Proteins are among the most complex organic compounds and are the fundamental constituents of all animal and plant cells. From a chemical point of view, proteins are polymers (also known as macromolecules) of amino acid residues. There are held together by a peptide bond, often with other molecules and/or other metallic ions (in which case they constitute a conjugated protein).
Proteins can be classified into two main families: globular proteins and proteins with an extended or fibrous structure. These two groups reflect the two broad functional separations that characterize them:
*Extended or fibrous proteins generally perform biomechanical functions, such as in the formation of nails, hair, of the corneous layer of the epidermis etc. providing a barrier against the outside world.
*Conversely, globular proteins perform a variety of critical biological functions. These include enzymes, respiratory pigments, numerous hormones and antibodies in the immune system.
The protein molecule consists of carbon, oxygen hydrogen and nitrogen atoms; sulphur is also often contained (such as in methionine, cysteine and cystine amino acids) and at times also phosphorous and/or metals such as iron, copper, zinc etc. may be present.
Protids are one of the fundamental components of cells. Their composition in terms of amino acids may vary and is genetically determined; hence their molecular weights may vary greatly, according to the number and type of amino acids (monomers) in each molecule (heteropolymer, of amino acids with an average molecular weight of 115). If the molecule has very few amino acid units (generally not more that 15-20) it is defined to be an oligopeptide. An oligopeptide generally lacks a well-defined conformation within a solution, changing continuously due to its rather high flexibility. Longer polymers are known as polypeptides. One or more polypeptides form a protein.
Living organisms contain a startling number of amino acids, yet only 20 are controlled genetically (as a result of evolutionary processes) and are thus contained in proteins:
1. aspartic acid (mono-amino di-carboxylic)
2. glutamic acid (mono-amino di-carboxylic)
3. alanine (mono-amino mono-carboxylic)
4. arginine (di-amino mono-carboxylic)
6. cysteine (mono-amino mono-carboxylic)
7. phenylalanine (mono-amino mono-carboxylic)
8. glycine (or glycine)
13. lysine (di-amino mono-carboxylic)
15. proline (iminoacid)
16. serine (mono-amino mono-carboxylic)
19. tryptophan (mono-amino mono-carboxylic)
Non-proteic amino acids include GABA (amino butyric acid, a chemical mediator of the nervous system), DOPA (3,4-dehydroxy-l-phenylalanine, a precursor of adrenaline) and others that have important specific biological properties. There are 10 essential amino acids for the human body (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine). Some of these are “conditionally essential”, meaning that they only become essential under certain physiological or pathological conditions (e.g. cysteine, tyrosine, taurin, glycine, arginin, glutamine, proline).
The quantity of proteins that is present in serum is defined as total proteinemia, with normal values between 6 and 8 grams per deciliter.
Considered in isolation, total proteinemia can only provide generic indications regarding pathological states; despite undergoing modifications individually, the various fractions of the proteins tend to compensate, seeking a certain biological equilibrium. Thus, total proteinemia displays considerable variations (both positively and negatively) only in very rare cases, such as in the presence of serious diseases.
For example, a proteinemia increase can be observed in the context of excessive sweating (prolonged physical exercise, intense physical activity) or dehydration whereby organic liquids are lost within a short period of time, such as cases of uncontrollable diarrhea or vomiting, uncompensated diabetes, shock or acute collapse.
A decrease in total proteinemia occurs upon a reduced protein intake, anorexia (eating disorder characterized by decreased appetite), intestinal absorption disorders, for failure or defective protein synthesis due to deficiency of vitamins and amino acids, for liver diseases of a chronic nature, for loss or destruction of proteins such as in hyperthyroidism, in certain kidney diseases, in extensive burns and haemorrhages.
PLASMA CELL DYSCRASIAS
A family of clinically and biologically different disorders whose aetiology is unknown, which are characterized by a disproportionate proliferation of a B cell clone; also, they are characterized by the presence of structurally and electrophoretically homogeneous immunoglobulins (monoclonal) or by a polypeptide subunit in serum or urine.
Pathogenesis and classification
The production of immunoglobulins is usually heterogeneous (polyclonal); over the course of its life, each plasma cell clone thereby secretes a single heavy chain (gamma, mu, alpha, delta or epsilon) and a light chain (kappa or lambda). Light chains are generally produced in slight excess, thus in the urine of healthy subjects, small quantities of light chains can be found (40 mg/24h). The disproportionate proliferation of a single clone implies an increase in the serum level of the secreted molecule, the monoclonal immunoglobulin (M protein). The M protein can be rapidly detected as a symmetric homogenous peak (M peak) with type 2-1-2 or serum or urine electrophoretic mobility. Immunofixation is required in order to identify the heavy and light chain protein classes. The magnitude of the M-component peak correlates with the number of cells in the body that synthesize it. Hence, these proteins are markers of B-cell clones. Most of the M proteins rather suggest being a normal product of a single cell clone that has undergone an intense proliferation. They are not qualitatively abnormal. Some of these M proteins display antibody activity, more commonly targeting autoantigens and bacterial antigens. A recent analysis suggests that the expression of immunoglobulin genes, leading to the production of Mproteins, appears to be antigenically determined. Serum levels of other non-monoclonal immunoglobulins commonly happen to be reduced. The difference in the production of immunoglobulins in multiple myeloma may be determined by the presence of a monocyte or macrophage that suppresses the maturation of normal B lymphocytes in plasma cells secreting antibodies. Plasma cell dyscrasias range from apparently stable asymptomatic conditions (in which only the protein is present) to neoplastic, clinically symptomatic forms (e.g. multiple myeloma). Transitory plasma cell dyscrasias have rarely been described in patients with hypersensitivity to drugs (sulfonamides, phenytoin, and penicillin), suspected of having viral infections and coincidentally to surgical operations.
Multiple myeloma is a form of cancer that affects plasma cells, a fundamental component of the immune system. Plasma cells are the result of the maturation of B lymphocytes; together with T lymphocytes (T-cells), these represent the two main cell types involved in the immune response. Plasma cells can mainly be found in the bone marrow and their role is to produce and release antibodies to fight infections. However, their growth occasionally proceeds in an uncontrolled manner, giving rise to a tumor. Myeloma cells produce large quantities of a protein known as monoclonal component (M Component), a particular type of antibody. The abnormal growth of plasma cells gives rise to problems to other blood cells (white blood cells, red blood cells and platelets) weakening the immune system, leading to anemia or defects in coagulation. Furthermore, myeloma cells produce a substance that stimulates osteoclasts, which are responsible for the destruction of the bone tissue; consequently, myeloma patients are often subject to bone fractures.
Types of myeloma
Myeloma is an alteration of the plasma cells, but can occur in different forms.
• Multiple myeloma: the most frequent. Tumor plasma cells are mainly located in the bone marrow and produce a monoclonal antibody that can be identified in large quantities within the serum of the patient.
• Light Chain myeloma: plasma cells only produce parts of immunoglobulins known as light chains.
• Non-secretory myeloma: plasma cells do not produce immunoglobulins, but are present in excessively large numbers.
• Solitary plasmacytoma: the tumor occurs in a single location, within a bone or at extra-medullary level.
• Plasma cell Leukemia: plasma cells are present in large numbers also in the blood.
• Indolent myeloma: the disease is asymptomatic and there are no lesions to bones or other organs.
Following the diagnosis, it is vital to define the stage of myeloma, according to which indications are also obtained regarding the prognosis of the disease. The Durie-Salmon system is the traditional method to assign a stage to myeloma. It identifies three stages taking into account four factors: amount of immunoglobulins in the blood or urine, the amount of calcium in the blood, the amount of hemoglobin in the blood and severity of bone damage (assessed by means of X-ray). However, the accuracy of this system is decreasing, as new diagnostic techniques are being introduced. The International System of staging of multiple myeloma is another staging system that has recently been introduced to define the three stages of myeloma. This system is mainly based on the levels of serum albumin and beta-2-microglobulin as well as on the renal function, on platelet count and age of the patient. In some cases, the tumor recurs after treatment: this is known as recurring myeloma, which may arise again in the bones or elsewhere in the body. Lastly, indolent myeloma is a tumor that displays neither active nor rapid growth, and thus causes no damage to bones or other organs. Because of the characteristics of this disease, patients with indolent myeloma generally do not undergo treatment, but are rather kept under careful observation.
Early diagnosis of multiple myeloma is difficult, since patients do not usually have any symptoms until more advanced stages. Similarly, they may have general symptoms, which could be caused by other diseases. Blood and urine tests provide a preliminary indication of the presence of a tumor of plasma cells. By means of electrophoresis on serum and urine proteins, high levels of immunoglobulins indicate the presence of the disease.
In addition to these techniques, analysis on other serum parameters may help to define the presence of myeloma, albeit not essential for the diagnosis. Specifically, hemoglobin and platelets levels are low in the presence of the disease, and at advanced stages of the tumor. The same is true for serum albumin levels. Also high levels of beta-2 microglobulin and calcium in the serum indicate the advanced stage of a myeloma. Bone marrow biopsy is a fundamental tool for the diagnosis of myeloma, which involves extracting and analyzing a fragment of bone and bone marrow. The bone marrow is aspirated with the use of a syringe (bone marrow aspirate) and analyzed for the presence of any cancer cells. Other techniques, including medical imaging techniques such as X-rays, CT, MRI and PET, are also used in order to complete and optimize the diagnosis of myeloma.
The term amyloidosis identifies a heterogeneous collection of disorders characterized by a deposit of abnormal proteins occurring in various organs and tissues. Such protein deposits occur in the form of fibrils and are referred to as amyloids. The damage caused by amyloids to the various organs and tissues is determined by its progressive accumulation. These forms of amyloidosis are rare pathologies, yet serious ones with a very high mortality rate. The distribution of amyloids can be either local or systemic. What determines the differences between different types of amyloidosis boils down to several proteins, which for reasons that are still unknown, can mutate their structure; furthermore, they are determined by the different points in which the amyloid tends to concentrate. Hence, it goes without saying that amyloidosis can occur under a variety of different clinical picture. Furthermore, it also follows that the mismatches between clinical picture and symptomatology may cause considerable difficulties in terms of allowing an early diagnosis; this timeliness is regarded as a key factor in addressing the pathology.
Amyloidosis: the main groups
The most important groups of amyloidosis are the following:
primary AL amyloidosis
secondary AA amyloidosis
Primary AL - AL amyloidosis (light-chain amyloidosis) is characterized by deposits of monoclonal immunoglobulin light chains, proteins that are produced within the bone marrow; these immunoglobulins are produced in order to protect the body from pathological processes. For yet unknown reasons, once these immunoglobulins have carried out their function, they do not dissolve but rather transform into amyloid fibrils, which are transported by the bloodstream and progressively build up in various organs and tissues.
Secondary AA amyloidosis - AA amyloidosis is a type of amyloidosis that is secondary to several other diseases. The protein that characterizes this type of amyloidosis is the SAA (Serum Amyloid A), a protein that is produced during acute phases of chronic diseases such as rheumatoid arthritis.
In some cases, primary amyloidosis treatment can slow down the progression of the disease, possibly even halting it.
The organs that are generally more seriously affected within this form of amyloidosis are the kidneys, with the possibility of proteinuria, nephrotic syndrome and renal failure. Other organs that may be affected by this type of amyloidosis, though less severely, are liver and spleen. In very rare cases, also the heart may be affected.
Hereditary amyloidosis–several proteins are involved in the development of hereditary amyloidosis; the mutation of the latter give rise to different types of hereditary amyloidosis. Among the different types of hereditary amyloidosis, transthyretin amyloidosis (TTR amyloidosis, MIM 176300) occurs with greater frequency. Transthyretin (TTR) is a protein that is mainly secreted by the liver. It is involved in the transport of thyroid hormones (thyroxine and RBP, retinol-binding protein). Clinically, this type of amyloidosis generally involves the peripheral nervous system as well as the autonomic nervous system. TTR amyloidosis has an autosomal dominant transmission (there is a 50% chance of disease transmission; if the children do not inherit the disease, there is no risk that they can pass it on to the next generation). TTR amyloidosis shares some similarities with AL amyloidosis: however, unlike the latter, TTR amyloidosis is less aggressive and chances of survival are greater. Another type of hereditary amyloidosis is apolipoprotein AI amyloidosis (AApoAI, MIM 107680). In this type of amyloidosis, amyloid accumulation generally involves organs such as heart, liver and kidneys; this leads to a clinical picture respectively of cardiomyopathy, liver disease and kidney disease.
Diagnosis of amyloidosis
In order to diagnose amyloidosis, amyloid deposits have to be identify by means of specific staining of a tissue sample. To this end, a peri-umbilical fine needle aspiration is generally performed, a simple and painless test with a sensitivity approaching 80% (in the systemic forms of amyloidosis). Where the needle aspiration fails to diagnose amyloidosis, a biopsy of the labial minor salivary gland may be performed in order to clear any remaining concerns. Depending on the situation, a biopsy may be required on organs suspected of being affected by amyloidosis. If the diagnostic tests confirm the presence of amyloid, it is necessary to identify the protein which causes deposits in order to determine which type of amyloidosis has affected the subject. The identification of the protein is a critical step with regard to the treatment, since the latter varies with the type of amyloidosis.
Cryoglobulins are a group of proteins that form a precipitate or gelify at lower temperatures, and solubileze at 37 ° C. They are present in a wide range of clinical manifestations and are represented by a heterogeneous group of immunoglobulins (Ig), displaying single or mixed form patterns in the immunochemical analysis after purification. Following the Brouet classification, the following may be distinguished: - type I cryoglobulinemia in which the cryoprecipitate is formed by a single complete monoclonal Ig (IgG, IgA, IgM) or, more rarely, by a single light chain. This can be identified more frequently in patients with multiple myeloma, Waldenstom macroglobulinemia. The cryoprecipitate in type II cryoglobulinemia consists of one or more monoclonal Ig and polyclonal Ig and is associated with lymphoproliferative diseases, autoimmune diseases and HCV. Type III cryoglobulinemia involves a cryoprecipitate consisting in polyclonal or oligoclonal Ig and can be found in patients with autoimmune diseases and chronic infections. Positive cryoglobulinemia is strongly influenced by methodological rigour within the preanalytical and analytical phases. Excluding or confirming their presence relies on strictly controlling the temperature at which processes are carried out (sampling, transportation and centrifugation at 37°C, purification and characterization).
Blood is collected without the use of anticoagulants, sampled in test tubes at 37 ° C and maintained at this temperature until it coagulates. The serum is separated by centrifugation at 37 ° C at 1500g for 15 minutes. It is aliquoted into a test tube and into a Wintrobe tube, both kept at 4°C. The presence of cryoglobulins is signaled after a period of incubation at 4 ° C for 7 days, generally showing up as a white precipitate or gel. The reversibility of cryoprecipitate has to be verified by heating the serum precipitate aliquot. Cryoglobulins are measured as a percentage ratio between the serum’s precipitate and volume after centrifugation at 1500 g for 10 min at 4°C. Precipitates are resuspended in physiological saline solution or in a solution with PBS at 4°C and washed three times. Immunofixation is performed on the dissolved cryoprecipitate using total human antiserum and specific antisera for γ, α, μ, κ, λ. Cryoglobulins can thereby be classified into the taxonomy outlined above. In the presence of cryoglobulinemia, the laboratory report must include cryocrit data along with Ig classification, according to Brouet’s system.
FREE LIGHT CHAINS (FLCs)
During immunoglobulin synthesis, plasma cells produce slightly more light chains compared to heavy chains. The proteins that remain unused in assembling an entire immunoglobulin, are called free light chains (FLC). FLC half-life is 2-4 hours for the kappa chains and 3-6 hours for lambda chains that are present in the serum as dimers, as their reduced size allows them to be easily filtered by the glomerulus. The difference in speed of renal filtration implies that the concentration of lambda chains in the serum can be greater, even if the synthesis favors kappa chains. Increased plasma concentration may be due to a variety of clinical situations such as immune depression or stimulation, kidney failure or plasma cell dyscrasias. With the exception of the latter condition, however, kappa and lambda chain ratios remain normal. The presence of lymphoproliferative disorders can cause an unbalanced ratio between the two light chains (kappa or lambda) to be observed. An immunometric assay for the measurement of light chains in the serum was introduced in the early 2000s; the test is capable of detecting the sole FLC by means of antibodies directed against the hidden epitopes within the whole immunoglobulin. This measurement is unable to distinguish between polyclonal and monoclonal FLC; nevertheless, as mentioned above, an alteration in the kappa/lambda ratio provides evidence of a monoclonal lymphoplasmacellular proliferation.
Reference intervals (mg/L)
κ FLC 3.3 – 19.4
λFLC 5.7 – 26.3
κ/λFLC 0.3 –1.2 0.26 – 1.65
Waldenström macroglobulinemia is a monoclonal neoplasia characterized by the proliferation of B cells. It is furthermore characterized by the presence of a plasma cell infiltrate in the bone marrow and by the presence of an M-component (M stands for monoclonal) brought about by the overproduction of a gamma globulin, which in this case belongs to the IgM class. However, unlike in myeloma, lytic lesions are not present. The predominant clinical manifestation is the hyperviscosity syndrome. Waldenström’s macroglobulinemia has long been considered to be a variation of the latter, due to the fact that it bears similarities with myeloma. The World Health Organization currently currently ranks it among low-grade malignancy lymphomas.
Monoclonal components are immunoglobulin ("Ig") molecules, or parts thereof. Normal plasma cells produce immunoglobulins consisting in the antibodies that are necessary to fight infections. Abnormal plasma cells - "myeloma cells" – are present in myeloma patients and do not produce antibodies in response to an infection. Instead, they produce monoclonal immunoglobulins, which cannot function as antibodies. This typical immunoglobulin is unique in each myeloma patient and can be formed by: heavy chains combined with light chains or by light chains alone, or by immunoglobulin fragments or combinations thereof.
Protein electrophoresis is a laboratory analysis based on the separation of proteins in the presence of an electric current. To perform this test, a solid support (such as agarose gel) can be used, as well as liquid-filled thin silica tubes (capillaries). When a sample containing a mixture of proteins is applied to the gel or into a capillary, the different proteins within the mixture are separated according to their electric charge. When searching for a specific monoclonal component, laboratories analyze serum and urine by protein electrophoresis as it is the only test that can confirm monoclonality unambiguously.
Immunoglobulins are proteins that have antibody functions. They are produced by special cells that belong to the immune system in response to the presence of external agents such as viruses, bacteria, protozoa, fungi, cancer cells or extraneous tissues which are recognized as such by the presence of antigen molecules on their surface. Antibodies are produced in the blood by a particular type of cells, plasma cells, resulting from the differentiation of B lymphocytes in the presence of the antigen. Antibodies are responsible for recognizing the external agents and neutralizing the threat, in a variety of different ways. The shape of the antibody molecule can be represented as a Y, formed by four protein chains (two 'heavy' and two 'light' chains). Some sections of this molecule are the same across all antibodies, while others are specific to each type of antibody and determine its particular properties. Antibodies, also known as immunoglobulins, are generally abbreviated to Ig; according to the range of different structures and functions, they are grouped into five classes, indicated by the letters A, D, E, G and M. IgM immunoglobulins are the first type of antibody produced by babies, as well as the first that are synthesized in adults upon detection of infective agents. IgGs, or gamma globulin antibodies, are predominant in the serum and are produced when the organism is exposed to a specific antigen for the second time. IgE antibodies are produced as a result of allergic reactions. IgAs are constantly present in saliva, in the digestive tract and in breast milk. The role of IgDs is unknown. Antigen recognition by an antibody occurs specifically due to fact that the structure of the antibody is complementary to that of the antigen. This enables the two molecules to bind, similarly to a key matching its corresponding lock. Upon recognizing and binding to the antigenic substances on extraneous cells, antibodies can neutralize these cells in two ways: 1) by activating the complement system, whereby plasma proteins drill the cell’s membrane; 2) by activating special blood cells that engulf and destroy the intruders with a phagocytosis process.
After having been in contact with a given antigen, the body continues to produce specific antibodies for a given number of days; after reaching a maximum value, the production decreases and finally stops.
In some cases, the body retains the antibodies for that antigen thereby remaining permanently immunized, as occurs in some cases such as chickenpox. The production of antibodies in the body can be stimulated with the inoculation of vaccines. The types of antibodies that any organism can synthesize is extremely high, as is the number of substances that behave as antigens. In fact, any substance that is introduced into the body is foreign to it and can therefore act as antigen.
In some diseases, the body loses its ability to recognize some of its own components, developing antibodies against them. These are typically defined as autoimmune diseases, and include systemic lupus erythematosus and multiple sclerosis.
Urine is the result of glomerular ultrafiltration and tubular reabsorption. In physiological conditions these processes prevent unwanted protein loss in the urine. Urinary protein detection is of fundamental importance at diagnostic level. As a fact, monoclonal free light chains testing (Bence-Jones protein, BJP) as well as the determination of the presence of intact monoclonal components, enables the identification of monoclonal gammopathies, and the prevention of problems related to the nephrotoxic action of BJPs. Plasma cells generally produce an excess of light chains compared to the respective heavy chain. When these proteins are produced in large quantities, such as in neoplastic diseases, they can harm the renal tubule leading to various degrees of renal impairment. BJPs can be detected by means of urinary electrophoresis, showing up as a well-defined band at varying locations according to the different degrees of protein polymerization, immunofixation thereby allowing its immunochemical characterization. The presence of BJP is a negative prognostic indicator of the progression of plasma cell dyscrasias and its follow-up is contained in all international guidelines.
HEAVY CHAIN DISEASES
Heavy chain diseases (HCDs) are rare lymphoplasmacytic B-cell proliferative disorders, characterized by the production of incomplete heavy chains or fragments thereof, with features resembling those of monoclonal immunoglobulin, lacking associated light chains. HCDs imply the presence of the three major classes of immunoglobulins that have been characterized as follows: the α-HCD form is the most common and presents the least amount of variations, whilst γ- and μ-HCD display considerable clinical and histopathological variability. HCDs can be thought of as a different types of non-Hodgkin lymphomas. α-HCD presents as lymphoma involving the extranodal marginal zone of the mucosa-associated lymphoid tissue; γ- HCD as non-Hodgkin lymphoplasmacytic lymphoma, and μ-HCD as small non-Hodgkin lymphocytic lymphoma or as chronic lymphocytic leukemia. HCDs diagnosis requires the detection of immunoglobulin heavy chain fragments in serum and urine in the absence of bound light chains. The prognosis may vary and there are no specific standardized treatment strategies with the exception of α-HCD, which may respond to antibiotic treatment in its early stages. Given the rarity of the disease, there have been cases in which the laboratory has helped clinicians in formulating a correct diagnosis. Immunofixation is a laboratory technique that employs specific antisera and reproducible methods in order to identify many proteins and their possible variants. The widespread use of monoclonal component characterization in serum and urine allows this method to be currently employed in most medium-large laboratories. Automation has contributed to make this electrophoretic technique simple and reproducible; nonetheless, the analyst’s protidological knowledge continues to play a key role in interpreting the various patterns and further steps to be taken in order to produce a report that may be compatible with the various associated pathologies. Heavy chain diseases are hard to identify among immunofixation patterns, thus requiring further tests in order to be validated. Methods aimed at highlighting the HCD are difficult to perform, requiring equipment that is often available only in university laboratories, or in those specialized in hematological disorders. Moreover, the presence of HCD always requires a multidisciplinary approach between the clinician and the clinical pathologist.
In fact, analysis of serum performed through electrophoretic techniques (capillary electrophoresis, gel electrophoresis) rarely yields monoclonal peaks, instead they often show up as increases in the gamma region or as slight increases in the beta1/beta2 zone.