3. Diagnostic approach

More than 80% of RA are hereditary and, therefore, have no curative treatment, exception made of palliative therapies such as blood transfusions or erythropoietic stimulating drugs (Erythropoietin). In clinical practice they may be some confusion between RA and the anaemias that appear in the course of non-haematological or systemic diseases, also called secondary anaemias. This confusion is due to the fact that anaemia is not a disease, but a clinical manifestation, and some Rare Diseases (RD) are associated with anaemia, moderate or severe. One example of this is the Rendu-Osler disease (hereditary telangiectasia), a relatively well known RD where anaemia, due to iron deficiency, is very common, and sometimes the first clinical manifestation of the disease. Furthermore, the anaemias due to rare chronic inflammatory diseases, vitamin deficiencies, immune diseases, malignancy, or other rare disorders, may probably be also considered RA, although they have not yet been included in this group.

Hereditary RA, as in other RD, the low number of patients creates the need to mobilize resources and their study can be efficient only if done in a coordinated European scene of action level. Among hereditary anaemias, haemoglobinopathies are the commonest genetic defect worldwide with an estimated 269 million carriers [7]. They are the consequence of mutations in the globin genes, which are responsible for the synthesis of haemoglobin, the main component of RBCs. These mutations are leading to abnormal proteins (haemoglobin variants) or to a decreased synthesis of globin chains (thalassaemias). In Europe, certain populations are particularly at risk of having a haemoglobinopathy. In Southern countries, their prevalence is higher than in central or northern Europe, but in all cases the prevalence is less than 1 per 2000 individuals. For this reason, in Europe, haemoglobinopathies and thalassaemias are considered a particular group of RD or RA. Whereas thalassaemia syndromes are inherent in the autochthonous European at risk groups (Mediterranean anaemia), other haemoglobinopathies have been imported by migration (Sickle-cell anaemia).

In general, the diagnosis of a RA is often prompted by pallor, noticed by the patient, the family, and/or the General Practitioner (GP). Severity of clinical manifestations is directly proportional to the acuteness of onset, and many patients do not notice any symptoms when anaemia occurs insidiously. At the laboratory level, the diagnosis of anaemia includes two main steps:

#### 3.1 General diagnostic tests

General diagnostic tests always include a Complete Blood Count (CBC), the reticulocyte count and the peripheral blood cell morphology examination. The CBC includes four main parameters: (a) haemoglobin concentration (Hb), the key for anaemia diagnosis, (b) RBC count or concentration of RBCs, given as number of cells per litre of blood, (c) haematocrit or packed cell volume (PCV), given as the percentage of blood by volume that is occupied by the RBCs and (d) RBC indices or calculations derived from (a), (b), and (c), of great help for the diagnosis and classification of anaemias. These indices are automatically measured by modern haematology full automated or semi-automated, analysers, and are mainly three: (1) the mean corpuscular volume (MCV) or average size of the RBCs expressed in femtolitres (fl), (2) the mean corpuscular haemoglobin (MCH) or average amount of haemoglobin inside a single RBC expressed in picograms (pg) and (3) the mean corpuscular haemoglobin concentration (MCHC) or average concentration of haemoglobin in the RBC expressed as a percent. Sometimes the RBC distribution width (RDW), a measure of the variation of RBC size, can be also used for anaemia classification. Usually RBCs have a standard size of about 6–8 μm, but in certain disorders, a significant variation in RBC size can be present. Here the RDW value is a relatively good indicator of RBC size heterogeneity. RDW is especially useful to differentiate iron deficiency (increased value) from thalassemia (normal value). Reticulocyte count or number of circulating young RBCs (reticulocytes) is an important complementary test which indicates the bone marrow capacity to overcome the severity of anaemia [8]. Accordingly, anaemias due to RBC destruction (haemolysis) are characterized by increased reticulocyte count (regenerative anaemias), whereas anaemias due to erythropoietic insufficiency (aplasia or dyserythropoiesis) are characterized by a lower than expected reticulocyte count from the severity of the anaemia (Non-regenerative anaemias). In thalassaemias, where erythropoietic insufficiency coexists with some degree of haemolysis, the reticulocyte count may be variable.

The reticulocyte count and MCV are, up to now, the most useful criteria for anaemia classification. According to MCV, anaemias are classified into microcytic

#### The Rare Anaemias DOI: http://dx.doi.org/10.5772/intechopen.86986

(low MCV), macrocytic (high MCV) and normocytic (normal MCV). The two main causes of microcytic anaemias are iron deficiency and thalassaemia and the two main causes of macrocytic anaemias are cobalamin (vitamin B12) and folic acid deficiencies. Normocytic anaemias can be due to several different causes, not related with nutritional defects or thalassaemia, being the most frequent haemolysis and erythropoietic failure. Here, the reticulocyte count is the most useful test to differentiate these two conditions. In clinical practice, the most frequent cause of anaemia is iron deficiency (ID), characterized by a low MCV (microcytic anaemia). In southern Europe countries with higher "at risk" thalassaemia population (Mediterranean basin), this hereditary disorder can be misdiagnosed as iron deficiency anaemia (IDA) because of the low MCV (<82 fL) or microcytosis. Accordingly, in a patient with microcytosis the first step is always to exclude ID. If present, iron supplementation has to be given until the MCV recovers its normal value. However, if after treatment, the MCV remains low, the coexistence of a thalassemic gene has to be investigated. It should be mentioned that there are a number of conditions where the MCV can falsely rose masking the main clue of thalassaemia diagnosis. This is the case in some patients with thalassaemia who co-inherit another cause of haemolytic anaemia leading to an increased reticulocyte count. This can falsely increase the value of MCV and masking the diagnosis of thalassemia if only the MCV is used for initial screening.

As part of the CBC, the blood film examination is sometimes very useful because it may provide a clue to the diagnosis of a particular RBC defect [9]. Despite the advances in automated blood cell counting, the blood film examination retains a crucial role in the diagnosis of RBC disorders. This is particularly important in haemolytic anaemias and in the differential diagnosis of macrocytic anaemias. RBC morphology examination provides in some cases (e.g. red blood cell membrane disorders, sickle-cell anaemia), a definitive diagnosis, but, more often, it suggests a differential diagnosis that indicates further study. Morphological changes such as basophilic stippling and target cells in the blood film are not definitively associated with a haemoglobinopathy, but would be helpful findings in patients with moderate or severe anaemia associated with low MCV (Thalassaemia intermedia, or Thalassaemia major). Finally, RBC morphology examination has also the advantage of speed that may be important in severe anaemias such as those mentioned before.

#### 3.2 Cause-oriented specific diagnostic tests

These tests are the next step for the identification of the cause of the anaemia or of its mechanism. They include a group of laboratory procedures depending on clinical or laboratory diagnostic orientation of the anaemia [8] and, when necessary, a final genetic identification of the cause of the disease [10]. In order to provide a first approach to the cause of the anaemia, several diagnosis oriented flowcharts can be found in the literature, mainly based on the morphological classification of the anaemia (microcytic, macrocytic and normocytic).

ENERCA website (www.enerca.org) provides practical flowcharts for the diagnostic orientation of anaemia. For this, three patient's data have to be provided: sex, Hb and MCV. If anaemia is detected, one of the three available flowcharts will appear, depending on the MCV value: low (microcytic anaemia), high (macrocytic anaemia) and normal (normocytic anaemia). These flowcharts are not exhaustive and the final diagnosis always requires the advice of a health professional, but they provide the basic information on how the investigation of anaemia causes can be undertaken in routine clinical practice. Using these flowcharts the most frequent RAs (haemoglobinopathies, thalassaemias and haemolytic anaemias) can be easily recognized. Depending on the results of the recommended basic tests, more specific tests (including molecular biology) can be performed. Some of these specific tests

can also be performed in general haematology laboratories but other tests require to be undertaken in specialized laboratories. In all the cases External Quality Assessment Schemes (EQAS) are necessary for assessing the quality of practice or for obtaining a technical qualification. Since the most specific tests are performed in few specialized laboratories, local (national or regional) EQAS organizations cannot establish a specific EQAS for these procedures due to its high cost. Accordingly, the EQAS for these procedures have to be promoted at European level as ENERCA 3 has done with some rare diagnostic tests for RA such as Pyruvate-kinase deficiency (PKD).

## 4. Rare Anaemias due to bone marrow defects

Bone marrow failure syndromes (BMFS) are multisystem diseases that are characterized by varying degrees of deficiency in the production of haematopoietic cells, which can range from the depletion of a single cell lineage (cytopenia) to that of multiple lineages or even of all lineages (pancytopenia). The most well-known acquired BMFS is aplastic anaemia (AA).This causes a deficiency of all three blood cell types (pancytopenia): red blood cells (anaemia), white blood cells (leukopenia), and platelets (thrombocytopenia) and aplastic refers to the inability of stem cells to generate mature blood cells. It is more frequent in people in their teens and twenties, but is also common among the elderly [11]. It can be caused by heredity, immune disease, or exposure to chemicals, drugs, or radiation. However, in about half the cases, the cause is unknown. The definitive diagnosis is by bone marrow biopsy; normal bone marrow has 30–70% blood stem cells, but in aplastic anaemia, these cells are mostly gone and replaced by fat. First line treatment for aplastic anaemia consists of immunosuppressive drugs, typically either anti-lymphocyte globulin or anti-thymocyte globulin, combined with corticosteroids and cyclosporine. Haematopoietic stem cell transplantation is also used, especially for patients under 30 years of age with a related matched marrow donor [11]. Congenital BMFS are, as AA, multisystem diseases characterized by varying degrees of deficiency in the production of haematopoietic cells, which can range from the depletion of a single cell lineage (cytopenia) to that of multiple lineages or even of all lineages (pancytopenia). In general they are monogenic diseases with high genetic heterogeneity and phenotypic overlapping, so a bone marrow and genetic analysis is required to reach a correct diagnosis [12]. They are ultra-rare diseases with a usual presentation during childhood and with an incidence of one to two cases per one million individuals. In almost all cases they are associated with morbidity and mortality, requiring lifelong blood transfusions, treatment of infections, growth factors and transplantation of haematopoietic progenitors. Likewise, they present a high risk of developing haematologic cancer or solid tumours and a high toxicity to treatment, which leads to a lower life expectancy. The most relevant aspects of some of these syndromes are the following:

#### 4.1 Fanconi anaemia (FA)

FA (OMIM 227650) is a hereditary disease with a predominantly autosomal recessive pattern and in one case linked to chromosome X. Its prevalence in the general population is estimated at two to five cases per one million individuals, with an estimated incidence of 1/131,000 births. 21 different gene mutations have been identified so far to be a cause of congenital aplasia [13]. In most cases, each of the parents carries one of the pathogenic variants, with three exceptions: male patients of the FA-B subtype (FANCB gene), patients of the FA-R subtype (RAD51 gene),

#### The Rare Anaemias DOI: http://dx.doi.org/10.5772/intechopen.86986

and the cases in which one of the two variants is de novo (not present in the parents or present in only some of the gametes of one of the parents). The gene FANCB is a gene linked to the X chromosome, so that women are asymptomatic carriers of the pathogenic variant. From the clinical point of view, FA is considered as a syndrome of chromosomal instability with a high spectrum of clinical manifestations that can be grouped into congenital malformations, endocrine dysfunction, haematological abnormalities such as severe cytopenias, myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML) Moreover, a predisposition to develop tumours and chromosomal fragility has been noted and in about 30% of the patients do not present congenital malformations and are only diagnosed at the time when the disease debuts with the haematological abnormalities [13]. To make a diagnosis, it is necessary to confirm the chromosomal fragility by cytogenetics [14] and until a few years ago, genetic testing was not part of the routine clinical analysis of AF patients, partly because it was considered sufficient with the cytogenetic confirmation of the diagnosis (chromosomal fragility) and the subtype of the patient, and on the other hand, because the mutational analysis was boring. However, with the implementation of new high-throughput sequencing technologies [15], subtyping and mutational study are now achieved at the same time, sequencing all AF genes in the same assay, and even identifying new genes involved (Table 2). However, this approach


#### Table 2.

Genetic subtypes and genes which mutation are implicated in the development of Fanconi anaemia.

can cause errors in these patients, due to the broad mutational spectrum of the disease and because all the genes related to the disease have not yet been described. For this reason, it is advisable to always start the diagnosis by the mutational study in patients with a positive chromosomal fragility for AF. With regard to treatment, in addition to correcting, as far as possible, some of the congenital malformations, it is essential to carry out a haematological follow-up in order to identify early signs and symptoms of bone marrow (BM) failure. Current guidelines recommend tracking blood counts every three to four months, and an annual bone marrow aspirate. Treatment should be initiated depending on the patient's clinical commitment and continue with it in accordance with the response to it. Support measures include transfusions of red blood cells or platelet concentrates, or the use of colony / cytokine stimulating factors. Currently, the only curative treatment for bone marrow failure in these patients is the allogeneic haematopoietic stem cell transplant (HSCT) from suitable donors. Finally, the correct and early diagnosis of AF does not only allow the discarding of other diseases, but fundamentally enables the proper management of their haematological alterations and genetic counselling to the individual and his family in case of successive pregnancies. In addition, in families with mutations in genes predisposing to cancer, it allows adequate monitoring and risk assessment in family members.
