polyclonal IgM * Clinical presentation * **systemic immune complex disease** * **palpable purpura** (leukocytoclastic vasculitis) * **arthralgia** * **hepatosplenomegaly** * **lymphadenopathy** * **anemia** * **sensorineural deficits** * **glomerulonephritis** * renal biopsies: MPGN, thrombotic microangiopathy * in all tissues there is **vasculitis** * **EM** shows subendothelial immune complex deposits with fibrillary or tubular structure in a fingerprintlike pattern * in blood smears, pale purple cloudy aggregates of protein

Question 31

Paroxysmal nocturnal hemoglobinuria Caused by

Answer 31

* PNH is an acquired clonal red cell disorder * membrane defects result from **decreased** glycosyl phosphatidyl inositol **(GPI) anchors** * Many GPI anchored proteins function to deflect complement mediated destruction * Affected cells have decreased * **decay accelerating factor** (**CD55**) * **membrane inhibitor reactive lysis** (**CD59**) * **acetylcholinesterase** (**AchE**) * **CD16** * **CD48** * GPI is encoded by the phosphatidyl inositol glycan class A (**PIG-A**) gene on **X** chromosome * PNH is a result of various PIG-A mutations

Question 32

PNH Clinical Presentation

Answer 32

* Can be episodic hemolysis, especially at night * More commonly, chronic hemolytic anemia * Thrombocytopenia and leukopenia * May evolve into aplastic anemia and/or AML * PNH may evolve from aplastic anemia * may present with thrombosis

Question 33

PNH lab findings

Answer 33

* Hams test and sucrose hemolysis test have low sensitivity * Flow cytometry of peripheral blood is test of choice * decreased GPI anchored proteins on neutrophils, monocytes, and red cells * Red cell analysis: **CD55 and CD59** * Type I - normal * Type II - partial deficiency * Type III - complete deficiency * **Granulocytes/monocyte analysis: CD14, CD15, CD16, and CD33** * Patients with \>20% type III RBCs or \>50% abnormal granulocytes are at high risk for thrombosis and hemolysis * fluorescent aerolysin **(FLAER)** obtained from **Aeromonas hydrophila** binds to GPI specifically and can be used instead of antibodies to detect GPI anchored antigens * **Leukocyte alkaline phosphatase (LAP)** score id decreased in PNH

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Question 36

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Question 37

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Question 39

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Question 42

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Question 49

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Question 51

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Question 53

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Question 55

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