Добавил:

Sekretar kiopkiopkiop18@yandex.ru t.me/Prokururor I Вовсе не секретарь, но почту проверяю Опубликованный материал нарушает ваши авторские права? Сообщите нам.

Вуз:

Ростовский Государственный Медицинский Университет

Предмет:

Медицина общая

Файл:

Книга_2617_Библиотеки_им_академика_М_И_Перельмана

.pdf

Скачиваний:

Добавлен:

30.08.2026

Размер:

59 Мб

Скачать

☆

<<< < Предыдущая 7 8 9 10 11 12 13 14 15 16 17 1819 / 5019 20 21 22 23 24 25 26 27 28 29 30 31 > Следующая >>>

Mitoferrin

Olotransferrin

15 Hematological Diagnostics

https://t.me/medicina_free

175

Table 15.7

Test Sideremia (Fe)

Transferrin saturation Ferritinemia

Liver biopsy By specic

MRI

Mutations in the gene HFE and other genes involved in iron regulation

Investigations for the diagnosis of hemochromatosis

Reference Range/ Methods Comment

12–32μmol/LM 10–30μmol/LF

18–40% Values above 45–50% are

18–250μg/LM 14–150μg/LF

coloring Iron accumulations and consequent brosis are normally absent

methods of image

Search for C282Y, H63D, S65C (E168X, W169X)

Not singularly diagnostic, it must be interpreted in association with transferrin saturation

indicative of iron overload Values above the limits indicate the risk of iron accumulation in the deposits Values above 1000 are diagnostic Note that ferritin behaves as an acute phase reactant In case of iron accumulation, semiquantitative measures of the concentration and distribution of iron in the liver lobules can be detected. The extent of brosis is assessed

few centers; considered superimposable to the biopsy RM is like SQUID, albeit less accurate Second level test The choice depends on the frequency and geographic distribution

Proton

pump

Apotransferrin

Fig. 15.13 Iron metabolism. Holotransferrin binds to TfR1 on the cell surface and the complex undergoes endocytosis in the clathrin-coated vesicle. The proton pump acidies the endosome and causes Fe release. Step 3 reduces Fe lent metal transporter (DMT-1). Iron is utilized in mitochondria via mitoferrin or deposited (nonerythropoietic cells) as ferritin. A fraction of intracellular Fe (redox) remains to constitute the Labile Iron Pool (LIP). Apotransferrin and TfR1 go to the membrane and dissociate at neutral pH. The apotransferrin cycle resumes. (Copyright EDISES

2021. Reproduced with permission)

Endosome

Steap3

Fe-S eme

centres

Clathrin-

coated

cavities

Use

Plasma

Ferritin

Deposit

Core

Mitochondrion

DMT-1

LIP

to Fe2+, which exits the endosome via diva-

• TIBC μmol/L S-Fe μmol/L=LIBC μmol/L

• TSat %=Fe/TIBC × 100

The transport of iron in the circulation is mediated by transferrin, a molecule circulating in four main forms in various proportions:

• Apotransferrin, an iron-free protein

• Monoferric transferrin with Fe bound to the carboxyl

terminus

• Monoferric transferrin with Fe bound to the

amino-terminal

• Holotransferrin, with two bound Fe atoms

Transferrin can be measured by two different methods. Specically, it can be assessed by an immunometric assay of transferrin protein, whose values are expressed in g/L (IR

1.9–3.7), or by a functional method, which evaluates the total iron-binding capacity (TIBC) and is obtained after saturation of serum or plasma with Fe3+ and measurement of bound iron. The latter provides diagnostic information for microcytic anemias, such as the latent iron-binding capacity (LIBC) and the percentage of saturation of transferrin (TSat %), through the following calculations:

The reference values are, respectively:

– S-Fe: 11.6–31.7μmol/L (M) and 10.0–30.4μmol/L (F) – TIBC: 45–66μmol/L – Transferrin: 1.9–3.7g/L – TSat: 20–50% (M) and 15–50% (F) – Absolute ratio of TIBC μmol/L over transferrin

(g/L)≈25.0

Unlike sideremia, the TIBC/transferrin ratio does not vary continuously in the blood; however, the diagnostic information of TIBC/transferrin in sideropenic anemia is delayed as it increases when iron stores are depleted.

Transferrin is mostly in the apo form (approximately 65–80%). Its function is to bind Fe3+, making it soluble to dampen its reactivity, and deliver it to cells via special transferrin receptors (TfRs). Figure15.13 shows the mechanism through which iron is incorporated into cells. Cells receive Fe via TfRs, which are located on the cell membrane and are particularly represented in erythropoietic cells. Their expression varies according to the intracellular Fe concentration. Two types of TfRs are known, 1 and 2. TfR1 shows a much

176

https://t.me/medicina_free

G. C. Guidi

greater afnity for Fe and is ubiquitous in cells, while TfR2 has been shown to perform different functions, not yet fully elucidated. Once its function has ceased, the extracellular portion of TfR is released into the circulation as a soluble truncated monomer (sTfR) complexed with transferrin. The major release of plasma sTfR occurs from erythroblasts, secondarily from reticulocytes. Levels of sTfR are determined by bone marrow erythropoietic activity, which can cause variations from eightfold to more than 20-fold of normal values. Therefore, sTfR has been used as a sensitive index of the recovery of erythropoiesis. It should be noted that sTfR concentration is not affected by inammation and thus may be useful in differentiating sideropenic anemia, in which sTfR is elevated, from chronic inammatory conditions, in which sTfR does not vary.

The reference values of sTfR are 2.2–5.0mg/L in males

and 1.9–4.4mg/L in females.

Ferritin is the main form of iron storage in the body. Its globular conformation with a high MW (474 kDa) and 24units of two types of chains (H and L) enclose within the shell over 4000 atoms of Fe3+ complexed with hydroxy phosphate in the form of ferrhydrite, so as to minimize any reactivity due to contact. Most organs have ferritin deposits, which vary greatly according to the general state of iron. The liver, spleen, and bone marrow are the organs with the largest iron deposits in the form of ferritin. The values of circulating ferritin are in constant equilibrium with those of the stores, with 1μg of circulating ferritin approximately corresponding to 7.5mg of iron in the stores. The exceptions are mainly due to inammatory and neoplastic diseases, as well as hepatopathies. In such conditions, ferritinemia values are unpredictably abnormally elevated and thus not reliable as biomarkers of the status of iron stores, even if the values are apparently in the normal range.

The reference values of ferritin are 18–250μg/L in males and 14–150μg/L in females.

Zinc protoporphyrin (ZnPP) is a surrogate biomarker of iron deciency. Zinc ts into the protoporphyrin ring in the absence of iron, and, therefore, its measurement in red blood cells can give an assessment of the extent of erythropoietic

iron deciency. Normally, the Fe/Zn concentration ratio is 30,000/l. In the case of iron deciency, a decrease in the ratio is observed. The test is not widely used but may be useful in some diagnostic conditions.

The differential diagnosis of iron deciency anemia and similar conditions is based on a reasoned combination of the tests described above. In Table 15.8, the diagnostic criteria that should be mainly considered in the conditions of microcytic anemia resulting from iron deciency, chronic inammatory conditions, and the combination of the two conditions, to which sometimes difculties in making a correct diagnosis are added, are presented. The iron overload condition is also reported in Table15.8.

Anemia DuetoThalassemia Syndromes

This is a very heterogeneous group of conditions that have a common feature: they are caused by mutations/deletions affecting the gene complexes responsible for the synthesis of globin chains. Due to its complexity, it is important to briey describe genetic-molecular and structural information concerning the three types of normal human hemoglobin, namely HbA (α2β2), HbF (α2γ2), and HbA2 (α2δ2), and the constituent globin chains, namely α, β, γ (in turn distinguished in Aγ and Gγ), and δ globins. In addition to these, two other globin chains, ε and ζ, are normally expressed in the embryonic period and, together with the α-globin, constitute the Portland hemoglobins (Hb), Gower 1 and Gower 2.

Gene Complexes, Molecular Expression, Globin Chains

Genes encoding for globin chains are organized into two gene complexes (clusters): the β complex, located on chromosome 11, and the α complex, located on chromosome 16 (Fig.15.14). At the cis position with respect to the β complex is the Locus Control Region (LCR), which regulates the expression of genes in the β complex together with other proximal gene elements (promoters, silencers, and enhancers). The LCR and proximal region are important as some forms of β-thalassemia are due to abnormalities involving them.

Table 15.8 Differential diagnosis of iron deciency anemia, chronic inammatory condition, and iron overload

Iron deciency anemia S-Iron Decrease Decrease Decrease Increase TIBC/Transferrin Increase Decrease/normal Decrease/normal Decrease Transferrin

saturation Ferritinemia Decrease (diagnosis if

sTfR Increase Increase Normal Decrease ZnPP Increase Increase Increase Decrease

Decrease Decrease/normal Decrease

<12μg/L)

Combined anemia (iron deciency and chronic-inammatory)

Normal (typically >100μg/L)

Anemia of chronic-inammatory conditions

Normal/increase Increase

Iron overload

δβ

Globin chains of

15 Hematological Diagnostics

https://t.me/medicina_free

the β gene complex on chromosome 11

εγ

177

HbF HbA

Globin chains of the

α gene complex on

chromosome 16

ζ2 ζ1 α2 α1

Fig. 15.14 Schematic diagram of the β and α gene complexes. In the β complex in 5′→3′ sequence, there are the ε, Gγ, and Aγ, δ, β genes. In the α complex in sequence, there are the genes ζ2, ζ1, α2, and α1. (Copyright EDISES 2021. Reproduced with permission)

HbA

Embryonic Hemoglobins, Fetal Hemoglobin (HbF), HbF/HbA Switches

As stated above, during the early part of the rst trimester of embryonic development, there is a consistent expression of an embryonic form of globin of the β gene complex, known as globin ε, in the erythropoietic cells of the yolk sac. Shortly

Fig. 15.15 Hemoglobin (Hb) synthesis during pre and postnatal development. Shown are the sites of erythropoiesis and the embryonic fetal and adult hemoglobins and their epochs of appearance and disappearance. As noted, the switch occurs shortly after birth at the equivalence of HbF and HbA. (Copyright EDISES 2021. Reproduced with permission)

thereafter, when production of the rst erythrocytes by erythropoietic cells of the fetal liver begins, the expression of globin ε ceases and globin γ becomes predominant. Therefore, HbF (α2γ2) appears in the 5th week of gestation and rapidly becomes the major Hb of the fetus (Fig. 15.15). After the 10th week, HbF is approximately 90% Hb and remains so until 32–36 weeks; then, adult HbA synthesis begins. At birth, the β/γ ratio is approximately 1.0. During fetal life, the Gγ/Aγ ratio is ≈ 3/1 and, starting at birth (HbF/HbA switch), it changes to 2/3 due to the progressive silencing of the two Gγ genes along with the change in erythropoiesis sites (yolk sac → liver → bone marrow), the decrease in erythrocyte volume from 120fL to 80fL, the change in RBC membrane antigens (i→I), and some changes in the isoenzyme prole. Table15.9 shows the normal hemoglobins and their respec-

Table 15.9

adulthood

Type of hemoglobin

Types of hemoglobin and normal variants at birth and in

Values Globine components

2β2

2γ2

α2δ

Values at birth

20– 25% 75– 80%

0.5% 2.5% High in trait

in the

adult Note

97% Values of the adult

at 1year of age

<1% Values for adults at

1year of age If it remains high, suspicion of thalassemia

β-thalassemia, of which it is a biomarker

tive values at birth and in adults.

Genetics ofThalassemias

α- and β-thalassemias are mostly inherited by Mendelian modality as autosomal recessive forms; therefore, the complete manifestation of the thalassemia is observed in children of both parents’ carriers. Given the heterogeneity of the conditions, thalassemias with double heterozygosity, in which the phenotype presents the combined expression of the two altered genes, are not uncommon. Rare, dominant modes of transmission are also described.

Molecular Alterations inThalassemias

Under normal expression conditions, the synthesis of the two major globin chains is balanced. Measurement of the α mRNA/β mRNA ratio shows that in α-thalassemias, this ratio correlates with the number of functional α genes and that in β-thalassemias, it is an indicator of disease severity. In thalassemias, there is an unbalanced expression of one or more chains, resulting in a reduction of intraerythrocytic cells and a decrease in MCH (Fig. 15.16). When MCH

178

Chains α

Chains β

Hb tetramers

Chromosome 16

Chromosome 11

α2 α1

https://t.me/medicina_free

G. C. Guidi

Fig. 15.16 Translation of genes for α and β globin chains and their expression under normal conditions. In α-thalassemias, there is usually deletion of one or more genes, whereas in β-thalassemias, there is usually reduced/altered transcription of the related gene, affecting translation and expression. (Copyright EDISES 2021. Reproduced with permission)

<27pg and after excluding iron deciency, it is appropriate to ascertain the existence of possible thalassemia. From a clinical point of view, the consequences for the patient are due to the excess presence of one or the other type of free globin chain.

Pathophysiological andGeneral Clinical Aspects ofα-Thalassemias

Most of the clinical consequences of α-thalassemias are due to excess β globin chains in the adult and γ in the newborn due to the deletion of 1–4 α genes. Indeed, excess β chains tend to aggregate into hemoglobin H (HbH) tetramers, in the case of β tetramers, or hemoglobin Bart (Hb Bart) tetramers, in the case of γ tetramers. Both HbH and Hb Bart tetramers show a high afnity for oxygen, are unstable, and tend to precipitate inside the red blood cell, leading to the formation of visible Heinz bodies. As they adhere to the cell membrane, they cause a loss of the cell’s plasticity characteristics and make it difcult to pass through the microcirculation. Eventually, this causes the early elimination of the erythrocytes by the reticuloendothelial system.

Clinical Forms ofα-Thalassemia

The α-thalassemias are responsible for very different clinical forms among them, ranging from apparently normal health conditions up to intense hemolytic anemia, which could be lethal, such as the fetal hydrops by Hb Bart (γ4) due to deletion of all four α globin genes.

fact that on the pair of chromosomes 16 of everyone there are a total of 4 α genes, and, therefore, the deletion can affect from 1 to 4 genes, with results that vary according to the

α2 α1

Genes

mRNA

Translation

rate

ααβ

α/β = 1

The heterogeneity of the syndrome is explained by the

Normal

thalassemia

heterozygote

α+homozygous

thalassemia

heterozygote

Hemoglobin

disease

Hydrops

fetalis

Fig. 15.17 Genotypes of α-thalassemias. (Copyright EDISES 2021. Reproduced with permission)

α2

α2-1

α2 α1

α2-1

α1

α2-1

genotype, as illustrated in Fig.15.17. HbH disease is due to the deletion of 3 α genes and, consequently, to the excess of β chains forming the β4 tetramers (HbH). In HbH disease, the anemia is due both to a reduced amount of Hb and to a reduced functionality of the residual Hb because the presence of HbH with an increased afnity for oxygen further reduces its functional adaptation capacity. It should be borne in mind that hemoglobin with increased afnity for oxygen is always disadvantageous for homeostasis because it yields smaller volumes of bound oxygen at the same gradient compared to normal hemoglobin.

α-Thalassemia Dueto−3.7 Deletion

In these forms, an α-thalassemic allele corresponds to the presence of only one gene on chromosome 16, while the other one has undergone a deletion of −3.7kb due to unequal crossing-over between the two genes on homologous chromosomes. Forms with a −4.2kb deletion are also known.

α-Thalassemias DuetoNondeletion

These are molecular forms whose clinical features do not differ or differ little from the deletion forms. They are due to mutations in the termination codon. The best known is the Hb Constant Spring (HbCS), in which the TAA → CAA mutation at position 142 (the usual position of the termination codon of the gene) causes an elongation of the expressed α globin by an additional 31 amino acids (from 142 to 173). As the related mRNA is less stable and degrades, a reduction in expression also occurs. In addition, HbCS is unstable and tends to precipitate on the membrane of not only the erythrocyte but also the bone marrow precursors. This causes both hemolytic anemia (circulating cells) and ineffective erythro-

15 Hematological Diagnostics

https://t.me/medicina_free

179

poiesis (bone marrow precursors). Other forms of α-thalassemia due to nondeletion are Hb Pakse, Hb Icaria, Hb Koya Dora, and Hb Seal Rock. All related mutations elongate the expressed α globin.

Pathophysiological andGeneral Clinical Aspects ofβ-thalassemias

From a pathophysiological point of view, the clinical consequences of β-thalassemia are due to an excess of α-chains, which are unable to associate and are highly unstable. In normal erythropoiesis, α-globins are associated with β-globins via AHSP (α-chain stabilization protein). Free α chains behave as active oxidants capable of causing apoptosis and thus inducing ineffective erythropoiesis. The main difference between α and β-thalassemias is that α-thalassemias are hemolytic diseases, while β-thalassemias are dyserythropoietic diseases. Furthermore, β-thalassemias are predominantly due to point mutations or short insertions/deletions of a few nucleotides, not extensive deletions like the α-thalassemic forms. From a translational point of view, the β-thalassemias are distinguished in β+ forms and β0 forms. In β+-thalassemias, mutated alleles encode for reduced amounts of mRNA and thus β-globin, with wide variations depending on the type of mutation and with normal or abnormal chain (e.g., HbE, Hb Knossos). In β0-thalassemias, the mutation completely prevents the production of mRNA, and therefore there is an absence of β globin.

The main molecular mechanisms underlying β-thalassemias

involve mutations affecting:

• Promoters, so the transcription efciency is decreased (the consequence is a β+-thalassemia).

• Splicing, which is completely abolished due to abnormalities of the exon–intron junction leading to β0-thalassemia.

• Splicing, in which the incorrect position of the splicing itself results in structural anomalies leading to β+-thalassemia; the typical example of this anomaly is HbE.

• Splicing, in which an alternative splicing sequence results in a premature stop codon leading to β+-thalassemia with expressed protein rapidly eliminated by proteolysis; the typical example of this anomaly is Hb Knossos.

• Introns (e.g., intron-1 at 110 G→A), with the creation of a new splicing acceptor site leading to β+-thalassemia. Another mutation creates a GT donor site that, interacting with the normal acceptor, unmasks a new acceptor that interacts with the normal donor, leading to the division of intron-2 into two separate introns with a reduction of chain production; also, in this case, β+-thalassemia is observed.

• Polyadenylation site, where there is inefcient cutting and defective polyadenylation at the normal site, resulting

in long and unstable mRNA and reduced production of normal mRNA leading to β+-thalassemia is observed.

• Translation, in which the cause is the formation of a nonsense codon; for example, at codon 36, the CAG→TAG mutation is typically present in Sardinia and is frequent in the Mediterranean; at codon 17, the AAG→TAG mutation is frequent in Asians and Chinese (such mutations do not allow the expression of globin, and therefore we are dealing with β0-thalassemias).

• Exons in which microdeletions or small insertions cause a frameshift and premature stop signal, with the expression of highly unstable globin that is oxidizing and toxic for the erythroid cell (the condition causes severe hemolytic anemia also in the heterozygote and is known as dominant thalassemia).

Among the about 200 defects causing β-thalassemia, 20

are due to deletion. These variants are quite rare, except for the 619bp one that is frequently found in south-eastern Asia. Among them, there are also two forms present with a certain frequency in the Mediterranean area:

• Hereditary persistence of fetal hemoglobin (HPFH), a deletion syndrome characterized by decreased/absent production of β-globin and variable compensatory increase of γ-globin, is present in Mediterranean and African subjects, sometimes with double HPFH/HbS heterozygosity. Some HPFH deletions are very large and can reach up to 100kb. There are also HPFH forms due to nondeletion that are classied as forms with only increased globin Aγ and forms with only increased globin Gγ.

• δβ-thalassemia: in the heterozygote state, it is very similar to the β-thalassemia, with the difference of increased levels of HbF included between 5% and 15% and a characteristic cellular distribution (F cells); the homozygotes and the double heterozygotes δβ/β-thalassaemia have a picture similar to thalassemia intermedia or major. The δβ-thalassemia is subdivided into two groups, within which there are further differences in the deletions:

– δβ-thalassemias with the production of Aγ and Gγ

chains, called AγGγ (δβ)0-thalassaemias; in them are included the Hb Lepore, hemoglobins whose globin chains are the result of unequal crossing-over δβ.

– δβ-thalassemias with the production of Gγ only, called

Gγ (Aγδβ)0-thalassemias.

HPFH and δβ-thalassemia are strictly related from a

molecular point of view, and the distinction has mainly clinical value; however, some forms of HPFH are due to punctiform mutations, which denotes their heterogeneity with respect to δβ-thalassemia. The heterozygote for HPFH has a

180

https://t.me/medicina_free

G. C. Guidi

normal hematological picture with normal indices, normal HbA2, and HbF between 15% and 30% with homogeneous pan-cellular distribution. The homozygote for HPFH is hematologically normal, except for a reduction of the MCV and MCH indexes, and so also appears the double heterozygote HPFH/β-thalassaemia.

Clinical Forms ofβ-thalassemia

The subjects with the minor forms of β-thalassemia (or β-thalassemic trait) do not generally present clinical conse-

quences but mainly of the laboratory:

• Low MCH.

• Low MCV.

• Minor morphological changes.

• Modest increase in HbA2.

• Sometimes a slight reduction in hemoglobin.

• Often an altered globin α/β ratio.

Patients with the major forms of β-thalassemia (homozygous or double heterozygous) always present, with different degrees:

• Severe hemolysis

• Ineffective erythropoiesis

• Iron overload

The intermediate forms of β-thalassemia represent a heterogeneous group with clinical manifestations intermediate between the two previous forms, in particular:

• Hemoglobin is between 60 g/L and 90g/L.

• Transfusions are sometimes necessary.

• From a hereditary point of view, homozygotes for mild

forms or double heterozygotes for forms of different

severity can be found.

• These forms could be present in subjects with a combina-

tion of other types of hemoglobinopathy (e.g., association

with unstable hemoglobin).

Diagnosis ofThalassemia

Molecular Diagnosis

In the case of α-thalassemias, the most common deletion forms are investigated by Gap-polymerase chain reaction (PCR). The rarer nondeletion forms are investigated by specic hybridization methods or using restriction enzymes, followed by sequencing.

In the case of β-thalassemia, two PCR-based techniques are preferably used:

• Hybridization with allele-specic oligonucleotide (ASO)

• Allele-specic priming

Larger deletions should be investigated by Gap-PCR.If PCR-based methods are not sufcient, DHPLC (to locate the mutation site region) and/or sequencing (to characterize the mutation) will be used in the presence of a suspected mutation.

Laboratory Diagnosis (Phenotypic) ofThalassemias

Screening methods based on the measurement of erythrocyte osmotic resistance in progressively hypoosmotic saline solutions have been abandoned as they are nonspecific and have poor sensitivity. In some less equipped realities, techniques based on the hemolysis time in isosmotic glycerol solutions, the so-called GLT50, and its commercial variants (Green test, Osmored A, and others), are still used.

The diagnostic process for thalassemia suspicion is based on the use of hematological methods, such as the CBC and the reticulocyte count, electrophoretic methods, and HPLC (with the use of dedicated instruments, which are today more common than electrophoretic systems), with eventual mass spectrometry in particular cases.

In some situations, the use of molecular biology techniques based on nucleic acids is appropriate. In the α-thalassemic trait, the erythrocyte indices show alterations characteristic of microcytic anemia, that is, decreased total hemoglobin, MCH <27 pg with a progressive decrease in relation to the number of deletions of α genes, MCV <80fL, erythrocyte count mostly increased, normal or little increased reticulocytes, and normal or decreased HbF.Indeed, there is no HbA2 marker as in the case of the β-thalassemia trait because it is also affected by α gene deletion. Moreover, the observable effects depend on the number of nonfunctioning genes and on the techniques used. For example, the presence of HbH (or rarely of Hb Bart) can already be evident in subjects with two deletions when sensitive methods like HPLC, and eventually mass spectrometry, are used, which is more difcult with electrophoresis. HbH is instead well evident on electrophoresis and HPLC in subjects with three nonfunctioning genes, together with HbH intraerythrocytic inclusion bodies.

In the β-thalassemic trait, the erythrocyte indices showing variations are as follows:

• Decreased hemoglobin (9–10g/L)

• Decreased MCH (<27pg)

• Decreased MCV decreased (<80fL)

• Increased erythrocyte count (>5.0T/L)

• Normal or slightly increased RDW, in contrast to sid-

eropenic anemia

• Normal or increased HbF

• Increased HbA2 due to increased relative to HbA

• Slightly increased reticulocytes

15 Hematological Diagnostics

https://t.me/medicina_free

Stained smears may highlight morphological features, such as microcytes, target cells, F cells (rich in HbF), and included bodies. These elements are not determinative per se but contribute to the diagnosis.

Electrophoresis at alkaline pH shows the relative increase in HbA2 and, where increased, HbF.

HPLC shows the relative increase in HbA2 and, where increased, HbF.

HPFH and δβ-thalassemia both present high HbF values. The distinction between the two forms lies in the distribution of intraerythrocytic HbF, homogeneously diffused in HPFH and concentrated in erythrocytes particularly rich in HbF (F cells) in δβ-thalassemia. The microscopic observation after staining with Kleihauer’s method or a more recent method with specic antibodies in ow cytometry can provide differential diagnostic elements.

Normocytic Anemias

Normocytic anemias include a series of manifestations with different etiologies. Some of them can be considered primitive because they are due to direct action in and/or on the erythrocyte, while others are secondary to concomitant pathologies and therefore depend on the evolution of such causal pathologies. Among the primitive forms, there are qualitative hemoglobinopathies that, with some exceptions (e.g., HbC), are to be considered normocytic anemias, even if sometimes accompanied by morphological alterations. Qualitative hemoglobinopathies involve molecular modications that affect the structural and functional characteristics of the hemoglobin involved.

This chapter is limited to sickle cell anemia and hemolytic anemias.

Sickle Cell Anemia (Hemoglobinopathy S, Sickle Cell Disease)

The term sickle cell anemia refers to a collection of autosomal recessive conditions with Mendelian transmission characterized by the presence of the HbS mutation, also in combination with other Hb variants. Mixed heterozygotes are dened as individuals who have one copy of HbS and

Punjab

one copy of another variant, for example, HbD

, or β-thalassemia. They usually present modest clinical manifestations, especially those with β-thalassemia, but there are mixed forms leading to sickle cell syndrome-like conditions, thus with severe clinical manifestations. Carriers have a single copy of HbS and a normal β gene (HbAS) or a variant that does not cause sickling (e.g., HbS/Hb Korle Bu); even in carriers, the clinical manifestations are usually mild or absent. The condition is referred to as sickle cell trait. In sickle cell anemia (HbSS genotype), the affected individual has two copies of the variant Hb, and this deter-

181

Fig. 15.18 Blood smear showing the presence of sickle cell erythrocytes

mines the greater severity of the condition. In circulation, the main form of hemoglobin is HbS, with variable levels of HbF and normal levels of HbA2. This is a qualitative hemoglobinopathy caused by a single missense substitution (GAG → GTG) that results in the insertion of a valine instead of a glutamic acid at position 6 of the β globin chain. The structural consequence of the substitution is the propensity of the molecule to form polymers under conditions of reduced oxygen pressure with a characteristic elongated and curved shape (sickle). This causes deformation of the erythrocytes that contain the polymerizing molecules, a phenomenon that occurs above all in the microcirculation, slowing down to the point of stopping the circulation (Fig.15.18). The main clinical features are as follows:

• Reduced erythrocyte survival

• Vaso-occlusion phenomena, which occur at the level of the cerebral vessels can cause strokes

• Painful seizures, respiratory complications, and organ damage

• Anemia

• Episodic hemolytic seizures

• Blood sequestration in organs like the spleen, lungs, and liver

• Tendency to infections of varying severity

From a clinical point of view, it is also important to deter-

mine the haplotype of the patient affected by sickle cell anemia. Indeed, some haplotypes favor the contextual expression of HbF, whose presence within the erythrocyte has an inhibiting effect on the polymerization of HbS and, therefore, on the sickling, with a consequently better prognosis. There are ve haplotypes of HbS, named according to the regions/ populations in which they have been observed (Senegal, Benin, Cameroon, Bantu, and Arab-Indians). The Senegal and Arab-Indian haplotypes favor a higher expression of HbF, while the Cameroon haplotype favors a low expression of HbF and is therefore associated with a less favorable prognosis than the previous one.

182

...

Electrophoresis

S A

https://t.me/medicina_free

G. C. Guidi

Double heterozygosity for other hemoglobin variants has different effects on sickling. For example, HbS/HbO HbS/HbD

Punjab

combinations are sickling, while HbS/Hb

Arab

and

Korle Bu delays sickling. The HbS/HbC combination tends to reduce sickling as HbC has a dehydrating effect on erythrocytes and thus causes an increased relative concentration of Hb with MCHC, which can reach 40%, and at that point, sickling is observed.

In areas where there is a higher prevalence of hemoglobinopathy S, infant mortality is high, with a peak between 1 and 3years, mainly due to infections. In the most developed areas, there is better survival, guaranteed by neonatal screening programs, early vaccinations, prophylaxis with oral penicillin, and, in general, better treatment of infections. In the United States, for example, the median life expectancy for individuals with HbS is 42years for males and 48years for females.

The laboratory diagnosis of HbS is based rst on the identication of the carrier (HbA/HbS), usually silent, by the following:

• Separation techniques:

– Cellulose acetate electrophoresis at alkaline pH and at

acid pH – Capillary electrophoresis – Isoelectrophoresis, if other hemoglobin fractions are

present – HPLC

• Conrmatory function tests: – Sick cell test – Solubility test for HbS

• Identication of the mutation by molecular biology tests

Sample

direction

–

Polypeptide β,

HbA

Sickle cell

polypeptide β,

HbS

Fig. 15.19 Electrophoresis at alkaline pH of Hb lysates belonging to samples from subjects with HbAA (left), HbAS (center), and HbSS (left). (Copyright EDISES 2021. Reproduced with permission)

βAβ

(Normal)

1 2 3 4 5 6 7

Val His Leu Thr Pro Glu Glu

H3N

Val ValHis Leu Thr Pro Glu

AβS

(Sickle

Cell Trait)

SβS

(Sickle cell

anemia)

(HbA)

(HbS)

Hb Hb

Hb A/Hb S

Hb A/Hb X

Fig. 15.20 Electrophoresis at acid pH of lysates from samples with different hemoglobin variants

Fig. 15.21 Isoelectrophoresis pattern at pH range of 6.0–9.0 for lysates from samples with different hemoglobin variants

Hb S/Hb X

Newborn

Hb A/Hb C

control

Hb A Hb F

Hb S

Hb E Hb

–

During electrophoresis at pH 8.5, HbS migrates more slowly than HbA (Fig.15.19). Noteworthy, other Hbs also migrate to the same position, such as HbD

Punjab

, which can

coexist in heterozygosis with HbS (see above).

Electrophoresis at an acidic pH (5.2) in agarose gels can provide additional diagnostic criteria to identify HbS and differentiate it from other hemoglobins (Fig.15.20).

Isoelectrophoresis can better dene the different fractions of hemoglobin (Fig.15.21).

The use of HPLC is favored by the fact that it is an automated method for measuring HbA1c, making it widespread in laboratories and useful for separating and quantifying the remaining hemoglobin fractions as well. Presumptive identication of variants is made based on retention time (Fig.15.22).

The invitro sickle cell test is performed by placing a drop of the patient’s blood on a slide and covering it with a coverslip. In a short time, the lowering of the oxygen level in the preparation causes the appearance of sickles visible under the microscope (Fig.15.18). The test is made more rapid and sensitive by adding a small amount of sodium dithionite or tetrathionate powder.

45,0

37,5

30,0

22,5

15,0

0123456

2- sample containing Hbs

15 Hematological Diagnostics

https://t.me/medicina_free

183

Hb A/Hb S

Hb A Hb S

7,5

0,0

Fig. 15.22 HPLC pattern of lysate from sample with HbA/HbS genotype

1- normal

Fig. 15.23 Solubility test for HbS.Evident turbidity in the sample on the right. (1) Normal and (2) sample containing HbS

The solubility test for HbS is done by adding a drop of blood to phosphate buffer pH6.8–2.8mol/L, deoxygenated with Na dithionite. A precipitate is observed in the presence of HbS alone (Fig.15.23). The test is specic but does not distinguish heterozygous from homozygous status.

Molecular biology tests involve amplication by polymerase chain reaction (PCR) of the DNA tract with the rst exon of the β-chain, the use of restriction enzymes, electrophoresis, and restriction fragment length polymorphism (RFLP) analysis. The A→T mutation eliminates a restriction site for various enzymes (e.g., MstII). Note that the method is not specic to HbS, as there may be other site mutations. PCR-ARMS (amplication refractory mutation system methods with allele-specic probes can also be used.

Hemolytic Anemias

Common features of hemolytic anemias are as follows:

• Increased rate of red blood cell destruction

• Variable degree of intensity of hemolysis

• Transient marrow response, increasing up to six- to eight-

fold on baseline erythropoiesis

• Progressive decrease in the marrow’s ability to respond to

demands

Table 15.10

Forms of hemolytic anemia Hereditary Acquired Membrane defects

Defects of enzymes of the glycolytic pathway Abnormality of nucleotide metabolism Defects of the pentose phosphate and glutathione cycle Defects of structure and synthesis of globins

Etiopathogenetic classication of hemolytic anemias

Immunohemolytic diseases Traumatic and microangiopathic anemias Anemia due to infectious agents Anemias from drugs, chemicals, poisons Physical agent anemias Hypophosphatemia anemia Paroxysmal nocturnal hemoglobinuria Anemias of liver disease (with spur cells) Vitamin E deciency anemia in infants

• Reduction in erythrocyte survival up to 15–20days without anemia (compensated state, limited in time and variable in magnitude)

The wide variety of hemolytic conditions makes it neces-

sary to proceed systematically, subdividing hereditary and acquired disorders on an etiopathogenetic basis (Table15.10). Membrane defects represent the most numerous groups of hereditary hemolytic conditions, and among them, hereditary spherocytosis is relatively more frequent. The main clinical features to be detected and evaluated in the hereditary forms are listed below.

• Degree of anemia:

– Absent, sometimes in crisis – Variable, moderate to mild (compensation) – Severe with cardiovascular syndrome

• Presence of jaundice:

– Neonatal – Persistent, episodic, absent – Acholuric, no itching

• Episodes of aplastic crisis:

– From human parvovirus type B19 (HPV)→inhibition

CFU-Es – Hb drop of 20–60g/L – Erythroid hypo/aplasia (folate deciency)

• Splenomegaly

• Cholelithiasis ➔ it is often the rst symptom

• Lower limb ulcers, especially in cases of spherocytosis and sickle cell trait

• Skeletal changes due to erythroid expansion in the marrow

Hemolytic crises can be characterized by acute onset

(e.g., after a noncompatible transfusion) similar to those of acute febrile illness with pain in the back, abdomen, head-

184

100

0.1

Hemolysis (%)

https://t.me/medicina_free

G. C. Guidi

Table 15.11

destruction

Test Results Comments RBC survival (

Endogenous CO >1.5% to 10–12%

Quantitative fecal urobilinogen Hemolytic index (urobilinogen/Hb mass) LDH-2in plasma Increased Not very specic Haptoglobin Hemolysis for values

Table 15.12

Test Results Comments Hemoglobinemia IR <10mg/L Test requiring

Hemoglobinuria Search for oxidized dimers

HbA1c Reduced Little/not used Sideruria with Prussian blue Methemalbumin, hemopexin in plasma

Hemolytic disorders: signs of increased red blood cell

Cr) Up to <30days Test performed in

(infants) Increased Little/not used

32–792mg/day/100g Hb (IR 11–21)

<250mg/L (sensitivity 83% and specicity 96%)

Hemolytic disorders: signs of intravascular hemolysis

of Hb

Hemosiderinuria in tubular cells Presence of spectral bands at 620–630nm that do not disappear with H

2O2

specialized centers Little/not used

Little/not used

Useful

technical precautions Rarely used

Little/not used

Table 15.13

Test Results Comments Count of reticulocytes Increase Used MCV Increase Used Circulating erythrocyte

precursors White blood cell count Increase Used Platelet count Increase Used Bone marrow Medullary erythroid

Fe plasma/Fe erythrocyte turnover Erythrocyte creatine Increase (IR

Erythrocyte enzymes (Hx, GOT, etc.)

Hemolytic disorders: signs of accelerated erythropoiesis

Presence Used

hyperplasia Increase Little/not

14–90mg/L) Increase Little/not

0.9 0.7 0.5 0.3

Specialist

used Little/not used

used

ache, vomiting, oligo/anuria, pallor, tachycardia, or subacute onset with gradual and insidious progression, with few symptoms, good compensation at least initially, jaundice, pallor, then crises with worsening. From the point of view of the clinical laboratory, the diagnostic indicators should be sought in different directions, as reported in Tables 15.11,

15.12, and 15.13.

In hemolytic conditions, direct and indirect antiglobulin

tests (the Coombs test) should always be performed. However, it should be considered that 2–5% of patients with anemia of immunohemolytic origin are falsely negative due to the reduced sensitivity of the test.

The evaluation of erythrocyte osmotic fragility is based

on tests that are parallel to those described for the screening of thalassemias; however, the mechanism and meaning are completely different, which makes these fragility tests useful for screening and therefore sensitive but little specic. In these tests, the principle is always that of hemolysis for osmotic stress, obtained either with progressively hypoosmotic solutions of NaCl or with solutions of glycerol in the acid buffer. Hemolysis begins when the erythrocyte surface/ volume ratio reaches a critical volume, which in a normal subject is ~165%, and occurs at a NaCl concentration of

0.45–0.50% to reach a 50% hemolysis value at 0.40–0.45%

After incubation

HS Normal

Fig. 15.24 Osmotic fragility test in progressively hyposmotic NaCl solutions of a sample from a normal subject and one from a subject with hereditary spherocytosis before and after incubation. (Copyright EDISES 2021. Reproduced with permission)

Before incubation

HS Normal

NaCl. If hemolysis is observed at higher NaCl concentrations, this is referred to as increased fragility due to the shift of the hemolysis curve to the left (Fig. 15.24). A normal erythrocyte swells when placed in 0.6% NaCl; hemolysis begins at 0.42% NaCl when the erythrocyte volume rises to ~145% of normal; in 0.35% NaCl, hemolysis is complete. In borderline cases, the test can be made more sensitive by performing it on a blood sample preincubated at 37°C for 24h under sterile conditions. The test is positive for conditions associated with hereditary spherocytosis. Evaluation of erythrocyte osmotic fragility is also performed with variants of the glycerol test, previously described for thalassemias, using an acidied glycerol solution. Additional commercial tests are the Pink Test and the Osmored B.

The self-hemolysis test, once widely used for the evalua-

tion of erythrocyte fragility but now less practiced, is based

<<< < Предыдущая 7 8 9 10 11 12 13 14 15 16 17 1819 / 5019 20 21 22 23 24 25 26 27 28 29 30 31 > Следующая >>>

Соседние файлы в папке Библиотека им академика М.И. Перельмана

#
30.08.202661 Мб0Книга_2609_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202660 Мб0Книга_2610_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202660 Мб0Книга_2611_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202660 Мб0Книга_2612_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202659 Мб0Книга_2616_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202659 Мб0Книга_2617_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202659 Мб0Книга_2618_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202658 Мб0Книга_2619_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202658 Мб0Книга_2620_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202657 Мб0Книга_2621_Библиотеки_им_академика_М_И_Перельмана.pdf
#
30.08.202657 Мб0Книга_2622_Библиотеки_им_академика_М_И_Перельмана.pdf