Ferritin in Iron Metabolism: Diagnosis of Anemias [2nd ed.] 978-3-211-82706-2;978-3-7091-4421-3

Table of contents :
Front Matter ....Pages I-IX
Introduction (Manfred Wick, Wulf Pinggera, Paul Lehmann)....Pages 1-2
Physiological Principles (Manfred Wick, Wulf Pinggera, Paul Lehmann)....Pages 3-17
Disturbances of Iron Metabolism/Disturbances of Erythropoiesis (Manfred Wick, Wulf Pinggera, Paul Lehmann)....Pages 18-30
Methods (Manfred Wick, Wulf Pinggera, Paul Lehmann)....Pages 31-57
Diagnostic Strategies: Ferritin in Iron Metabolism (Manfred Wick, Wulf Pinggera, Paul Lehmann)....Pages 58-88
Diagnosis of Anemias (Manfred Wick, Wulf Pinggera, Paul Lehmann)....Pages 89-98
Back Matter ....Pages 99-115

Citation preview

M. Wick· W. Pinggera . P. Lehmann

Storage

Ferritin in Iron Metabolism Diagnosis of Anemias Second edition

Excretion

Hb Synthesis

Blood loss on

Transport

Absorption

Storage Springer-Verlag Wien GmbH

With compliments Diagnostics

M. Wick, W. Pinggera, and P. Lehmann

Ferritin in Iron Metabolism Diagnosis of Anemias Second edition

Springer- Verlag Wien GmbH

Dr. Manfred Wick Institute of Clinical Chemistry, Klinikum Grosshadern, University of Munich, Germany

Prim. Univ.-Prof. Dr. Wulf Pinggera Medical Department, General Hospital, Amstetten, Austria

Dr. Paul Lehmann Boehringer Mannheim GmbH, Mannheim, Germany

Translation of Ferritin im Eisenstoffwechsel und Diagnostik der Anamien. Zweite, erweiterte Auflage Wien-New York: Springer-Verlag 1994 © 1994 Springer-Verlag Wien Urspliinglich erschienen bei Springer-VerlaglWien 1994 ISBN 978-3-211-82706-2 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machines or similar means, and storage in data banks. © 1991 and 1995 Springer-Verlag Wien Originally published by Springer-Verlag/Wien 1995 Product Liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting and Printing: Adolf Holzhausens Nachfolger, A-1070 Wien Printed on acid-free and chlorine free bleached paper

With 31 Figures

ISBN 978-3-211-82706-2

ISBN 978-3-7091-4421-3 (eBook)

DOI 101.1007/978-3-7091-4421-3

Foreword This book is based on papers of Dr. Wick and Prof. Pinggera. It is a joint work by three authors. It seemed appropriate to divide the work, in view of the rapid development of clinical chemistry, together with the knowledge that medicine as a science is constantly in a state of flux and that it influences clinical practice. We have endeavored to describe clearly the relevance of the principal analytes of iron metabolism to early recognition, diagnosis, and the monitoring of therapy, as well as to provide useful bedside support for the doctor. Diagnostics, and in particular the methods of clinical chemistry, have advanced so much in recent years that it seems worthwhile also extending the range of routine diagnostics. The authors have therefore decided to update the book. The authors are grateful to Dr. Axel Manke and Cheryl Byers of Boehringer Mannheim and to Michael Katzenberger of Springer-Verlag for their committed cooperation and their expert support in the publication of this book. Doris Raab of Boehringer Mannheim has made a valuable contribution in preparing the diagrams. M. Wick W. Pinggera P. Lehmann May 1995

Table of Contents Introduction Physiological Principles ................................................................ . Absorption of Iron ...................................................................... . Iron Transport ............................................................................. . Transferrin and Iron-Binding Capacity .................................. . Transferrin Saturation ............................................................. . Iron Storage ................................................................................ . Ferritin and Isoferritins ........................................................... . Iron Distribution ......................................................................... . Iron Requirement ........................................................................ . Iron Losses ................................................................................. . Erythropoiesis ............................................................................. . Physiological Cell Maturation ................................................ . Hemoglobin Synthesis ............................................................ . Erythrocyte Degradation ......................................................... . Hemoglobin Degradation ....................................................... .

Disturbances of Iron Metabolism / Disturbances of Erythropoiesis ........................................................................... . Iron Deficiency ........................................................................... . Iron Overload ............................................................................. . Disturbances of Iron Distribution .................... ............................ Disturbances of Iron Utilization ................................................. . Non-Iron-Induced Disturbances of Erythropoiesis ..................... . Disturbances of Stem Cell Proliferation ................................. . Vitamin B 12 and Folic Acid Deficiency ....... ......................... . Hemoglobinopathies ............................................................... . Disturbances of Porphyrin Synthesis ...................................... . Pathologically Increased Hemolysis ........................................... . General Features of Hemolysis ............................................... . Haptoglobin ............................................................................ . Features of Severe Hemolysis ................................................ . Corpuscular Hemolysis .............................................................. .

3 3 5 5 7 7 9 11 13 13 13 13 14

16 16 18 18 19 20 21 22 22 22 23

25 28 28 28 29

30

VIII

Table of Contents

Extracorpuscular, Intravascular Hemolysis ........................... ......

30

Methods .......................................................................................... Determination of Iron .................................................................. Methods ................................................................................... Sample Material ....................................................................... Determination of the Iron Saturation = Total Iron-Binding Capacity (TIBC) and of the Latent Iron-Binding Capacity (LIB C) ................................................................................. Determination of the Iron-Binding Proteins: Ferritin and Transferrin ............................................................................... Immunoassay Methods ................................................... ..... .... Ferritin ......................................................................................... Methods ................................................................................... Reference Range ............................... ............................... ........ Transferrin in Serum.......................................................... .......... Transferrin Saturation .............................................................. Methods ................................................................................... Haptoglobin ........... ... ... .......................................................... ...... Methods ................................................................................... Determination of Vitamin B 12 and Folate .................................. Vitamin B12 ................................................................................ Methods ................................................................................... Folic Acid .................................................................................... Methods ................................................................................... Erythropoietin (EPO) .......... ......................................................... Diagnostic Strategies: Ferritin in Iron Metabolism .................... The Body's Iron Balance ............................................................. Iron Absorption Test .................................................................... Clinical Significance of the Determination of Ferritin ................ Clinical Interpretation: Decreased Ferritin Concentration ........... Prelatent Iron Deficiency .................................. ..... ... ... .... ... ..... Latent Iron Deficiency ................................. ............................ Manifest Iron Deficiency: Iron Deficiency Anemia ................ Differential Diagnosis of Iron Deficiency............................... Clinical Pictures of Iron Deficiency........... ............................. Clinical Interpretation: Elevated Ferritin Concentration ............. Representative Ferritin Increase .......................... ....... ............. Primary Hemochromatosis ...................................................... Secondary Hemochromatosis .................................................. Non-representative Ferritin Increase ....................................... Macrocytic Anemia ................................ .....................................

31 33 34 34 37 37 38 41 42 45 46 47 47 49 49 51 51 52 53 54 56 58 58 59 62 64 65 65 66 67 68 72 72 74 75 75 79

Table of Contents

IX

Folic Acid Deficiency .............................................................. Vitamin B12 Deficiency .......................................................... Normocytic Anemia .................................................................... Extracorpuscular Hemolytic Anemias ..................................... Corpuscular Anemias .............................................................. Uremic Anemia ....................................................................... Erythropoietin .......................................................................... Erythropoietin as Tumor Marker ............................................. Other Factors Influencing Uremic Anemia .............................

79 81 82 83 85 85 86 87 88

Diagnosis of Anemias .................................................................... Representative Ferritin Levels ..................................................... Transferrin and Transferrin Saturation in Disturbances of Iron Distribution ...................................................................... Deficiencies in the Cofactors of Erythropoiesis ..........................

89 89 91 93

References ......................................................................................

99

Recommended Reading ................................................................. 100 Subject Index ..... ............................................................................ 111

Introduction Disturbances of iron metabolism, particularly iron deficiency, are among the most commonly overlooked or misinterpreted diseases. This is due to the fact that the determination of transport iron in serum or plasma, which used to be the test in conventional diagnosis, does not allow a representative estimate of the body's total iron reserves. A proper estimate was formerly possible only by the costly and invasive determination of storage iron in the bone marrow, however, sensitive, well-standardized immuno-chemical methods for the precise determination of the iron storage protein ferritin in plasma are now available. Since the secretion of this protein correctly reflects the iron stores in the majority of cases, these methods permit fast and reliable diagnoses, particularly of iron deficiency conditions. The fact that iron deficiency is so common and is usually simple to treat ought to be well known in the medical world. Even non-iron-determined causes of anemia can now be identified rapidly by highly sensitive, well standardized immuno-chemical methods. This book is intended to contribute to a better understanding of the main pathophysiological foundations and diagnostic principles (Fig. 1).

Introduction

2

Absorption

Hb synthesis

Blood loss

Dietary Iron

r.:T+"l ~ ---

Bone marrow Erythroblasts Ferritin

Hel reduction

Heme

Bile

+ Feces

Hepatocytes Ferritin

"'--"""---..,.

+--"'T"'--_,_--

Excretion

Transport

Storage

Fig. 1. Physiological Fundaments of Iron Metabolism

Physiological Principles Iron, as a constituent of hemoglobin and cytochromes, is one of the most important biocatalysts in the human body.

Absorption of Iron The absorption of iron by the human body is limited by the physico-chemical and physiological properties of iron ions, and is possible only through protein binding of the Fe2+ ion (Fig. 2).

Cells of the intestinal mucosa Lumen of the intestine

Blood

Fe'" -------.. j

Apetransferrin

Apoferritin

jt

~50%

Methods There is (at present) no reference method for the determination of transferrin. Because of the relatively high transferrin concentration (23--45 ).lmol/l), direct immunological precipitation methods [e.g. radial immunodiffusion (RID), nephelometry, turbidimetry] are suitable. Turbidimetric and nephelometric methods are widely used as routine methods [33,49,55]. As in the case of ferritin, it is difficult to make any general recommendation for one particular test, in view of the many commercial methods available. Table 14. Historical Survey

Year

Milestone

Refs.

1958

Determination of transferrin by radial immunodiffusion, RID (Ramsay et a1.)

[120]

1965

Quantitative determination by RID (Mancini et al.)

[102]

1976

Turbidimetric determination of transferrin (Kreutzer)

[92]

1978

Nephelometric determination (Buffone et al.)

[16]

Haptoglobin

49

Transferrin, like ferritin, but unlike iron, shows no pronounced circadian rhythm. In view of the effect of an upright body position on high molecular weight blood components, the blood collection conditions for transferrin determinations must again be stand-ardized with regard to body position and vein constriction. Preferably the same sample should be used for the transferrin determination as for iron and ferritin.

Haptoglobin The haptoglobin detectable in the serum binds the hemoglobin as a result of pathologically increased hemolysis in a fixed haptoglobin-hemoglobin (Hp-Hb) complex. A decrease in the free haptoglobin thus serves as an indicator of intravascular hemolysis. The 1: 1 Hp-Hb complex is incorporated into the hepatocytes with a half-life time of less than 10 min. There, the hemoglobin is enzymatically metabolized. The haptoglobin released is passed back into the serum with a half-life the of about 3 days. The formation of the fixed Hp-Hb complex and its extremely rapid elimination from the bloodstream prevents hemoglobinuria with severe renal iron loss. Haptoglobin is a glycoprotein structurally related to the immunoglobins, and is made up of 2 light «(X- )chains with a molecular weight of 9000 daltons and two heavier (~- )chains (molecular weight: 16,000 daltons). Three phenotypes with different molecular weight are known: Hp 1-1, Hp 2-1 and Hp 2-2. Hp 1-1 has a molecular weight of 100,000 daltons. Hp 2-1 and Hp 2-2 are high-molecular-weight polymers with a molecular weight ranging from 200,000 to 400,000 daltons.

Methods There is (at present) no reference method for the determination of haptoglobin. Because of the relatively high haptoglobin concentration in the serum (7-32 pmol/l), direct immunological precipitation methods (e.g. radial immunodiffusion, nephelometry,

Methods

50

Table 15. Historical Survey Year

Milestone

Refs.

1960

Haptoglobin-hemoglobin binding test; determination of free haptoglobin in serum (Nyman)

[110]

1965

Radial immunodiffusion (RID) (Fahey et al.)

[41]

1979

Nephelometric determination of haptoglobin (van Leute et al.)

[151]

1987

Turbidimetric test for haptoglobin

[80]

Table 16. Reference Range 0.6-2.7 [gil] 60--270 [mg/dl]

Adults

7-32 [mmolll]

turbidimetry) are suitable. Turbidimetric and nephelometric methods are widely used as routine methods [80, 155]. Haptoglobin concentrations in the serum of healthy persons. There are no major differences based on age or sex.

Remarks - Neonates have no measurable haptoglobin levels in the first 3 months; the reference range for adults applies from the 4th month. - The reference range is dependent on the phenotype: [gil]

Hp 1-1 Hp 2-1 Hp 2-2

0.7-2.7 0.9-3.6 0.6-2.9

[mg/dl] 70-230 90-360 60-290

Like the ferritin and transferrin level, that of haptoglobin shows

Detennination of Vitamin B 12 and Folate

51

no appreciable circadian rhythm. In view of the effect of an upright body position on high molecular weight blood components, the blood collection conditions must be standardized with regard to body position and vein constriction. The haptoglobin determination should preferably be performed using the same serum sample as for the other iron metabolism parameters. In presence of massive hemolysis-no haptoglobin concentration is detectable-hemopexin (Hpx) should be measured [110],

Determination of Vitamin B12 and Folate The diagnostic value of determining vitamin B12 and folic acid in anemic states-in addition to serum ferritin-is being increasingly appreciated, and is beyond dispute. All modern vitamin B12 and folate determinations in the serum are based on an immunological analysis method. The principle and measuring techniques are described on p.52 and 54. Because the indication is the same in virtually all cases, it is customary and often useful to determine folic acid and vitamin B12 in the plasma simultaneously as a double assay.

Vitamin B12 Vitamin B 12 has a molecular weight of 1355 daltons and, as cyanocobalamin, belongs to a biologically active substance group which has a common structural element of a prophyrin ring with cobalt as the central atom [20]. The vitamin B12 taken in with food or synthesized by intestinal bacteria ("extrinsic factor") forms a complex with the "intrinsic factor", a glycoprotein formed in the gastric mucosa. Formation of this complex serves to protect the vitamin from degradation in the intestine and facilitates its receptor-dependent absorption by the mucosa of the small intestine. After dissociation of the vitamin B 12 "intrinsic-factor" complex the vitamin can be transported to the liver, where it is stored. In the cells the vitamin is present mainly as 5' deoxyadenosyl-cobalamin,

Methods

52

Table 17. Historical Survey Year

Milestone

Refs.

1950

Microbiological detennination of Lactobacillus leichmannii (Mathews)

[101]

1961

Radioimmunological detennination (Bavakat, Ekins)

[5]

1978

Use of highly purified intrinsic factor as binding protein (Kolhouse et al.)

[88]

1980

Microbiological detennination of vitamin B12 (recommended as reference by NCCLS)

[108]

1985

Automated determination of B 12/folate (Henderson et al.)

[65]

whereas methylcobalamin predominates in the plasma. Transcobalamin II (TC-II) serves as the most important transport protein for vitamin B 12 in the plasma.

Methods A microbiological assay is recommended by the NCCLS as reference method for the research laboratory. The biological activity of vitamin B 12 is measured in relation to the growth of the microorganisms E. gracilis or L. Leischmannii. It is not suitable for wider use because of the apparatus required and the specificity of the technique. A considerable advance in methods was made in 1962 with the introduction of a radioimmunoassay. The determination is based on the competitive binding of radio labeled vitamin B12 and the free vitamin B12 in the sample to the intrinsic-factor. Before the determination is performed, vitamin B 12 is released from the endogenous binding proteins in a thermal stage or by pretreatment in an alkaline solution. False normal results were observed by conventional methods. The method described by Kolhouse et al. in 1978 makes it possible to exclude binding of endogenous cobalamin

Folic Acid

53

analogs in the serum to nonspecific R proteins in the immunological test. Serum/plasma vitamin B 12 must be determined in a very low concentration range (50-1500 pg/ml). This calls for a sufficiently sensitive method. The list of milestones shows that the first generation of determination methods was based exclusively on indirect measurement with a radioimmunoassay. In 1986 Henderson, Friedman et al. introduced a non-radioactive immunoassay [65]. Table 18. Reference Range

Adults

200-900 pg/ml (150-670 pmol/l)

Vitamin B12 concentrations in the serum of healthy persons. There are not found major differences based on age or sex. Remarks - Parenteral vitamin B12 intake (e.g. during absorption tests) often leads even after some months to excessive cobalamin concentrations, caused by the extremely low daily requirement of 1 pg vitamin B 12 . - In about 20% of pregnant women, the vitamin B12 concentration in the serum falls to values of < 125 pmol/l despite adequate depots. - Some of the reference ranges for vitamin B12 given in the literature differ considerably. This is undoubtedly due to the considerable differences in methods used in the past.

Folic Acid Folic acid is a pteridine derivative and is present as a conjugate with several glutaminic acid molecules (n = 2-7). After the ingestion of food it is initially hydrolyzed enzymatically to pteroylmonoglutaminic acid (PGA, molecular weight 441 daltons) in the mucosal epithelium of the small intestine. Reduction and

Methods

54

Table 19. Historical Survey

Year

Milestone

Refs.

1966

Microbiological determination of N-5-methyltetrahydrofolic acid (MTHFA) with Lactobacillus casei (Herbert) Determination of folic acid by radioimmunoassay (Dunn et aI., Rothenberg et aI.) Simultaneous determination of folate and Bl2 (Gutcho, Mansbach)

[66]

1973 1979

[34, 129] [9]

methylation then takes place in the intestinal wall; the resultant N-5-methyl-tetrahydrofolic acid (MTHFA, molecular weight 459 daltons) is released into the bloodstream. Tetrahydrofolic acid (THFA, molecular weight 445 daltons) is formed from METHFA under the influence of vitamin B l2 • It is involved in numerous reactions as a coenzyme.

Methods The microbiological assay recommended as reference method is unsuitable for widespread use [66]. Measurements are made of the biological activity ofN-5-methyltetrahydrofolic acid (MTHFA) in relation to the growth of Lactobacillus casei. An HPLC method developed by Gregory in 1987 [53] is not suitable for the quantitative determination of folic acid in the serum, since it is not sufficiently sensitive. A major advance in methods was achieved in 1973 with the introduction of the radioimmunological competitive proteinbinding test. The determination is based on the competitive binding of radiolabeled N-5-methyltetrahydrofolic acid (MTHFA) 25 I-MTHFA) and the N-5-methyltetrahydrofolic acid (MTHFA) of the sample to the binding protein ~-lactoglobulin. Before the determination is performed, MTHFA is released from the endogenous binding proteins in a thermal stage or by pretreatment in

e

Folic Acid

55

an alkaline solution. The false normal results observed in individual cases which, compared with the microbiological reference method, did not show a folic acid deficiency, can be corrected by using a chromatographically highly purified B-Iactoglobulin free from nonspecific binding proteins [52]. Serum/plasma folic acid must be determined in a very low concentration range (0.5-20 ng/ml). This calls for a sufficiently sensitive method. The list of milestones shows that the first generation of determination methods was based exclusively on indirect measurement with a radioimmunoassay. Immunoassays have recently become commercially available which do not use radioisotopes but employ an elegant, simple reaction for separating folic acid from the endogenous binding proteins (Magic Lite) [19] Table 20. Reference Range

Adults

2.5-19 ng/ml (6-43 nmol/l)

There are no major differences based on age or sex. Remarks - Because of the close connection between vitamin Bl2 and folic acid in the metabolism and the difficulty of hematological and clinical differentiation between the two vitamin deficiency states, it is advisable to determine both parameters simultaneously if the corresponding vitamin deficiency symptoms appear. - Parenteral folic acid intake (e.g. during absorption tests) often leads even after some weeks to excessive folic acid concentrations, caused by the extremely low daily requirement of about 200 pg, with a total folic acid content of the body of about 70 mg. - The reference ranges for folic acid given in the literature differ considerably. This is undoubtedly due to the considerable differences in methods used in the past.

56

Methods

Erythropoietin (EPO) Erythropoietin (EPO) is a glycoprotein with a molecular weight of 30,400 daltons. As hematopoetic growth factor, the hormone regulates the rate of red cell-production keeping the circulation of red cell mass constant. EPO is produced in response to a hypoxy signal as a result of oxygen tension in hemoglobin. This hormone which is primarily produced in the kidney stimulates the proliferation of the bone marrow erythroid progenitor cells, facilitating their differentiation into mature erythrocytes, promoting hemoglobin synthesis and stimulating the release of reticulocytes from the bone marrow.

•

Bone Marrow

EPO

Reticulocyte

~

Erythrocytes

Fig. 20. Stimulation and Regulation of Erythrocyte Formation by Erythropoietin

Erythropoietin (EPO)

57

In clinical diagnosis, EPO measurement is mainly used in erythrocytes differential diagnosis. The relevance of anemia differential diagnosis is constantly increasing method. There is no reference method for the determination of EPO. Only extremely sensitive methods are suitable for its measurement because of the extremely low concentrations in serum « 0.05 gil or < 1 )lmol/l). Radioimmunoassays are mainly used in the routine laboratory but enzyme-based immunoassays are also available for the past 5 years. The measured EPO concentrations can only be used in conjunction with the hemoglobin or hematocrit levels and not with the EPO reference ranges which are clinically irrelevant.

Diagnostic Strategies: Ferritin in Iron Metabolism Knowledge of physiological principles and the availability of clinical laboratory results are tools used by the doctor. It is becoming increasingly difficult to interpret the mass of data from the clinical laboratory and to correlate the findings with the patient's clinical status. How should the laboratory results be interpreted to enable their use by the clinician?

The Body's Iron Balance The iron balance is controlled entirely by absorption. The uptake of iron is therefore of great importance to the iron metabolism as a whole (Fig. 21). The daily iron requirement depends on age and sex. It is increased in puberty and in pregnancy. Under physiological conditions, the total iron pool is more or less constant in adults. The quantity of iron absorbed is about 10% (l mg) of the quantity consumed daily in the normal diet, but this fraction can increase to 20 to 40% (2 to 4 mg) in conditions of iron deficiency. Physiological loss of iron is low, and comparable to the quantity absorbed. It takes place as a result of the shedding of intestinal epithelial cells and through excretion in bile, urine and perspiration. Since the absorption and the loss of iron are limited under physiological conditions, the quantity of storage iron can only be increased by massive supplies from outside the body. Thus, every transfusion of one liter of blood increases the quan-

The Body's Iron Balance

59

ENTERAL ABSORPTION

I

~ ACTIVE IRON 10%

~

1 ERYTHROCYTE

DEPOT IRON 10%

I

70%

Fig. 21. Utilization of Iron

tity of storage iron in the body by about 250 mg. A continually increased supply of iron also increases this quantity, provided that there is no compensating blood loss, e.g. via the gastrointestinal tract. Disturbances in iron absorption can be detected by an ironabsorption test. This is of particular interest in iron deficiency of unknown origin and to demonstrate excessive iron reabsorption in primary hemochromatosis. Iron Absorption Test The iron absorption test should be carried out as follows: The blood sample should be collected from a fasting patient to determine the baseline value for the serum iron concentration. Oral administration of a bivalent iron preparation (200 mg Fe++).

60

Diagnostic Strategies: Ferritin in Iron Metabolism

Under routine conditions: A second blood collection after 3 hours. Alternative where there is a definite suspicion of disturbed absorption kinetics: Blood collection after 1, 2, 3,4 and 5 hours. Interpretation of the iron absorption test: With normal iron absorption, there must be an increase of 2-3 times the baseline value during the observation period. A reduced or delayed rise indicates secondary iron overload or a disturbance of iron absorption which may result in an iron deficiency. By contrast, an accelerated or increased rise is found in forms of iron deficiency which are not due to a disturbance of absorption, and in primary hemochromatosis. The iron balance is held constant ± 1 to 2 mg per day, and is regulated via the absorption capacity of the intestinal mucosa. The body generally stores enough iron to compensate for sudden extreme blood losses. The excess that is not used for the synthe-

c! 4 ¥

5 g (100%)

3.5 - 4 g (100%)

Depot Iron (20%)

Active Iron 80% Hemoglobin 65% Myoglobin )0% Enzymes 5'10 Catalase Peroxidase Cytochrome

Ferritin Ihmoslderln

Tnnsport IrOn (0.1- 0.2%) Transferrin

Hemoglobin

Fig. 22. Total Content of Iron in the Body

The Body's Iron Balance

61

Table 21. Distribution of Iron in the Human Body 70 kg mg Active iron

Hemoglobin Myoglobin Enzymes

Transport iron

Transferrin

Depot iron

Ferritin Hemosiderin

L~

60 kg %

mg

%

2800 400 200

66

10 4

2500 350 150

70 10 4

5

0.1

5

0.1

800

20

500

16

4200

100

3500

100

sis of hemoglobin, myoglobin, or iron enzymes is stored in the depot proteins ferritin and hemosiderin. The total iron content of the healthy human body is about 3.5 to 4 g in women and 4 to 5 g in men [121]. About 70% of this is stored in hemoglobin, 10% is contained in iron-containing enzymes and myoglobin, 20% in the body's iron depot, and only 0.1 to 0.2% is bound to transferrin as transport iron. However, this distribution applies only under optimum nutritional conditions (Fig. 22). Hemoglobin accounts for approximately 85% of the active iron. An iron content of 3.4 mg is calculated per gram of hemoglobin. Since the life cycle of the red blood cells is 120 days, an adult requires about 16 to 20 mg of iron daily in order to replace these vital cells [13]. Most of the iron required for this purpose comes from lysed red blood cells. During pregnancy, birth, and breast-feeding, the additional iron requirement is far in excess of the quantity of absorbable iron contained in the food. The loss of iron in normal menstruation is about 15 to 30 mg (l00 ml of blood contains about 50 mg of iron). The additional iron requirement during pregnancy is between 700 and 1000 mg.

62

Diagnostic Strategies: Ferritin in Iron Metabolism

The second largest component in active iron is myoglobin, at about 10%. Like hemoglobin, myoglobin is an oxygen-binding hemoprotein with a molecular weight of about 17,100 daltons. It is formed in the striated muscle (skeletal muscle and myocardium). Because it has a higher affinity for oxygen than hemoglobin, myoglobin is responsible for the transportation and storage of oxygen in the striated muscles. The diagnostic specificity of the serum myoglobin determination is limited, since it is not possible to differentiate between skeletal muscle myoglobin or myocardium myoglobin. A increase in the serum is observed in all forms of muscle disease and injuries, and in myocardial infarction. The diagnostic value in the assessment of impaired kidney function is limited, since myoglobin is degraded to monomers and is filtered in the glomeruli and completely reabsorbed in the tubuli mortially. The determination of myoglobin in sports medicine for the assessment of performance is worthy of mention. As a result of menstruation and pregnancy, the quantity of depot iron in women decreases through blood loss to 250 mg, i.e. only 5 to 10% of the total iron content. According to a WHO estimate, 50% of all fertile women in western countries suffer from hypoferremia.

Clinical Significance of the Determination of Ferritin Massive disturbances of the iron balance can be detected by measurement the reserve iron stores (deficiency or overload). Disturbances of the distribution of iron between active iron, depot iron, and transport iron can also be observed. The ferritin concentration is an index of the iron reserve stored. Additional indices (transferrin, transferrin saturation, and hematological investigations) are required for the diagnosis of disturbances of distribution. Ferritin is the most important iron storage protein. Together with the quantitatively and biologically less important hemosiderin, it contains about 15 to 20% of all the iron in the body. Ferritin

Clinical Significance of the Detennination of Ferritin

63

occurs in nearly all organs. Particularly high concentrations are found in liver, spleen, and bone marrow. A direct correlation is found for healthy adults between the plasma ferritin concentration and the quantity of available iron stored in the body. Comparative studies with quantitative phlebotomies and the histochemical assessment of bone marrow aspirates have shown that ferritin provides accurate information on the iron reserves available to the body for hemoglobin synthesis in iron deficiency and in primary or secondary stages of iron overload [81]. If more iron is supplied than the body can store as ferritin, iron is deposited as hemosiderin in the cells of the reticuloendothelial system. Unlike ferritin, hemosiderin is insoluble in water, and it is only with difficulty that its iron content can be mobilized. The value of ferritin was demonstrated more than 10 years ago in a comparison of the various parameters available for the determination of the body's iron stores [103]. Ferritin is well suited to the assessment of the iron metabolism of a patient population having no primary consumptive disease or chronic inflammation. The determination of ferritin is particularly useful in the diagnosis of iron metabolism, in the monitoring of iron therapy, for the determination of iron reserves

Table 22. Merits of the Various Parameters for the Detection of Iron Deficiency (according to Mazza J, et al. [103]) Sensitivity (%) Specificity (%) Efficiency (%) Serum iron transferrin saturation

84 84

43 63

52 67

Serum ferritin

79

96

92

Serum iron + serum ferritin

84

42

51

84

50

64

Transferrin saturation + serum ferritin

64

Diagnostic Strategies: Ferritin in Iron Metabolism OVERLOAD

DEFICIENCY

Ferritin ng/mI Transferrin mg/dl

ECONDARY

PRELATENT

LATE T

MA I FEST

< 20

360

»

normiil) -

normal

nornul

microcyti c

normal

normtll

40 - 60

< 10

< 10

40 - 60

40 - 60

360

PRIMARY

.

> 400 norm .. 1 -

,

Iron absorption

Erythrocytes

SlderobIasts %

Fig. 23. Iron Status

in high-risk groups, and in the differential diagnosis of anemias [96] (Fig. 23).

Clinical Interpretation: Decreased Ferritin Concentration In the case of a negative iron balance, the iron depot is reduced first; this can be seen from the measurement of the ferritin. The (transferrin-bound) iron concentration decreases only at a relatively late stage (as shown by the lower transferrin saturation). Diagnosis of non-manifest iron deficiency is practically impossible by clinical methods. The physical findings and the patient's subjective condition do not reveal any definitely attributable changes. The familiar clinical symptoms such as pallor, weakness, loss of concentration, exertional dyspnea, and reduced resistance to infection appear only with the development of anemia, i.e. after depletion of tissue iron has already taken place. The relatively specific changes in the skin and mucous membranes, atrophic glossitis and gastritis, cracks at the corners of the mouth, and atrophy of the hair and nails are late symptoms.

Clinical Interpretation:Decreased Ferritin Concentration

65

An important observation is that iron deficiency very often accompanies serious, life-threatening conditions, and can therefore serve as an indicator of such diseases. Only laboratory tests can confirm a diagnosis of iron deficiency, and depending on the pattern of the results, it is possible to distinguish between prelatent, latent, and manifest iron deficiencies. In prelatent iron deficiency, the body's iron reserves are reduced. Latent iron deficiency is characterized by a reduced supply of iron for erythropoiesis. This gives way to manifest iron deficiency, which is characterized by typical iron deficiency anemia (Fig. 23).

Pre latent Iron Deficiency Prelatent iron deficiency, which is equivalent to depot iron deficiency, is characterized by a negative iron balance. The body's reaction is to increase the intestinal absorption of iron. Histochemically, the iron content of bone marrow and of liver tissue decreases. The concentration of the iron transport protein transferrin in the blood increases as a measure of the increased intestinal absorption. The consumption of depot iron is most easily detected by quantitative determination of the ferritin concentration, which typically falls to less than 20 ng/ml. The ferritin that can be detected in the circulating blood is directly related to the iron reserves, and can thus be taken as an index. The determination of ferritin has practically replaced the determination of iron in bone marrow for the assessment of stocks of reserve iron [147].

Latent Iron Deficiency With increasing consumption of reserve iron, or when the iron depot is completely empty, the replenishment of iron for erythropoiesis becomes negative. The total loss of storage iron is indicated by a decrease in the ferritin concentration to less than 12 ng/ml. An indication of the inadequate supply of iron for

66

Diagnostic Strategies: Ferritin in Iron Metabolism

erythropoiesis is a decrease in the transferrin saturation to less than 15%. As a morphological criterion, the number of sideroblasts in the marrow falls to less than 10%. There are no noticeable changes in the red blood count in this stage of latent iron deficiency.

Manifest Iron Deficiency: Iron Deficiency Anemia In addition to the decrease of the ferritin concentration to less than 12 ng/ml, the laboratory results in this stage are characterized by a fall in the hemoglobin concentration to less than 12 g/dl. A morphological effect of manifest iron deficiency is that the erythrocytes become increasingly hypochromic and microcytic. The mean cell volume (MCV) decreases to less than 85 fl, the mean cellular hemoglobin (MCH) is less than 27 pg, and the mean cellular hemoglobin concentration (MCHC) may fall to less than 31 g/dl. The erythrocytes in the peripheral blood count are smaller than normal, and pale in the center. The altered

•

Fig. 24. Anulocytes

Clinical Interpretation:Decreased Ferritin Concentration

67

erythrocytes are known as anulocytes. The morphological change in the blood count does not appear immediately, but only after the normochromic erythrocyte population has been replaced by the hypochromic microcytic erythrocytes in the course of the natural process of renewal. Iron deficiency is by no means ruled out by normochromic normocytic anemia. Pronounced hypochromia and microcythemia indicate that the iron deficiency has already been in existence for at least several months (Fig. 24). A hypochromic microcytic anemia is not necessarily an iron deficiency anemia. However, a hypochromic microcytic anemia accompanied by a ferritin value of less than 12 ng/ml can always be identified as an iron deficiency anemia, and there is then, for the present, no need for further tests.

Differential Diagnosis of Iron Deficiency It is extremely important in differential diagnosis to distinguish between the common iron deficiency anemias and other hypochromic anemias, since this has considerable implications in relation to therapy. Only iron deficiency anemia responds to iron substitution. For patients suffering from other hypochromic anemias, unindicated administration of iron would lead to a risk of iron overload. Differential diagnostic possibilities to consider for hypochromic anemia are chronic anemias due to infections or tumors, sideroblastic anemia, or thalassemia. The most important distinguishing feature is the quantity of reserve iron. These forms of anemia can be identified on the basis of the ferritin concentration, since they all differ from true iron deficiency anemia in that the depot iron concentrations are normal or increased (Fig. 25). It is possible for an infection-related or tumor-related anemia or a thalassemia to be combined with iron deficiency anemia. In all such cases, the ferritin determination shows that the iron stores are low, and that iron therapy is justified.

68

FERRITIN Dlfml

HEMOGLOBIN Ifdl

TRANSFERRIN mg/dl

Diagnostic Strategies: Ferritin in Iron Metabolism

PRELATENT < 20

¥>12 6> IS

~

MANIFEST

IS

¥ 400 ng/ml

[ FERRITIN> 400 ng/ml

I TRANSFERRIN SATURATION> 50% ]

I TRANSFERRIN SATURATION ~ [TRANSFERRIN.

ICONFIRMATION OF DIAGNOSIS

U

I CONFIRMATION ()F DIAGNOSIS I

by:

by:

CLINICAL EXAMINATION CASE HISTORY BIOPSY INTESTINAL IRON ABSORPTION HLA TYPING

CLINICAL EXAMINATION CASE HISTORY

DI TURB HEMOCHROMATOSES

CE OF 01 TRlB TIO

RELEASE INCREA ED YNTHESIS?

~ = reduced

Fig. 27. Differential Diagnosis of Conditions with Elevated Ferritin

Iron storage diseases can be divided into a primary HLA-associated form, known as idiopathic hemochromatosis, and various secondary forms, or acquired hemochromatoses. The secondary forms are also known as hemosideroses .

74

Diagnostic Strategies: Ferritin in Iron Metabolism

Primary Hemochromatosis Primary, hereditary, and HLA-associated hemochromatosis [3,44] is an autosomal recessive disturbance of iron metabolism, which leads to severe parenchymal iron overload. It is mainly characterized by iron deposition in the parenchymal cells of the liver, the heart, the pancreas, and the adrenal glands. Primary hemochromatosis, which is about ten times as likely to occur in men as in women, usually becomes clinically manifest between the 35th and 55th years, and consists of disturbances of liver function, diabetes mellitus, dark pigmentation of the skin, and cardiomyopathies and consequent arrhythmias. Joint trouble and symptoms of secondary hypogonadism are also found, as well as adrenocortical insufficiency. Development of a hepatocellular carcinoma is observed in about 15% of those affected. This corresponds to a 300-fold increase in the risk of occurrence of a primary hepatocellular carcinoma. By means of HLA typing the hereditary path and the danger for family members of patients with hemochromatoses can be identified at an early stage. A biopsy is necessary in order to confirm a diagnosis of primary hemochromatosis. Liver biopsies are most useful. Bone marrow, skin, and mucous membrane biopsies are unsuitable for the determination of the degree of iron overload in this form of hemochromatosis. Even in the presence of a massive iron overload, the iron content of marrow is normal. Careful monitoring by imaging methods (sonography, CT, NMR) and regular alpha-fetoprotein determinations are necessary in view of the high incidence of hepatocellular carcinomas. The increase in the ferritin concentration is a relatively late occurrence in primary hemochromatosis. The parenchymal cells of the liver, the heart, the pancreas, and other organs first become overloaded with iron, and only then is the reticuloendothelial system filled up. Ferritin values above 400 ng/ml and a transferrin saturation of more than 50% are evidence of an existing iron overload. In the manifest stage of primary hemochromatosis, the ferri-

Clinical Interpretation: Elevated Ferritin Concentration

75

tin concentration is usually higher than 700 ng/ml. Transferrin is almost completely saturated. Venesection is still the treatment of choice. The therapeutic objective is substantially to empty the iron depot. Measurements of the body's iron status should be performed at the shortest possible intervals. The determination of ferritin has proved to be useful in hereditary hemochromatosis. The intensity and the frequency of the venesection are determined according to the changes in the clinical parameters. Bloodletting may be required weekly, monthly, or every three months. The treatment should never be completely terminated because the objective is to prevent progression of the disease.

Secondary Hemochromatoses Secondary or acquired hemochromatoses include disturbances of hematopoiesis accompanied by ineffective or hypoplastic erythrocytopoiesis, such as thalassemia major, sideroblastic anemia, or aplastic anemia. Acquired hemochromatoses can also develop from alimentary iron overload, parenteral administration of iron, or other chronic diseases with ineffective erythrocytopoiesis. Erythropoietin was introduced in 1988 as a therapeutic substance for patients with renal insufficiency on hemodialysis; transfusion-induced iron overload should therefore soon be a thing of the past for this group. Unlike primary hemochromatosis, the cells of the reticuloendothelial system first become overloaded with iron in the secondary forms. Organic lesions occur at a relatively late stage. They result from redistribution of iron from the cells of the reticuloendothelial system to parenchymal cells. The duration of a chronic iron overload is therefore a determining factor in the secondary iron storage diseases.

Non-representative Ferritin Increase Whereas a correlation exists between the ferritin value and the body's total iron reserves in primary and secondary hemochroma-

76

Diagnostic Strategies: Ferritin in Iron Metabolism

toses, in certain diseases this correlation does not exist. Increased ferritin levels are found in cases of infectious or toxic hepatocellular damage, as a result of ferritin release through liver cell necroses, as well as in latent and manifest inflammations or infections and in rheumatoid arthritis. An increase in the ferritin concentration is also observed in physical and mental stress situations, for example following severe traumas. In critically ill patients, an increase in the ferritin concentration accompanies the deterioration of the clinical condition, probably as a result of increased liberation from the macrophage system. Ferritin thus satisfies the criteria of an acute phase protein [11, 98]. Other situations in which the ferritin value is not representative of the body's total iron reserves are shortly after oral or in particular after parenteral iron therapy and in the presence of malignant growths. It may be accepted as a basic rule than an elevated ferritin level must be expected if the blood sedimentation rate is raised and/or in the presence of pathological C-reactive protein levels [98].

Macrocytic Anemia A non-representative ferritin increase may indicate disturbances in the proliferation and maturation of bone marrow cells induced by vitamin deficiency. These macrocytic forms of anemia are caused by disturbed DNA synthesis. As non-iron-determined disturbances of erythropoiesis, they influence not only the proliferation of cells in erythropoiesis, but above all that of the gastrointestinal epithelial cells. Since considerable numbers of macrocytic cells are destroyed while still in the bone marrow, they are also included under the heading ineffective erythropoiesis. Most macrocytic forms of anemia are due to a deficiency in either vitamin B 12 or folic acid, or both [37, 134]. The most common causes of folic acid and vitamin B12 deficiency are listed in Tables 25 and 26. Drug-induced macrocytic anemia is now common (Table 27).

Macrocytic Anemia

77

Table 25. Causes of Folic Acid Deficiency 1. Inadequate intake - alcoholics - unbalanced diet 2. Increased requirement -

pregnancy adolescence malignoma increased cell turnover (hemolysis, chronic exfoliative skin diseases) hemodialysis patients?

3. Malabsorption - sprue - drugs (barbiturates, phenytoin) 4. Disturbed folic acid metabolism - inhibition of dihydrofolic acid reductase (e.g. methotrexate, pyrimethamin" triamterene) - congenital enzyme defects

Drugs which disturb DNA synthesis are now part of the standard therapeutic armamentarium of chemotherapy [137]. There are also rare metabolic disturbances which cause macrocytic anemia, plus megaloblastic anemia with as yet unknown causes, such as the congenital dyserythropoietic anemias or anemias as part of DiGuglielmo syndrome (Table 28). Acute severe macrocytic anemia is a rarity; it can be observed in intensive care patients who require multiple transfusions, hemodialysis or total parenteral nutrition. This form of acute macrocytic anemia may be limited mainly to patients who already had borderline folic acid depots before they fell ill.

78

Diagnostic Strategies: Ferritin in Iron Metabolism

Table 26. Causes of Vitamin B 12 Deficiency 1. Inadeqnate intake

- vegetarians (rare) 2. Malabsorption

deficiency of intrinsic factor pernicious anemia gastrectomy congenital (extremely rare) - diseases of the terminal ileum sprue Crohn's disease extensive resections selective malabsorption (lmerslund syndrome-extremely rare) - parasite-induced deficiences fish tapeworm bacteria ("blind loop syndrome") drug-induced PAS, neomycin

Table 27. Drug-induced Forms of Macrocytic Anemia 1. Drugs which act via folic acid malabsorption alcohol, phenytoin, barbiturates - metabolism alcohol, methotrexate, pyrimethamine, triamterene, pentamidine 2. Drugs which act via vitamin B12 - PAS, colchicine, neomycin 3. Inhibitors of DNA metabolism - purine antagonists (azathioprine, 6-mercaptopurine) pyrimidine antagonists (5-fluorouracil, cytosine arabinoside) - others (procarbazine, acyclovir, zidovudine, hydroxyurea)

Macrocytic Anemia

79

Table 28. Symptomatic Forms of Macrocytic Anemia 1. Metabolic diseases - aciduria (orotic acid) 2. Uncertain origin - Di Guglielmo syndrome - congenital dyserythropoietic anemia - refractory megaloblastic anemia

Folic Acid Deficiency Patients with a folic acid deficit are usually in a poor nutritional state, and often have a range of gastrointestinal symptoms such as diarrhea, cheilosis and glossitis. By contrast, with advanced vitamin B 12 deficiency no symptoms of neurological deficit are found. Folic acid deficiency can be attributed to three main causes: inadequate intake. increased requirement, and malabsorption. Special attention must be devoted to various groups in the population who commonly have inadequate folic acid intake: chronic alcoholics. the elderly, and adolescents. In chronic alcoholics. the alcohol which is the main source of energy (beer and wine) contains practically no folic acid or only small quantities. Alcohol also leads to disturbances in the processing of absorbed folic acid [141, 144]. The folic acid deficit in the elderly seems to be caused mainly by an extremely unbalanced diet ("coffee and a sandwich"). Unbalanced diets have a considerable attraction for adolescents. Those who consume fast food are particularly at risk. An increased folic acid requirement is present during the growth phase in childhood and adolescence, as well as during pregnancy. Since the bone marrow and the intestinal mucosa have an increased folic acid requirement because of a high cell prolifera-

80

Diagnostic Strategies: Ferritin in Iron Metabolism

tion rate, patients with hematological diseases, especially those with increased erythropoiesis, may not be able to meet their increased folic acid requirement from their dietary intake. Disturbances in the absorption of folic acid occur in both tropical sprue and gluten enteropathy. Manifest macrocytic anemia may develop in both clinical pictures. Other signs of malabsorption may also occur. Alcohol-induced folic acid deficiency may to a certain extent also be caused by malabsorption. Diseases of the small intestine may also prevent the absorption of folic acid. The folic acid absorption test [31] is used as a general, practical means of detecting malabsorption.

Folic Acid Absorption Test Folic acid is absorbed in the entire small intestine, so that the substance is suitable for a global test of small-intestine absorption. Data on normal serum concentrations vary slightly from laboratory to laboratory, but do not diverge to any great extent [31, 104].

Reference Range in the Serum 4-34 nmol/l (2-15 )lg/l) Procedure The patient is given 1 mg folic acid i.v. or Lm. per day on the 4 days preceding the examination. This serves to make up any folic acid deficiency in the tissue which could affect the absorption test and produce a false positive (no measurable increase in the serum folic acid concentration after oral ingestion). On the day of the examination the fasting patient is given 40 pg/kg folic acid orally, and the serum folic acid concentration is determined at the following times: 0, 60, 120 minutes. In normal patients the serum concentration rises to over 170 nmol/l (75 pg/l).

Macrocytic Anemia

Vitamin

B12

81

Deficiency

The symptoms of vitamin B 12 deficiency are partly hematological, partly gastroenterological [94]. Neurological manifestations are often observed independently of the duration of vitamin B12 deficiency. There is usually tachycardia with an enlarged heart; the liver and spleen may also be slightly enlarged. Fissures in the mucous membrane, a red tongue, anorexia, weight loss and occasional diarrhea indicate that the gastrointestinal system is involved. It may be very difficult to classify the neurological symptoms. Irrespective of the duration of the vitamin B 12 deficit, in extreme cases they may range from demyelinization to neurone death. The earliest neurological manifestations consist of paresthesia, but also weakness, ataxia and disturbances of fine coordination. Objectively, disturbance of deep sensibility usually occurs early and Rhomberg and Babinski are positive [94]. The eNS symptoms range from forgetfulness to severe forms of dementia or psychoses. These neurological conditions may precede the hematological manifestations by a long way, but as a rule hematological symptoms predominate in the average patient [68]. The main cause of the vitamin B 12 deficiency, the so-called pernicious anemia, which is caused by a deficiency of intrinsic factor. It is generally accompanied by atrophy of the gastric mucosa [12] .

The disease shows geographical clustering in Northern Europe. It is generally a disease affecting the elderly beyond the age of 60; it is less common in children younger than 10 years old. It is found with striking frequency in black patients. The current view about the pathogenesis of pernicious anemia is that it is produced by an auto-immune process directed against the parietal cells of the stomach. It is therefore most marked in patients with clinical pictures attributed to auto-immune diseases, such as immune hyperthyroidism, myxedema, idiopathic adrenal insufficiency, vitiligo and hypoparathyroidism. Antibodies against parietal cells can be detected in 90% of patients with pernicious

82

Diagnostic Strategies: Ferritin in Iron Metabolism

anemia. The detection of these antibodies does not however mean that the pernicious anemia is necessarily manifest. The incidence of antibodies to intrinsic factor is about 60%. Given the pathological mechanism involved, hypoacidity or anacidity is the rule, patients often have gastric polyps, and the incidence of stomach cancer is about double that in the normal population [37]. If the source of the intrinsic factor is destroyedsuch as by total gastrectomy or by extensive destruction of the gastric mucosa (for example by corrosion)-megaloblastic anemia may develop. It must also not be forgotten that a number of bacteria in the intestinal flora require vitamin B\2. Deficiency syndromes may thus develop after anatomical lesions as a result of massive bacterial proliferation. Deficiency symptoms may also occur as a result of strictures, diverticula and blind loop syndrome. They may also occur with pseudo obstruction in diabetes mellitus as a result of amyloid deposits, or in scleroderma. Vitamin B\2-deficiency-anemia is also known to occur as a result of tropical sprue and the fish tapeworm. Most of these clinical pictures are also accompanied by malabsorption syndromes, often with steatorrhea. Regional enteritis, Whipple's disease and tuberculosis may be accompanied by disturbances of vitamin B \2 absorption. This also applies to chronic pancreatitis, in rare instances to Zollinger-Ellison syndrome and segmental diseases of the ileum [126]. Defects of absorption of vitamin B12 were usually detected by the Schilling-Test. This test has been replaced by determination of parietal cell antibodies and antibodies against the intrinsic factor. Hereditary megaloblastic anemia is extremely rare; it is caused by congenital disturbances of orotic acid metabolism or in LeschNyhan syndrome, by the disturbance of enzymes involved in folic acid metabolism.

Normocytic Anemia Normocytic anemia generally occurs during acute blood loss in hemolysis and as a result of renal failure or endocrine disturbances.

Normocytic Anemia

83

Besides the classification of anemia based on formal pathological aspects, into microcytic, macrocytic and normocytic, differentiation by origin has now become established, especially in disturbances of erythropoiesis. The hemolytic anemias (cell trauma, membrane abnormality), enzyme disturbances (glucose6-phosphate-dehydrogenase defect) and disturbances of hemoglobin synthesis have become clinically the most important.

Extracorpuscular Hemolytic Anemias Hemolytic anemia is generally acquired autoimmune hemolytic anemia. A common form of differentiation is based on the thermal behavior of the antibodies, as warm or cold antibodies. The most common clinical pictures of symptomatic hemolytic anemia are summarized in the Table 29. In the detection of hemolysis, the determination of haptoglobin and LDH in particular have proved to be diagnostically useful tests. After the occurrence of intravascular hemolysis the hapTable 29. Antibody-induced Hemolytic Anemia 1. Warm antibodies -

idiopathic lymphomas SLE drugs neoplasms (rare)

2. Cold antibodies - cold agglutinins infections (generally acute) lymphoma idiopathic - paroxysmal cold hemoglobinuria 3. Alloantibodies - blood transfusions - pregnancies

84

Diagnostic Strategies: Ferritin in Iron Metabolism Table 30. Mechanically Induced Forms of Hemolytic Anemia

1. Microangiopathies -

splenomegaly hemolytic-uremic syndrome (HUS) thrombotic-thrombocytopenic purpura (TTP) disseminated intravascular coagulopathy (DIC) cirrhosis of the liver eclampsia

2. Heart-valve protheses 3. Extracorporeal pump systems - hemodialysis - hemofiltration - extracorporated oxygenation

toglobin concentration drops rapidly. This is attributable to the very short half-life of the haptoglobin-hemoglobin complex of only about 8 minutes. In its function as a transport (and acutephase) protein it binds intravascular, free hemoglobin and transports it extremely rapidly to the reticulo-endothelial system for degradation. The hemoglobin is metabolized there. Haptoglobin is therefore eminently suitable for the detection of hemolysis, i.e. for the diagnosis and assessment of the course of hemolytic diseases. Sharply decreased haptoglobin levels are the indicator of intravascular hemolysis which may have immunohemolytic, microangiopathic, mechanical, drug (G-6-P-dehydrogenase deficiency) and infectious causes (e.g. malaria). Extravascular hemolysis (e.g. ineffective erythropoiesis, hypersplenism), on the other hand, shows a drop in haptoglobin only in hemolytic crises. Reduced haptoglobin levels may also be congenital (albeit rarely in Europe). They may also be observed in other, nonhemolytic diseases, e.g. in liver diseases and in malabsorption syndrome.

Normocytic Anemia

85

Since the LDH concentration in the erythrocytes is about 360 times higher than in the plasma, with hemolytic processes there is a rise in LDH. The LDH increase is directly dependent on erythrocyte destruction. A particularly large rise in LDH can be observed in hemolytic crises. Hemolytic anemia caused by a circulatory trauma is characterized by the occurrence of burr and helmet cells. The most important clinical occurrences are listed in Table 30.

Corpuscular Anemias The most important of the enzyme defects is glucose-6-phosphate dehydrogenase deficiency with all its genetically fixed variants, of which fauvism is now clinically the best known form. The hemoglobinopathy that has become important is sicklecell anemia in all its variants; M-hemoglobin is also worthy of mention as the cause of familial cyanosis. All variants of thalassemia are also hemoglobinopathies, clinical pictures which are gaining importance with the increasing mobility of the world's population. These hemoglobinopathies generally present a variable morphological picture with target cells. A clear diagnosis can be made only by hemoglobin electrophoresis or by highpressure liquid chromatography (HPLC) [28]. All forms of hemolytic anemia are characterized by a greater folic acid requirement because of the increased cell and iron turnover.

Uremic Anemia Uremic anemia is an important special form of normochromic normocytic anemia [39]. It roughly correlates with the course of azotemia. By contrast, the cause of the kidney failure clearly plays a more secondary role, although patients with polycystic kidney disease for a long time have a normal blood count or less pronounced anemia than patients with kidney disease of a different origin. The striking fact is that uremic anemia is amazingly well tolerated by patients, and hemoglobin levels of up to 5 g/dl

86

Diagnostic Strategies: Ferritin in Iron Metabolism

are borne with relatively few symptoms. Most patients' reticulocyte count is low and the survival time of the red blood cells is only moderately reduced. The anemia is thus the result of a massive disturbance of erythrocyte production in the bone marrow. The main cause of uremic anemia is inadequate erythropoietin production. Plasma erythropoietin-which is today relatively simple to determine using commercially available immunoassays-is lower in uremic patients compared with nonuremic patients with a comparable degree of anemia.

Erythropoietin Erythropoietin is a glycoprotein with a molecular weight of 30,400 daltons and consists of 193 amino acids. Its protein component comprises two disulfide bonds which are among other things responsible for the biological activity. The tertiary structure of erythropoietin is unknown. Oxygen shortage stimulates erythropoietin production. Goldberg et al. [54] have postulated that a hemoprotein serves as renal oxygen sensor for this mechanism. Fischer et al. [45] discuss the possibility of a reduction in oxygen tension. This leads to the release of adenosine which, as messenger substance, activates adenylate cyclase via a cascade which leads to increased erythropoietin production and erythropoietin secretion. Also shunts in the renal cortex take influence on erythropoietin production [139]. The site of erythropoietin production in the kidney is also unknown: All working groups agree that erythropoietin is produced in the renal cortex but not the glomerular system. More recent studies have shown that a sUbpopulation of macrophages produces erythropoietin outside the kidney. A difference between the erythropoietin production of the kidney and that of the macrophages could be due to a difference in oxygen sensitivity [122,123,152]. The clinical finding that plasma erythropoietin is lower in uremic patients than in nonuremic patients with a comparable degree of anemia suggests that the kidney is the main producer of

Normocytic Anemia

87

erythropoietin. Studies of iron kinetics have revealed a disturbance in iron incorporation. It is therefore legitimate to talk of ineffecti ve erythropoiesis. In this connection the question arises of whether the determination of erythropoietin can be sensibly used in clinical diagnostics [22]. The determination of serum erythropoietin can in principle be used in the differential diagnosis of erythrocytosis, to check paraneoplastic erythropoietin production, in the differential diagnosis of anemia and finally also for diagnosis in patients who apparently have non-renal anemia and have adequate iron depots. The determination of erythropoietin can thus be used in the differential diagnosis of polycythemia of uncertain etiology and anemia. In particular, diagnosis to differentiate between polycythemia vera, where suppression of erythropoietin is expected, and secondary polycythemia (lung diseases, cardiac decompensation) where increased erythropoietin production is expected, is useful. But there is evidence that determination in certain patients is without consequences. Extremely high erythropoietin levels have been observed in aplastic anemia and other forms of medullary hyperplasia [38, 78].

Erythropoietin as Tumor Marker Autonomous erythropoietin production has also been observed in renal-cell carcinoma and as a paraneoplastic phenomenon. However, these phenomena are also seen in Wilms' tumors, hepatocellular cancer [86] and cerebellar angioblastomas [79]. In a few cases a drop in the erythropoietin level has been observed after surgical procedures, and a rise when the tumor developed metastases or there was a local recurrence. There are no known major differences in secretion between malignant and nonmalignant producing sites. Therefore measurement of erythropoietin levels in erythropoietin-producing tumors is limited to monitoring and checking the outcome of treatment using it as a tumor marker.

88

Diagnostic Strategies: Ferritin in Iron Metabolism

Other Factors Influencing Uremic Anemia Although uremic anemia is obviously multifactorial in origin and can be partially improved by appropriate hemodialysis therapy, the role played by the extra corporeal hemolytic component must not be underestimated. Some patients have developed a defect in the hexose monophosphatase shunt; in addition, influences in hemodialysis such as mechanical intravascular hemolysis or pump trauma are playing an increasing role. If there are adequate ferritin depots, uremic anemia can be slightly improved by hemodialysis. In the past it has generally been fully corrected in a very short time by a successful kidney transplant. The polycythemia occasionally observed following transplantation is considered to be a sign of an incipient rejection reaction [17]. The treatment of renal anemia was revolutionized by the development and introduction of recombinant human erythropoietin [40, 56J. If sufficient depot iron is available, which clinical experience shows as requiring ferritin levels of at least 100 ng/ml, uremic anemia can be corrected. As mentioned before, transferrin saturation should be at least 30% as indication of functional avaiable iron. The adjustment of the erythropoietin dose to the individual needs of the patient and subcutaneous administration [100], with the aim of achieving a hemoglobin level of between 10 and 11 g/dl, prevents the unpleasant side effects observed initially, such as hypertension and dialysis shunt thrombosis. Since the folic acid deficiency in dialysis patients can contribute to the development of anemia, care should be taken to maintain sufficiently high depot iron reserves and to avoid a folic acid deficit. Changed iron kinetics or aluminum accumulations may delay or inhibit the action of erythropoietin [29, 138].

Diagnosis of Anemias The determination of ferritin is the most appropriate method for assessment of the iron metabolic situation [81]. The transferrin concentration and the transferrin saturation are used as additional tools in differential diagnosis [135].

Representative Ferritin Levels In clinical practice, the ferritin concentration is representative of the stored iron reserve, especially at the beginning of therapy. A Table 31. Clinical Significance of Ferritin I. Representative result

Detection of pre latent and latent iron deficiency Differential diagnosis of anemias Monitoring of high-risk groups Monitoring of iron therapy (oral) Determination of the iron status of dialysis patients and patients who have received multiple blood transfusions Diagnosis of iron overload Monitoring of bloodletting therapy or chelating agent therapy 2. Non-representative result

Destruction of hepatocellular tissue Infections Inflammations (collagen diseases) Malignancies Iron therapy (parenteral)

90

Diagnosis of Anemias

deficiency in the stores of the reticuloendothelial system (RES) can be detected at a particularly early stage. A value of 20 ng/ml has been found to be a suitable clinical limit for prelatent iron deficiency. This value reliably indicates exhaustion of the iron reserves that can be mobilized for hemoglobin synthesis. A decrease to less than 12 ng/ml is defined as latent iron deficiency. Neither of these values calls for further laboratory confirmation, even where the blood count is still morphologically normal. They are indications of therapy, though it is still necessary to look for the cause of the iron deficiency. If the reduced ferritin concentration is accompanied by hypochromic microcytic anemia, the patient is suffering from manifest iron deficiency. If the ferritin level is elevated and a disturbance of distribution can be ruled out by determination of the transferrin saturation and/or of the C-reactive protein and by investigation of the blood sedimentation rate and the blood count, the raised ferritin level indicates that the body is overloaded with iron. A ferritin value of 400 ng/ml is taken as a limit. The transferrin saturation is massively increased (over 50%) in these cases. If there is no evidence of any other disease, primary or secondary hemochromatosis must be suspected. Differential diagnosis must be pursued through history-taking, liver biopsy, bone-marrow puncture, or NMR tomography. Diagnosis of a primary

Table 32. Ferritin Concentrations for Healthy Individuals and for Patients with Iron Deficiency and Iron Overload

ng/ml Reference range - Men and women over 50 years of age - Women under 50 years of age

= )lg/l

30-300 10-160

Prelatent iron deficiency (storage iron deficiency)

400

Transferrin and Transferrin Saturation

91

hemochromatosis calls for further investigations for organic lesions.

Transferrin and Transferrin Saturation in Disturbances of Iron Distribution Raised non-representative ferritin values are ambiguous, and are found in a number of inflammatory diseases, in the presence of malignant growths, and in the presence of damage to the liver parenchyma. Elevated ferritin levels are also found in a number of anemias arising from various causes, in some cases with true iron overload. Recently ended oral or, in particular, parenteral iron therapy can also lead to raised non-representative ferritin values. These non-representative increases in the ferritin concentraTable 33. Transferrin in the Differential Diagnosis of Disturbances of Iron Metabolism 1. Normal to slightly reduced transferrin concentrations Primary hemochromatosis-exception: late stage

Secondary hemochromatosis 2. Decreased transferrin concentrations Infections Inflammations/collagen diseases Malignant tumors Hemodialysis patients Cirrhosis of the liver-disturbances of synthesis Nephrotic syndrome-losses Ineffective erythropoiesis (e.g. sideroachrestic and megaloblastic anemias) Thalassemias 3. Increased transferrin concentrations Iron deficiency Estrogen-induced increase in synthesis (pregnancy, medication) Ineffective erythropoiesis (some forms, e.g. myelodysplastic syndromes)

92

Diagnosis of Anemias

tion are generally due to disturbances of distribution, and differential diagnosis is possible by determination of the transferrin concentration and of the transferrin saturation. In all these processes, the transferrin value is reduced or close to the lower limit of the reference range. The transferrin saturation is low to normal, and hypochromic anemia can often be diagnosed on the basis of the blood count. Elevated transferrin values are found in the presence of iron deficiency and particularly in pregnancy. The transferrin level may also be raised by drug-based induction (administration of oral contraceptives). Detailed patient history is essential [127]. A number of rare anemias with hyperferritinemia and low transferrin levels belong to the group of sideroachrestic diseases, and are congenital hypochromic microcytic anemias (atransferrinemia, autotransferrin antibodies, receptor defects). Increased ferritin concentrations together with low transferrin levels point to anemias with ineffective erythropoiesis (thalassemias, megaloblastic, sideroblastic, and dyserythropoietic anemias). Myelodysplastic syndromes, on the other hand, may be accompanied by elevated values for both transferrin and ferritin [149]. Elevated ferritin concentrations unrelated to the iron depot are often found in patients with malignancies. Possible reasons that have been suggested for this phenomenon are increased ferritin synthesis by neoplastic cells, release of ferritin on decomposition of neoplastic tissue, and blockage of erythropoiesis as a result of chronic inflammatory processes in and around the tumor tissue. Ferritin determinations on patients with malignant tumors sometimes also record high concentrations of acidic isoferritins [97]. In the presence of inflammations, infectious diseases, or malignant tumors, low transferrin concentrations and low transferrin saturation values point to disturbances of iron distribution. Low transferrin concentrations may be caused either by losses (renal, intestinal) or by reduced synthesis (compensation, liver damage).

Deficiencies in the Cofactors of Erythropoiesis

93

Low transferrin values found for patients with cirrhosis of the liver are usually due to the defective protein metabolism. In nephrotic syndrome, so much transferrin is lost via the urine that low transferrin levels are the rule. The excretion of transferrin in the urine is used to determine the selectively of proteinuria [35, 145]. The transferrin concentration and the transferrin saturation are valuable aids in the differential diagnosis of raised ferritin concentrations. True iron overload is accompanied by increased transferrin saturation. Non-representative ferritin values in patients with disturbances of distribution are characterized by low transferrin saturation and low transferrin concentrations (Fig. 28).

Deficiencies in the Cofactors of Erythropoiesis Deficiencies in vitamin B l2 , folic acid and erythropoietin have been found to be crucial factors in non-iron-deficiency-induced forms of anemia [34]. A initial diagnosis can be made with a high degree of accuracy by recording the patient's history or from knowledge of the primary disease. Given the known interaction between vitamin B12 and folic acid, the determination of these two cofactors by immunoassay should now be standard clinical practice. Deficiency in folic acid or vitamin B 12 is the main feature of diseases accompanied by macrocytic anemia. Sternal puncture or bone biopsies also offer clear histological and morphological pictures. In the differential diagnosis of macrocytic anemia, an elevated level of LDH with simultaneous reticulocytosis and hyperbilirubinemia (both to be interpreted as hyperregeneratory anemia) directs attention to a vitamin B 12 deficit. Macrocytic anemia without these components makes a genuine folic acid deficiency likely (pregnancy, alcohol). Normocytic forms of anemia draw attention to the hemolytic component of erythrocyte destruction, with haptoglobin playing

94

Diagnosis of Anemia

Ferritin

..

1.2 mg/dl

IBone marrow biopsy

.] , ~

~ ~

E.g. Myelodysplasia Toxic bone marrow damage

Renal anemia

Further lab clarification not necessary

Ferritin> 100 ng Iml Transferrin saturation> 30 % Erythropoietin Zn protoporphyrin

Kidney insufficiency

Corresponding to initial diagnosis

~i5 Commensurate with diagnosis

Erythropoietin therapy (Iron substitution if Ferritin < 100 nglml and/or Transferrin saturation < 30 %

Fig. 29. Normocytic Forms of Anemia

Deficiencies in the Cofactors of Erythropoiesis

97

I

Reticulocytes> 15 %

Haptoglobin> 50 mg/dl LDH 210 U/l Bilirubin > I m~/dl (Free hemoglobin t )

Blood loss

Hemolysis

Further lab clarification

I

Erythrocyte morphology

not necessary

Characteristic forms

Hb-electrophoreaia Oamotic resistance Erythroc. enzymes

Corresponding to initial diagnosis

Blood transfusion Regular monitoring of ferritin

e.g. Thalesamia Mechanic Hemolysis Spherocytosis

Uncharacteristic forms Coombs test

/

\

positive negative

positive:

~e:

I:'"

Toxic hemo-

Auto-

lytic

anema

lysis

Therapy of underlying illness Avoidance of noxae

I

Diagnosis of Anemia

98

Table 34. Deficiencies in the Cofactors of Erythroppoiesis Microcytic anemia

Macrocytic anemia

Normocytic anemia

Iron metabolism disturbances

Folic acid deficiency (Tab.2S)

Renal anemia (erythropoietin

Hemoglobinopathies BI2 deficiency (Tab.26) Drug-induced (Tab.27) Metabolic disease (Tab. 28) Uncertain origin (Tab.29)

deficiency) Hemolytic anemia Hemoglobinopathies Bone-marrow diseases Toxic bone-marrow damage

the crucial role as the key to diagnosis. Erythrocyte morphology provides a means to exclude mechanical hemolysis or to look for hemoglobinopathy (Hb electrophoresis) or an enzyme defect. Proof of impaired renal function makes it likely that the most important deficiency of a cofactor in Hb synthesis is present, a deficiency in erythropoietin. The determination of erythropoietin which can now be performed by immunoassay at least forms a good base for a prognosis of the likely outcome of treatment. Before giving any treatment with erythropoietin, it is important to determine the iron depots, since otherwise action by the parenterally administered erythropoietin, which is today genetically engineered, is impossible. In summary it can be said that, given adequate iron depots, normocytic forms of anemia with normal or reduced reticulocyte counts (= disturbance of erythrocyte production) justify looking for erythropoietin deficiency.

References 1. Addison GM, et al (1972) An immunoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. J Clin Pathol 25: 326-329 2. Arosio P, Levi S, Gabri E, et al (1984) Heterogeneity of ferritin II: immunological aspects. In: Albertini A, Arosio P, Chiancone E, Drysdale J (eds) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford, pp 33-47 3. Basset ML, Halliday JW, Bryant S, Dent 0, Powell LW (1988) Screening for hemochromatosis. Ann NY Acad Sci 526: 274-289 4. Batho A (1983) Clinical applications of atomic spectroscopy. Instrumentation Laboratory: 3.9-3.l1; Warrington, UK 5. Bavakat RM, Ekins P (1961) Assay vitamin B 12 in blood: a simple method. Lancet i: 25-26 6. Bently DP (1985) Iron metabolism and anemia in pregnancy. Clin Haematol 14: 613-628 7. Bernard A, Lauwerys R (1984) Turbidimetric latex immunoassay for serum ferritin. J Immunol Methods 71: 141-147 8. Bernat 1(1981) Eisenresorption. In: Bernat I (Hrsg) Eisenstoffwechsel. Gustav Fischer, Stuttgart New York, S 36-37 9. Bernat I (1981) Der Eisentransport. In: Bernat I (Hrsg) Eisenstoffwechsel. Gustav Fischer, Stuttgart New York, S 68-84 10. Berson SA, Yalow RS, et al (1958) Isotopic tracers in the study of diabetes. Adv BioI Med Phys 6: 349 11. Bobbio-Pallavicini F, Verde G, Spriano P, Losi R, etal (1989) Body iron status in critically ill patients: significance of serum ferritin. Int Care Med 15: 171-178 12. Borch K (1986) Epidemiologic, clinicopathologic and economic aspects of gastroscopic screening of patients with pernicious anemia. Scand J Gastroenterol21: 21-30 13. Bothwell TH (1979) Iron metabolism in man. Blackwell Scientific Publications, Oxford 14. Bothwell TH, Baynes RD, MacFarlane BJ, MacPhail AP (1989)

100

References

Nutritional iron requirements and food iron absorption. J Intern Med 226: 357-365 15. Braun HJ (1971) Eigenschaften, Funktion und Serumkonzentration des mensch lichen Hamopexins. Klin Wochenschr 49: 445 16. Buffone GJ, Lewis SA, Josefsohn M, etal (1978) Chemical and immunochemical measurement of total iron-binding capacity compared. Clin Chern 24: 1788-1791 17. Bunn HF (1991) Anemia associated with chronic disorders. In: Harison's principles of internal medicine, 12th ed. McGraw-Hill, New York, pp 1529-1531 18. Cavanna T, et al (1983) An immunoreactive assay for human heart ferritin using a specific monoclonal antibody. Clin Chern 29: 1245-1246 19. Chanarin J (1987) Megaloblastic anemia, cobalamin and tolate. J Clin Pathol 40: 978-984 20. Chen JW, Sperling MJ, Heminger LA (1989) Vitamin B12. In: Pesce AJ, Kaplan LA (eds) Methods in clinical chemistry. CV Mosby, St. Louis Washington Toronto, pp 1127-1129 21. Chiancone E, Stefanini F (1984) Heterogeneity offerritin I structural and functional aspects. In: Albertini A, Arosio P, Chiancone E, Drysdale J (eds) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford, pp 23-31 22. Cotes PM, Dore CJ, Yin Yal (1986) Determination of serum immunoreactive protein in the investigation of erythrocytosis. N Engl J Med 315: 283-287 23. Covell AM, Worwood M (1984) Isoferritins in plasma. In: Albertini A, Arosio P, Chiancone E, Drysdale J (eds) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford, pp 49-65 24. Dallmann PR (1989) Iron deficiency: does it matter? J Intern Med 226: 367-372 25. Dati F, Sauder U (1990) Immunchemische Methoden im klinischen Labor. GIT Labor Medizin 7-8: 357-372 26. Deinhard AS, List A, Lindgren B, Hund JV, Chang PN (1986) Cognitive deficits in iron-deficient and iron-deficient anemic children. J Pediatr 108: 681-689 27. de Jong G, von Dijk IP, van Eijk HG (1990) The biology of transferrin. Clin Chim Acta 190: 1-46 28. Dietzfelbinger H (1993) Korpuskulare hamolytische Anamien. In: Begemann H, Rastatter J (Hrsg) Klinische Hamatologie, 4. Auf!. Thieme, Stuttgart New York, S 248 29. Donnelly SM, Smith EK (1990) The roll of aluminium in the func-

References

30. 3l. 32. 33. 34. 35. 36. 37. 38. 39. 40. 4l. 42. 43.

44. 45.

101

tional iron deficiency of patients treated with erythropoietin: case report of clinical characteristics and response to treatment. Am J Kidney Dis 16: 487-490 Doss M (1992) Porphyrie. In: Thomas L (Hrsg) Labor und Diagnose. Medizinische Verlagsgesellschaft, Marburg, S 549 Deutsch E, Gever G, Wenger R (1992) Folsaure-Resorptionstest. In: Laboratoriumsdiagnostik. Wissenschaftliche Buchreihe, Schering, S 91 Drysdale JW (1977) Ferritin phenotypes: structure and metabolism. In: Jacobs A (ed) Iron metabolism. Ciba Foundation Symposium 51 (excerpta medica). Elsevier, Amsterdam, pp 41-57 Dubois S, McGovern M, Ehrhardt V (1988) EisenstoffwechselDiagnostik mit Boehringer Mannheim/Hitachi-Analysensystemen: Ferritin, Transferrin und Eisen. GIT Labor-Medizin 9: 468-471 Dunn RT, Foster LB (1973) Radioassay of serum folate. Clin Chern 19: 1101-1105 Ebrich J, Foellmer H, Krull F, Winnecken H, Wurster U (1984) Aussagen, Fehlerquellen und Grenzen der Proteindiagnostik im Urin. Monatsschr Kinderheilk 132: 136 Engvall E, Perlmann P (1971) Enzyme linked immunosorbent assay (ELISA): a quantitative assay of immunoglobin G. Immunochemistry 8: 871-874 Eriksson S (1981) Pernicious anemia as a risk factor in gastric cancer. The extent of the problem. Acta Med Scand 210: 481 Erslev AJ (1991) Erythropoietin. N Engl J Med 324: 1339-1344 Eschbach JW, Haley NR, Adamson JW (1990) The anemia of chronic renal failure: pathophysiology and effects of recombinant erythropoietin. Contrib Nephrol 78: 24-37 Eschbach JW, Adamson JW (1989) Guidelines for recombinant human erythropoietin therapy. Am J Kidney Dis 14: 2-8 [No 2, Suppl 1] Fahey JL, McKelvey EM (1965) Quantitative determination of serum immunoglobulins in antibody-agar plates. J Immunol 94: 84-90 Fateh-Moghadam A, Wick M (personliche Mitteilung) Finch CA, Huebers A, Cazzila M, Bergamaschi G, Bellotti V (1984) Storage iron. In: Albertini A, Arosio P, Chiancone E, Drysdale J (eds) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford, pp 3-21 Finlayson NDC (1990) Hereditary (primary) hemochromatosis. BMJ 301: 350-351 Fisher J, Nathashima J (1992) The role of adenosine hypoxic regu-

102

References

lation of kidney production of erythropoietin. In: Pagel H, Weiss Ch, Je1kman W (eds) Pathophysiology and pharmacology of erythropoietin. Springer, Berlin Heidelberg New York Tokyo, pp 29-51 46. Ferro chern, Serum iron and total iron binding capacity. Analyzer, Bedford, MA, Environmental Sciences Associated, Inc 47. Forman DT, Parke SL (1980) The measurement and interpretation of serum ferritin. Ann Clin Lab Sci 10: 345-349 48. Forman DT, Vye MV (1980) Immunoradiometric, serum ferritin concentration compared with stainable bone marrow iron as indices to iron stores. Clin Chern 26: 145-147 49. Franco RS (1987) Transferrin. In: Pesce AJ, Kaplan LA (eds) Methods in clinical chemistry. CV Mosby, St. Louis Washington Toronto, pp 1279-1280 50. Franco RS (1987) Ferritin. In: Pesce AJ, Kaplan LA (eds) Methods in clinical chemistry. CV Mosby, St. Louis Washington Toronto, pp 1240-1242 5l. Garry PJ (1984) Ferritin. In: Hicks JM, Parker KM (eds) Selected analytes in clinical chemistry. American Association for Clinical Chemistry Press, Washington, pp 149-153 52. Givas JK, Gutcho S (1975) pH dependence of the binding of folates to milk binder in radio assay of folates. Clin Chern 21: 427--428 53. Gregory JF (1989) Folacin. Chromatographic and radiometric assays. In: Pesce AJ, Kaplan LA (eds) Methods in clinical chemistry. CV Mosby, St. Louis Washington Toronto, pp 1131-1132 54. Goldberg MA, Dunning SP, Bunn HF (1988) Regulation of the erythropoietin gene: evidence, that the oxygen sensor is a hemo protein. Science 24w: 1412-1415 55. Gressner AM (1990) Entwicklungstendenzen nephelometrischer und turbidimetrischer Immunoassays. GIT Labor-Medizin 9: 419--429 56. Griitzmacher P, Ehmer B, Messinger D, et al (1991) Therapy with recombinant human erythropoietin (rEPO) in hemodialysis patients with transfusin dependent anemia. Report of an European multicenter trial. Nephrologia 11: 58-65 57. Gosling JP (1990) A decade of development in immunoassay methodology. Clin Chern 36: 1408-1427 58. Gundermann KD (1978) Chemiluminescence: current status. In: Schramm E, Stanley P (eds) Proceedings of the International Symposium on Analytical Applications of Bioluminescence and Chemi-

References

59. 60. 6l. 62.

63.

64. 65. 66. 67. 68. 69.

70.

7l. 72. 73.

103

luminescence. State Printing and Publishing Westlake Village, California, p 37 Gutcho S, Mansbach L (1977) Simultaneous radio assay of serum vitamin B12 and folic acid. Clin Chern 23: 1606-1614 Hammilii J (1985) Fluoroimmunoassays and immunofluorometric assays. Clin Chern 31: 359-370 Harjn E, Pakarmen A, Larmi T (1984) A comparison between serum concentration and the amount of bone marrow stainable iron. Scand J Clin Lab Invest 44: 555-556 Heidelberger M, Kendall FE (1935) The precipitin reaction between type III pneumococcus polysaccharide and homologous antibody III. A quantitative study and theory of the reaction mechanism. J Exp Med 61: 563-591 Heinrich HC (1980) Diagnostischer Wert des Serumferritins flir die Beurteilung der Gesamtkorper-Eisenreserven. In: Kaltwasser JP, Werner E (Hrsg) Serumferritin. Springer, Berlin Heidelberg New York, S 58-95 Helman AD, Darnton-Hill I (1987) Vitamin and iron status in new vegetarians. Am J Clin Nutr 45: 785-789 Henderson DR, Friedman SF, Harris JD, Manning WB, Zoccoli MA (1986) CEDIA TM, a new homogeneous immunoassay system. Clin Chern 32 (9): 1637-1641 Herbert V (1966) Aseptic addition method for actobacillus cassei assay of folate activity in human serum. J Clin Pathol 19: 12-16 Herbert V (1977) The five possible causes of all nutrietent deficiency, illustrated by deficiencies of vitamin B 12 and folic acid. Am J Clin Nutr 26: 17 Herbert V (1985) Megaloblastic anemias. Lab Invest 52: 3-19 Hershko Ch, Konijin AM (1981) Serum ferritin in hematologic disorders. In: Albertini A, Arosio P, Chiancone E, Drysdale J (eds) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford, pp 143-158 Hermann U (1990) (personliche Mitteilung) [siehe auch: Del Cura J, Maside C, Rus A, Ruiz R, Borque L, et al (1990) Evaluation of an improved colorimetric iron assay on Boehringer Mannheiml Hitachi analyzers. Poster presented at the 4th ANT, Barcelona] Hytten FE, Cheyne CA, Klopper AI (1964) Iron loss at menstruation. J Obstet Gynaecol Br Cwlth 71: 255 International Committee for Standardization in Hematology (1988) Recommendations for measurement of serum iron in blood. Ind J Hematol6: 107-111 International Committee for Standardization in Hematology (1978)

104

References

Recommendations for measurement of serum iron in blood. Ind J Hemato138: 291-294 74. International Committee for Standardization in Hematology (1985) Proposed international standard of human ferritin for the serum ferritin assay. Br J Haematol 61: 61 75. Jacobella C, Ghielmi S, Belloli S, et al (1984) Use of a reference standard to improve the accuracy and precision of seven kits for determination of ferritin in serum. Clin Chern 30: 298-301 76. Jacobs A, Hodgetts J, Hoy TG (1984) Functional aspects of isoferritins. In: Albertini A, Arosio P, Chiancone E, Drysdale J (eds) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford, pp 113-127 77 . Jacobs A, W orwood M (1975) Ferritin in serum. N Engl J Med 292:951-956 78. Jelkmann W, Wiedemann G (1990) Erythropoietinbestimmung im Serum. Klin Lab 37: 229-234 79. Johannsen H, Gross AJ, Jelkman W (1989) Erythropoietinproduction in malignancy. In: Jelkman W, Gross JA (eds) Erythropoietin. Springer, Berlin Heidelberg New York Tokyo, pp 80-91 80. Johnson AM (1987) Nephelometric immunoassay. J Pharm Biomed Anal 5: 803-809 81. Kaltwasser JP, Werner E (eds) (1980) Serumferritin: Methodische und klinische Aspekte. Springer, Berlin Heidelberg New York 82. Kaltwasser JP, Werner E (1980) Serumferritin als Kontrollparameter bei der Therapie des Eisenmangels. In: Kaltwasser JP, Werner E (Hrsg) Serumferritin: Methodische und klinische Aspekte. Springer, Berlin Heidelberg New York, S 137-151 83. Karlsson FA, Burman P, Loof L (1987) Enzyme-linked immunosorbent assay of H+/K+ ATPase, the parietal cell antigen. Clin Exp Immunol 70: 604 84. Keller H (1986) Klinisch-chemische Labordiagnostik fUr die Praxis. Analyse, Befund, Interpretation. G Thieme, Stuttgart New York 85. Keller H (1980) Einfliisse auf klinisch-chemische Mefigrofien. In: Lang H, Rick W, B iittner H (Hrsg) Validitat klinisch-chemischer Befunde. Springer, Berlin Heidelberg New York 86. Kero WC, Fisher JW (1986) Serum, erythropoietin concentrations in patients with hepatocellular carcinoma. Cancer 58: 2485-2488 87. Kleeberg UR (1975) Pathophysiologie und Diagnostik hamolytischer Anamien. Dtsch Med Wochenschr 100: 1400 88. Kolhouse JF, Kondo H, Allen NC (1978) Cobalamin analogous are present in human plasma and can mask cobalamin deficiency

References

89. 90. 91. 92. 93. 94. 95.

96. 97. 98. 99. 100.

101. 102. 103.

105

because current radioisotope dilution assay are not specific for true cobalamin. N Engl J Med 299: 785-792 Knoll E (1989) Radioimmunoassay. In: Borsdorf R, Fresenius W, Gtinzler H, et al (Hrsg) Analytiker-Taschenbuch, Bd 8. Springer, Berlin Heidelberg New York Tokyo, S 93-125 Konijn AM, et al (1982) Serum ferritin employing a flu orogenic substrate. J Immunol Methods 54: 297-307 Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495 Kreutzer HJH (1976) An immunological turbidimetric method for serum transferrin determination. J Clin Chern Clin Biochem 14: 401-406 Levy A, Vitacce P (1961) Direct determination and binding capacity of serum iron. Clin Chern 7: 241-248 Lindenbaum J (1988) Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N Engl J Med 318: 1720-1728 Linke R, Ktippers R (1989) Nicht-isotopische Immunoassays - Ein Dberblick. In: Borsdorf R, Fresenius W, Gtinzler H, et al (Hrsg) Analytiker-Taschenbuch, Bd 8. Springer, Berlin Heidelberg New York Tokyo, S 127-177 Linkesch W (1979) Serumferritin - diagnostische und klinische Bedeutung. Acta Med Austr 5: 169-171 Linkesch W (1986) Ferritin bei malignen Erkrankungen. Springer, Wien New York Linkesch W (1984) Physiologie und Pathophysiologie des Eisenstoffwechsels. Wien Med Wochenschr 134: 59-63 Lipschitz DA, Cook JD, Finch CA (1974) A clinical evaluation of serum ferritin as an index of iron stores. N Engl J Med 290: 1213-1218 Max Dougall IC, Roberts DE, Neubert P, et al (1989) Pharmacokinetics of intravenous, intraperitoneal and subcutaneous recombinant erythropoietin in patients on CAPD-A rationale for treatment. Contrib Nephrol 76: 112-121 Matthews DM (1962) Observations on the estimation of serum vitamin B 12 using Lactobacillus leichmannii. Clin Sci: 101-ll1 Mancini G, Carbonara AO, Heremans JF (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2: 235-243 Mazza J, Barr RM, MacDonald JWD, Valberg LS (1978) Usefulness of the serum ferritin concentration in the detection of iron deficiency in a general hospital. CMA 19: 884-886

106

References

104. McNeely MDD (1987) Folic acid. In: Pesce AJ, Kaplan LA (eds) Methods in clinical chemistry. CV Mosby, St. Louis Washington Toronto, pp 1131-1134 105. Metzmann E (1989) Plasmaproteinbestimmungen. In: Borsdorf R, Fresenius W, Giinzler H, et al (Hrsg) Analytiker-Taschenbuch, Bd 8. Springer, Berlin Heidelberg New York Tokyo, S 179-198 106. Miles LEM, et al (1974) Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Biochem 61: 209-224 107. Milmann N, Sondergaard M, Sorensen CM (1985) Iron stores in female blood donors evaluated by serum ferritin. BLUT 51: 337-345 108. National Committee for Clinical Laboratory Standards (1980) Guidelines for evaluating a B 12 (cobalamin) assay. Villanova, PA 109. Nickerson HJ, Holubets MC, Weiler BR, Haas RG, et al (1989) Causes of iron deficiency in adolescent athletes. J Pediatr 114: 657-663 110. Nyman M (1959) Serum haptoglobin methodological and clinical studies. Scand J Clin Lab Invest 11 [SuppI39] 111. O'Connor B, Manganello J, Pacholski J, et al (1989) Performance of the Kodak iron and total iron-binding capacity methods [Tech Brief]. Clin Chern 35: 2250-2251 112. Oellerich M (1984) Enzyme-immunoassays: a review. J Clin Chern Clin Biochem 22: 895 113. O'Neil-Cutting MA, Crosby WH (1986) The effect of antacids on the absorption of simultaneously ingested iron. JAMA 255: 1468-1470 114. Perrotta G (1987) Iron and iron-binding capacity. In: Pesce AJ, Kaplan LA (eds) Methods in clinical chemistry. CV Mosby, St. Louis Washington Toronto, pp 1258-1261 115. Pollitt E, Saco-Pollitt C, Leibel RL, Viteri FE (1986) Iron deficiency and behavioral development in infants and preschool children. Am J Clin Nutr 43: 555-565 116. Packungsbeilage, Enzymun-Test®-Ferritin, Boehringer Mannheim, Mannheim, Germany. Immundiagnostica fUr ES 600, ES 300, ES 22, ES Il1manuell, Februar 1989 117. Packungsbeilage, Tina-quant®-a Transferrin, Boehringer Mannheim, Mannheim, Germany. September 1988 118. Packungsbeilage, Eisen ohne EnteiweiBung, Boehringer Mannheim, Mannheim, Germany. Juni 1988 119. Packungsbeilage, Tina-quant®-a Ferritin, Boehringer Mannheim, Mannheim, Germany. Marz 1990 120. Ramsey WNM (1958) Plasma iron. Adv Clin Chern 1: 1

References

107

121. Refsum AB, Schreiner BBI (1984) Regulation of iron balance by absorption and excretion. Scand J Gastroenteroll9: 867-874 122. Rich IN (1991) Erythropoietin production-a personal view. Exp Hematol 19: 985-990 123. Rich IN, Noe G, Vogt C, Lemke C (1990) Erythropoietin is produced in the tubules of the renal cortex (Abstr). Blood 76 [Suppl I: 162a] 124. Ritchey AK (1987) Iron deficiency in children. Update of an old problem. Postgrad Med 82: 59-63 125. Robinson SH (1990) Degradation of hemoglobin. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA (eds) Hematology, 4th ed. McGraw-Hili, New York 126. Rosenberg IH, Alpers DH (1983) Nutritional deficiencies in gastrointestinal disease. In: Sleienger MH, Fordtran JS (eds) Gastrointestinal disease, 3rd ed. Saunders, New York, pp 1810-1819 127. Rosenmund A, Caponovo F, Koechli HP (1986) Der EinfluB hormoneller Kontrazeptiva und der Schwangerschaft auf Eisenstoffwechsel, Plasmaactoferrinkonzentration und Granulozytenzahl der Frau. Schweiz Med Wochenschr 116: 1411-1414 128. Rossander L (1987) Effect of dietary fiber on iron absorption in man. Scand J Gastroenterol [SuppI129]: 68-72 129. Rotheberg SP, DaCosta M, Rosenberg BS (1972) A radio assay for serum fotale: use of a two-phase sequential incubation, ligandbinding system. N Engl J Med 185 (25): 1335-1339 130. Rowland TW, Kelleher JF (1989) Iron deficiency in athletes. Insights from high school swimmers. Am J Dis Child 143: 197-200 131. Rowland TW, Black SA, Kelleher JF (l987)1ron deficiency in adolescent endurance athletes. J Adolesc Health Care 8: 322-326 132. Ruggeri G, Jacobello C, Albertini A, et al (1984) Studies of human isoferritins in tissues and body fluids. In: Albertini A, Arosio P, Chiancone E, Drysdale J (eds) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford, pp 67-78 133. Sassa S (1990) Synthesis of heme. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA (eds) Hematology, 4th ed. McGraw-Hill, New York, p 332 134. Savage D, Lindenbaum J (1986) Anemia in alcoholics. Medicine 65:322 135. Schrocksnadel W, Gabl F (1984) Die Diagnostik des gestorten Eisenstoffwechsels. Wien Med Wochenschr 3/4: 63-68 136. Schultz BM, Freedman ML (1987) Iron deficiency in the elderly. Baillieres Clin Haematol 1: 291-313

108

References

137. Scott JM, Weir DG (1980) Drug induced megaloblastic change. Clin Haematol 9: 587-606 138. Scigalla P, Ehmer B, Woll EM, et al (1990) Zur individuellen Ansprechbarkeit terminal niereninsuffizienter Patienten auf der RH-EPO-Therapie. Nieren-Hochdruckerkrankungen 19: 178-183 139. Schurek HJ (1992) Oxygen shunt diffusion in renal cortex and its physiological link to erythropoietin production. In: Pagel H, Weiss C, Jelkmann W (eds) Pathophysiology and pharmacology of erythropoietin. Springer, Berlin Heidelberg New York Tokyo, pp 53-55 140. Selby GB, Eichener ER (1986) Endurance swimming, intravascular hemolysis, anemia and iron depletion. Am J Med 81: 791-794 141. Shackleton PJ, Fish DI, Dawson DW (1989) Intrinsic factor antibody tests. J Clin Pathol42: 210 142. Short LF, Murray GF, Uptografft WR, et al (1984) Transferrin levels derived from total iron-binding capacity: is it a reliable relationship? Am J Surg 148: 621-623 143. Siedel J, Wahlefeld AW, Ziegenhorn J (1984) A new iron Ferro Zine-reagent without deproteinisation. Clin Chern 30: 975 (AACCMeeting-Abstract) 144. Strant PW (1975) Alkoholwirkung auf das Blut. Schweiz Med Wochenschr 105: 1072 145. Thaler E, Balzar E, Kopsa H, Pinggera WF (1978) Erworbener Antithrombin III-Mangel bei Patienten mit glomerularer Nephropathie. Haemostasis 7: 257 • 146. Thomas AJ, Bunker VW, Stansfield MF, Sodha NK, Clayton BE (1989) Iron status of hospitalized and house bound elderly people. QJ Med 70: 175-184 147. Tompson WG (1988) Comparison of tests for diagnosis of iron depletion in pregnancy. Am J Obstet Gynaecol159: 1132-1134 148. Tosker PP, Deren JJ (1973) Vitamin B12 absorption and malabsorption. Gastroenterology 65: 662 149. Uchida T, Kokubun K, Abe R, Kimura H, et al (1988) Ferrokinetic evaluation of erythropoiesis in patients with myelodysplastic syndroms. Acta Haematol (Basel) 79: 81-83 150. Valcour AA, Krzymowski G, On oro ski M, et al (1990) Proposed reference method for iron in serum used to evaluate two automated iron methods. Clin Chern 36: 1789-1792 151. VanLente F, Marchand A, Galen RS (1979) Evaluation of a nephelometric assay for haptoglobin and its clinical usefulness. Clin Chern 25: 2007 152. Vogt C, Noe G, Rich IN (1990) Normal steady-state haemopoiesis

References

109

assay of erythropoietin by regulator molecules in the bone marrow. In: Sachs L, Abraham NG, Wiedermann CJ, Levine AS, Konwalinka G (eds) Molecular biology of haematopoiesis. Intercept, Andover, pp 401-412 153. Wagner HA (1987) Haufigkeit und Frtiherkennung des Eisenmangels bei Blutspendem. A.rztl Lab 33: 93-98 154. Weigert M (1990) (personliche Mitteilung) 155. Whicher JT, Price CP, Spencer K (1983) Immunonephelometric and immunoturbidimetric assays for proteins. CRC Crit Rev Clin Lab Sci 18: 213-260 156. Weiss TL, Kavinsky CJ, Goldwasser E (1982) Characterization of a monoclonal antibody to human erythropoietin. Proc Natl Acad Sci USA 79: 5465-5469 157. Wick M, Pinggera W (1994) (personliche Mitteilung) 158. Wognum AW, Landsdorp PM, Eaves AC (1989) An enzymelinked immunosorbent assay for erythropoietin using monoclonal antibodies, tetra metric immune complexes, and substrate amplification. Blood 74: 622-628 159. Worwood M (1980) Serum ferritin. In: Jacobs A, Worwood M (eds) Iron in biochemistry and medicine II. Blackwell, Oxford, p 204 160. Yanagawa S, Hirade K, Ohnota H (1984) Isolation of human erythropoietin with monoclonal antibodies. J BioI Chern 259: 2707-2710 Relative molecular weight of Apotransferrin = 79,570 daltons: 161. Haupt H, Baudner S (1990) Chemie und klinische Bedeutung der Human Plasma proteine. Behring Institut Mitteilungen 86: 1-66

Recommended Reading Albertini A, Arosio P, Chiancone E, Drysdale J (eds) (1984) Ferritins and isoferritins as biochemical markers. Elsevier, Amsterdam New York Oxford Basset ML, Halliday JW, Bryant S, Dent 0, Powell LW (1988) Screening for hemochromatosis. Ann NY Acad Sci 526: 274-289 Begemann H, Rastetter J (1993) Klinische Hamatologie, 4. Aufl. Thieme, Stuttgart New York Bernat 1(1981) Eisenstoffwechsel. G Fischer, Stuttgart New York de Jong G, von Dijk JP, von Eijk HG (1990) The biology of transferrin. Clin Chim Acta 190: 1-46 Finlayson NDC (1990) Hereditary (primary) hemochromatosis. BMJ 301:350-351 Forth W (1993) Iron, bioavailability, absorption, utilization. Wissenschaftsverlag, Mannheim Garry PJ (1984) Ferritin. In: Hicks JM, Parker KM (eds) Selected analytes in clinical chemistry. American Association for Clinical Chemistry Press, Washington, pp 149-153 Greiling H, Gressner AM (eds) (1989) Lehrbuch der klinischen Chemie und Pathobiochemie, 2. Aufl. Schattauer, Stuttgart New York Huber H, Loffler H, Pastner D (1992) Diagnostische Hamatologie Laboratoriumsdiagnose hamatologischer Erkrankungen, 3. Aufl. Springer, Berlin Heidelberg New York Tokyo Kaltwasser IP, Werner E (eds) (1980) Serumferritin: Methodische und klinische Aspekte. Springer, Berlin Heidelberg New York Keller HL (1986) Klinisch-chemische Labordiagnostik fiir die Praxis. Analyse, Befund, Interpretation. Thieme, Stuttgart New York Linke R, Kiippers R (1989) Nicht-isotopische Immunoassays - Ein Oberblick. In: Borsdorf R, Fresenius W, Giinzler H, et al (Hrsg) Analytiker-Taschenbuch, Bd 8. Springer, Berlin Heidelberg New York, S 127-177 Schrocksnadel W, Gabl F (1984) Die Diagnostik des gestOrten Eisenstoffwechsels. Wien Med Wochenschr 3/4: 63-68 Thomas L (ed) (1988) Labor und Diagnose, 3. Aufl. Medizinische Verlagsgesellschaft, Marburg Williams WJ, Beutler E, Ersler AJ, Lichtman MA (eds) (1990) Hematology, 4th ed. McGraw-Hili, New York

Subject Index Anemias 89 hemolytic 82,83,84,85 hypochromic microcytic 67 macrocytic 76,77 normocytic 82 renal 87 pernicious 81, 82 sideroachrestic 27, 92 tumour related 20, 67,77 Antibodies 37 monoclonal 38 polyclonal 38 Antigen-antibody reaction 38,40 Antigen excess 41 Anulocytes 66 Auto-antibodies 30 against intrinsic factor 82 against parietal cells 81 cold antibodies 30 warm antibodies 30 Bilirubin 16,28,97 Blood transfusion 72,75 Bone-marrow diseases 93 Burr cells 23,85 Cell necroses 76 CRP (C-reactive protein) 76 Co factors of erythropoiesis 93 Erythropoiesis 13 ineffective 72, 76

co-factors 22,93 deficiencies 22 physiological 13 Erythropoietin 56,86 as tumor marker 86 Erythrocytes 13 degradation 15,17 maturation 13 morphology 22,25 Ferritin 9, 10, 41 apoferritin 8 isoferritins (acidic, basic) 9, 10 organ-specific 10 structure 9 Ferritin concentration decreased 64 increased, non-representative 73,75,89 increased, representative 89, 72,73 merits, according to Mazza 64 Ferritin, determinations automation 44 reference range 45 Ferro Zine Method 33 Folic acid 58 absorption test 80 deficiency 79 requirement 55 reference range 55 Haptoglobin 28, 49, 83, 84

112

Subject Index

Heidelberger and Kendall curve 40 Hematological Diagnosis 94, 95, 96 Heme Iron 11 Hemochromatosis 73 idiopathic 75 primary, hereditary 74 secondary, acquired 75 Hemoglobin 14 degradation 17 free 29 HbAo, A 2 , Hb Bart's 14,24 HbF, HbH, HbS 14,24 synthesis 14 Hemoglobinopathies 23 Hemolysis 28 auto-immune 30 corpuscular 30 extracorpuscular 30

intravascular 30 mechanical 30 toxic 30 Hemopexin 29# Hemosiderin 8, 10, 64 Hepatocytes 2, 9 Hypermenorrhea 69,70 Immunoassay methods 38 direct 39 indirect 39 Immunoassays 39 enzyme 39, 54 fluorescent latex immunoagglutination 39, 43 luminescent 39, 43, 54 nephelometric 39 radial immunodiffusion 39 turbidimetric 39, 48, 50 Iron 3 active iron 61 day-to-day fluctuations 36

depot iron 61 Transport iron 61 Iron binding capacity (TIBC) 32 Iron determination 33 in bone marrow aspirate 63 determination without deproteinization 33,34 reference range 36 Iron deficiency 17, 65, 66, 70, 71 latent 65 manifest 66 prelatent 65 blood donation 17, 71 breast-feeding 68 competitive athletes 69 growth 18,68 menstruation 18, 68 pregnancy 18, 68 unbalanced diet 69 Iron distribution in the body 12,

62 Iron fraction in serum 35, 36, 51 Iron metabolism 2, 35 absorption 2,59 distribution 10,62 disturbances 18 losses 12,63 requirement 12 transport 4, 11 Iron saturation 6, 36 Iron status 61 Iron storage 63, 64 Iron storage proteins 62, 64 Iron transport proteins 62,64 Iso-electric focusing 5 Lead poisoning 26 LDH 83,85,97 Macrocytes 25,78 Malignant neoplasia 76 Myelodysplastic syndromes 21, 95

Subject Index Myoglobin 60, 62 Porphyrias 25,26,27 Reticulo-endothelial system (RES) 8, 16, 75 Schilling-Test 82 Sickle cells 25 Sickle cell anemia 25,27 Spherocytes 25,30 Spherocytosis 30 Target cells 25 Thalassemia 23,24 Transferrin 5,47 isotransferrin 46,47 Transferrin methods 48

clinical interpretation 91 clinical significance 91, 92 reference range 47 Transferrin receptor 8 Transferrin saturation 47, 48 Transferrin, TIBC 47 Tumors 95 Uremic anemia 85, 88 Vitamin B6 deficiency 27 Vitamin BI2 51,52 absorption 82 deficiency 81, 82 reference range 53 requirement 53 Zinc-protoporphyrin 21, 27

113

Springer-Verlag and the Environment WE AT SPRINGER-VERLAG FIRMLY BELIEVE THAT AN

international science publisher has a special obligation to the environment, and our corporate policies consistently reflect this conviction. WE ALSO EXPECT OUR BUSINESS PARTNERS- PRINTERS,

paper mills, packaging manufacturers, etc. - to commit themselves to using environmentally friendly materials and production processes. THE PAPER IN THIS BOOK IS MADE FROM NO-CHLORINE

pulp and is acid free, in conformance with international standards for paper permanency.