1. Running the Ironman Brad Lewis SFGH
Blood alone moves the wheels of history.
Benito Mussolini
Blood will tell, but often it tells too much.
Don Marquis

2. An Approach to Anemia
Anemia ?

3. An Approach to Anemia Anemia ? Smear LDH Bilirubin Iron Studies B12
Coombs

4. Evaluating Hemolysis The Bucket with The Hole

5. Evaluating Hemolysis The Bucket with The Hole
Retic
Hemoglobin Level
Loss or Hemolysis

6. Corr Retic = Retic x hgb/nl hgb
Reticulocytes
Retic #=1/mm
Retic %= 20%
Corr Retic = Retic x hgb/nl hgb
Retic # = 1/mm
Retic % = 30%
RPI = corrected retic. count/Maturation time
(Maturation time = 1 for Hct=45%, 1.5 for 35%, 2 for 25%, and 2.5 for 15%.)

7. An Approach to Anemia
Anemia
Retic Hi
Retic Low

8. An Approach to Anemia
Anemia
Retic Hi
Retic Low
MCV Lo
MCV Nl
MCV Hi

9. An Approach to Anemia Anemia Retic Hi Retic Low Loss Destruction
MCV Lo
MCV Nl
MCV Hi
Tissue
On Floor
Occult
Intrinsic
Extrinsic
Splenic
Mechanical
Recovery
Iron
(Lead)
Thal
Frags
Chronic Disease
Renal
Mixed
Mild/Treated
Early
Transfused
Endocrine
Intrinsic BM
Dilution
B12
Folate
Liver
ETOH
Thyroid
Toxic
MDS

10. Anemia Retic Hi Retic Low Loss Destruction MCV Lo MCV Nl MCV Hi Iron
(Lead)
Thalassemia
Fragmentation
Sideroblastic Anemia
acquired
congenital

11. Diagnostic Tests Low Retic Microcytic
Iron/TIBC vs. Ferritin
Hemoglobin Electropheresis
GENETIC SCREENING OF FAMILY
The “Normal” Electropheresis
Smear?
Value of MCV and RDW
Lead?

12. 23 yo Chinese Woman 13 wks Pregnant Hgb 11, MCV 72
What Tests?

13. 23 yo Chinese Woman 13 wks Pregnant Hgb 11, MCV 72
What Tests?
Iron Studies first
may mask beta-Thal by decreasing Hgb A2
What if Hemoglobin Electropheresis is normal?
If iron nl, then not beta-thal
BUT alpha-thal carrier state has normal HPLC

14. Diagnostic Tests Low Retic Microcytic
Iron/TIBC vs. Ferritin
Hemoglobin Electropheresis
GENETIC SCREENING OF FAMILY
The “Normal” Electropheresis
Smear?
Value of MCV and RDW
Lead?

15. Body Iron Distribution and Storage
Dietary iron
Utilization
Duodenum
(average, 1-2 mg
per day)
Muscle
(myoglobin;
300 mg)
Liver
(1000 mg)
Bone
marrow
(300 mg)
Circulating
erythrocytes
(hemoglobin;
1800 mg)
Reticuloendothelial
macrophages
(600 mg)
Sloughed mucosal cells
Desquamation/menstruation
Other blood loss
(average, 1-2 mg per day)
Storage
iron
Plasma
Iron loss
transferrin
(3 mg)
Andrews NC. N Engl J Med. 1999;341:1986–1995.

16. Iron Metabolism Spleen Liver RBC Plasma Fe-Tf Bone Marrow Duodenum
Increased Hepcidin in inflammation blocks the marked iron movements
Bone Marrow
Duodenum
Tomas Ganz ASH 2006

17. Iron Metabolism 20 mg/d Spleen RBC Plasma Fe-Tf Bone Marrow Duodenum
Increased Hepcidin in inflammation blocks the marked iron movements
Bone Marrow
Duodenum
Tomas Ganz ASH 2006

18. Iron Metabolism Spleen RBC Plasma Fe-Tf Bone Marrow Duodenum
Increased Hepcidin in inflammation blocks the marked iron movements
Bone Marrow
Duodenum
Tomas Ganz ASH 2006

19. Hepcidin Small molecule which blocks iron movement
Evolutionary conservation
Problems with assays
Regulation
Increased by dietary iron <1day
Congenital absence>>juvenile hemochromatosis
Decreased by anemia, hypoxia

20. Hepcidin Regulation Hepcidin Adequate Iron Intake Inflammation Hypoxia
Increased
Inflammation
IL-6
Hypoxia
Hepcidin
Anemia?
In irradiated mice, anemia does not decrease hepcidien, it is the rbc turnover (phenylhydraxine hemolysis in mice)
Decreased
RBC turnover
Hemochromatosis

21. Hepcidin and Inflammation
Suppressed in hours by IL-6 (?others)
Not in IL-6 deficient mice
Plasma Iron turnover q3hrs
30% drop in 1 hour if recycling blocked
“Anemia of Acute Disease”
??Role in host defense
Plasma
Fe-Tf
3 mg
RBC
Spleen
20 mg/d
Bone Marrow
Tomas Ganz ASH 2006

22. Infectious Risk of Iron Overload
Bacterial
Hepcidin, lactoferrin, transferrin bacteriostatic in vitro
Listeria, Yersenia, Aeromonus
Cunninghamella bertholletiae
Fungal
Increased growth in vitro
Case reports of increased Mucor in MDS pts
? Increased risk with chelation with streptomyces pilosis
Both transferrin and the structurally related protein, lactoferrin, are bacteriostatic in vitro for a number of bacteria. Lactoferrin is a prominent component of the granules of polymorphonuclear leukocytes. The protein is released at high concentrations by the cells in areas of infection. There is also evidence that iron overload per se compromises the ability of phagocytes to kill microorganisms. A combination of problems likely contribute to the increase in susceptibility to infection in these patients. Therefore, iron overload does not produce the susceptibility to infection seen with defects in more central systems (e.g., chronic granulomatous disease). Nonetheless, a number of infections, often with unusual organisms, have been reported in patients with iron overload (Bullen, Spaulding et al. 1991); (Abbott, Galloway et al. 1986); (Christopher 1985).
•Abbott, M., A. Galloway, et al. (1986). Haemochromatosis presenting with a double Yersinia infection. Journal of Infection 13: •Boelaert, J., G. van Roost, et al. (1988). The role of desferrioxamine in dialysis-associated mucormycosis: report of three cases and review of the literature. Clinical Nephrology 29: •Bullen, J. J., P. B. Spaulding, et al. (1991). Hemochromatosis, iron and septicemia caused by Vibrio vulnificus. Archives of Internal Medicine 151: •Christopher, G. (1985). Escherichia coli bacteremia, meningitis, and hemochromatosis. Archives of Internal Medicine 145: 1908.•Daly, A., L. Velzquez, et al. (1989). Mucormycosis: association with deferoxamine therapy. American Journal of Medicine 87: •Robins-Browne, R. and J. Prpic (1985). Effects of iron and desferrioxamine on infections with Yersinia enterocolitica. Infection and Immunity 47: •Windus, D., T. Stokes, et al. (1987). Fatal Rhizopus infections in hemodialysis patients receiving deferoxamine. Annals of Internal Medicine 107:

23. Hepcidin and Iron Transport
Low Hepcidin
High Hepcidin
Iron
Iron
DMT1
ferritin
ferritin
lysosome
Fpn
This works well in the enterocytes, which are then sloughed q2-3 days, but leads to BM iron stores artefactually elevated.
Fpn
Enterocytes
Macrophages
Hepcidin
Iron release into Plasma

24. Erythropoiesis Signal
Iron Metabolism
Iron Signal
Spleen
Hepcidin
Hepcidin
RBC
Plasma
Fe-Tf
Hepcidin
Increased Hepcidin in inflammation blocks the marked iron movements
Hepcidin
Bone Marrow
Duodenum
Erythropoiesis Signal
Tomas Ganz ASH 2006

25. Erythropoiesis Signal
Iron Metabolism
Iron Signal
Spleen
Hepcidin
Hepcidin
RBC
Plasma
Fe-Tf
Hepcidin
Increased Hepcidin in inflammation blocks the marked iron movements
Hepcidin
Bone Marrow
Duodenum
Erythropoiesis Signal
Tomas Ganz ASH 2006

26. Evaluating Iron Stores vs. Response
Ferritin
Sensitive/specific
Except increased in inflammation, liver disease, malignancy
Fe/TIBC (Transferrin) and Saturation
Decreased in inflammation, malignancy
THEREFORE:
Iron Trial
Serum (soluble) Transferrin Receptor
Mediates iron transfer into cell
Increased in Fe-def, rapid cell production
CHR-Retic Hemoglobin Concentration?
Follow-up GI Eval
10 -15% with malignancy
?Only if ferritin <100?
Anemia of chronic disease and iron deficiency anemia, the most common forms of anemia, are differentiated primarily by estimates of iron status. Standard measures of iron status, such as ferritin, total iron-binding capacity, and serum iron are directly affected by chronic disease. In contrast, soluble transferrin receptor (sTfR) is elevated in iron deficiency but is not appreciably affected by chronic disease. sTfR is elevated in subjects with hyperplastic erythropoiesis (eg, hemolytic anemia, beta-thalassemia, polycythemia, etc) and depressed in subjects with hypoplastic erythropoiesis (eg, chronic renal failure, aplastic anemia, or post-transplant anemia). Transferrin receptor (TfR) is the major mediator of iron uptake by cells. TfR is a transmembrane, disulfide-linked dimer of two identical subunits that binds and internalizes diferric transferrin, thereby delivering iron to the cell cytosol. When a cell needs iron, TfR expression is increased to facilitate iron uptake. Since the major use of iron is for hemoglobin synthesis, about 80% of total TfR is on erythroid progenitor cells. Soluble transferrin receptor arises from proteolysis of the intact protein on the cell surface, leading to monomers that can be measured in plasma and serum. Thus, the concentration of sTfR in plasma or serum is an indirect measure of total TfR. The serum level of sTfR reflects either the cellular need for iron or the rate of erythropoiesis. The concentration of sTfR in plasma or serum is elevated in iron deficiency. The concentration of sTfR in plasma or serum is correlated with erythron transferrin uptake, a ferrokinetic measure of erythropoietic activity.
Postgraduate Medical Journal 2004;80: risk of colon CA with iron deficiency

27. Colon CA in Iron Deficiency
Am J Gastroenterol ;102(1):82-88.

28. Evaluating Iron in Inflammation
Bone Marrow Iron Stores??
Saturation (Fe/Transferrin) <8-10%
Iron Trial
?IV repletion, check 1 month
% Hypochromic RBC’s
Nl. <2.5%, Fe-deficient >10% correlates with Fe response
Reticulocyte Hgb Concentration?
Sensitive, specific for diagnosis in dialysis pts
Responds to iron in 48 hours
Serum (soluble) Transferrin Receptor
Increased in Fe deficiency or increased RBC turnover

29. Treatment of Iron Deficiency
Oral always preferred
?low dose equally effective (325 mg FeSO4)
?role for Vitamin C
When to use IV iron
Recent decreased risk of anaphylaxis
Poor compliance
Side-effects, etc
Poor Absorption
Jejeunal/duodenal disease
Sprue
“Chronic Disease”
Anemia of Malignancy
Iron replacement at low doses: Am J Med Volume 118, Issue 10, Pages (October 2005)

30. Iron Overload Iron overload
Serum transferrin iron binding capacity exceeded
NTBI circulates in the plasma
Insoluble iron complexes are deposited in body tissues
Excess iron promotes free radical formation
Cardiac
Liver
Pancreas
Reproductive
Endocrine
NTBI = non-transferrin bound iron
Adapted from: Olivieri NF, et al. Blood. 1997;89: ; Olivieri NF. N Engl J Med. 1999;341:

31. Basic Causes of Iron Overload
Acquired iron overload1
Transfusional
Ineffective erythropoiesis
Toxic ingestion (very rare in adults)
Hereditary
HFE hemochromatosis
Homozygous C282Y mutation in HFE gene2
Defective regulatory receptor in intestine results in increased absorption of iron
Other genetic mutations
1. Porter JB. Br J Haematol. 2001;115:239–252.
2. Feder JN, et al. Nat Genet. 1996;13:399–408.

32. Diseases With High Risk of Iron Overload
Diseases requiring frequent or repeated transfusions
-Thalassemia (major and intermedia)
Sickle cell anemia
Myelodysplastic syndromes (MDS)
Aplastic anemia
Rare chronic anemias
Blackfan-Diamond anemia (red cell aplasia)
Fanconi anemia (hypoplastic anemia)
Others

33. Iron Loading From Blood Transfusions
1 unit of blood contains 200 mg of iron1
Chronic transfusion-dependent patients have an iron excess of ~0.4 to 0.5 mg/kg/day2
There is no physiologic mechanism to remove excess iron
Therefore, iron accumulates with repeated blood transfusions
Signs of iron overload can be seen anywhere between 10 and 20 transfusions1
Iron overload can result in iron-related dysfunction of key organs1,2
1. Porter JB. Br J Haematol. 2001;115:239–252.
2. Kushner JP, et al. Hematology. 2001;47–61.

34. Erythropoiesis Signal
Iron Metabolism
Iron Signal
Spleen
Hepcidin
Hepcidin
RBC
Hepcidin
Plasma
Fe-Tf
Increased Hepcidin in inflammation blocks the marked iron movements
Bone Marrow
Duodenum
Erythropoiesis Signal
Tomas Ganz ASH 2006

35. Hereditary Hemochromatosis
Autosomal recessive
HFE gene in 90% (hepcidin deficiency)
Rare Transferrin Receptor 2 defect
Variable penetrance, caucasions only
Severe Disease
Hemojuvelin
HAMP (hepcidin)
Autosomal dominant
Rare, ferroportin defect
Severe, early onset, Hepatocytes only
Other rare defects
DMT1(microcytosis), atransferrenemia, ceruloplasmin

36. Hemochromatosis Diagnosis
Consider in :
Chronic fatigue
Arthropathy
Impotence
Hyperpigmentation
Cirrhosis DM
Cardiomyopathy
Screening elevated Fe sat or Ferritin

37. Hemochromatosis Diagnosis
Fe/TIBC >60%
Decreased in early, family-hx diagnosis
Decreased with inflammation
HFE testing (C282Y)
Compound hetero C282Y/H63D
Rarely a problem, unless ETOH
Ferritin to quantify iron overload
IF confusing, consider MRI

38. Hemochromatosis Management
Ferritin >1000 associated with sx
Fe/TIBC saturation >75%
Unstable/labile iron with increased risk of oxidant damage
Urgent phlebotomy
IF sx or end organ damage
Weekly to <1000 ferritin as tolerated
Target ferritin <50
? Role of deferasirox (Exjade)
Rarely in hemochromatosis
intolerance