Iron, porphyrin and metabolism

Tamás Nagy UP, Dept. of Laboratory Medicine 2019 Email: [email protected] Hemoglobin Disorders Type 1. Decreased production - aplastic (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) - (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 (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 • • 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 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 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 Alpha Thalassemia - 4 allels/ person – severity depends on the number of allels damaged - excess beta globin: forms Hgb H (beta4) - jaundice, fatigue, anemia, splenomegalia

- 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 . 20. Hemoglobinopathies: disorders of the porphyrin metabolism. 21. Laboratory findings in the disorders of iron metabolism.