Haematinics Flashcards
(34 cards)
Iron absorption
Iron is absorbed in the gut, primarily in its Fe2+ form
It is exported out of the intestine and into the plasma by ferroportin under the regulatory control of hepcidin
In the plasma it is carried by transferrin
Ferritin is the major storage molecule for intracellular iron
NO physiologically regulated means of iron secretion, THUS dietary iron absorption highly regulated
Iron storage in body
Total 3-4g
75% in red cells
25-30% in liver
Other in spleen, losses (ie mensuration, bleeding, mucosal sloughing) , muscle tissue
Recycling Fe from senescent rbc curcial to maintain Fe homeostasis
Ferritin
Ferritin sequesters Fe providing storage form that protects cells from toxicity (limits ROS generation)
Found in almost all cells, MOST ferritin is stored in:
1. hepatocytes and
2. macrophages in BM&spleen
In healthy statues - plasma ferriitn DIRECTLY proportional to total Fe stores. As low Fe = suppression of ferritin synthesis
Hepcidin
Master (negative) regulator of Fe balance
Synthesized in liver
Hepcidin BLOCKS ferroportin function –> therefore blocks Fe export from hepatocytes, macrophages and enterocytes and triggers its subsequent degradation
IL-6 –> increase hepcidin, so you get a functional Fe deficiency anaemia of chronic disease
Serum iron
- colorimetric
A colorimetric assay using Ferrozine, which forms a magenta-coloured solution with ferrous (Fe2+) iron.
Acid liberates iron from transferrin –> ascorbate promotes reduction FE3 to Fe2
Fe2 + ferrozine –> colored complex which is measured and directly proportional to Fe
Serum Fe interpretation
Serum Fe NOT helpful in assessing Fe status
- wide biological variability
- diurnal variation (AM peakl; evening nadir)
- effect of food intake (ingestion causes increase)
- negative acute phase reactant
CLINICAL utility:
- Used in conjunction with the TIBC to calculate TSAT, a screening tool for haemochromatosis/iron overload
- Diagnosis of iron poisoning
How should iron studies be collected
Serum
Fasting
Iron containing supplements avoided for 24h prior to draw
Transferrin Method
- Immunoturbidimetric
Anti-transferrin antibodies react with antigen to form an antigen/antibody complex
Following agglutination, this is measured turbidometrically
Interference - paraprotiens ie IgM and turbidity in samples
Interpretation of transferrin results
- Increased in iron deficiency
- Increased with higher estrogen (pregnancy, HRT, OCP)
- Decreased in inflammation (negative acute phase reactant)
TIBC and Transferrin saturation calculation
**% Transferrin saturation = IRON/TIBC x 100
**
TIBC (total iron binding capacity) = transferrin x constant
- constant can vary between labs and variation due to MW chosen for transferrin
Transferrin Saturation interpretation
% Transferrin saturation = IRON/TIBC x 100
HIGH transferrin sats >45% useful as first sign of Fe overload, however if serum Fe is high saturation may be high as Fe is the numerator –> note the high biological variability up to 40% of serum fe
Ferritin Method
- immunoturbidmetric assay
- sandwich assay
Immuniturbidimetric assay:
- ferritin binds to anti-ferritin polyclonal Ab coated latex partciles
- precipitate is measured turbidmoetrically
Sandwich assay:
Primary and secondary anti-ferritin antibodies bind to ferritin (with the ferritin sandwiched between), each with its own detection method
The primary antibody is biotinylated
The secondary antibody is ruthenium-conjugated
Streptavidin magnetic particles bind the biotinylated antibody; unbound substances are washed away
After washing, voltage is applied which induces chemiluminescent emission
Ferritin Analytical issues
A positive acute phase reactant
Elevated in chronic disease states, liver disease, chronic renal failure, and some cancers
Gross haemolysis results in liberation of ferritin (falsely high results)
Sandwich assay:
-High dose biotin therapy causes falsely low ferritin (preferentially binds streptavidin, ferritin is washed away)
-Human anti-mouse antibodies can cause false results
-Gross haemolysis results in release of RBC ferritin
Complex and variable molecule, manufacturers use different Abs directed to different ferritin epitopes.
No reference measurement procedure.
Important that reference materials are traced to WHO reference standard so results are equivalent among procedures and to avoid calibration bias.
variability in ferritin diagnostic cut-offs in lab
Hyperferritinaemia
Fe accumulation
* Hereditary haemochromatosis
* Ineffective erythropoiesis (sideroblastic anaemia, some MDS)
* Secondary iron overload (blood transfusion/excessive Fe intake)
* Thalassaemias
* Hereditary acaeruloplasminaemia (rare)
* Atransferrinaemia (rare)
* Ferroportin disease
No significant Fe accumulation
* Malignancy
* Acute or chronic liver disease (hepatotoxicity)
* Alcohol
* Acute or chronic inflammation (var. aetiology)
* CKD
* Metabolic syndrome
* MAS/Adult onset Still’s Disease/HLH
* Gaucher disease
* Benign L-hyperferritinaemia (AD)
* Hyperferritinaemia cataract syndrome…
Glycosylated ferritin
Marker of HLH/MAS (expressed as % of total ferritin)
- activated macrophages secrete HYPOGLYCOSYLATED ferritin
- performed at StVincents pathology
Glycosylated ferritin <20% = suspicious for HLH
Soluble transferrin receptor
Marker of total erythropoietic activity
> Iron def causes overexpression of STFr levels
BM erythropoiesis = major determinant of [solTfR] which increases when erythropoiesis is stimulated.
SolTfR increases in:
- Fe deficiency (inflammation independent and not an acute phase reactant
- haemolytic anaemia
- EPO stimulating agents
SolTfR decreases in aplastic anaemia.
Promising utility in distinguishing true Fe deficiency
in complicated anaemia (ACD)
However evidence base still emerging
And relationship between raised solTfR and Fe def. may be
confounded in other conditions where erythropoiesis is
increased and in malignant states (eg. CLL)
Analytical issues, availability and expense currently preclude routine use
SolTfR Measurement
* No reference measurement procedure
* No standardisation of assays
» However currently there is wide analytical variation in absolute measured values
* Limited availability
* Non-Medicare rebatable
Hereditary haemochromatosis
Autosomal recessive inheritance
Systemic iron overload of genetic origin caused by hepcidin deficiency:
* reduced production of hepcidin
* reduced activity of hepcidin-ferroportin binding (hepcidin resistance)
HH should be suspected when there is a TSAT >45% or ferritin >200 in females or >300 in males, and/or evidence of liver iron deposition on MRI or biopsy
Not all individulas with geneit predisposition to HHC will develop iron overload, has an incomplete penetrance
Classification of HH - four groups
HFE-related: homozygosity for C282Y or compound heterozygosity with a rare non-HFE variant
C282Y/H63D is recognized as insufficient for the HH phenotype in the absence of an additional susceptibility factor for iron overload such as fatty liver, alcohol or HCV
In the event of iron overload, these susceptibility factors should be sought and treated; venesection can be used as an adjunct
Non-HFE related: e.g. HJV, HAMP, TFR2
Digenic: double heterozygosity and/or double homozygosity/heterozygosity for mutations in 2 different genes involved in iron metabolism (HFE or non-HFE)
> Most commonly, the C282Y mutation in the HFE gene coexists with mutation in other genes
Molecularly undefined
Main clinical, biochemical and imaging elements for suspicion of HC
Varying HFE genotypes and risk of iron overload
Anaemia of chronic disease
Impaired production of erythrocytes associated with chronic inflammatory states.
May also occur in severe acute illness or mild persistent inflammation.
Immune driven –> disturbed iron homeostasis
> increased uptake and retention of iron with cells of RES –> diverts iron from erythroid progenitor cells
> Inflammatory cytokines promote erythrophagocytosis and reduced iron recycling
> IL-6 expression –> Increases Hepcidin WHICH reduces Fe absoprtion from gut and inhibits erythropoiesis.
Common disease associated with ACD :
- infections
- Cancer
- Autoimmune
- CKD
- Chronic rejection after solid-organ transplantation.
Investigations of AoCD
Normochromic, normocytic anaemia
>Characteristically mild-mod 85-95 g/L
CRP increased
serum EPO decreased
Low reticulocyte count
Iron studies – essential to differentiate from IDA or to diagnose concurrent IDA
In both serum Fe/transferrin sats reduced – reflecting absolute iron deficiency in IDA and hypoferritinaemia due to RES acquisition in ACD
T/f Sat is reduced in both – in IDA this is largely secondary to increased transferrin, whereas in ACD t/f levels remain normal or are decreased.
Ferritin levels are increased/normal in ACD secondary to immune activation/RES storage
The soluble t/f receptor is a truncated fragment of the membrane receptor that is increased in IDA when the availability of iron for erythropoiesis
This essentially mirror normal transferrin but it is decreased in ACD whereas transferrin may be normal in ACD
Ferroportin disease
Autosomal dominant
Mutation in SLC40A1 (which encodes for ferroportin) leading to abnormal iron accumulation in body.
Dietary absorption of B12
Water soluble vitamin
Vitamin B12 is essential for carbohydrate, fat and protein metabolism as well as nucleic acid synthesis.
Free B12 binds to R proteins (also called transcobalamin I /haptocorrin) in the saliva or gastric secretions
Protein bound B12 undergoes cleavage by pepsin
In the duodenum, proteases degrade the R proteins, and B12 binds to intrinsic factor (which is made by parietal cells of the stomach) allowing it to travel safely to the terminal ileum
B12 is absorbed in the terminal ileum
In the serum B12 is transported to tissues by transcobalamin II (the transcobalamin II-vitamin B12 complex is also known as holotranscobalamin)
Deficiency of TCII leads to megaloblastic anaemia
Transcobalamin I (TCI) is also present in the serum and >70% of total B12 is bound to TCI, but is not responsible for B12 uptake in tissues; TCI deficiency therefore usually has no clinical sequelae, but results in low total B12 levels
