College Learning Series Update on Anaemia

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[Title] [Presenter name]

Update on Anaemia

College Learning Series

Dr Giselle Kidson-Gerber

Prince of Wales Hospital & Royal Hospital for Women

University of New South Wales

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Key learning points

1. Anaemia is common, universal but with different aetiologies across specialities, ages, ethnicities, continents.

2. Classify anaemia according to MCV.

Use the blood film to explore the aetiology of anaemia.

3. Find and treat the cause of

anaemia. Anaemia is not ‘normal’.

4. Update on pathophysiology and treatment options

Knowledge guide:

Haematology

• Anaemia

• Haemoglobin structure and function

• Haemoglobinopathies, such as

thalassaemia and sickle cell disease

• Iron, B12, and folate metabolism

• Investigations

• FBC, blood film

• Iron studies

• Serum B12 & folate levels

• Tests of haemolysis

• Social factors influencing anaemia

Curricula standards

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Aims & Key Concepts

Anaemia

Reduced capacity of blood to carry oxygen from lungs to tissues.

Measured by a reduction in the haemoglobin level.

 Females <115 g/l (non-pregnant)

 Males <120 g/L Key Concepts to be covered

• Causes of anaemia: classify according to cell size

• Iron regulation and iron deficiency

• Haemoglobinopathies

• Haemolysis

• Macrocytic anaemia

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Causes of Anaemia

Microcytic (MCV: <80fl) Normocytic (MCV: 80-100fl) Macrocytic (MCV: >100fl)

Iron deficiency ↓ Production

- BM failure

- Chronic disease

Megaloblastic

- B 12 deficiency

- Folate deficiency Thalassaemia,

Haemoglobinopathy

Sideroblastic Anaemia ↑ Red Cell Loss

- Haemolysis

- Bleeding (acute)

Non-Megaloblastic

- Myelodysplasia

- Liver disease

- Alcohol

- Pregnancy

- Hypothyroidism

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Microcytic Anaemia

Microcytic (MCV: <80fl) Normocytic (MCV: 80-100fl) Macrocytic (MCV: >100fl)

Iron deficiency ↓ Production

- BM failure

- Chronic disease

Megaloblastic

- B 12 deficiency

- Folate deficiency Thalassaemia,

Haemoglobinopathy

Sideroblastic Anaemia ↑ Red Cell Loss

- Haemolysis

- Bleeding (acute)

Non-Megaloblastic

- Myelodysplasia

- Liver disease

- Alcohol

- Pregnancy

- Hypothyroidism

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38yo female

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Iron Deficiency Anaemia

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Body Iron Distribution and Storage

Dietary iron

Utilization Utilization

Duodenum (average, 1 - 2 mg

per day)

Muscle (myoglobin)

(300 mg)

Liver (1,000 mg)

Bone marrow (300 mg) Circulating

erythrocytes (hemoglobin)

(1,800 mg)

Reticuloendothelial macrophages

(600 mg) Sloughed mucosal cells

Desquamation/Menstruation Other blood loss

(average, 1 - 2 mg per day) Storage

iron

Plasma transferrin

(3 mg)

Iron loss

(Ferritin)

(TIBC)

Andrews NC. Disorders of iron metabolism. N Engl J Med. 1999; 341:1986-1995.

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Vaulont, S. et al. J. Clin. Invest. (2005) 115:2079-2082

Regulation of systemic iron homeostasis by hepcidin

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Iron Deficient?

Appropriate cut-offs for diagnosis Interpretation of Iron Studies Iron

Deficiency Anaemia of chronic

disease

Serum iron Low Low-Normal

Transferrin High Low-Normal

Transferrin

saturation Low Low-Normal

Ferritin Low Normal-High

Recommended Ferritin for diagnosis:

<30 μg/L for adults (both male and female)

<20 μg/L for children and adolescents

When inflammatory conditions: a higher cut off level should be used (typically <100μg/L for adults & <70 μg/L for children). Use transferrin saturation in conjunction (TSAT <20% is supportive of iron deficiency when ferritin levels are above 100 μg/L).

NB: WHO

• <12 μg/L for apparently healthy < 5 years;

• <15 μg/L for apparently healthy adolescents & adults

• <15 μg/L for women in the T1 pregnancy

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‘Iron Studies’ parameters considerations

(Serum iron)

• Diurnal variation

• Dependent in recent iron intake (diet/supplementation) (Transferrin)

• Increased in iron deficiency

• Decreased in iron overload

• Decreased in inflammation, infection, malignancy, cirrhosis Ferritin

• Acute phase reactant

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Stages of Iron Deficiency: ID vs IDA

Adequate iron stores Falling iron stores Iron stores depleted

Iron deficient for

red cell production Iron deficiency

anaemia

Bloodsafe elearning

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Iron Deficiency Anaemia

Causes

 ↓ Intake: Diet

Malabsorption: antacids, resection, Coeliac

 ↑ Loss / use: Blood loss (*occult GI bleeding) Pregnancy

Menstrual loss GI parasite Investigate

FBC, blood film, iron studies Cause

Treatment

 Replace!

Sub-populations at risk of Iron Deficiency

• Premature or low birth weight babies, toddlers and preschool children

• Adolescents

• Menstruating, pregnant and breastfeeding women

• Some Aboriginal and Torres Strait Islander populations

• Hospitalised and institutionalised patients, including elderly people in aged-care homes

• Refugees and recent migrants from economically poor countries

• Regular blood donors

• Endurance athletes

• People with restrictive diets, such as vegetarians and

vegans, or malabsorption disorders, such as Coeliac disease

Australian Red Cross Lifeblood, “Risk Groups and causes of iron deficiency”, updated 6 February 2018.

https://transfusion.com.au/transfusion_practice/ anaemia_management/iron_deficiency_anaemia/risk_groups_and_causes

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Haemoglobinopathies

Quantitative disorders Beta Thalassaemia

Alpha Thalassaemia Qualitative disorders

HbS: NM_000518.4(HBB):c.[20A>T]

HbE: NM_000518.4(HBB):c.[79G>A]

HbD: HBB: c.364G>C

Point mutations, deletions, frame shifts Autosomal recessive

7% world population

Altered amino acid sequence

• Inadequate Hb production

• Distorted α:β ratios

unbalanced accumulation

ineffective erythropoiesis & haemolysis

Variable affect on 2°, 3° & 4° structure Reduced globin chain synthesis

Slideshare.net. RP Saravanan

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(Old World) Distribution of Haemoglobinopathies

Increasing ‘at risk’ ethnicities in Australia

β globin variants

Weatherall DJ Nat Rev Genetics 2:245, 2001

α globin variants

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Genetic Locus

https://quizlet.com/509514039/heme-l4-hemoglobin-flash-cards/

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Alpha Thalassaemia

in cis

in trans

Piel FB, Weatherall DJ. N Engl J Med 2014;371:1908-1916.

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Spectrum of clinical phenotype

Genotype Anaemia MCV Clinical

β Thalassaemia (2 genes)

Minor (trait) β

+,

°, β 1 abnormal gene None-mild ↓ (N) Asymptomatic Intermedia β

+

, β

+

β°, β 2 mildly abnormal genes

1 severely abnormal gene Mild-moderate ↓ Variable

Major β°, β° 2 abnormal genes Severe ↓ Transfusion

dependent α Thalassaemia (4 genes)

Silent - α, αα 1 missing gene None N Asymptomatic

2-gene Minor - α, - α

- - ,αα 2 missing genes None-mild N/↓ Asymptomatic

HbH disease - - ,- α 3 missing genes Mod - severe ↓ Variable Barts disease - - , - - 4 missing genes Incompatible with life – death in utero

Sickle Cell (2 genes)

Trait β

s

, β 1 abnormal gene None N Absent to mild

Disease β

s

, β

s

β

s

, β°/β

+

2 abnormal genes Mild to severe N/↓ Vaso-occlusive

phenomena and

haemolysis

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Diagnostic Methods

FBC

Hb, MCV, MCH, RCC, reticulocyte count Blood film

Measure haemoglobins

HbEPG* (Electrophoretic method) HPLC (Chromatographic method)

CE (Electrophoretic method)

> confirm with a second method

• Ancillary tests (HbH bodies, HbBarts ICT, Sickle tests)

• Molecular testing (sequencing, MLPA, Gap PCR)

• Mass spectrometry

• Iron Studies

MCV MCH Haemogl

obins HbH / ICT Heterozygous β

thalassaemia (trait) Low Low HbA

2

Elevated Not detected Heterozygous α

thalassaemia (trait) Low Low HbA

2

Normal Detected sometimes Heterozygous HbS

(Sickle cell trait) Low-

Normal Low-

Normal HbS

elevated Not detected Variant (eg HbE) Low-

Normal Low-

Normal Variant

elevated Not detected

Simplified Diagnostic Criteria

*HbEPG: Haemoglobin Electrophoresis

HPLC: High Performance Liquid Chromatography

CE: Capillary Electrophoresis

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Clinical spectrum in thalassaemia

Musallam et al, Haematologica:2013; 98(6):833

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Management Options in Transfusion Dependent Thalassaemia (TDT)

Established

• Regular PRC transfusion

• Iron chelation: Jadenu (deferasirox), deferiprone, desferrioxamine

• MDT: endocrinopathies, fertility, cardiac

• Chronic disease management Phase 2-3

• HSCT (risks, donors, age)

• Luspatercept

• Gene therapy Early development

• Target hepcidin /erythropoeitic pathways, induce HbF, reduce α-globin synthesis

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Non-Transfusion Dependent Thalassaemia (NTDT)

Musallam et al, Haematologica:2013; 98(6):833

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Proposed pathophysiology for NTDT

Musallam et al, Haematologica:2013; 98(6):833

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Sickle Cell Anaemia

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Sickle Cell anaemia

Haemoglobin S (HbSS, HbSC, HbS-β-thalassaemia) β globin gene defect: Glu-Val substitution

• Deoxy-Hb S is less soluble than deoxy-Hb A, and forms a gelatinous network of fibrous polymers

• Red cells ‘sickle’ at sites of low pO2

• Distorted inflexible red cells adhering to vascular endothelium

• Occlusion in the microvascular circulation

• Infarction (spleen, marrow, brain, kidney, lung, bone)

https://thestrangeandspectacularworldofbiochemistry.wordpress.com/2013/03/24/protein-mutations-and-sickle-cell-anemia/

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Normocytic Anaemia

Microcytic (MCV: <80fl) Normocytic (MCV: 80-100fl) Macrocytic (MCV: >100fl)

Iron deficiency ↓ Production

- BM failure

- Chronic disease

Megaloblastic

- B 12 deficiency

- Folate deficiency Thalassaemia,

Haemoglobinopathy

Sideroblastic Anaemia ↑ Red Cell Loss

- Haemolysis

- Bleeding (acute)

Non-Megaloblastic

- Myelodysplasia

- Liver disease

- Alcohol

- Pregnancy

- Hypothyroidism

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71 yo man

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Bone Marrow Failure

Aplastic

• Idiopathic (autoimmune) ‘Aplastic Anaemia’

• Medications: chloramphenicol, chemotherapy

• Viral

• Inherited (Fanconi, DBA, novel..) Infiltration

• Haematological malignancies

o Acute and chronic leukaemia, myelofibrosis, plasma cell dyscrasias

• Other malignancies: breast, prostate, lung, thyroid, kidney…

Dysfunction

• Myelodysplasia

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Chronic Disease

Causes Pathophysiology?

• Inflammation

• Infection

• Malignancy

• Diabetes

• Renal impairment

• Trauma

• Acute or chronic immune activation

Predominantly a reduction in RBC production by the bone marrow

Hepcidin-induced alterations in iron metabolism

• Reduced absorption of iron from the GI tract

• Trapping of iron in macrophages

• Inability to increase erythropoiesis in response to anemia

• A relative decrease in EPO production

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Haemolysis

Intracorpuscular (intrinsic) defects

 Enzyme deficiency: PK, G6PD

 Membrane defect: hereditary spherocytosis

 Hb synthesis: sickle cell disease

 Acquired: Paroxysmal nocturnal haemoglobinuria Extracorpuscular (extrinsic) defects

 Immune: AIHA

 Microangiopathic: DIC, HUS, prosthetic valve

 Infections: Malaria, Clostridium welchii

 Chemicals (Pb), burns

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Immune haemolytic anaemia

antibodies and / or complements

Primary

• Idiopathic (autoimmune)

• Drugs

• Viral

Associated with other disorders

• Connective tissue disorders (eg SLE)

• Lymphoproliferative disorders

Direct antiglobulin test (DAT)

Wardrop KJ, doi.org/10.1016/j.cvsm.2011.09.005

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Hereditary Spherocytosis

• Spectrin deficiency / unable to assemble spectrin onto the membrane protein

• Variable inheritance

• Lifelong haemolysis (usually compensated)

• Loss of red cell membrane causes spherocytosis

• Most detected by reduction in EMA fluorescence

Kim et al, Blood Research. 52. 84-94, 2017

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Paroxysmal Nocturnal Haemoglobinuria

Acquired clonal disease in multipotent haematopoietic stem cell

• Somatic mutation in PIGA gene:

• Unable to synthesize the glycosyl-

phosphatidyl-inositol (GPI) anchor which attach proteins to cell membrane

• Absent complement inhibitors on cell surface

• Unable to block complement activation

Increased complement-mediated haemolysis

Intravascular haemolysis (hence dark urine) & extravascular

haemolysis

Additional findings

• Aplastic anaemia

• Thrombosis in atypical locations;

venous & arterial

• Smooth muscle dystonia Specific investigation

Flow cytometry to assess GPI-linked

proteins (CD59, CD55)

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Microangiopathic Haemolytic Anaemia

Endothelial disorders

• Haemolytic uraemic syndrome

• Thrombotic thrombocytopenic purpura

• Pregnancy-related: Preeclampsia, HELLP

Disseminated Intravascular Coagulopathy

Prosthetic valve

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Haemolysis Investigations

Anaemia:

• low Hb

• MCV

• Blood film Reticulocytosis LDH raised

Bilirubin raised

• Indirect, unconjugated Haptoglobin low

Urine for haemoglobin or haemosiderin (intravascular haemolysis)

According to cause

• Blood film: spherocytes, fragments, bite &

blister cells

• Direct antiglobulin test

• Abnormal coagulation tests

• Cr, stool

• Enzyme levels

• Loss of CD59, CD55

• EMA fluorescence

• Family studies

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Macrocytic Anaemia

Microcytic (MCV: <80fl) Normocytic (MCV: 80-100fl) Macrocytic (MCV: >100fl)

Iron deficiency ↓ Production

- BM failure

- Chronic disease

Megaloblastic

- B 12 deficiency

- Folate deficiency Thalassaemia,

Haemoglobinopathy

Sideroblastic Anaemia ↑ Red Cell Loss

- Haemolysis

- Bleeding (acute)

Non-Megaloblastic

- Myelodysplasia

- Liver disease

- Alcohol

- Pregnancy

- Hypothyroidism

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Macrocytic Anaemias

Megaloblastic

• B12/folate/homocysteine pathways; GI B 12 requirements

• Commonest causes:

• B 12 : Pernicious anaemia, vegan

• Folate: diet Myelodysplasia

• Clonal bone marrow disorder

• Requires blood film, BMAT, molecular studies for diagnosis

• Prognostic scoring system

• Supportive care, Azacitdine, others…

https://quizlet.com/164061348/gi-system-flash-cards/

https://path.upmc.edu/cases/case428/dx.html

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Investigation of Anaemia summary

• Full blood count o Haemoglobin

o Platelets, white cell count o MCV

• Blood film

• Reticulocyte count

• Iron studies

• B 12 & Folate Studies

• Haemolysis markers

• Liver & renal function tests

Use the MCV to evaluate the cause

Ferritin is the best measure generally

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Update on Anaemia Part 2:

Multiple Choice Questions

Dr Annmarie Bosco

Haematologist, Prince of Wales Hospital University of New South Wales

Multiple choice questions refer to CLS Anaemia lecture by Dr Giselle Kidson-Gerber

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Microcytic anaemia

Which of the following is not a cause of microcytic anaemia?

A. β thalassemia trait B. Alcohol excess

C. Sideroblastic anaemia D. Lead poisoning

E. Iron deficiency

Ref CLS Anaemia Slide 4

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Microcytic anaemia

• Which of the following is not a cause of microcytic anaemia?

A. β thalassemia trait B. Alcohol excess

C. Congenital sideroblastic anaemia D. Lead poisoning

E. Iron deficiency

Ref CLS Anaemia Slide 4

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Key points

• Hemoglobin consists of 2 alpha and 2 β globin chains

• Microcytic anaemia occurs due to reduced production of hemoglobin

• β thalassemia trait is due to a mutation in the beta globin chain

• Alcohol excess is toxic to developing red cells and stops them dividing normally. This causes a macrocytic anaemia.

• Congenital sideroblastic anemia results in reduced hemoglobin due to defects in the heme synthesis pathway

• Lead poisoning interferes with heme synthesis and RBC survival

• Iron is an important component of hemoglobin. Iron deficiency

results in reduced hemoglobin production.

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• Which of the following iron study profiles is most consistent with iron deficiency anemia?

Serum Iron Transferrin Transferrin saturation

A. Normal Normal Low

B. Low Low High

C. Low High Low

D. Low High High

E. Low High Normal

Ref: CLS Anaemia Page 10

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• Which of the following iron study profiles is most consistent with iron deficiency anemia?

Serum Iron Transferrin Transferrin saturation

A. Normal Normal Low

B. Low Low High

C. Low High Low

D. Low High High

E. Low High Normal

Ref: CLS Anaemia Page 10

Transferrin is the carrier protein for iron in the serum. The levels increase in

iron deficiency anaemia and the transferrin saturation reduces. Transferrin

levels decrease during inflammation or in anaemia of chronic disease.

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A 65-year-old Caucasian woman is on prednisolone for polymyalgia

rheumatica. She has active shoulder girdle pain and stiffness. She now

presents with lethargy and indigestion.

Her results are below:

Hb 108g/L (115-165) MCV 78 fL (80-100)

WBC 12.4 x 10 9 /L (3.5 – 11) Platelets 454 x 10 9 /L

ESR 50 mm/hr (0-20)

The iron studies show:

Iron level 11.6 umol/L (5.0 – 30.4 umol/L) Transferrin saturation 16% (20-51)

Transferrin 1.6 (1.8-3.5) Ferritin 200 ug/L (30-300)

What is the most likely cause of her anaemia?

• A. Inflammation

• B. Alpha thalassemia trait

• C. Prednisolone

• D. Iron deficiency

• E. Immune-mediated haemolysis

Ref CLS Anaemia Slide 10, 11

(46)

A 65-year-old Caucasian woman is on prednisolone for polymyalgia

rheumatica. She has active shoulder girdle pain and stiffness. She now

presents with lethargy and indigestion.

Her results are below:

Hb 108g/L (115-165) MCV 78 fL (80-100)

WBC 12.4 x 10 9 /L (3.5 – 11) Platelets 454 x 10 9 /L

ESR 50 mm/hr (0-20)

The iron studies show:

Iron level 11.6 umol/L (5.0 – 30.4 umol/L) Transferrin saturation 16% (20-51)

Transferrin 1.6 (1.8-3.5) Ferritin 200 ug/L (30-300)

What is the most likely cause of her anaemia?

• A. Inflammation

• B. Alpha thalassemia trait

• C. Prednisolone

• D. Iron deficiency

• E. Immune-mediated haemolysis

Ref CLS Anaemia Slide 10, 11

(47)

A 65-year-old Caucasian woman is on prednisolone for polymyalgia

rheumatica. She has active shoulder girdle pain and stiffness. She now

presents with lethargy and indigestion.

Her results are below:

Hb 108g/L (115-165)

MCV 78 fL (80-100) microcytic

WBC 12.4 x 10 9 /L (3.5 – 11) inflammation Platelets 454 x 10 9 /L inflammation

ESR 50 mm/hr (0-20) inflammation

The iron studies show:

Iron level 11.6 umol/L (5.0 – 30.4 umol/L) Transferrin saturation 16% (20-51)

Transferrin 1.6 (1.8-3.5) Ferritin 200 ug/L (30-300)

What is the most likely cause of her anaemia?

A. Inflammation

B. Alpha thalassemia trait C. Prednisolone

D. Iron deficiency

E. Immune-mediated haemolysis

Ref CLS Anaemia Slide 10, 11

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Key points

• A low serum transferrin can be an indicator of inflammation

• A high serum transferrin suggests iron deficiency

• There are other markers of inflammation in the question that are hints, such

as the elevated white cell count, platelet count and ESR

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What is the pathophysiology of the anaemia in the previous question?

A. Prednisolone has decreased ferroportin and reduced iron absorption and transferrin saturation B. Ineffective erythropoiesis has increased red blood cell breakdown and hemolysis

C. Cytokine-induced hepcidin increase has decreased ferroportin, reduced iron absorption and transferrin saturation

D. Bleeding has caused a depletion in her iron stores and reduced red cell synthesis

E. Anti-gliadin antibodies have caused gastrointestinal inflammation leading to reduced iron absorption

Ref: CLS Anaemia Slide 9, 12, 14

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What is the pathophysiology of the anaemia in the previous question?

A. Prednisolone has decreased ferroportin and reduced iron absorption and transferrin saturation B. Ineffective erythropoiesis has increased red blood cell breakdown and hemolysis

C. Cytokine-induced hepcidin increase has decreased ferroportin, reduced iron absorption and transferrin saturation

D. Bleeding has caused a depletion in her iron stores and reduced red cell synthesis

E. Anti-gliadin antibodies have caused gastrointestinal inflammation leading to reduced iron absorption

Ref: CLS Anaemia Slide 9, 12, 14

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Ref CLS Anaemia slide 18, 22 A 21-year-old Indonesian woman has been referred for investigation of anaemia. Her family history is significant for an older sibling who has anaemia, leg ulcers and splenomegaly requiring occasional

transfusions.

The blood counts and iron studies are as follows:

• Haemoglobin 78 g/L [115-165]

• Haematocrit 0.29 [0.32-0.42]

• Red blood cells 4.14 x10 9 /L [3.60-5.30]

• Mean corpuscular volume 71 fL [80-100]

• Reticulocytes 200 x10 9 /L [20-80]

• Serum ferritin 378 ug/L [15-200]

• Results of her hemoglobin electrophoresis are:

• HbA2 1.9 % [2.0-3.5]

• HbF 0.3 % [<1.1]

• No abnormal bands seen

Which of the following is the most likely diagnosis?

A. beta0-thalassaemia trait B. beta+-thalassaemia trait C. Sickle-beta thalassemia

D. alpha+-thalassaemia trait (α-/αα)

E. alpha0-thalassaemia trait (--/αα)

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Ref CLS Anaemia slide 18, 22 A 21-year-old Indonesian woman has been referred for investigation of anaemia. Her family history is significant for an older sibling who has anaemia, leg ulcers and splenomegaly requiring occasional

transfusions.

The blood counts and iron studies are as follows:

• Haemoglobin 78 g/L [115-165]

• Haematocrit 0.29 [0.32-0.42]

• Red blood cells 4.14 x10 9 /L [3.60-5.30]

• Mean corpuscular volume 71 fL [80-100]

• Reticulocytes 200 x10 9 /L [20-80]

• Serum ferritin 378 ug/L [15-200]

• Results of her hemoglobin electrophoresis are:

• HbA2 1.9 % [2.0-3.5]

• HbF 0.3 % [<1.1]

• No abnormal bands seen

Which of the following is the most likely diagnosis?

A. beta0-thalassaemia trait B. beta+-thalassaemia trait C. Sickle-beta thalassemia

D. alpha+-thalassaemia trait (α-/αα)

E. alpha0-thalassaemia trait (--/αα)

(53)

Key points

• The patient’s sibling has features of non-transfusion dependent thalassemia

• The patient has a hypochromic microcytic anemia with an elevated ferritin

• Her HbA2 is normal – this excludes beta-thalassemia

• There are no abnormal bands seen on her Hb EPG – this excludes sickle cell disease

• α thalassemia with one gene deletion is usually silent, meaning that the patient’s Hb is not reduced and the MCV is normal

• α thalassemia with two gene deletions causes a hypochromic microcytic

anaemia

(54)

A 20 year old student from Greece presents for a check up and is found to have mild anemia. His blood count is a below:

• Hb 115 g/L [128-175]

• Mean cell volume 62 fL [80-97]

• Platelets 390 x 10 9 /L [150-400]

• Serum ferritin 295 μg/L [15-325]

The blood film reports microcytes and occasional target cells

• A hemoglobin electrophoresis is arranged which shows:

• HbA2 5.2% [1.8-3.5]

• HbF 1.2% [0-2.0]

• No abnormal Hb bands seen

• HbH preparation: no HbH bodies seen

The most likely explanation for his anaemia is:

A. Iron deficiency anaemia

B. Heterozygous β thalassaemia C. Homozygous β thalassemia C. Homozygous sickle cell disease D. α-thalassemia trait

Ref: CLS Anaemia slides 18, 19

(55)

A 20 year old student from Greece presents for a check up and is found to have mild anemia. His blood count is a below:

• Hb 115 g/L [128-175]

• Mean cell volume 62 fL [80-97]

• Platelets 390 x 10 9 /L [150-400]

• Serum ferritin 295 μg/L [15-325]

The blood film reports microcytes and occasional target cells

• A hemoglobin electrophoresis is arranged which shows:

• HbA2 5.2% [1.8-3.5]

• HbF 1.2% [0-2.0]

• No abnormal Hb bands seen

• HbH preparation: no HbH bodies seen

The most likely explanation for his anaemia is:

A. Iron deficiency anaemia

B. Heterozygous β thalassaemia C. Homozygous β thalassemia C. Homozygous sickle cell disease D. α-thalassemia trait

Ref: CLS Anaemia slides 18, 19

(56)

Key points

• The patient has a hypochromic microcytic anaemia with normal ferritin levels

• This is not suggestive of iron deficiency

• The HbA2 is increased. This is suggestive of β thalassemia

• β thalassemia major is diagnosed in early childhood, not in adolescence

• The HbA2 is not increased in α thalassemia trait

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• Which one of the following combinations would cause the least concern to a couple presenting for genetic counselling?

CLs anaemia slide 14, 18, 19

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• Which one of the following combinations would cause the least concern to a couple presenting for genetic counselling?

CLs anaemia slide 14, 18, 19 Answer: E

Key point: The imbalance between α and β chains is the key determinant of disease severity in thalassemia.

Co-inheritance of α and β gene abnormalities reduces the imbalance as it reduces the excess of one type of chain over the other

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Sickle cell anemia

What is the cause of vaso-occlusive crises in sickle cell disease?

A. Red cell membrane fragments occluding microvasculature

B. Increased solubility of HbS compared to HbA at low temperatures C. Red cells clumping at sites of low pO2

D. Obstruction of microvascular circulation due to inflexible red cells E. Hemoglobin not releasing oxygen due to high affinity

Ref CLS Anaemia Slide 25

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Sickle cell anemia

What is the cause of vaso-occlusive crises in sickle cell disease?

A. Red cell membrane fragments occluding microvasculature

B. Increased solubility of HbS compared to HbA at low temperatures C. Red cells clumping at sites of low pO2

D. Obstruction of microvascular circulation due to inflexible red cells E. Hemoglobin not releasing oxygen due to high affinity

Ref CLS Anaemia Slide 25

(61)

41-year-old man presents with fatigue, bruising, dyspnoea. He has been well until a few weeks ago. He has a complete diet, does not consume alcohol and is not on any prescribed medications.

His blood count is as follows:

• Hb 107 g/L (130-180)

• MCV 85 fL (80-100)

• WBC 2.67 x 10 9 /L (3.5 -11.0)

• Platelets 48 x 10 9 /L (150 – 450)

Which of the following results would confirm your suspicion of bone

marrow failure?

A. HbH inclusion bodies in red cells B. Fragments on blood film

C. Hypersegmented neutrophils D. Low platelet count

E. Low reticulocyte count

Ref: CLS Anemia Slide 27

(62)

41-year-old man presents with fatigue, bruising, dyspnoea. He has been well until a few weeks ago. He has a complete diet, does not consume alcohol and is not on any prescribed medications.

His blood count is as follows:

• Hb 107 g/L (130-180)

• MCV 85 fL (80-100)

• WBC 2.67 x 10 9 /L (3.5 -11.0)

• Platelets 48 x 10 9 /L (150 – 450)

Which of the following results would confirm your suspicion of bone

marrow failure?

A. HbH inclusion bodies in red cells B. Fragments on blood film

C. Hypersegmented neutrophils D. Low platelet count

E. Low reticulocyte count

Ref: CLS Anemia Slide 27

(63)

Key points

• A low reticulocyte count is seen in cases of bone marrow hypoproduction

• Aplastic anemia is a condition where the normal marrow tissue is replaced by fat

• It presents with pancytopenia

• Reticulocytes < 1% or absolute reticulocytes < 40% are suggestive of

severe aplastic anemia

(64)

Pathophysiology of anaemia of chronic disease – all except

• A. Reduced absorption of iron from GI tract

• B. Increased production of immature red cells

• C. Reduced erythropoeitin production

• D. Sequestration of iron in macrophages

• E. Increased hepcidin

Ref: CLS anemia slide 29

(65)

Pathophysiology of anaemia of chronic disease – all except

• A. Reduced absorption of iron from GI tract

• B. Increased production of immature red cells

• C. Reduced erythropoeitin production

• D. Sequestration of iron in macrophages

• E. Increased hepcidin

Ref: CLS anemia slide 29

(66)

A 55-year-old woman with a history of systemic lupus

erythematosus presents with a 2- week history of dyspnea and

fatigue. Clinically there is pallor and a tippable spleen.

Her results are as below:

• Hb 70g/L (115-165)

• Neutrophils 11.20 x 10 9 /L (3.5-11)

• Platelets 443 x 10 9 /L (150-450)

• LDH 800 U/L (150-250)

• Total bilirubin 89 umol/L (0-20)

• Spherocytes are reported on her blood film

What is the next best test to demonstrate the cause of anaemia?

A. Urinary hemosiderin B. Serum haptoglobin

C. Unconjugated bilirubin fraction

D. Direct and indirect antiglobulin (Coombs) tests

E. Peripheral blood flow cytometry

Ref: CLS Anemia Slide 31; https://en.wikipedia.org/wiki/Spherocytosis

(67)

A 55-year-old woman with a history of systemic lupus

erythematosus presents with a 2- week history of dyspnea and

fatigue. Clinically there is pallor and a tippable spleen.

Her results are as below:

• Hb 70g/L (115-165)

• Neutrophils 11.20 x 10 9 /L (3.5-11)

• Platelets 443 x 10 9 /L (150-450)

• LDH 800 U/L (150-250)

• Total bilirubin 89 umol/L (0-20)

• Spherocytes are reported on her blood film

What is the next best test to demonstrate the cause of anaemia?

A. Urinary hemosiderin B. Serum haptoglobin

C. Unconjugated bilirubin fraction

D. Direct and indirect antiglobulin (Coombs) tests

E. Peripheral blood flow cytometry

Ref: CLS Anemia Slide 31; https://en.wikipedia.org/wiki/Spherocytosis

(68)

A 22-year-old man has been noted to have yellowing of his eyes on occasion. This has happened for years when he gets sick. His medical history is otherwise unremarkable. His conjunctivae are mildly icteric but otherwise the examination is normal.

Diagnostic testing results are shown below:

Hb 122 g/L (130-180) MCV 88 fL (80-100)

Platelets 295 x 10 9 /L (150-450)

White cell count 9.0 x 10 9 /L (2.0 – 7.5) Reticulocytes 5% (0.2-2.0)

Reticulocytes absolute 95 x 10 9 /L (20-80)

Peripheral blood film report: Anisocytosis, polychromasia, spherocytes Bilirubin 53 umol/L (0-20)

Which of the following tests would most likely help confirm the diagnosis?

A. Hemoglobin electrophoresis B. Red cell EMA quantification

C. Direct and indirect antiglobulin (Coombs) tests D. Bone marrow aspiration and biopsy

E. Red cell flow cytometry for CD 59 and CD 55 Ref CLS Anemia slide 32, 33

https://doi.org/10.7939/R3HM53096

(69)

A 22-year-old man has been noted to have yellowing of his eyes on occasion. This has happened for years when he gets sick. His medical history is otherwise unremarkable. His conjunctivae are mildly icteric but otherwise the examination is normal.

Diagnostic testing results are shown below:

Hb 122 g/L (130-180) MCV 88 fL (80-100)

Platelets 295 x 10 9 /L (150-450)

White cell count 9.0 x 10 9 /L (2.0 – 7.5) Reticulocytes 5% (0.2-2.0)

Reticulocytes absolute 95 x 10 9 /L (20-80)

Peripheral blood film report: Anisocytosis, polychromasia, spherocytes Bilirubin 53 umol/L (0-20)

Which of the following tests would most likely help confirm the diagnosis?

A. Hemoglobin electrophoresis B. Red cell EMA quantification

C. Direct and indirect antiglobulin (Coombs) tests: As the haemolysis is not immune mediated, the direct Coombs test will be negative

D. Bone marrow aspiration and biopsy: This test is for Paroxysmal Nocturnal Haemoglobinuria

E. Red cell flow cytometry for CD 59 and CD 55 Ref CLS Anemia slide 32, 33

https://doi.org/10.7939/R3HM53096

(70)

Which of these statements is true about the condition in the previous question?

A. The red cells have an abnormality in a membrane protein B. The red cells are deficient in a metabolic enzyme defect C. It is an X-linked condition

D.The red cells have an abnormally high surface:volume ratio

E. The red cells have an increased sensitivity to lysis by complement

Ref CLS Anemia Slide 32, 33

(71)

Which of these statements is true about the condition in the previous question?

A. The red cells have an abnormality in a membrane protein B. The red cells are deficient in a metabolic enzyme defect C. It is an X-linked condition

D.The red cells have an abnormally high surface:volume ratio

E. The red cells have an increased sensitivity to lysis by complement

Ref CLS Anemia Slide 32, 33

(72)

The following are all causes of

microangiopathic hemolytic anaemia except:

A. Paroxysmal nocturnal hemoglobinuria B. Disseminated intravascular coagulation C. Prosthetic valve-related hemolysis

D. Hemolytic uremic syndrome

E. Thrombotic thrombocytopenic purpura

Ref: CLS anaemia Slide 33, 34

(73)

The following are causes of microangiopathic hemolytic anaemia except:

A. Paroxysmal nocturnal hemoglobinuria B. Disseminated intravascular coagulation C. Prosthetic valve-related hemolysis

D. Hemolytic uremic syndrome

E. Thrombotic thrombocytopenic purpura

Ref: CLS Anaemia Slide 33, 34

(74)

Which is most specific for intravascular hemolysis?

A. Low haptoglobin B. High reticulocytes C. Positive DAT for IgG

D. Positive urinary hemosiderin E. Positive cryoglobulins

Ref CLS Anaemia Slide 35

(75)

Which is most specific for intravascular hemolysis?

A. Low haptoglobin B. High reticulocytes C. Positive DAT for IgG

D. Positive urinary hemosiderin E. Positive cryoglobulins

Ref CLS Anaemia Slide 35

(76)

22-year-old woman is brought to the emergency

department after having a witnessed tonic-clonic seizure.

She had appeared confused for the preceding few hours.

She has a history of vitiligo but is otherwise well with no prior medical history. Her results are as follows:

WCC H 11.50 x10^9/L (4.5 – 11) Hemoglobin L 92 g/L (115-165) Platelets L 19 x10^9/L (150-450) MCV 78.0 fL (80-100)

Blood film: Anisocytosis, poikilocytosis, schistocytes Creatinine 101 umol/L (38-82)

Bilirubin 37 umol/L (0-20) LDH 662 U/L(<250)

PT second(s) 14 (11 – 15) INR 1.2

APTT 35.8 second(s) (31-39)

What is the most diagnostic test result that fits with her clinical presentation?

A. Low ADAMTS13 level

B. Low EMA by flow cytometry C. Low complement levels D. Low fibrinogen

E. Low reticulocytes

Ref CLS Anaemia Slide 34

(77)

22-year-old woman is brought to the emergency

department after having a witnessed tonic-clonic seizure.

She had appeared confused for the preceding few hours.

She has a history of vitiligo but is otherwise well with no prior medical history. Her results are as follows:

WCC H 11.50 x10^9/L (4.5 – 11) Hemoglobin L 92 g/L (115-165) Platelets L 19 x10^9/L (150-450) MCV 78.0 fL (80-100)

Blood film: Anisocytosis, poikilocytosis, schistocytes Creatinine 101 umol/L (38-82)

Bilirubin 37 umol/L (0-20) LDH 662 U/L(<250)

PT second(s) 14 (11 – 15) INR 1.2

APTT 35.8 second(s) (31-39)

What is the most diagnostic test result that fits with her clinical presentation?

A. Low ADAMTS13 level

B. Low EMA by flow cytometry C. Low complement levels D. Low fibrinogen

E. Low reticulocytes

Ref CLS Anaemia Slide 34

(78)

An 78-year-old Caucasian female with a background of thyroiditis presents with bruising and fatigue. On examination, there is a low body mass index and pallor. You suspect vitamin B12

deficiency. Her blood results are as below:

WCC 3.10 x10^9/L Haemoglobin 108 g/L Platelets 58 x10^9/L MCV 103.0 fL

Blood film report: pancytopenia, hypersegemented neutrophils, oval macrocytes

The initial Vitamin B12 level is 196 pmol/L (150-700)

Which of the following confirms your suspicion?

A. Low folate level B. Low homocysteine

C. Low methylmalonic acid D. Low holotranscobalamin E. Low pyridoxine

Ref CLS Anaemia Slide 37

http://www.seals.health.nsw.gov.au/SiteFiles/sesiahshealthnswgovau/CHEMAI_VitaminB12_Testing_Fact_Sheet_V3_March_19.pdf

(79)

An 78-year-old Caucasian female with a background of thyroiditis presents with bruising and fatigue. On examination, there is a low body mass index and pallor. You suspect vitamin B12

deficiency. Her blood results are as below:

WCC 3.10 x10^9/L Haemoglobin 108 g/L Platelets 58 x10^9/L MCV 103.0 fL

Blood film report: pancytopenia, hypersegemented neutrophils, oval macrocytes

The initial Vitamin B12 level is 196 pmol/L (150-700)

Which of the following confirms your suspicion?

A. Low folate level B. Low homocysteine

C. Low methylmalonic acid D. Low holotranscobalamin E. Low pyridoxine

Ref CLS Anaemia Slide 37

http://www.seals.health.nsw.gov.au/SiteFiles/sesiahshealthnswgovau/CHEMAI_VitaminB12_Testing_Fact_Sheet_V3_March_19.pdf

(80)

Key points

• Holotranscobalamin (holoTC) is the active form of B12 taken up into cells

• Measurement of holoTC is a more reliable method of detecting B12 deficiency

• MMA and homocysteine are functional markers of vitamin B12 status. The levels increase when vitamin B12 deficiency is present

Ref CLS Anaemia Slide 37

http://www.seals.health.nsw.gov.au/SiteFiles/sesiahshealthnswgovau/CHEMAI_VitaminB12_Testing_Fact_Sheet_V3_March_19.pdf

https://www1.health.gov.au/internet/main/publishing.nsf/Content/7C28F0B6D06F9FFCCA257EB9001D769B/$File/Vitamin%20B12%20testing%20Protocol.pdf

(81)

What is the most specific test to determine the underlying cause in the patient above?

A. Anti parietal cell antibodies B. Anti intrinsic factor antibodies C. Anti transglutaminase antibodies D. Anti gliadin antibodies

E. Anti mitochondrial antibodies

Check on pernicious anemia testsing

Ref CLS Anaemia Slide 37

(82)

What is the most specific test to determine the underlying cause in the patient above?

A. Anti parietal cell antibodies B. Anti intrinsic factor antibodies C. Anti transglutaminase antibodies D. Anti gliadin antibodies

E. Anti mitochondrial antibodies

Ref CLS Anaemia Slide 37

https://www.rcpa.edu.au/Manuals/RCPA-Manual/Clinical-Problems/V/Vitamin-B12-deficiency

(83)

A 77-year-old man is evaluated for

exertional dyspnea and fatigue. There has been a progressive macrocytic

anaemia over the last 3 years. Most

recently, his blood counts are as follows:

Hb 85 g/L (130-180) MCV 108 Fl (80-100)

White cell count 3.43 X 10 9 /L (4.5 – 11) Neutrophils 1.07 X 10 9 /L (1.7-7.0)

Platelets 136 X 10 9 /L (150-450) Reticulocytes 1.6% (0.2 – 2.0)

Blood film: mild polychromasia, round macrocytes, hypogranular neutrophils, hypogranular platelets

Which of the following genetic changes is likely to be detected in his bone

marrow biopsy?

A. t(9, 22)

B. JAKV617F mutation

C. abnormalities of chromosome 7 D. C282Y mutation

E. HBB 6 Glu>Val substitution

Ref: CLS Anemia slide 37

(84)

A 77-year-old man is evaluated for

exertional dyspnea and fatigue. There has been a progressive macrocytic

anaemia over the last 3 years. Most

recently, his blood counts are as follows:

Hb 85 g/L (130-180) MCV 108 Fl (80-100)

White cell count 3.43 X 10 9 /L (4.5 – 11) Neutrophils 1.07 X 10 9 /L (1.7-7.0)

Platelets 136 X 10 9 /L (150-450) Reticulocytes 1.6% (0.2 – 2.0)

Blood film: mild polychromasia, round macrocytes, hypogranular neutrophils, hypogranular platelets

Which of the following genetic changes is likely to be detected in his bone

marrow biopsy?

A. t(9, 22)

B. JAKV617F mutation

C. abnormalities of chromosome 7 D. C282Y mutation

E. HBB 6 Glu>Val substitution

Ref: CLS Anemia slide 37

MDS most commonly manifests as isolated macrocytic anemia.

This can evolve to include pancytopenia over several years.

(85)

College Learning Series Page 39

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