Iron, porphyrin and hemoglobin metabolism
Tamás Nagy UP, Dept. of Laboratory Medicine 2019 Email: [email protected] Hemoglobin Disorders Type 1. Decreased production - aplastic anemia (toxins, viral infection, autoimmun, radiation) - EPO insufficiency (renal failure) - Hem synthesis disorders - B12 vit., folic acid deficiency (megaloblastic anemia) - Iron deficiency - Porphyrias - Globin synthesis disorders - Thalassemia - Bone-marrow disorders - Myelodysplasia, malignant tumors
Type 2. Increased elimination - Hereditary RBC morphologic disorders - Spherocytosis, elliptocytosis - Enzyme deffects (piruvate-kinase, Glc-6-P dehydrogenase) - Hemoglobinopathies (sicle-cell anemia) - Paroxysmal nocturnal hemoglobinuria (GPI-linker deficiency <- complement reaction) - Antibody mediated lysis (autoimmun disease) - Warm agglutinin (IgG) – unkown cause, or SLE, CLL - cold agglutinin (IgM) - Rh incompatibility
- mechanical damage - DIC
Diagnostic markers • Automated hematology (blood picture, cell counter instruments) • Blood smear • Reticulocyte number, % • Reticulocyte Hgb • Serum Iron • Transferrin (CRP!) • Transferrin saturation • Ferritin • Soluble transzferrin receptor (sTfR) • sTfR/log ferritin • Haptoglobin • Hgb electrophoresis/chromatography • Mentzer index (MCV/RBC: talassemia > 13 > Iron deficiency) • Coombs –test • Porphyrin metabolit levels
Complete Blood Count
2. sz. ábra: Vashiányos anaemia White blood cells 4.0 - 10 G/l
Neutrophil 37 - 80 %
Limfocyte 10 - 50 %
Monocyte 0 - 12 %
Eosinophil 0 - 7 %
Basophil 0 - 2.5 %
Red blood cells 4.0 - 5.5 T/l
Hemoglobin 120 - 170 g/l
Hematocrit 37.7 - 50 %
MCV 80 - 97 fl
MCH 27 - 31.2 pg
MCHC 318 - 354 g/l
RDW 11.6 - 16 %CV
Trombocyte 142 - 424 G/l
MPV 6 - 12 fl MCV (Mean Cell Volume) = Htc / RBC
Unit: fl.
MCHC (Mean Cell Hemoglobin Concentration) = Hgb / Htc
Unit: g/l.
MCH (Mean Cell Hemoglobin) = Hgb / RBC
Unit: pg.
RDW (Red Cell Distribution Width) Standard deviation of MCV devided the mean Unit: % Case 1.
White blood cells 3,770 ! 4,000-10,000 Giga/l Neutrophil % 45,8 34,0-71,1 % Neutrophil (abs) 1,73 1,56-6,13 Giga/l Limfocyte % 29,0 19,3-51,7 % Limfocyte (abs) 1,09 ! 1,18-3,74 Giga/l Monocyte % 15,1 ! 4,7-12,5 % Monocyte (abs) 0,570 0,240-0,860 Giga/l Eosinophil % 2,6 0,0-5,8 % Eosinophil (abs) 0,100 0,000-0,360 Giga/l Basophil % 0,5 0,0-1,2 % Bazophil (abs) 0,020 0,000-0,080 Giga/l Not identified WBC 6,800 ! <5,000 % Red blood cells 2,21 ! 3,90-5,30 T/l Hemoglobin 79 ! 120-157 g/l Hematocrit 23,0 ! 34,1-44,9 % MCV 103,7 ! 80,0-95,0 fl MCH 35,5 ! 26,0-33,0 pg MCHC 343 310-360 g/l RDW 21,8 ! 11,6-14,4 %CV Platelets 33,0 ! 140,0-440,0 Giga/l MPV 9,20 ! 9,40-12,40 fl Smear – morphology
1. Normal RBC 2. Macrocyte 3. Megalocyte 4. Hypersegmented granulocyte Case 2.
White blood cells 12,500 ! 4,000-10,000 Giga/l Neutrofil % 59,1 34,0-67,9 % Neutrofil (abs) 7,36 ! 1,78-5,38 Giga/l Limfocita % 18,2 ! 21,8-53,1 % Limfocita (abs) 2,26 1,32-3,57 Giga/l Monocita % 7,4 5,3-12,2 % Monocita (abs) 0,925 ! 0,300-0,820 Giga/l Eozinofil % 13,7 ! 0,0-7,0 % Eozinofil (abs) 1,700 ! 0,000-0,540 Giga/l Bazofil % 1,6 ! 0,0-1,2 % Bazofil (abs) 0,205 ! 0,000-0,080 Giga/l Red blood cells 4,53 4,50-6,00 T/l Hemoglobin 101 ! 137-175 g/l Hematokrit 31,7 ! 40,1-51,0 % MCV 70,1 ! 80,0-95,0 fl MCH 22,4 ! 26,0-33,0 pg MCHC 319 310-360 g/l RDW 19,4 ! 11,6-14,4 %CV Platelets 202,0 140,0-440,0 Giga/l MPV 10,30 9,40-12,40 fl Smear – morphology
Iron deficiency
1. Ring-shaped RBC 2. microcyte 3. Target cell 4. limfocyte 5. Normal RBC Serum Iron • > 95% binds to transferrin • Low iron level: • Iron deficiency • 10-28 µmol/L • Acute or chronic inflammation; chronic • diurnal variation; highest at noon, bleeding ~ 30% fluctuation • Elevated iron levels: • hereditary • excess dietary iron • multiple blood transfusion (< pH2.0) 1. Transzferrin-Iron apotransferrin + Fe3+
Ascorbate 2. Fe3+ Fe2+
3. Fe2+ + FerroZine Color complex Transferrin • 77 kDa molecular weight • Elevated serum transferrin: • empty iron storages • produced by the liver • Decreased serum transferrin: • saturated iron storages • Binds two Fe3+ (oxidated)-ion • Other causes: • • Elevated: elevated oestrogen (pregnancy, oral Saturation is ~ 15 - 45% contraceptives) (Calculated from serum iron and • Decreased: malnutrition, chronic liver disease, transferrin) malignancies, chronic protein loss • Decreased: in newborns • Serum concentration: 2-3.4 g/l • Acute inflammation (transferrin is a negative acute phase protein)
measurement: Immuno-turbidimetric test
Ferritin
• Iron storage (main organ: liver) • 460,000 Da, apoferritin, 22 subunit, binds 4.500 iron atom / complex • A small amount leaks into the circulation (< 1% of the serum iron is ferritin bound) • Reference range in the serum: 30-400 ug/l • Low levels suggest iron deficiency
Measurement: "ECLIA" (elektrochemiluminescent immunoassay) Soluble transferin receptor
• Transferrin receptor takes iron from transferrin and delivers it inside the cell
• Recognizes and binds transferrin
• The‚ soluble’s form is the truncated, shedded portion of the receptor in the serum
• Elevated serum level in iron deficiency
• Decreased serum level in iron overload
sTfR/log ferritin, Reticulocyte Hgb
1. Ret-Hb normal, sTfR/log Ferritin normal: Normal
2. Ret-Hb normal, sTfR/log Ferritin increased: Latent iron deficiency
3. Ret-Hb low, sTfR/log Ferritin normal: Functional iron deficiency (due to chronic dieseases, e.g. inflammation)
4. Ret-Hb low, sTfR/log Ferritin increased: Manifest iron deficiency
Hepcidin
- only hormone regulating iron metabolism - Hepatic bacteriocidal protein - Inactivates ferroportin Stops iron getting out of gut cells and RES cells - Diagnostic value: still to be decided
Porphyrias
A group of rare disorders caused by deficiencies of enzymes of the heme biosynthetic pathway.
The majority of the porphyrias are inherited in an autosomal dominant fashion
Affected individuals have decreased level of the enzymes, but can still synthesize some heme molecules.
Affected individuals have an accumulation of heme precursors (porphyrins), which are toxic at high concentrations.
Attacks of the disease are triggered by certain drugs, chemicals, and foods, and also by exposure to sun.
Smear – morphology
Sideroblastic anemia (hem synthesis disorder, hereditary (x-linked) or acquired) Acute Porphyria Chronic porphyria e.g. acute intermittent porphyria e.g. Porphyria cutanea tarda nervous system: skin: - abdominal pain, photosensitivity - tachycardia skin rash - muscle weakness bullous dermatitis - seizure - coma
Triggered by several drugs or lead poisoning
laboratory analysis: Sample: blood, Urine, 24h collected Urine
Detection: direct photometry (uro- coproporphyrin) photometry after Ehrlich reaction (DALA, PBG) Globin disorders
Thalassemia major
1. erythroblast 2. Target cells 3. Polychromatic RBC 4. Howell-Jolly body 5. Limfocyte 6. granulocyte Hemoglobin electrophoresis
Hemoglobin A: 2 alpha and 2 beta Hemoglobin A2: 2 alpha and 2 delta Hemoglobin F: 2 alpha and 2 gamma Normal Hgb Sickle cell disease Alpha Thalassemia - 4 allels/ person – severity depends on the number of allels damaged - excess beta globin: forms Hgb H (beta4) - jaundice, fatigue, anemia, splenomegalia Beta Thalassemia
- jaundice, fatigue, microcytic anemia, splenomegalia - minor (only 1 allel involved), intermediate and major forms (homozygous, both allel involved) - Hbg A2 increased Further diagnostic options
• Genetic screening for hereditary disorders; hemoglobinopathies, enzyme defects • Kidney functional tests • Inflammation markers (CRP, sedimentation, interleukins) • Bilirubin, liver function • haptoglobin Related Exam questions:
19. Laboratory diagnostic approaches in anemias. 20. Hemoglobinopathies: disorders of the porphyrin metabolism. 21. Laboratory findings in the disorders of iron metabolism.