Haem Flashcards
Blood composition and haematopoesis
- Composition
a. RBC (haematocrit) = 45%
b. Platelets/WBC = 1%
c. Plasma = 55%
i. Water, metabolites (CHO, FFA etc), proteins, electrolytes, hormones, gases, clotting factors
ii. Serum = plasma without clotting factors and fibrinogen
iii. Plasma proteins = albumin (liver), fibrinogen (liver), globulins (plasma cells) - Haematopoeisis
a. All cells arise from pleuripotent stem cell
i. Promegakaryocyte = platelets
ii. Pro-erythroblast = RBC
iii. Myeloblast = neutrophils, basophils, eosinophils
iv. Monoblasts = monocytes
v. Lymphoblasts = lymphocytes
b. Committed cells have receptors for the colony stimulating factors
c. Myeloid stem cell -> promegakaryocyte, pro-erythroblast, myeloblast, monoblast
d. Lymphoid stem cell -> lymphoblasts - Stages of haematopoeisis
a. Mesoblastic (extraembryonic structures – yolk sac)
i. Starts at 10-14 days
ii. Ceases around 10-20 weeks
iii. Taken over by liver
b. Hepatic – in liver
i. Start sat 6-8 weeks
ii. Continues for remainder of gestation, diminishes in second trimester
iii. Predominantly erythropoietic
c. Marrow
i. Increases in second trimester
ii. Produces erythrocyte and neutrophils
Erythrocytes - general physiology
a. Biconcave disc shaped cells
b. Stages in development:
i. Proerythroblast
ii. Normoblast (basophilic polychromatic orthochromatic )
iii. Reticulocyte (following extrusion of the nucleus)
iv. Mature erythrocyte
c. Regulation = EPO
d. Energy production
i. NO mitochondria
ii. Produces energy via anaerobic metabolism/glycolysis (the Embden-Meyerhodd pathway)
iii. Results in production of 2,3 DPG
1. Binds with greater affinity to deoxygenated blood than oxygenated blood
2. Interacts with deoxygenated Hb by decreasing affinity to oxygen – so allosterically promotes the release of remaining oxygen molecules bound to Hb
3. Ie. results in Hb curve shift to the RIGHT
e. Lifespan = 120 days
f. Broken down in reticuloendothelial system of the spleen
i. Globin hydrolysed to free amino acids
ii. Heme iron released and transferred to transferrin
iii. Heme converted to biliverdin by macrophages binds to bilirubin
g. Cytoskeleton
i. Unique – maintains shape but is flexible to squeeze through microvasculature
ii. Maintained by key proteins: spectrin, Ankyrin, protein 4.1, actin
h. Enzymes
i. Carbonic anhydrase – conversion of Co2 + H2O = H2CO3 (CO2 binds to globin not heme)
i. Development with age
i. Produced in the fetal liver during 1st and 2nd trimesters
ii. Fetal EPO binds to immature RBC and stimulates differentiation
iii. Cells often larger in size than adult RBC
j. Haemaglobin = 33% of cytoplasm
Platelets - physiology
a. Cell development
i. Regulated by TPO
ii. Formed as megakaryocytes in the bone marrow
iii. Megakaryocytes ‘fragment ‘ into minute platelets in the bone marrow/after entering the blood
iv. Normal life span in circulation is 10-14 days
v. 25-40% stored in the spleen, removed by macrophages in the reticuloendothelial system
b. Development with age
i. Production increases from 22-40 weeks gestation
ii. Production is regulated by TPO (coded for by long arm of chromosome 3)
c. Key characteristics
i. No nucleus
ii. Contain contractile actin + myosin molecules
iii. Have residuals of endoplasmic reticulum + Golgi apparatus that synthesize enzymes + store Ca++
iv. Mitochondria that can form ATP / ADB
v. Enzyme synthesize
vi. Receptors for: vWF, fibrinogen, agonists that trigger platelet aggregation (thrombin, collagen, ADP)
Neutrophils - physiology
a. Characteristics = 3-5 lobes
b. Normal development
i. Start as granulocytes
ii. Develop under influence of SCF/ GMCSF/ GCSF, IL3/6/11
iii. Survive 4-5 days in the circulation
c. Actions
i. Phagocytosis of bacteria
ii. Release antimicrobial chemicals via NADPH oxidation pathway
iii. Key processes: touches endothelium, starts rolling via selectin interaction, increases expression of adhesion molecules CD18 – adheres to endothelium, undergoes diapedesis, emigration and chemotaxis
d. Development with age
i. Macrophages appear first in yolk sac/ liver/ lung and brain
ii. Neutrophils observed from about 5 weeks
iii. Increase when developing in marrow space
e. ↑ in bacterial infection, lymphoproliferative disease
Eosinophils - physiology
a. Characteristics = 2 large nuclei, pink granules in cytoplasm
b. Normal development
i. Starts as granulocyte
ii. Accounts for 2-4 % BC
iii. Survives for 4-5 days
c. Actions
i. Phagocytosis of antigen-antibody complex, allergens and inflammatory chemicals
ii. Release oxidizing enzymes that destroy parasites/worms, degranulate to release membrane toxic granules (this is mostly extracellular)
iii. Limit action of histamine and other proinflammatory chemicals
d. ↑ in CHINA
i. Churg Straus
ii. Hereditary eosinophilia
iii. Infection (helminth/parasites)
iv. Neoplasm
v. Atopic conditions (atopic dermatitis being the most prominent; ABPA)
Basophils - physiology
a. Characteristics = violet granules
b. Normal development
i. Starts as a granulocyte
ii. Accounts for 0.5-1% normal WBC in the peripheral system
iii. Survive for 4-5 days
c. Functions
i. Secrete histamine to promote blood flow
ii. Secrete heparin
iii. Release tryptase, heparin, histamine, proteoglyons, chondroitin (does NOT release interferon)
d. ↑ in VAV, DM, myxedema, sinusitis, polycythaemia
Lymphocytes - physiology
a. Characteristics = single nucleus with dimple on the side, usually minimal cytoplasm
b. Development
i. From lymphoid progenitor
ii. Accounts for 25-33% WBC – 85% T cell, 15% B cells, 5% NK cells
iii. Can survive for years
iv. Key signaling: IL2 – T/ B/ NK cells, IL7/15: T and NK cell development
c. Function
i. Dependent on cell type
ii. Perforin punches holes in membrane, granzymes inserted in side
d. ↑ viral infection, lymphoproliferative disease
Monocytes - physiology
a. Characteristics = kidney shaped nucleus, abundant cytoplasm with small granules
b. Functions
i. Differentiate to macrophages (depending on tissue)
ii. Phagocytosis
iii. Can differentiate into APC
iv. Survive for years
c. ↑ with viral infection, inflammation
Anisocytosis - definition
= RBC with increased variability in size (increased RDW on FBE)
Poikilocytosis - definition
= increased proportion of RBCs of abnormal shape (e.g. IDA, myelofibrosis)
Discocyte - RBC morphology
Biconcave disc = normal
Spherocyte - RBC morphology
Spherical RBC due to loss of membrane
Smaller, lack central pallor
- Hereditary spherocytosis
- Immune haemolytic anaemia
- Post splenectomy
- Liver disease
Eliptocyte/ovalocyte - RBC morphology
Oval shaped, elongated RBC
- Hereditary elliptocytosis
- Megaloblastic anaemia
- Myelofibrosis
- IDA
- MDS
Schistocyte - RBC morphology
Helmet cell
Fragmented cells due to traumatic disruption of membrane
- Microangiopathic haemolytic anaemia (eg. HUS/TTP, DIC)
- Vasculitis
- GN
- Prosthetic heart valve
Sickle cell - RBC morphology
Sickle (scythe) shaped RBC due to polymerization of HbS
• Sickle cell disorders
Target cell - RBC morphology
Bullseye on dried film
- Liver disease (macrocytic)
- Thalassaemia (microcytic)
- Hb SC
- IDA
- Asplenia
Tear drop cell - RBC morphology
Looks like teardrop
- Myelofibrosis
- Thalassaemia major
- Megaloblastic anaemia
Spur cell - RBC morphology
Distorted RBC with irregularly distributed thorn-like projections (due to abnormal membrane lipids)
- Severe disease (spur cell anaemia)
- Starvation/anorexia
- Post-splenectomy
- Vitamin E deficiency
- Burns
Burr cell - RBC morphology
RBC with numerously regularly spaced, small spiny projections
- Uraemia
- HUS
- Burns
- Cardiopulmonary bypass
- Post-transfusion
- Storage artefact
Rouleaux formation - RBC morphology
Aggregates of RBC resembling stacks of coins
Due to increased plasma concentrations of high MW proteins
- Inflammatory conditions – due to polyclonal Ig
- Plasma cell dyscrasias – due to monoclonal paraproteinaemias eg. multiple myeloma, macroglubinaemia
Blister/bite cell - RBC morphology
Abnormally shaped RBC with semicircular portions removed from the cell margin
Bites from removal of denatured Hb by macrophages in the spleen
• Oxidative haemolysis, most commonly G6PD
Acanthocytes - RBC morphology
RBC with spiked membrane
Similar to spur cell
- Abetalipoproteinaemia
- Malabsorption
RBC inclusions - general
Nucleus
Present in erythroblasts (immature RBCs)
• Hyperplastic erythropoiesis (hypoxia, haemolytic anaemia)
• Extramedullary haematopoeisis (BM infiltration)
Heinz bodies Denatured and precipitated Hb • Oxidative stress • G6PD deficiency (post exposure to oxidant) • Thalassaemia • Unstable Hb
Howell-Jolly bodies Small nuclear remnant resembling a pyknotic nucleus • Post-splenectomy • Hyposplenism (sickle cell) • Neonates • Megaloblastic anaemia
Basophilic stippling
Deep blue granulations indicating ribosome aggregation • Thalassaemia
• Heavy metal poisoning
• Megaloblastic anaemia
• Hereditary (pyrimidine 5’nucleotidase deficiency)
Sideroblasts Erythrocytes with Fe containing granules in the cytoplasm • Hereditary • Idiopathic • Drugs • Hypothyroidism
RBC colour - hypochromasia vs polychomasia
a. Hypochromic = increased size of central pallor (normally <1/3 RBC diameter)
i. IDA
ii. Anaemia of chronic disease
iii. Haemolytic anaemias
iv. Sideroblastic anaemia
b. Polychromasia = increased reticulocytes (pinkish-blue)
i. Increased RBC production by the marrow
WBC morphology - general
a. Critical to assess morphology – machine may miss blasts, cannot identify left shift
b. Lymphocytes
i. Reed-Sternberg cell = giant, multinucleated B lymphocyte
ii. Smudge cell = lymphocyte damaged during preparation of blood smear indicating cell fragility, seen in CLL and other lymphoproliferative disorders
c. Neutrophils
i. Blasts promyelocyte myelocyte metamyelocyte band mature neutrophil
ii. Normally only mature neutrophils (2-4 lobed nucleus) and band neutrophils (immediate precursor with horseshoe-shaped nucleus) are found in circulation
iii. Hyper segmented neutrophil = >5 lobes
1. Megaloblastic process – B12 or folate deficiency
iv. Left shift
1. Increase in granulocyte precursors (bands, metamyelocytes, myelocytes, promyelocytes, blasts) in circulation
2. Acute infections, hypoxia, shock, CML
v. Other
1. IT ratio – used in neonates (immature forms/total neutrophils)
2. Neutrophil toxic changes – bacterial infection
PRBC transfusion - background
- PRBC = RBC with plasma removed
a. + anticoagulant + citrate
b. + phosphate + dextrose - Indications
a. Should be based on need to optimise tissue delivery
b. O2 delivery = Hb x CO (HR x CV) x oxygen saturation x 1.34
c. Consider:
i. Expected trajectory of Hb – haemolysis vs. blood loss
ii. Ability to cope with tachycardia - General rules
a. Hb <70g/L = although lower thresholds may be acceptable in patients without symptoms and where specific therapy (eg iron) is available.
b. Transfusion may be indicated at higher thresholds for specific situations:
i. Hb <70-100g/L during surgery associated with major blood loss or if evidence of impaired oxygen transport
ii. Hb <80g/L; patients on a chronic transfusion regimen or during marrow suppressive therapy (for symptom control and appropriate growth)
iii. Hb <100g/L; only for very select populations (eg. neonates) - Transfusion equation = 0.5 x weight x (target Hb – current Hb)
• The increase in hemoglobin from 1 unit of RBCs will be approximately 1 g/dL; the increase in hematocrit will be approximately 3 percentage points
PRBC treatments
a. Leukodepletion (all products in Victoria automatically leukodepleted)
i. Involves ‘filtering’ of blood product
ii. Removes most WBC (by size) but not all
iii. Reduces risk of non-haemolytic transfusion reactions, cytokine based reactions and infections such as CMV
b. Washing
i. Blood product washed in normal saline
ii. Removes plasma proteins
iii. Reduces risk of allergic reactions
c. Irradiation
i. Kills T-lymphocytes
ii. Reduces risk of GVHD (AND risk of similar haplotypes reacting to each other eg related donors)
iii. Not required for acellular products
d. CMV negative
i. Requires CMV negative donors
ii. For CMV -ve transplant patients
RBC antigens and incompatability
- RBC typing
a. Goal = to reduce risk of immune reactions
b. Typing done for:
i. ABO group
ii. Rh group
iii. Common antigens – Kell, Duffy - Kell = most common antibody implicated in haemolytic disease of the newborn, take special care in females pre child birth
a. Antibodies to RBC antigens can occur to
i. Natural exposure – carbohydrates that mimic blood antigens
ii. Autoantibodies – against autologous blood group antigens
iii. Allogeneic exposure – from transfusion/ pregnancy
1. IgG mediated can cross the placenta
b. ABO system
i. Oligosaccharide (CHO) antigen on RBC surface
ii. Genes determining ABO type found on chromosome 9
iii. A and B alleles are codominant so both A and B antigens will be expressed on the RBC whenever either allele is present
iv. Blood groups = A (AA, AO), B (BB, BO), AB, O
v. H antigen – required to bind AB antigens to RBC surface, if not present then patient is O blood group despite genotype A, B, AB = Bombay phenotype
vi. Production of antibodies
1. Natural production of antibodies against antigens not on cell surface
a. E.g. A + blood group – then natural production of Anti-B
2. ABO incompatibility – IgM production to sensitization against Ag not present on own RBC
a. IgM cannot cross the placenta
3. AB antibodies begin to appear 2-8months after birth and occur without external exposure
c. Rhesus system
i. Rh system comprises 61 antigens
1. D antigen = most immunogenic and important Rh antigen autosomal dominant
a. RH +ve refers to presence of Rh D protein (15% of the population lack D)
2. Others = C, E
ii. Routine Rh typing of doors and patients only tests for the presence/absence of D
iii. Antibody production
1. Development of antibodies to Rhesus only occurs due to prior sensitisation – NOT naturally occurring eg. prior transfusion, pregnancy
2. Results in the production of IgG antibodies (cf. ABO antibodies IgM)
3. IgG can cross the placenta
iv. In a negative person exposed to +ve blood
1. Spontaneous reactions rarely occur - person needs to be exposed to lots of Rh antigen before agglutinins develop to cause a significant transfusion reaction
2. Usually causes a mild delayed transfusion reaction, which is increased on second exposure
Group and Hold vs Crossmatch
G&H
Also known as Group and Screen or Group and Hold.
Group and Save is the sample processing that determines the patient blood group (ABO and RhD) and screens for any atypical antibodies.
The process takes around 40 minutes and no blood is issued.
If patient blood have atypical red cell antibodies, the laboratory will do additional tests to identify them.
CROSSMATCH
A crossmatch is the final step of pretransfusion compatibility testing, to request blood from the laboratory.
Crossmatching involves physically mixing of patient’s blood with the donor’s blood, in order to see if any immune reaction occurs
After ensuring that donnor blood is compatible, the donor blood is issued and can be transfused to the patient.
This process takes around 40 minutes, in addition to the 40 minutes required to G&S the blood.
It is not possible for the laboratory to provide crossmatched blood without having processed a G&S sample first.
Transfusion/RBC - screening (infection)
a. In Australia, blood is tested for 5 transmissible disease: HIV, HBV, HCV, HTLV (human T-cell lymphotropic virus), syphilis
b. Specifically:
i. HBV surface Ag; HCV antibody; HIV-1 and HIV-2 antibody; HTLV-I and HTLV-II antibody; syphilis Ab
ii. ALSO now test for HIV-1 and HCV RNA – looks for the actual presence of the virus, not just the immune response – this reduced the ‘window period’ (time between exposure to a virus and appearance of detectable antibodies) – see table below
iii. Also test for malaria in donors who have resided in or travelled to a malarial area
c. Donors are notified of abnormal result
RBC transfusion reactions/complications
a. Infection
i. HIV = 1/2 million – last occurred in late 90s
ii. HCV - 1/1.5-2 million
iii. Bacterial contamination = 1/5 million – usually Yersinia and gram negatives
iv. Transfusion related sepsis 1/17,000
b. Acute
i. Volume overload
ii. Non-haemolytic febrile transfusion reactions
1. Anti-HLA antibodies directed against contaminated leukocytes cytokine release
2. Results in fever/chills/rigors
3. Reduced by giving leukocyte depleted blood (all blood in Aus)
iii. Acute haemolytic transfusion reaction
1. Recipient with preformed antibodies against donor blood = destruction of donor RBC entering the circulation
2. Can be due to ABO/ Rh/ Kell / Duffy
3. Clinical features = chest/ flank pain, fever, tachycardia, hypotension, AKI
4. Rx = stop transfusion, give saline. Check Coombs/ haptoglobin, free Hb and repeat X match
iv. Allergy
1. Urticaria (1%)
2. Anaphylaxis (beware IgA deficient patients with anti-IgA antibodies)
v. Transfusion related acute lung injury (TRALI)
1. 1/5000
2. Donor plasma has anti-HLA / polymorph antibodies that bind to WBC cytokine production
3. Cause pulmonary oedema/ARDS fever, tachycardia, hypoxia
4. Within 2-8hrs of transfusion
5. V uncommon in kids (more common in elderly females)
vi. Citrate toxicity (the anticoagulant in blood products)
vii. Hyperkalaemia : RBC lysis, in irradiated cells
c. Delayed
i. Refractory thrombocytopaenia after multiple transfusions (due to sepsis, DIC, antibodies against HLA types / human platelet antigens)
ii. Delayed haemolytic transfusion reaction
1. Low level alloantibodies not picked up on cross match (often against Rh/ Kidd)
2. Bind to donor RBC, instigate extravascular haemolysis
3. 2-10 days post transfusion: fever, haemolysis, jaundice, positive Coomb’s test
iii. Transfusion related GVHD = 8-10 days post transfusion
1. Results in rash, fever, abnormal LFTs, TENs
2. Reaction between HLA types = viable T cells in transfusion attack patient cells
3. 100% fatal
4. Does not occur in immunocompetent recipient as they have far greater WBC
5. Prevented by using irradiated products
6. Risk factors
a. Any immunodeficiency
b. Neonates
c. HLA matched donor (i.e. the donor has 2 haplotypes that are the same, and the recipient has 1 of those haplotypes but not the other recipient won’t mount a response as it recognizes the 2 haplotypes as same, but the graft will attack the host as it recognizes an abnormal haplotype)
7. Treatment = IVIG/immunosuppressants
iv. Iron accumulation
Platelets for transfusion - general
- Key points
a. Kept at room temperature = highest risk of infection - Amount
a. 5-20 ml/kg
b. 5-10 ml/kg will raise platelet count by 50—100 x109/L - Methods of treating
a. Washing
b. Single donor apheresis (for adults, all pediatric packs single donor)
c. HLA matched - Refractoriness
a. Repeated transfusions/ pregnancy allo-immunization
b. Try apheresis/ HLA platelets - Risks
a. Plts are stored at room temperature for 5 days only - HIGHEST risk of bacterial contamination!!! *** - EXAM Q
• The platelet count increase from 5 to 6 units of whole blood-derived platelets or 1 unit of apheresis platelets (200-300mL) will be approximately 30,000/microL in an average sized adult
Blood products for coagulopathy - general
Platelets - separate card
- Fresh frozen plasma
a. Plasma is separated from whole blood contains all components except for RBC, slightly diluted
b. Contains
i. All clotting factors
ii. Some fibrinogen
c. Indications
i. Warfarin
ii. Liver disease with abnormal coagulation
iii. Acute DIC when there is bleeding or abnormal coagulation
iv. Massive transfusion or cardiac bypass
v. Deficiencies of clotting factors 2, 5, 10, 11
d. Neonate indications
i. Massive transfusion
ii. Haemorrhage due to vit K deficiency
iii. DIC with bleeding
e. Cross matching = needs to be plasma compatible - Cryoprecipitate
a. FFP is frozen and thawed, the bit that thaws on top = cryo
b. Contains
i. Fibrinogen
ii. vWF/ factor VIII/XIII
c. Indications
i. Dysfibrinogenemia
ii. DIC
iii. vWD if no other options available
- haemophilia A
d. Note much smaller volume required than with FFP - Cryo deplete plasma
a. FFP with cryo removed
b. Has less vWF/ factor 8
c. Good for TP if replacing ADAMST13 - Prothrombinex
a. 2, 9, 10, 7 (vit K dependent factors)
b. For Warfarin reversal - Note - for adequate clotting, one needs:
a. 25-30% normal clotting factors
b. Fibrinogen 75-100 mg/dL
c. No inhibitors
Polycythaemia - definitions
- Definition = RBC count, Hb level and total RBC volume all exceed the upper limits of normal
- Postpubertal = total RBC mass >25% over the mean normal value (based on BSA) or Hb >185g/L (in males) or >165g/L (females) indicate absolute erythrocytosis
- Haemoconcentration/relative polycythaemia = RBC mass is not increased and normalization of the plasma volume restores Hb to normal levels ie. decrease in plasma volume ie acute dehydration or burns
Neonates: HCT >0.65
Children: Depends on age
Adults: HCT >0.49 (men) >0.48 (women)
Blood viscosity and hct have a linear relationship when the hct is <60 percent [6,9]. This relationship becomes exponential when the hct exceeds 65 percent, such that a small increase in hct is associated with a dramatic increase in viscosity.
Polycythaemia rubra vera - general
= clonal/primary polycythaemia
- Key points
a. Rare in children
b. Acquired clonal myeloproliferative disorder
c. Primarily manifests as erythrocytosis +/- thrombocytosis, leukocytosis
d. Risk factors = FHx, autoimmune disorder (eg. Crohn’s) - Genetics + pathogenesis
a. Gain of function mutation of JAK2 (a cytoplasmic tyrosine kinase) is found in more than 90% of adult patients, but in <30% of children
b. The erythropoietin receptor is normal, and serum erythropoietin levels are normal or low
c. In vitro cultures do not require added erythropoietin to stimulate growth of erythroid precursors - Clinical manifestations
a. Hepatosplenomegaly
b. +/- hypertension
c. +/- headache
d. +/- SOB
e. +/- neurologic symptoms
f. Agranulocytosis – may cause diarrhea or pruritis from histamine release
g. Thrombocytosis (+/- platelet dysfunction) may cause thrombosis or haemorrhage - Diagnostic criteria
a. Major
i. Hb >185g/L (men) or >165g/L (women)
OR Hb/HCT>99th percentile or reference range for age, sex, altitude of resistant
OR Hb >170 g/L (men) or Hb >150 g/L (women) if associated with a sustained increase of ≥2 g/dL from baseline that cannot be attributed to correction of iron deficiency
OR elevated red cell mass >25% above mean normal predicted value
ii. Presence of JAK2 or similar mutation
b. Minor
i. Bone marrow trilineage myeloproliferation
ii. Subnormal serum erythropoietin level
iii. Endogenous erythropoietin level
iv. Endogenous erythroid colony growth
c. Diagnosis
i. Both major criteria and one minor criteria OR first major criteria and 2 minor criteria - Treatment
a. Phlebotomy = alleviate symptoms of hyperviscosity and decrease risk of thrombosis
b. Iron supplementation = prevent viscosity problems from iron-deficiency microcytosis or thrombocytosis
c. Marked thrombocytosis = antiplatelet agents (ie aspirin) may reduce risk of thrombosis and bleeding
d. Anti-proliferative treatments (hydroxyurea, anagrelide, interferon- α)
i. If unsuccessful or progressive hepatosplenomegaly from above therapies
e. JAk-2 inhibitors = active area of investigation - Prognosis
a. Transformation of the disease into myelofibrosis or acute leukemia is rare in children
b. Prolonged survival is not unusual
Non-clonal polycythaemia - general
• Definition = when polycythaemia is caused by physiologic process that is not derived from a single cell
- Congenital
a. Lifelong or familial polycythaemia should trigger search for congenital
b. May be transmitted as dominant or recessive disorders
c. Aetiology
i. Autosomal dominant - Hemoglobins that have increased oxygen affinity (P50 <20 mm Hg)
- Erythropoietin receptor mutations resulting in an enhanced effect of erythropoietin
- Mutations in the von Hippel–Lindau gene that result in altered intracellular oxygen sensing.
- Hemoglobin M disease (autosomal dominant) causes methemoglobinemia and can lead to polycythemia.
a. Cyanosis may occur in the presence of as little as 1.5 g/dL of methemoglobin
ii. Autosomal recessive - 2,3-diphosphoglyceric acid deficiency (rare) – left shift of oxygen dissociation curve, increased oxygen affinity and consequence polycythaemia
- Congenital methemoglobinemia – autosomal recessive deficiency of cytochrome b5 reductase may cause cyanosis and polycythemia
d. Clinical manifestations
i. Asymptomatic
ii. Neurologic abnormalities - present in patients whose enzyme deficits are not limited to hematopoietic cells
iii. Cyanosis in Haemoglobin M disease – uncommon in other hemoglobin variants unless hyperviscosity results in localized hypoxemia - Acquired
a. Usually due to chronic arterial oxygen desaturation.
b. Aetiology
i. Hypoxic - Cardiovascular defects involving R-L shunts and pulmonary diseases interfering with proper oxygenation – most common cause hypoxic polycythemia
- Living at high altitudes –Hb level increases 4% for each rise of 1,000 m in altitude
ii. Partial obstruction of a renal artery rarely results in polycythemia.
iii. Benign and malignant tumors that secrete erythropoietin.
iv. Exogenous or endogenous excess of anabolic steroids also may cause polycythemia.
v. A common spurious cause is decrease in plasma volume such as in mod-severe dehydration
c. Clinical manifestations
i. Cyanosis
ii. Hyperemia of the sclera and mucous membranes
iii. Clubbing
iv. As the hematocrit rises to >65%, clinical manifestations of hyperviscosity, such as headache and hypertension, may require phlebotomy - Treatment
a. Mild disease – observation
b. Phlebotomy – prevent or treat symptoms of headache, dizziness, exertional dyspnoea
i. When haematocrit >65-70% (Hb>23g/L), blood viscosity markedly increases
ii. Apharesed blood should be replaced with plasma or saline to prevent hypovolaemia in patients accustomed with a chronically elevated blood volume
c. Iron supplementation
i. Increased demand for RBC production may cause iron deficiency
ii. Iron deficient microcytic red cells are more rigid - increasing the risk of intracranial and other thrombosis
iii. Periodic assessment of iron status, with treatment of iron deficiency should be performed
Neonatal polycythaemia - general
- Key points
a. Defined at haematocrit >65% during first week of life – venous sample
b. Relationship between viscosity and Hct is linear below 60-65% but increases exponentially above this level
c. Hyperviscosity is related to increased resistance to blood flow therefore increased risk circulatory impairment - Physiology
a. Polycythemia results from increased red cell mass, with decreased, normal or increased plasma volume
b. Haematocrit peaks at 4-6 hours of life, then drops slowly to value at birth and stays stable - Aetiology
a. Chronic intrauterine hypoxia
i. SGA
ii. Post-dates
iii. Maternal hypertension / smoking
b. Excessive transfusion of blood
i. Placental transfusion during cord clamping
ii. Twin to twin transfusion syndrome
iii. Maternofetal transfusion
c. Endocrine
i. Infants of diabetic mothers
ii. Congenital adrenal hyperplasia
iii. Neonatal thyrotoxicosis
d. Syndromic
i. Down’s syndrome
ii. BWS - Clinical manifestations
a. Lethargy/poor feeding
b. Plethora
i. Hyperbilirubinaemia
c. Hyperviscosity
i. Respiratory – pulmonary hypertension, RDS
ii. CVS – tachypnea, cyanosis, tachycardia, cardiomegaly
iii. CNS – apnea, irritability, lethargy, convulsions
iv. GIT – NEC
v. Renal – impaired renal function
vi. Metabolic –thrombocytopenia, hypoglycaemia, hypocalcaemia - Treatment
a. Goal – decrease Hct to 50-55%
b. Partial exchange transfusion
i. If symptomatic or Hct >75%
ii. Volume exchanges (mL) = Observed Hct – desired Hct / observed Hct
iii. Reverses physiological abnormality and decreases symptoms but has not been shown to improve long term outcomes
Acanthocytosis - general
- Definition
a. Characterized by RBCs with irregular circumferential pointed projections - Aetiology
a. Liver disease + vitamin E deficiency – due to alterations in cholesterol:phospholipid ratio
b. Congenital Abetalipoproteinaemia or hypoprebetalipoproteinaemia
c. Fat malabsorption
d. Neuromuscular abnormalities
e. Retinitis pigmentosa
f. Normoproteinemic neuroacanthocytosis
i. Chorea-acanthocytosis
1. Autosomal recessive
2. The production of acanthocytes in chorea-acanthocytosis appears related to altered Lyn kinase activity with increased tyrosine phosphorylation and altered linkage of band 3 to other RBC membrane proteins.
ii. McLeod syndrome
1. X-linked recessive, rare
2. Absence of the KX (Kell) antigen, late-onset myopathy, peripheral neuropathy, chorea, splenomegaly, and hemolysis with acanthocytosis.
3. There is usually >3% acanthocytes on peripheral smear and caudate atrophy noted on MRI.
iii. Pantothenate kinase-associated neurodegeneration
1. Autosomal recessive
2. Dystonia, rigidity, chorea, dysarthria, spasticity, retinopathy)
iv. Huntington disease–like 2
1. Autosomal dominant
Spleen - anatomy and physiology
- Anatomy
a. Splenic precursor recognizable by 5 weeks gestation.
b. Size
i. Birth = 11g
ii. Puberty = 135g
iii. Diminishes in size during adulthood
c. 15% have an accessory spleen
d. The major splenic components
i. Lymphoid compartment (white pulp) = periarterial lymphatic sheaths of T lymphocytes with embedded germinal centres containing B lymphocytes
ii. Filtering system (red pulp) = skeleton of fixed reticular cells, mobile macrophages, partially collapsed endothelial passages (cords of Billroth), and splenic sinuses.
iii. Marginal zone separates the red pulp from the white pulp = rich in dendritic APCs
iv. Splenic capsule = smooth muscle and contracts in response to adrenaline
e. Vascular
i. 10% of the blood delivered to the spleen flows rapidly through a closed vascular network
ii. 90% flows more slowly through an open system (the splenic cords), where it is filtered through 1-5 µm slits before entering the splenic sinuses - Function
a. Haematopoiesis (fetal)
i. Major splenic function at 3-6 mo of fetal life but then disappears.
ii. Splenic haematopoiesis can be resumed in patients with myelofibrosis or severe haemolytic anaemia
b. Reservoir
i. Factor VIII and 1/3 of circulating platelet mass are sequestered in the spleen can be released by stress or adrenaline injection.
ii. Thrombocytosis and leucocytosis occur with loss of the splenic reservoir function
c. Filtering
i. Spleen receives 5-6% of the cardiac output, but normally contains only 25 mL of blood - Can retain much more when it enlarges cytopenias
ii. Filtering function facilitated by slow blood flow past macrophages and through small openings in the sinus walls
iii. Removal of excess membrane on young RBCs - Loss of this function target cells, poikilocytosis, decreased osmotic fragility
iv. Main site for destruction of old RBCs – assumed by other reticuloendothelial cells post splenectomy
v. Removes damaged/abnormal RBCs (ie spherocytes, Ab-coated RBCs) and damaged platelets
vi. Intracytoplasmic inclusions may be removed from RBCs without cell lysis - Howell-Jolly bodies = nuclear remnants
- Denatured Hb = Heinz bodies
d. Host defence
i. Largest lymphoid organ in the body
ii. Contains nearly half of the body’s total Ig-producing B lymphocytes
iii. Processes foreign material to stimulate production of opsonizing antibody
iv. Produces properdin and tuftsin
v. Phagocytosis to trap and destroy intracellular parasites
vi. Minor role in antibody response to IM or s/cut injected antigens but is required for early Ab production after exposure to IV antigens
vii. The spleen may be an important site of antibody production in ITP
viii. Encapsulated bacteria
Splenomegaly and pseudosplenomegaly - background
- Key points
a. Splenic edge >2 cm below the left costal margin abnormal
b. In most the spleen must be 2-3 x normal size before it is palpable
c. Soft, thin spleen palpable 15% of neonates, 10% normal children, 5% adolescents. - Investigations
a. FBE
b. Peripheral smear
c. US, CT, or technetium-99 sulfur colloid scan (also assesses splenic function) - Pseudosplenomgaly
a. Abnormally long mesenteric connections may produce a wandering or ptotic spleen
b. An enlarged left lobe of the liver
c. LUQ mass
d. Splenic hematoma
e. Splenic cysts may contribute to splenomegaly or mimic
i. Congenital (epidermoid)
ii. Acquired (pseudocyst) after trauma or infarction
iii. Usually asymptomatic, found on imaging
f. Splenosis after splenic rupture – most not palpable
g. Accessory spleen (present in 15% of normal individuals) – most not palpable
h. Congenital polysplenism syndrome – cardiac defects, left-sided organ anomalies, bilobed lungs, biliary atresia, and pseudosplenomegaly
Splenomegaly - differentials
Anatomical • Cysts • Hamartomas • Polysplenia • Haemangioma
Haematological – hyperplasia
• Acute and chronic haemolysis
• Chronic iron deficiency
• Extramedullary haematopoeisis
Storage disease
• Lipidosis, mucopolysaccharidoses, mucolipidoses, defects in CHO metabolism etc.
Immunological/ inflammatory
• All autoimmune conditions
Infections
• Almost all infections can cause splenomegaly
Malignancies
• Primary – leukaemia, lymphoma, angiosarcoma, Hodgkin
• Metastasis
Other
• Heart failure
• Portal hypertension
Hypersplenism - general
- Key points
a. Increased splenic function (sequestration or destruction of circulating cells) peripheral blood cytopenias, increased bone marrow activity, splenomegaly
b. Usually secondary to another disease
c. Treatment
i. Underlying condition
ii. Splenectomy - Congestive splenomegaly (Banti syndrome)
a. Secondary to obstruction in the hepatic, portal or splenic veins hypersplenism
b. Aetiology
i. Hepatic inflammation, fibrosis and vascular obstruction secondary to Wilson disease, galactosemia, biliary atresia, and α1-antitrypsin deficiency
ii. Congenital abnormalities (absence or hypoplasia) of the portal or splenic veins
iii. Splenic venous flow may be obstructed by masses of sickled erythrocytes leading to an infarction
iv. Obliteration of these vessels secondary to septic omphalitis or thrombophlebitis (spontaneous or as a result of umbilical veins catheterization in neonates)
c. Management
i. If spleen site of vascular obstruction splenectomy cures hypersplenism
ii. Obstruction usually is in the hepatic or portal systems portocaval shunting may be more helpful, because both portal hypertension and thrombocytopaenia contribute to variceal bleeding
Hyposplenism - general
- Aetiology
Congenital absence
• Associations – complex cyanotic heart defects, dextrocardia, bilateral trilobed lungs, heterotropic abdominal organs (Ivemark syndrome)
Sickle cell disease
• May have splenic hypofunction as early as 6 months of age
• Initially caused by vascular obstruction reversed with RBC transfusion or hydroxyurea
• The spleen eventually autoinfarcts and becomes fibrotic and permanently nonfunctional
Functional hyposplenism
• Normal neonates, especially if premature
• Malaria
• Post-radiation
• Reticuloendothelial function overwhelmed – severe haemolytic anaemia, metabolic storage disease
Other
• Autoimmune disorders = SLE, RA, GN etc
• Oncohaematological disorders
• GI disorders
• Hepatic disorders
• Infectious
• Iatrogenic = methyldopa, steroids, TPN, irradiation
• Amyloidosis - Investigations
a. Peripheral film
i. RBC inclusions (Howell-Jolly bodies or Heinz bodies)
ii. ‘pits’ on interference microscopy
b. Poor uptake on technetium or other spleen scans
c. +/- reduced IgM memory B cells may also be detected and is a risk factor for overwhelming sepsis
- IgM memory B cells dependent on spleen for suvival, produced in marginal zone
Splenic trauma - general
- Clinical manifestations
a. Small splenic capsular tears -> abdominal or referred left shoulder pain (diaphragmatic irritation by blood)
b. Larger tears with severe blood loss -> similar pain and signs of hypovolaemic shock - Risk factors
a. Enlarged spleens (ie infectious mononucleosis) -> more likely to rupture with minor trauma
b. Patients with splenomegaly should avoid contact sports/other activities that increase risk of splenic trauma - Investigations
a. FBE – serial tests
b. CT IV contrast – best imaging modality to assess splenic trauma - Treatment
a. Small capsular injury
i. Observation
ii. PRBC if required
iii. Restricted activities
b. More marked abdominal bleeding
i. Laparotomy +/- splenectomy if clinical instability/deterioration, if other organ damage suspected
ii. Partial splenectomy if possible
Splenectomy - general
- Indications
a. Splenic rupture
b. Anatomic defects
c. Severe transfusion dependent haemolytic anaemia
d. Immune cytopaenias
e. Metabolic storage disease
f. Secondary hyposplenism - Complications
a. Sudden, overwhelming post-splenectomy infections (sepsis or meningitis)
b. Thromboembolic complications
i. Not dependent on indication for splenectomy or platelet count
iii. Proposed mechanisms – loss of filtering function of the spleen, allowing abnormal RBCs to remain in the circulation and activate the coagulation cascade. - Splenosis
a. Splenosis refers to implants of splenic tissue resulting from spillage of cells following abdominal trauma or surgery.
b. Up to 50% of children whose spleen is removed because of trauma have spontaneous splenosis
c. Surgical splenosis (distributing small pieces of spleen throughout the abdomen) may decrease the risk of sepsis in patients whose splenectomy is necessitated by trauma
d. The splenic tissue that regrows frequently has poor function
Post splenectomy (/hypo/asplenia) - overview, sepsis
- Overview
a. Register with Spleen Australia
b. Education
c. Vaccination
d. Antibiotic prophylaxis
e. Rapid treatment of infections - Post-splenectomy sepsis
a. Increased risk if children <5 years at the time of surgery
i. Lifelong risk 5% - most occur within 2 year after splenectomy
b. Risk impacted by indication
i. Lower risk (2-4%) = trauma, RBC membrane defects, immune thrombocytopaenia
ii. High risk (8-30%) = haemaglobinopathies, pre-existing immune deficiency (eg. Wiskott-Aldrich), reticuloendothelial blockade (eg. storage disease, severe haemolytic anaemia)
c. Organisms
i. Encapsulated bacteria = 80% (SHiNE SKiS = Strep pneumo, Haem influenzae, Neisseria, E. coli, Salmonella, Klebsiella, GBS) - Streptococcus pneumoniae >60%
- Haemophilus influenzae
- Neisseria meningitides
ii. Protozoal infections – malaria and babesiosis
iii. Capnocytophaga canimorsus or C. cynodegmi following animal bite
d. Management
i. Rapid treatment if febrile – emergency antibiotics at home
ii. Broad spectrum cephalosporin (cefotaxime or ceftriaxone)
iii. +/- vancomycin (to cover penicillin-resistant pneumococci)
Post splenectomy (/hypo/asplenia) vaccination and treatment of infection
a. Vaccination
i. Timing
1. Elective = two weeks before scheduled operation
2. Emergency = >1 week after surgery
ii. Pneumococcal
1. Prevenar 13
a. Primary course as per immunisation schedule
b. One additional dose at >=12 months of age
2. Prevenar 23
a. One dose at 4-5 years of age
b. Booster 5 years post initial dose
iii. Meningococcal
1. Quadrivalent – MenA, C, W, Y135
2. Meningococcal B
iv. Haemophilius
1. Primary course as per immunisation schedule
2. No booster required
v. Annual influenza vaccine = influenza is a risk factor for secondary pneumococcal infections
b. Prophylaxis
i. Options
1. Oral amoxicillin 20 mg/kg daily
2. Oral phenoxymethylpenicillin (penicillin V)
a. <5 years = 125 mg twice daily
b. >5 years = 250 mg twice daily
3. Start prophylaxis in children with sickle cell as soon as diagnosed
ii. Duration
1. Until 16 years of age
2. Minimum
a. Up to the age of 5 years in children with Asplenia
b. Up to the age of 5 years in patients who have hyposplenism due to sickle cell anaemia or other congenital haemaglobinopathy
c. At least 3 years after splenectomy
3. Lifelong
a. Severely immunosuppressed
b. Splenectomy for haematological malignancy, particularly those with ongoing immunosuppression
c. Episode of severe sepsis – particularly after 2nd
c. Emergency antibiotics
i. Augmentin 22.5 mg/kg orally twice daily
Anaemia - overview/background
- Definition = any cause of reduced haemoglobin below normal range for age and sex
- Consequences
a. ↓ O2 carrying capacity
b. Tissue hypoxia
c. Compensation
i. Cardiac overactivity
ii. Cardiorespiratory failure
iii. Vasoconstriction with redistribution of blood flow to tissues with high oxygen dependence - Symptoms/signs suggestive of anaemia
a. Pallor
b. Pale conjunctivae
c. Flow murmur
d. Lethargy
e. Poor growth
f. Signs of cardiac failure
g. Weakness
h. Listlessness
i. Shortness of breath - Classification
a. Microcytic, normocytic or macrocytic
b. Decreased production, increased destruction or blood loss
i. Reticulocyte % or absolute number helpful in making distinction
ii. Normal reticulocyte percentage of total RBCs= 1% - Investigations
a. Every child
i. FBE + Film
ii. Reticulocyte count = indicates whether marrow is responding appropriately
b. Further investigations dependent on MCV
Microcytosis - Iron deficiency - Thalassemia - Hereditary spherocytosis - Rare – sideroblastic anaemia (XLR, lead)
Normocytic, normochromic
- Blood loss
- Mixed nutritional deficiency
- TEC
- Anaemia chronic disease
Macrocytosis - B12/folate deficiency - Brisk reticulocytosis/ haemolysis - MDS - Fanconi’s/ Diamond Blackfan Anaemia - Congenital dyserythropoetic anaemia, osteoporosis
Reduced production • Haematinic deficiency • Marrow failure • Marrow replacement • Anaemia of chronic disease • Ineffective erythropoiesis • Dyserythropoiesis
Increased destruction • Immune o Autoimmune o Alloimmune • Non-immune o Inherited = Hb, membrane, enzyme o Other Physical damage = MAHA, thermal, cardiac defects Infectious agents = malaria
Increases losses
• Bleeding – occult or massive
Anaemia of inadequate production - differentials
- Bone marrow failure
a. Congenital pure red cell aplasia
i. Diamond-Blackfan anaemia
ii. Aase syndrome
b. Acquired pure red cell aplasia
i. Autoimmune
ii. Infections
iii. Drugs
iv. Transient erythroblastopaenia of childhood
c. Malignancy
d. Bone marrow failure syndromes
i. Aplastic anaemia
ii. Fanconi’s anaemia
iii. Others
e. Myelofibrosis
i. Renal failure
ii. Vitamin D deficiency
iii. Hypoparathyroidism
f. Bone disease = osteopetrosis - Impaired EPO production
a. Hypothyroidism
b. Starvation
c. Chronic renal disease
d. Anaemia of chronic disease - Disorders of erythroid maturation/ ineffective erythropoiesis
a. Iron deficiency anaemia
b. Sideroblastic anaemia
c. Lead toxicity
d. Megaloblastic anaemia
e. Congenital dyserythropoetic anaemia
Microcytic anaemia - ddx
TAILS
Thalassaemia Anaemia of chronic disease Iron deficiency Lead poisoning Sideroblastic anaemia
Iron deficiency anaemia - background, physiology
- Epidemiology
a. Most common nutritional deficiency in children + most common cause of anaemia in children
i. IDA caused by diet, most commonly occurs at 9-24 months in term infants
b. Usually nutritional (insufficient red meat, fish, chicken, green vegetables, pulses; excessive cow’s milk); rarely due to malabsorption or GI bleeding
c. Can lead to reduced cognitive and psychomotor performance in the absence of anaemia - Physiology
a. Most iron in neonates is in circulating Hb
b. As the relatively high Hb concentration of the newborn infant falls during the first 2-3 months of life considerable iron is recycled – iron stores sufficient for blood formation in first 6-9 months of life
c. Stores are depleted sooner in LBW infants, or infants with perinatal blood loss
d. Delayed cord clamping (1-3 minutes) improves iron status and reduces risk of iron deficiency anaemia
e. Iron absorption
i. Absorbed in the duodenum in Fe 2+ (ferrous form), active transport across apical via DMT1 transporter (1-2 mg/day)
ii. Once inside enterocyte, leaves via ferroportin transporter
f. Regulation
i. Enhanced by gastric acid, vitamin C, breast milk
ii. Decreased by bovine milk proteins, egg whites, phytates, bran, calcium, zinc and lead
iii. Absorption upregulated if liver stores are low – via hepcidin: if liver stores are full, hepcidin increases binds to ferroportin and stops iron absorption
g. Transport + storage
i. Transported in blood bound to transferrin
ii. Stored in liver and bone marrow (reticuloendothelial macrophages)
iii. Body has minimal means of LOSING iron aside from blood loss, sloughed mucosal cells, menstruation
iv. Iron stores last 5-6 months - Daily requirements
a. Term infant body has 0.5 g of iron (compared to adult total 5g)
b. Infants require 1 mg daily (daily intake of 8-10 mg as < 10% absorbed)
c. Breast milk contains 1 mg/L of iron - Phases of iron deficiency
a. Iron depletion = low ferritin, normal Hb and indices
b. Iron deficiency = low ferritin and indices, Hb normal
c. Iron deficiency anaemia
Iron deficiency anaemia - manifestations, aetiology, RFs
- Clinical manifestations
a. Most children are asymptomatic and are identified through screening
b. Pallor most important clinical sign – only apparent when Hb falls to 70-80 g/L
c. Irritability, anorexia, lethargy and flow murmurs – when Hb falls to <50 g/L
d. Consequences
i. Impaired neurocognitive function in infancy
ii. Possible link with seizures, strokes, breath holding and exacerbation of RLS
iii. Poor growth
iv. Exercise intolerance
v. Pica - Aetiology
a. Low stores
i. Prematurity
b. Low intake
i. Prolonged breast feeding without introduction of solids >6months
ii. Cow’s milk in first year – increase intestinal blood loss, decrease iron absorption
c. Blood loss
i. Focal lesion – peptic ulcer, Meckel’s, haemangioma
ii. GIT disease – IBD, Celiac, cow’s milk protein enteropathy
iii. Hookworm infestation - Risk factors based on age
a. Perinatal
i. Maternal iron deficiency
ii. Prematurity
iii. Administrating of EPO for anaemia of prematurity
iv. Perinatal haemorrhagic events (eg. TTTS or fetal-maternal haemorrhage, placenta praevia)
b. Infancy
i. Dietary factors - Lack of iron supplements for breastfed infants
- Use of low iron infant formula
- Feeding of unmodified (non-formula) cow’s milk, goat’s milk or soy milk
- Insufficient iron rich complementary foods – should be introduced by 6 months
ii. Other risk factors - Disorders with GI blood loss – eg. milk protein induced proctocolitis
- Malabsorptive disease
c. 1 to 12 years
i. Dietary risk factors - Excessive intake of cow’s milk – no more than 500ml per day
- Insufficient iron in foods
ii. Other risk factors - Disorders with GI blood loss (eg. inflammatory bowel disease or chronic gastritis)
- Malabsorptive disease eg. celiac, worms
- Obesity
Iron deficiency anaemia - ix/ddx
- Investigations
a. FBE = low MCV, elevated RDW, common to have thrombocytosis
b. Film = ‘cigar cells’, target cells , tear dropping, anisocytosis
c. Iron studies
i. L ferritin -> L iron -> H transferrin -> L transferrin sat -> L Hb -> L MCV
ii. Serum iron is biphasic and unreliable except to monitor compliance with replacement therapy
iii. Ferritin - If low = deficient
- If normal = can reflect acute phase reaction
iv. Transferrin usually elevated in Fe deficiency
v. Soluble transferrin receptors (present on erythroid cells) - Ratio of transferrin receptor: ferritin > 2 suggestive of iron deficiency anaemia
- Useful In patients with chronic inflammation
d. Bone marrow – iron stain can also be performed - DDx
a. Lead poisoning
i. Elevated whole blood lead
ii. Film = basophilic stippling
b. Alpha/beta trait
i. Diagnosis of exclusion
ii. If <6months – will NOT be iron deficiency or B thal (no B chains yet) so microcytic anaemia either A thal or blood loss
iii. Thal vs IDA - RCC – normal or elevated in thalassaemia, low in IDA
- MCV – low MCV out of proportion to degree of anaemia in thalassaemia
- RDW – normal in thalassaemia, elevated in IDA (anisocytosis)
c. Chronic disease
i. Ferritin usually elevated
ii. Serum transferrin receptor levels may be useful – not affected by inflammation, increased in iron deficiency (very sensitive), normal in anaemia of chronic disease
Iron deficiency anaemia - rx/prevention
- Management
a. Iron supplementation
i. Aim for iron supplementation at 2 - 6mg/kg/day of elemental iron
ii. 2mg/kg/day is the preventative dose for iron deficiency and also effective in mild-moderate deficiency - The higher range doses are usually only necessary for severe deficiency, and iron studies should be monitored carefully to prevent overload
- Higher doses should be divided (up to tds) to reduce gastric irritation
iii. Supplementation usually = reticulocyte response in 48-72 hours
iv. IV therapy = poor compliance, inability to tolerate due to GI issues
v. Assessing response - 12-24 hours = subjective improvement
- 72 hours = reticulocytosis
- 7-10 days = Hb levels increase
- 1-3 months = repletion of stores – always treat for 3 months
vi. Cause of poor response - Non-compliance (++++)
- Ongoing losses
- Insufficient duration
- High gastric pH
- Inhibitors of absorption
- Incorrect diagnosis – thalassaemia, chronic disease, sideroblastic anaemia
b. Prevention
i. Encourage breast feeding – then transition to additional source of iron (E.g. cereals)
ii. In non-breast fed infants use iron-fortified formula
iii. Encourage vitamin C intake
iv. Introduce iron rich foods from 6 months (E.g. meats)
v. Avoid unmodified cow’s milk until 12 months
Iron metabolism disorders - brief summary
- Defect in iron absorption
a. Iron refractory iron deficiency anaemia
b. Defect in transmembrane proteins, autosomal recessive
c. Unresponsive to oral iron, partially responsive to IV - Defects of iron recycling
a. Aceruloplasminemia – iron cannot be appropriately transported from macrophages to plasma - Defects in mitochondrial iron utilization = sideroblastic anaemia
Sideroblastic anaemia - general
- Definition = ring sideroblasts are present on the BMA stained for iron
- Aetiology
a. Hereditary
i. XLR – most common form ALAS gene X chromosome - Presents during late childhood
- Splenomegaly
- Respond to B6
ii. Autosomal = dominant or recessive
iii. Pearson Syndrome - Refractory sideroblastic anemia – MACROCYTIC not microcytic
- Exocrine pancreatic dysfunction
b. Acquired
i. Myelodysplasia
ii. Nutritional (copper, B6 deficiency) or toxins (lead, zinc)
iii. Drugs (EtOH, isoniazid, chloramphenicol)
iv. Hypothermia
v. Uremia
vi. Hyperthyroidism - Congenital sideroblastic anaemia
a. Germline mutation in nuclear or mitochondrial genes
b. 1/3 do not have an identifiable gene
c. Genetics + pathogenesis
ii. Impaired heme synthesis - Defective steps in heme synthesis in cytoplasm = porphyria
- Defective steps of heme synthesis in mitochondrion = sideroblastic anaemia
a. d-ALA or ferrochelatase incorporation of iron into porphyrin ring
b. Accumulation of iron in mitochondria of nucleated RBC (sideroblasts)
d. Clinical manifestations
i. Severe anaemia– infancy, milder forms – early adulthood
ii. Pallor, icterus
iii. Iron overload without transfusion history
iv. Moderate splenomegaly and/or hepatomegaly
e. Investigations
i. FBE – hypochromic, microcytic, high RDW (microcytic RBC mixed with normal)
ii. Film – ringed sideroblasts
iii. Iron studies – ↑ serum iron ↑ ferritin ↑ transferrin saturation; ↓ transferrin
f. Treatment
i. Severity of anaemia varies – some require no treatment, some regular RBC transfusions
ii. Stem cell transplant in transfusion dependent
iii. Vitamin B6 – X linked form responsive
iv. Management of Fe overload
Anaemia d/t lead poisoning - general
• Interferes with heme synthesis including D-ALA and ferrochelatase -> microcytic hypochromic anaemia • Coarse basophilic stippling • Elevated Lead levels + protoporphyrins • Clinical features o Anaemia o Discolouration of gums o Abdominal pain o Peripheral neuropathy
Macrocytic anaemia - list of causes
- Normal newborn
- Massive reticulocytosis
- Megaloblastic anaemia
a. Nutritional
b. IEM
c. Drug effect eg. azathioprine - Liver disease
- Hypothyroidism
Megaloblastic anaemia - general
- Overview
a. Megaloblastic anaemia= group of disorders cause by impaired DNA synthesis
b. RBCs are larger than normal at every developmental stage and there is maturational asynchrony between the nucleus and cytoplasm
c. Delayed nuclear development becomes increasingly evidence as cell divisions proceed
d. Myeloid and platelet precursors are also affected - Investigations
a. FBE: MCV >100fL, +/- thrombocytopaenia, leukopenia
b. Film
i. Large RBCs (often oval)
ii. Hypersegmented neutrophils, many have >5 lobes
c. Bone marrow: +/- metamyelocytes and neutrophil bands - Aetiology
a. Majority – folic acid or vitamin B12 deficiency (cobalamin) – vitamins essential for DNA synthesis
b. Rarely – inborn errors of metabolism
B12 - physiology
a. Source
i. Found in animal products only (meat, eggs, fish and milk)– synthesized by microorganisms
ii. B12 is a generic term encompassing all biologically active cobalamins
iii. Methylcobalamin and adenosylcobalamin are the metabolically active derivatives
b. Absorption
i. Stomach
1. Chief cells pepsinogen pepsin breaks down protein to release B12
2. B12 binds to R protein (also called haptocorrin = HC, salivary glycoprotein)
ii. Duodenum
1. Lipase/amylase/protease degrade protein R
2. Gastric antrum parietal cells intrinsic factor
3. IF + B12
iii. Ileum = absorption of B12 + IF via receptor mediated endocytosis
c. Transport
i. Transported in plasma bound to transcobalamin II (required to transport cobalamin into cells)
d. Roles
i. Serve as cofactors in 2 essential metabolic reactions
1. Methylation of homocysteine to methionine (via methionine synthase)
2. Conversion of methyl-malonyl-coenzyme A (CoA) to succinyl CoA (via L-methyl-malonyl-CoA mutase)
ii. Product and byproducts of these enzymatic reactions are critical to DNA, RNA and protein synthesis
iii. Deficiency = high homocysteine, high MMA causes neurological regression
e. Requirements + Stores
i. Require 6-9 mcg / day
ii. Older children and adults usually have 3-5 year stores of vitamin B12
iii. Young infants may manifest symptoms as early as 6- 18 months
B12 deficiency - aetiology
2. Aetiology by age Birth-6 months • Severe maternal deficiency • Metabolic causes 6 months- mid childhood • Dietary deficiency • Maternal deficiency (deficiency in BF infants) • Malabsorption Mid childhood onwards • Juvenile pernicious anaemia • Gastritis • Malabsorption • Medication
- Aetiology
a. Reduced intake = breastmilk in vitamin B12 deficient mothers, vegan diet
b. Impaired absorption
i. Gastric abnormalities - Pernicious anaemia – Ab to IF or gastric parietal cells
- Hereditary intrinsic factor deficiency
- Gastrectomy/ bariatric surgery
- Gastritis
- Autoimmune atrophic gastritis
ii. Small bowel disease - Malabsorption syndrome
- Ileal resection or bypass
- IBD eg. Crohn’s
- Celiac disease
- Bacterial overgrowth
- Tapeworm
iii. Drugs which block or impair absorption = neomycin, Biguanides (metformin), PPI + H2 antagonists
iv. Pancreatic disease = insufficiency
c. Impaired transport = inherited transcobalamin II deficiency
d. Impaired utilisation
i. Methylmalonic acidurais
ii. Methylcobalamin deficiency
B12 deficiency - ix/rx
- Investigations
a. Film
i. Hyper segmented neutrophils (>5 neutrophils with >5 lobes)
ii. Oval macrocytes
iii. Macrocytic anaemia +/- leukopenia +/- thrombocytopenia
iv. +/- teardrop cells
b. Active (holotranscobalamin) = measures TCII/B12
i. Most sensitive
ii. Measurement of total B12 is NOT sensitive or specific
c. If B12 deficiency is confirmed
i. Urinary methylmalonic acid (MMA) and serum homocysteine - One or both are elevated in almost all patients with clinical deficiency, but decrease immediately after treatment
- Homocysteine (ONLY) may also be elevated in folate deficiency
d. Haemolysis screen – LDH often very high
e. Iron studies + red cell folate – for coexistant deficiency
f. Bone marrow aspirate usually not necessary
i. If one shows hypercellular – left shift
ii. Megaloblasts + giant metamyelocytes - Management
a. For infants and those with neurological involvement – standard replacement is IM B12 (cyanocobalamin)
b. Oral doses are poorly absorbed (0.5-4%)
c. High doses can be effective for lower risk cases (ie. older children/adults WITHOUT evidence of tissue deficiency ie. no clinical features and normal homocysteine/MMA)
d. Regimens vary
i. Infants with clinical deficiency (macrocytic anaemia or neurological involvement) - 250 - 1000mcg intramuscular B12, on alternate days for 1-2 weeks, then 250mcg weekly
- Short-term parenteral therapy is often sufficient, especially if maternal deficiency is proven
- Switch to oral supplements once child is well, no diarrhoea, feeding improved, and maternal stores replaced
ii. Older children with mild disease an alternative would be: 1000mcg oral daily
iii. Subclinical, dietary deficiency - 50-200 mcg oral daily (generally as 100mcg tablets, also available as sublingual sprays/tablets).
- Increase dietary intake
iv. Supplement (not deficient, no dietary intake) - 50-100mcg daily or alternate daily
v. Pernicious anaemia – oral B12 1000 ug/ day
Transcobalamin II deficiency - general
• AR failure to absorb and transport B12
• Serum B12 levels are normal – need to check “active B12”
o >80% of serum cobalmin is bound to haptocorrin (HC)
• Manifests first week of life – FTT, diarrhoea, vomiting, glossitis, neurologic abnormalities + megaloblastic anaemia
• Diagnosis
o Severe megaloblastic anaemia
o Normal serum B12 and folate levels
o No evidence of any other inborn errors of metabolism
• Treatment
o Large parenteral doses of B12 – ‘overcomes’ transcobalamin deficiency
o Death in infancy if untreated
Folate - physiology
- Background
a. Folates are essential for DNA replication and cellular proliferation
b. Biologically active folates are derived from folic acid (pteroic acid conjugated to glutamic acid) and serve as donors and acceptors in biosynthetic pathways
c. To form functional compounds – folates must be reduced to tetrahydrofolates (by enzyme – dihydrofolate reductase) - Physiology
a. Source
i. Widely available from food – 1/3 meat and fish, 1/3 cereals and bread, 1/3 fruit and vegetables (may be less in cow’s milk)
ii. Folates are heat labile and water soluble – decreased amounts of vitamin with heating/boiling
iii. NO folate in goat’s milk
b. Folic acid monoglutamated, naturally occurring folates polyglutamated
c. Absorption
i. Polyglutamated form – less efficiently absorbed than monoglutamated
ii. Dietary folate polyglutamates are hydrolysed to simple folates
iii. Primarily absorbed in the proximal small intestine
iv. Folates travel in blood stream and are taken into cells as unconjugated methyltetrahydrofolate
v. Subsequently reconjugated/polyglutamated in the cell
d. Function
i. Folates act in numerous single carbon reactions - Synthesis of methionine from homocysteine
- Purine and pyrimidine metabolism
ii. Circulates in plasma as 5-methyl THF
iii. Body folate stores limited to several weeks - Acute folate deficiency may develop in hospitalized patients
Folate deficiency - aetiology
a. Inadequate folate intake
i. Increased requirements
1. Pregnancy
a. Supplementation recommended to prevent NTD
b. Folate-deficient mothers generally do not give birth to infants with folate deficiency due to selective transfer of folate to the fetus via placental folate receptors
2. Accelerated growth after birth
a. Increases demands for folic acid
b. Premature, unwell infants and those with certain hemolytic disorders will have particularly high folate requirements.
c. Dietary sources – goat’s milk inadequate
3. Haemolysis
ii. Malnutrition
1. Most common cause in older children
2. Increased risk – patients with hemoglobinopathies, infections, and/or malabsorption
3. Body stores of folate are limited; deficiency can develop quickly in malnourished individuals
a. On a folate-free diet, megaloblastic anaemia will occur after 2-3 months
b. Decreased folate absorption
i. Chronic diarrheal states – chronic infectious enteritis
ii. Diffuse inflammatory disease
iii. Coeliac disease
iv. Enteroenteric fistulas
v. Previous intestinal surgery
vi. Certain anticonvulsant drugs – phenytoin, primidone, phenobarbital
vii. Alcohol overuse
c. Acquired and congenital disorders of folate metabolism or transport
i. Hereditary folate malabsorption (HFM)
b. Inability to absorb folic acid and derivatives
ii. Enzyme deficiencies (extremely uncommon)
1. Functional methionine synthase deficiency
2. Dihydrofolate reductase deficiency
3. NOTE: methylenetetrahydrofolate (MTHFR) deficiency is the MOST common inborn error of folate metabolism
iii. Drug induced
1. Methotrexate – prevents formation of active form of folate
2. Pyrimethamine, trimethoprim – folic acid deficiency
Folate deficiency - sx/rx
- Incidence
a. Megaloblastic anaemia as a consequence of folate deficiency is rare
b. Peak incidence 4-7 months (earlier than iron deficiency) - Clinical manifestations
a. Irritability (older – depression, dementia, psychosis)
b. Chronic diarrhea
c. Poor weight gain
d. Advanced – haemorrhages from thrombocytopaenia - Treatment
a. MUST exclude vitamin B12 deficiency before treating
b. Folic acid – oral or parenteral for 3-4 weeks, 0.5-0.1mg/day
c. Haematological response in 72 hours
d. Maintenance therapy – multivitamin
e. Preconception folate supplements for prevention NTD
i. Fefol/FGF inadequate in pregnant women with increased folate requirements
Folate deficiency - ix
a. FBE = macrocytic anaemia, low reticulocytes +/- neutropaenia +/- thrombocytopaenia
i. Note: acute folate deficiency not macrocytic
b. Film = nucleated RBC with megaloblastic morphology, variation in RBC size/shape, neutrophils large some with hypersegmented nuclei
c. Bone marrow
i. Hypercellular (due to erythroid hyperplasia)
ii. Megaloblastic changes prominent
iii. Large, abnormal neutrophilic forms (giant metamyelocytes) with cytoplasmic vacuolation
d. Serum folic acids levels = < 3ng/ml (normal 5-20ng/ml)
e. RBC folate= is a better indicator of chronic deficiency (normal 150-600ng/ml)
f. Iron = normal/elevated
g. Vitamin B12 = normal/elevated
h. LDH (marker of ineffective erythropoiesis) = markedly elevated
i. Homocysteine = elevated
j. MMA = normal
Orotic aciduria - general
a. Rare AR disorder of megaloblastic anaemia
b. Presents in 1st yr of life
c. Most common metabolic error in the de novo synthesis of pyrimidines and therefore affects nucleic acid synthesis
d. Clinical manifestations
i. Growth failure
ii. Developmental retardation
e. Investigations
i. Severe megaloblastic anemia
ii. Normal serum B12 and folate levels
iii. No evidence of TC deficiency
iv. Increased urinary orotic acid
f. Treatment
i. Responds promptly to administration of uridine (refractory to B12 or folic acid)
Roger syndrome - general
Thiamine-responsive megaloblastic anaemia
a. Very rare autosomal recessive disorder
b. Clinical manifestations
i. Megaloblastic anemia
ii. Sensorineural deafness
iii. Diabetes mellitus
iv. Thiamine-responsive megaloblastic anemia usually presents in infancy
c. Investigations
i. Bone marrow – megaloblastic changes, ringed sideroblasts.
d. Treatment
i. Thiamine supplementation = reverses the anemia and diabetes but not existing hearing defect
Diamond Blackfan Anaemia - background
Inherited red cell aplasia
Ribosomopathy
- Key points
a. Rare, congenital bone marrow failure syndrome
b. Up to 50% of affected individuals have additional extrahematopoietic anomalies - Genetics + pathogenesis
a. Predominantly AD
d. Mutations in 1 of the 10 ribosomal protein genes (RP) identified in 50-70% of cases
e. DBA is a ribosomopathy - mechanism by which RP haploinsufficiency leads to DBA is unclear
Key features
- anaemia (normochromic, macrocytic)
- reticulocytopenia
- insufficient/absent RBC precursors in BM
- congenital anomalies
Diamond Blackfan Anaemia - sx/ix
- Clinical manifestations
a. Usually becomes symptomatic in early infancy, >90% of cases recognized before 1 year of life
b. Anaemia (normochromic and macrocytic) + reticulocytopaenia
i. Profound anaemia evident by 2-6 months of age
ii. 25% of patient’s anemic at birth and hydrops fetalis occurs rarely
c. Congenital abnormalities = 50%
i. Craniofacial (50%) – hypertelorism, snub nose, high arched palate
ii. Skeletal anomalies (30%) – mostly upper limb and hand - Thumb abnormalities – flattening of thenar eminence and triphalangeal thumb
- Radial pulse may be absent
iii. Genitourinary (38%) = absent kidney, horseshoe kidney, hypospadias
iv. Cardiac (30%) = VSD, ASD, CoA, complex cardiac disease
v. Ophthalmologic
vi. Musculoskeletal = growth retardation, syndactyly
d. Short stature common – unclear if disease related vs treatment
e. Malignancy
i. Cancer predisposition syndrome
ii. Increased risk of myelodysplastic syndrome, AML, colon carcinoma, osteogenic sarcoma, female genetic cancers - Investigations
a. FBE = normochromic macrocytic anaemia
i. +/- thrombocytosis, thrombocytopenia (rare) and occasionally neutropenia
ii. Low reticulocyte percentages
b. Blood film = red cell patterns similar to foetal population – increased fetal Hb and expression of ‘I’ antigen
c. Bone marrow = erythrocyte precursors markedly reduced in most
i. NORMAL myeloid/megakarocyte lines
d. Serum iron levels = elevated
e. HbF = often elevated
f. Erythrocyte adenosine deaminase (ADA) activity = increased in most
Diamond Blackfan Anaemia - ddx
a. Anaemia with low reticulocyte count transient erythroblastopaenia of childhood (TEC)
i. Relatively late onset, but occasionally onset <6mo of age
ii. Don’t typically see macrocytosis, congenital anomalies, fetal red cell characteristics, elevated erythrocyte ADA
b. Macrocytic bone marrow failure syndromes = FA, SDS, DC, MDS
c. Haemolytic disease of the newborn
i. Can mimic features of DBA if protracted course and coupled with markedly reduced erythropoiesis
ii. Anaemia usually resolves spontaneously 5-8 weeks of age
d. Aplastic crisis in chronic haemolytic disease
i. Reticulocytopaenia and decreased numbers of RBC precursors
ii. Often occurs after first several mo of life
iii. Often caused by parvovirus B19 in utero = pure RBC aplasia in infancy, hydrops fetalis at birth
Diamond Blackfan Anaemia - rx/prognosis
- Treatment
a. Mainstay = corticosteroids
i. 80% of patients initially respond
ii. RBC precursors in BM within 1-3 weeks; followed by reticulocytosis
iii. Hb can reach normal limits in 4-6 weeks
iv. Adequate response = Hb >9
v. Aim lowest dose to maintain Hb
vi. Side effects – reduced BMD, cataracts/glaucoma, avascular necrosis fem head
vii. Other – Bactrim prophylaxis, PPI, stress steroid regime when unwell
b. Transfusions
i. For non-responders OR fail to tolerate side effect profile
ii. Transfusions every 3-5 weeks
c. Spontaneous remission has been reported
d. Haematopoietic stem cell transplantation (HSCT) can be curative
i. Best outcomes in HLA matched sibling donors < 9 years of age - Prognosis
a. Actual survival – 75% at 40 years (87% maintained on steroids) and 57% for transfusion dependent patients
b. Deaths
i. Treatment related (67%)
ii. Malignancy or severe aplastic anaemia (22%)
DBA vs TEC
Diamond Blackfan Anaemia versus Transient erythroblastopenia of childhood
DBA Age: <1 year Antecedent history: None Physical anomalies: 1/3 MCV: Increased Haemaglobin F: Increased i-Antigen: Increased
TEC Age: >1 year Antecedent history: Viral illness Physical anomalies: None MCV: Normal HbF: Normal i-Ag: Normal
Pearson Marrow Pancreas Syndrome - general
- Background
a. Rare mitochondrial disorder which presents with hypoplastic anaemia - Genetics + pathogenesis
a. Mitochondrial DNA deletion of variable size and location
b. Variable clinical picture – heterogeneity in different tissues and patients
c. Proportion of deleted mtDNA in BM correlated to severity of hematological picture - Clinical
a. Marrow failure typically appears in neonatal period
b. Multi-organ involvement
i. FTT
ii. Exocrine pancreas dysfunction
iii. Liver and renal tubular defects
iv. Malabsorption
v. Myopathy - Investigations
a. FBE = macrocytic anaemia, occasionally neutropaenia and thrombocytopaenia
b. Bone marrow = vacuolated erythroblasts and myeloblasts, ringed sideroblasts (unique variant of congenital sideroblastic anaemia)
c. Elevated haemoglobin F level - DDx
a. Diamond Blackfan anaemia
b. Transient erythroblastopaenia of childhood
c. Kearns-Sayre syndrome – similar mitochondrial DNA deletion - Treatment
a. Supportive
b. Red cell transfusions to correct anaemia
c. Granulocyte colony stimulating factor to reverse episodes of severe anaemia
Transient erythroblastopenia of childhood - general
- Key points
a. Mainly occurs previously healthy children between 6 mo-3 years
b. More prevalent than congenital hypoplastic anaemia (Diamond-Blackfan) - Aetiology
a. Suppression of erythropoiesis linked to IgG, IgM and cell mediated mechanism
b. Familial cases reported – suggesting hereditary component
c. Often follows viral illness (no specific virus implicated)
d. Rarely caused by parvovirus induced RBC aplasia in children with hemolytic anaemia or congenital or acquired immunodeficiencies - Clinical manifestations
a. Severe, transient hypoplastic anaemia
b. Most older than 12mo at onset
c. Anaemia develops slowly, significant symptoms only with severe anaemia - Investigations
a. FBE
i. Reticulocytopaenia, mod-severe normocytic anaemia (due to temporary suppression of erythropoiesis), MCV normal for age
ii. Some degree of neutropaenia in 20%
iii. Platelets normal or elevated - thrombocytosis presumably caused by increase erythropoietin, which has some homology with thrombopoietin
b. HbF levels = normal before recovery phase
c. RBC adenosine deaminase levels = normal (cf elevated in congenital hypoplastic anaemia) - Differential diagnosis
a. Congenital hypoplastic anaemia
i. Differences in age at onset, age-related MCV, HbF and adenosine deaminase
b. Iron deficiency anaemia in infants receiving milk as main caloric source
i. Peak occurrence at similar age
ii. Differences in MCV - Prognosis
a. Virtually all children recover within 1-2 months - Treatment
a. RBC transfusions if severe anaemia
Parvovirus B19 anaemia/red cell aplasia - bg, sx, ix
- Key points
a. Best-documented viral cause of RBC aplasia in patients with chronic haemolysis, immunocompromised and fetuses in utero
b. Does NOT cause significant anaemia in immunocompetent individuals with normal red cell life spans - Pathogenesis
a. Small, non-enveloped single-stranded virus is particularly infective and cytotoxic to marrow erythroid progenitor cells – binding to red cell P antigen - Clinical manifestations
a. Causes erythema infectiosum (fifth disease)
b. Usually transient with recovery occurring in <2 weeks
c. Anaemia either not present or not appreciated in otherwise normal children whose peripheral RBC life span is 100-120 days - Investigations
a. Parvovirus IgG/IgM + PCR
b. Decreased/absent erythroid precursors
c. Bone marrow
i. Characteristic nuclear inclusions in erythroblasts
ii. Giant pronormoblasts
Red cell aplasia aw parvo B19 - complications
a. Chronic haemolysis
i. RBC lifespan is shorter in patients with haemolysis (eg. spherocytosis, immune haemolytic anaemia, sickle cell)
ii. Brief cessation of erythropoiesis can cause severe anaemia – aplastic crisis
iii. Parvovirus-induced aplastic crisis usually occurs only once in children with chronic haemolysis
iv. Recovery from mod-severe anaemia usually spontaneous, heralded by a wave of nucleated RBCs and subsequent reticulocytosis in the peripheral blood
v. Treatment = RBC transfusion if symptomatic from anaemia
b. Immunodeficiency
i. Chronic parvovirus infection in immunosuppressed children
ii. Resultant pure RBC aplasia may be severe, and affected children may be thought to have TEC – no spontaneous recovery and >1 transfusion required
iii. Note PCR usually required for diagnosis as serology impaired
iv. Treatment = high dose IVIG – contains neutralizing Ab to parvovirus and is effective in short term
c. Miscarriage and hydrops fetalis
i. Parvovirus infection and destruction of erythroid precursors in utero
ii. Associated with increased fetal wastage in the first and second trimesters
iii. Born with hydrops fetalis and anaemia
iv. Investigations
1. Immunologic tolerance to the virus can prevent usual development of specific Ab
2. Persistent congenital parvovirus – detected by PCR blood and/or bone marrow
Acquired red cell aplasia - chloramphenicol, CKD
- Drugs = chloramphenicol
a. Can inhibit erythropoiesis in a dose-dependent manner
b. Reversible reticulocytopenia, erythroid hypoplasia, and vacuolated pronormoblasts in the bone marrow
c. Distinct from the idiosyncratic and rare development of severe aplastic anemia in chloramphenicol recipients. - Chronic kidney disease
a. Acquired Ab-mediated pure red cell aplasia rare complication in CKD patients treated with erythropoiesis-stimulating agents
b. Treatment
i. Discontinue EPO
ii. RBC transfusion
iii. Immunosuppression
iv. Renal Tx
Anaemia of chronic disease - background
- Key points
a. Conditions where there is ongoing immune activation – infection, malignancy, autoimmunity, GVHD
b. A similar anemia is associated with CKD
c. Normocytic, normochromic, hypoproliferative anemia
d. Associated ↓ serum iron and ↓ transferrin saturation - Pathogenesis
a. Decreased red cell life span = cytokines (eg. IL-1) may increase macrophages ability to destroy RBC
b. Impaired erythropoiesis = immune cell/cytokine driven EPO production + BM suppression
c. Increased uptake of iron in the reticuloendothelial system
i. Diversion of iron from the circulation into the reticuloendothelial system results in functional iron deficiency impaired heme synthesis and iron-restricted erythropoiesis
ii. Inflammation-associated excess synthesis of hepcidin (protein which controls intestinal absorption + tissue distribution); hepcidin synthesized by hepatocytes and expressed in other cells (monocytes) - Functions by binding to and initiating the degradation of the iron exporter, ferroportin.
Anaemia of chronic disease - sx/ix/rx
- Clinical manifestations
a. Signs and symptoms associated with underlying disease
b. Mild-mod anaemia can affect QoL - Investigations
a. FBE = normochromic, normocytic anaemia (Hb 60-90)
i. Modest hypochromia and microcytosis in some patients, particularly if concomitant iron deficiency
ii. Low or normal absolute reticulocyte counts
iii. Leukocytosis
b. Iron studies
i. Serum iron level = low - NO increase in serum transferrin that occurs in iron deficiency (low to normal)
ii. Ferritin level = normal or increased
c. Bone marrow = normal cellularity; RBC precursors decreased or adequate, marrow hemosiderin may be increased, and granulocytic hyperplasia may be present. - Treatment
a. Treatment of underlying disorder = anemia will improve or resolve
b. Transfusions rarely required
c. Erythropoietic stimulating agents (ESAs) ie EPO
i. Increase Hb level and improve activity
ii. Concurrent treatment with iron usually necessary for optimal effect
iii. Response variable
d. Iron deficiency patients
i. Difficult to identify iron deficiency in patients with an inflammatory disease
ii. May be no response to iron therapy as persistent inflammation impairs iron absorption and utilization
iii. IV iron may further increase hepcidin production.
Anaemia of renal disease - background
- Key points
a. Usually normocytic, and the absolute reticulocyte count is normal or low
b. Most common in ESRF
c. Associated with incidence of LVH, impaired physical activity, reduced QoL + hospitalisation/ mortality - Pathogenesis
a. Decreased EPO production = MAIN CAUSE
b. Absolute or functional iron deficiency from chronic blood loss – blood sampling, surgery, dialysis
c. Disturbances in the iron metabolic pathway
d. Higher hepcidin levels
i. Hepcidin is filtered by the glomerulus and excreted by the kidney
ii. Serum concentrations are increased in patients with decreased GFR
e. Inflammation
f. Hyperparathyroidism and deficiencies of vitamin B12, folate, and carnitine
Anaemia of renal disease - ix/rx
- Investigations
a. Anemia in children with CKD is defined by age
i. Hemoglobin (Hb) - <110 g/L (0.5-5 yr)
- <115 g/L (5-12 yr)
- <120 g/L (12-15 yr)
- <130 g/L (males older than 15 yr)
- <120 g/L (females older than 15 yr).
b. FBE
i. Normocytic and normochromic (unless concomitant Fe deficiency or vitamin deficiency)
ii. Absolute reticulocyte count – low
iii. White cell and platelet counts – normal
iv. Ferritin – low if iron deficiency, high if inflammation
c. EPO = low - Treatment
a. Oral iron therapy = all CKD patients with anaemia
i. Oral iron at 3-6 mg of elemental iron/kg of target dry weight once daily for 3 mo
ii. Consider IV iron if no improvement in transferrin saturation and/or ferritin
b. IV iron therapy = consider in patients on dialysis
c. ESAs = mainstay of therapy
i. All children with CKD when Hb concentrations are at 90-100 g/L, with a goal of 110-120 g/L
ii. Dosing varies with age and dialysis modality
iii. Continue with Fe supplementation
iv. Note: subset of patients is hyporesponsive to ESAs
v. Complication = Ab mediated pure red cell aplasia - Must STOP ESA (antibodies can target endogeneous EPO as well)
Congenital dyserythropoetic anaemias (CDAs) - general
- Overview
a. Heterogeneous class of inherited disorders resulting from abnormalities of late erythropoiesis.
b. Rare conditions characterized by variable degrees of anemia, ineffective erythropoiesis, and secondary hemochromatosis
c. Dyserythropoiesis is the major cause of anemia but a shortened half-life of circulating red cells may also contribute.
d. The CDAs have historically been classified into 3 major types (I, II, and II) based upon distinctive bone marrow morphology and clinical features, although additional subgroups and variants have also been identified - Type 1 CDA
i. AR
ii. Causative gene (CDAN1) encodes codanin-1
i. Most cases recognized in childhood/adolescence – rarely diagnosed in utero
i. Treatment primarily supportive – do not respond to erythropoietin - Type II aka HEMPAS
a. Hereditary erythroblastic multinuclearity with a positive acidified serum test
b. Most common form of CDA – families mostly from Europe + Middle East
c. Erythroblast multinuclearity and circulating RBCs that are sensitive to lysis by acidified normal serum.
i. AR
i. Diagnosis is usually made later in life (cf CDA 1), milder anaemia and semiology
g. Prognosis = usually normal life expectancy
h. Ddx = hereditary spherocytosis - CDA III
a. Extremely rare, ill-defined entity manifested by a mild-to-moderate macrocytic anemia.
i. AD
Neonatal anaemia - general
- Normal Hb
a. Increases with gestational age
i. Term Hb 170 g/L
ii. VLBW Hb 150-160 g/L
b. Physiologic ↓ in Hb
i. Term 8-12/52 – 110 g/L
ii. Preterm 6/52 - 70-100 g/L - Blood volume at birth
a. 90mL/kg = 3.5kg infant = 300mL - Early vs late cord clamping
a. Controversy
b. Benefits delayed cord clamping – improved blood volume, reduced iron deficiency in childhood
c. Disadvantages delayed cord clamping – increasing plethora/polycythemia, increasing hyperbilirubaemia
d. Current recommendation clamp at 1 minute in uncompromised infants - Causes of neonatal anaemia
a. Physiological
b. Anaemia or prematurity
c. Haemorrhage (external)
i. APH
ii. Fetomateral transfusion
iii. Twin-twin transfusion
d. Neonatal internal haemorrhage
i. Traumatic – E.g. sub-galeal, umbilical cord snap
ii. Coagulopathy
iii. Thrombocytopenia
iv. Haemorrhagic disease newborn
e. Haemolysis
i. Inherited
ii. Acquired
a. Congenital aplasia
At birth
- Haemorrhage – APH, FMH, TTTS
- Traumatic haemorrhage – sub-galeal, umbilical cord snap
First few days
- Haemolysis
- Coagulopathy / HDN
Later
- Physiological
- Prematurity
- Haemorrhagic disease newborn
- Haemolysis
- Congenital aplasia
Physiologic anaemia of infancy - general
- Key points
a. Full term infants have higher Hb + larger RBC than older children + adults
b. Decline in Hb from first week of life to 6-8 weeks = physiologic anaemia of infancy
c. Term infants – Hb reaches nadir of 11 g/dL at 8-12 weeks after birth (EPO prevents further decline)
d. Preterm infants – more pronounced nadir, usually occurs at 3-12 weeks after birth in infants <32 weeks
e. Onset inversely proportional to gestational age, resolves by 3-6 months - Pathogenesis
a. Increase in blood oxygen content and delivery at birth
i. Respiration = more O2 for binding to Hb Hb–oxygen saturation increases from 50% to >95%
ii. Fetal to adult Hb synthesis after birth replacement of high-oxygen-affinity fetal Hb with lower-affinity adult Hb, capable of delivering more oxygen to tissues
b. The increase in blood oxygen content and delivery downregulation of EPO production suppression of erythropoiesis aged RBCs that are removed from the circulation are not replaced Hb level decrease
c. Hb concentration declines until tissue oxygen requirement > oxygen delivery – occurs at 8-12 weeks of age when the Hb concentration is about 110 g/L EPO production increases and erythropoiesis resumes
d. The supply of stored reticuloendothelial iron, derived from previously degraded RBCs, remains sufficient for this renewed Hb synthesis, even in the absence of dietary iron intake, until approximately 20 weeks of age - Investigations
a. Normocytic and normochromic RBC
b. Low reticulocyte count
c. Serum EPO low - Triggers
a. Some dietary factors, such as folic acid deficiency, can aggravate physiologic anemia
b. Unless significant blood loss, iron stores should be sufficient to maintain erythropoiesis early on
c. Vitamin E deficiency does NOT play a role in anemia of prematurity - Management
a. Iron supplementation
b. Monitoring
c. Transfusion
d. EPO = LIMITED efficacy in decreasing the number of blood donors to which infant exposed to; therefore not generally recommended
e. Restrict phlebotomy
Physiologic anaemia of prematurity - general
i. More extreme + rapid – nadir of 70-90 g/L reached by 3-6 weeks of age – lower in very small babies
ii. Additional factors for premature neonates
1. Blood loss from repeated phlebotomies
2. Suboptimal erythropoietic response
a. Increased demand of erythropoiesis shortened RBC life span (40-60 days) and accelerated expansion of RBC mass that accompanies rapid rate of growth
b. Plasma EPO levels are lower than would be expected for the degree of anemia
i. Switch from liver (normal fetal source of EPO) to kidney synthesis is not accelerated in prematurity reliance on the liver as the primary site for synthesis (less sensitive than kidney) reduced responsiveness to anemia
iii. Treatment
- Transfusions – optimal Hb not established
a. Note decline in Hb in VLBW infants associated with abnormal clinical signs NOT benign and requires transfusions
b. Beneficial effect NOT documented when administered for poor weight gain, respiratory difficulties and abnormal HR
c. Negative associations
i. Neurodevelopmental outcomes poorer in liberally transfused
ii. NEC (late exposure to PRBC)
iii. IVH (early transfusions)
iv. Donor exposure
d. In early preterm infants’ half-life of transfused RBCs is about 30 days - Recombinant human EPO if symptomatic (? Increased risk of ROP)
Pancytopenia - general overview
- Definition
a. Reduction below normal values of all 3 peripheral lineages – leucocytes, platelets and erythrocytes
b. Pancytopaenia requires microscopic examination of a bone marrow biopsy specimen and a marrow aspirate to assess overall cellularity and morphology - Aetiology
a. Hypocellular marrow
i. Inherited (‘constitutional’) marrow failure syndromes
ii. Acquired aplastic anaemia of various aetiologies
iii. Hypoplastic variant of myelodysplastic syndrome (MDS)
iv. Some cases of paroxysmal nocturnal haemoglobinuria with pancytopaenia
b. Cellular marrow
i. Primary bone disease ie acute leukaemia and MDS
ii. Secondary to systemic disease, such as autoimmune disorders (SLE), vitamin B12 or folate deficiency and storage disease (Gaucher and Niemann-Pick diseases), overwhelming infection, sarcoidosis, hypersplenism
c. Bone marrow infiltration
i. Metastatic solid tumours
ii. Myelofibrosis
iii. Haemophagocytic lymphiohistiocytosis
iv. Osteoporosis
Aplastic anaemia - differentials
Inherited/constitutional pancytopenia
- Key points
a. Aplastic anaemia = pancytopaenia + hypocellular bone marrow
b. 30% of causes of paediatric marrow failure – Fanconi most common - Aetiology
a. Inherited
i. Fanconi anaemia
ii. Schwachman-Diamond syndrome
iii. Dyskeratosis congenital
iv. Congenital amegakaryocytic thrombocytopenia
v. Reticular dysgenesis
vi. Unclassified inherited bone marrow failure syndromes
vii. Other genetic syndromes = Down syndrome, Dubowitz syndrome, Seckel syndrome, Schimke immunoosseous dysplasia, Cartilage-hair hypoplasia, Noonan syndrome
b. Acquired
i. Drugs, chemicals, radiation
ii. Viral infection, immune disorder
iii. MDS
iv. PNH
Fanconi anaemia - genetics, pathogenesis
a. AR – one uncommon form X-linked
b. Sibling discordance in clinical + haematological findings (even monozygotic twins)
c. Mutation in FA (FANC) gene – resulting in chromosomal fragility
i. Wild-type FANC gene product recognise and repair DNA
ii. Mutant gene proteins lead to genomic instability and chromosome fragility.
iii. Corrective effect (complementation) – cell fusion of FA cells with normal cells/cells from unrelated patients with FA produces a corrective effect on chromosomal fragility – allows subtyping
iv. Multiple different subtypes (complementation groups) – A-P - genes prefixed with FANC (FANCA, FANCB); NOTE FANCD1 is identical to the breast cancer susceptibility gene, BRCA2
d. Consequences
i. Inability to remove oxygen free-radicals oxidative damage
ii. Leukocyte telomerase length reduced + telomerase activity increased high proliferative rate of marrow progenitors that ultimately leads to their premature senescence
iii. Increased marrow cell apoptosis occurs and is mediated by Fas
Fanconi anaemia - manifestations
a. Presentation
i. 75% of patients are 3-14 years at diagnosis
ii. At presentation, patients with FA may have
1. Typical physical anomalies and abnormal hematologic findings (majority of the patients)
2. Normal physical features but abnormal hematologic findings (about one-third of patients)
3. Physical anomalies and normal hematologic findings (unknown percentage)
b. Haematological
i. Marrow failure usually in the first decade of life
ii. Initially – thrombocytopaenia, RBC macrocytosis
iii. Subsequent onset of granulocytopenia, anaemia
iv. Months to years – severe aplasia
c. Skin (most common, 55%)
i. Hyperpigmentation of the trunk, neck, and intertriginous areas
ii. Café-au-lait spots and vitiligo, alone or in combination
d. Short stature (51%) – may be aggravated by abnormal GH secretion or hypothyroidism
e. Skeletal (upper limb 43%, lower limb 10%)
i. Absence of radii
ii. Thumbs that are hypoplastic, supernumerary, bifid, or absent are common
iii. Anomalies of the feet
iv. Congenital hip dislocation
v. Leg abnormalities
vi. The “r” radial pulse may be weak or absent
f. Genitourinary (35%, mostly male)
i. +/- underdeveloped penis; undescended, atrophic, or absence of the testes; hypospadias or phimosis
ii. Malformations of the vagina, uterus, and ovary
g. Facies
i. Microcephaly
ii. Small eyes, epicanthal folds
iii. Abnormal shape, size, or positioning of the ears, deafness (9%)
h. Renal = ectopic, pelvic, or horseshoe kidneys (21%)
i. Cardiovascular and gastrointestinal malformations also occur (11%)
j. Malignancy
i. Solid tumours = SCC of the head, neck, upper oesophagus, vulva and/or anus, cervix, lower oesophagus; HPV suspected in pathogenesis
ii. Therapy related oral cancer post BMT
iii. Androgen therapy = benign and malignant liver tumors occur (adenomas, hepatomas)
1. Peliosis hepatis (blood-filled hepatic sinusoids) – reversible when androgen therapy is discontinued, and tumors may regress.
iv. Clonal marrow cytogenetic abnormalities – advanced MDS and AML (15% leukaemia by age 35)
Fanconi anaemia - ix
a. FBE
i. Marrow failure usually in the first decade of life
ii. Initially – thrombocytopaenia, RBC macrocytosis
iii. Subsequent onset of granulocytopenia, anaemia
iv. Months to years – severe aplasia
b. AFP = stable elevation
c. BMA = hypocellular, fatty
d. Chromosome fragility = fragility (spontaneous chromatid breaks etc)
i. Lymphocyte chromosomal breakage study using DEB or mitomycin C = unique to Fanconi Anaemia
ii. Skin fibroblasts = testing of skin fibroblasts instead of lymphocytes confirms diagnosis
1. 10-15% have somatic mosaicism – therefore lymphocytes may not show chromosomal fragility due to mixed population
e. Prenatal diagnosis = abnormal chromosome breakage analysis and genetic testing can be performed in amniotic fluid cells or in tissue from a chorionic villus biopsy
f. Genetic testing
i. Large number of FANC genes - genetic diagnosis has traditionally with complementation testing.
ii. Determining whether cellular hypersensitivity to crosslinking agents (e.g., mitomycin C or radiation) or immunoblotting for FANCD2 is restored after generating hybridoma of the patient cells with known genetic complementation cells or after transducing the cells with a known FANC gene
iii. The mutant gene or the complementation group is deduced when a specific wild-type FANC gene corrects the abnormal chromosome fragility
Fanconi anaemia - rx, prognosis
- Treatment
a. Surveillance
i. Endocrinology - Monitor growth velocity
- Screening for glucose intolerance + hyperinsulinism – annually or biannually
ii. Cancer - FBE 1-3 monthly
- BMA + biopsy annually to surveil for leukaemia and MDS
- Annual assessment for solid tumours including gynaecological cancers in pubertal females
b. HSCT
i. Only curative therapy
ii. Patients <10 years with FA – undergo transplantation with HLA-identical sibling donor >80% survival - Survival rates lower for patients >10 yr old
iii. May do MUD for children without HLA-matched sibling donor – 50% survival
c. Androgens
i. Oral oxymetholone daily
ii. Response in 50% of patients - Reticulocytosis and a rise in Hb within 1-2 months WBC platelets
iii. May take months to achieve the max response – then taper androgen dose but not ceased
iv. ? Add prednisolone - may counter androgen-induced growth acceleration and prevent thrombocytopenic bleeding by promoting vascular stability
v. Usually becomes refractory as the disease progresses
vi. AE = masculinization, elevated hepatic enzymes, cholestasis, peliosis hepatis, and liver tumors. - Require screening
d. GCSF + EPO
i. Increases ANC +/- platelets and Hb levels
ii. Possible increased risk of marrow cells with clonal cytogenetic abnormalities such as monosomy 7
iii. Combined with EPO to boost Hb
iv. Short-term treatment – patients lose response after 1 year due to marrow failure
e. Future = gene therapy - Prognosis
a. Cases reported in the 1990s, the projected median survival was >30 yr of age
b. Careful surveillance has improved survival
Shwachman-Diamond Syndrome - genetics, ddx
- Genetics + pathogenesis
a. AR
b. Mutant gene SBDS (chromosome 7q11) in 90%
i. The wild-type gene protein product is involved in ribosomal biogenesis
ii. Found in 80-90% of patients
iii. Results in ribosomal dysfunction in 90-95% of patients
c. Pancreatic insufficiency is a result of failure of pancreatic acinar development – fatty replacement
d. BM failure - dysfunctional stem cells, accelerated apoptosis of marrow progenitors and a defective marrow microenvironment that does not support and maintain normal hematopoiesis - Differential diagnosis
a. Fanconi Anaemia
i. Similarities – marrow dysfunction, growth failure
ii. Differences – SDS has pancreatic insufficiency, lacks characteristic skeletal abnormalities, normal chromosomal breakage study with DEB.
b. Pearson syndrome
i. Refractory sideroblastic anemia, cytoplasmic vacuolization of bone marrow precursors, lactic acidosis, exocrine pancreatic insufficiency, and a diagnostic mitochondrial DNA mutation is similar
ii. Differ in clinical course, morphologic features of the bone marrow, and gene mutation
iii. Severe anemia requiring transfusion (cf neutropenia) is present from birth to 1 yr of age.
Shwachman-Diamond Syndrome - sx
a. Pancreatic insufficiency
i. 2nd most common cause of pancreatic insufficiency in children
ii. Extensive lipomatous changes in the pancreas
iii. Variable abnormality of enzyme secretion
iv. 50% of patients show an age-related improvement (4 years) in pancreatic function
v. Despite adequate pancreatic replacement and correction of malabsorption, poor growth commonly continues
b. Haematological
i. Neutropenia (may be cyclic, neutrophil chemotaxis defects) occurs in at least 85-100%
1. Common cause of congenital neutropenia
2. Neutrophils may have a defect in mobility, migration, and chemotaxis owing to alterations in neutrophil cytoskeletal or microtubular function
3. Recurrent pyogenic infections (otitis media, pneumonia, OM, dermatitis, sepsis) = frequent and are common cause of death
4. Can have neonatal sepsis and early death
ii. Thrombocytopenia found in 25% of patients
iii. Anaemia in 50%
iv. Pancytopenia in 10-25%
v. Myelodysplastic syndromes in 30% - 10% acute myeloid and other leukaemias
c. Skeletal defects
i. Often not apparent until patient >2 years
ii. Delayed bone maturation
iii. Metaphyseal dysostosis/chondrodysplasia
iv. Flared ribs
v. Thoracic dystrophy
d. Failure to thrive + short stature
i. Birth weight low and by 6/12 are typically below the 5th centile for weight and height
ii. Independent of nutrition
e. Oher
i. +/- hepatomegaly and elevation of liver enzymes
ii. +/- neurocognitive problems and poor social skills
iii. Dental disease
Shwachman-Diamond Syndrome - ix
- Investigations
a. FBE = neutropenia, thrombocytopenia, anaemia, pancytopenia
i. There is no increased chromosomal breakage after DEB testing of SDS lymphocyte
b. Pancreatic function tests
i. Impaired enzyme secretion, but with preservation of ductal function
ii. Serum trypsinogen and isoamylase levels are reduced
iii. 72 hour stool collection – fat malabsorption
c. Immunoglobulins
i. +/- B cell defects with 1 or more of the following: low IgG or IgG subclasses, low % of circulating B lymphocytes, decreased in vitro B-cell proliferation, and lack of specific antibody production
ii. +/- low percentage of circulating T cells, subsets, or NK cells, and decreased in vitro T-cell proliferation
d. Bone marrow = varying degrees of marrow hypoplasia and fat infiltration
e. CT/US = fatty replacement of pancreatic tissue - Treatment
a. Surveillance
i. Regular FBE, 3 monthly
ii. For malignant myeloid transformation - Serial bone marrow aspirations for smears and cytogenetics and marrow biopsy.
- One recommendation is to perform marrow testing every 1-2 yr
b. Treatment of pancreatic insufficiency = replacement as for CF
c. G-CSF = for profound neutropenia ? predisposition to MDS/acute leukaemia
d. Transfusion = for management of severe anemia or thrombocytopenia
e. Allogeneic HSCT = only curative option
i. Traditional myeloblastic HSCT resulted in treatment-related mortality in 35-50% of the patients
ii. HSCT for severe marrow failure has produced 50-70% survival rate
iii. Fludarabine-based protocols using reduced-intensity conditioning - safer and effective for SDS HSCT
f. +/- androgens + steroids (some evidence that blood counts have improved) - Prognosis
a. Median age of survival 35 years
b. With pancytopenia age of survival reduced to 24 years
c. 50% of patients experience spontaneous conversion from pancreatic insufficiency to pancreatic sufficiency as a result of improvement in pancreatic enzyme secretion
