Nasledstvennaya gemoliticheskaya anemia, due to the activity of pyruvate kinase deficiency of red blood cells

Nasledstvennaya gemoliticheskaya anemia, Related erythrocyte pyruvate kinase deficiency - a relatively rare form of hemolytic anemia, which is inherited in an autosomal recessive manner, It gives clinical manifestations in homozygotes, proceeds with signs of intracellular hemolysis, macrocytosis of erythrocytes, low content of potassium. There are more than 200 cases of homozygous carriers of deficit piruvatkinaznoy activity.

Pathogenesis

Pyruvate kinase is one of the enzymes, concluding glycolysis. This link glycolysis and ATP formed nikotinamiddinukleotid. The majority of patients with deficiency of pyruvate kinase activity is reduced in the content of the red blood cells of these two metabolites, In some cases, the content of ATP is normal, but disturbed the stability of ATP during incubation. Normal levels of ATP found at significant reticulocytosis. This is due to the presence of reticulocytes and the possibility of mitochondrial ATP not only through glycolysis, but also due to the oxidative phosphorylation.

The consequence of pyruvate kinase deficiency It is also the accumulation of the products of glycolysis, formed at previous stages - fosfofenolpiruvata, 3-phosphoglycerate, and especially 2,3-diphosphoglycerate. Content piruvatanlaktata decreases in red blood cells.

The lack of ATP in erythrocytes pyruvate kinase deficiency It leads to disruption of the function of ATP-aznogo pump red blood cells and to the loss of potassium ions, wherein the concentration of sodium ions in the erythrocytes of non-increasing. This causes a decrease in the content of monovalent ions in the erythrocytes, dehydration of the cell and its shrinkage. This phenomenon is called dessikotsitoz. The decrease in red blood cells of water complicates the process of oxygenation and oxygen release by hemoglobin. In this regard, certain compensatory role played by a significant accumulation in erythrocytes of 2,3-acid difosfoglitserinovoy.

The affinity of hemoglobin for oxygen is reduced by its interaction with 2,3-diphosphoglycerate. Hence, at higher concentrations for 2,3-diphosphoglycerate returns oxygen to the tissues is facilitated.

Clinical manifestations of the activity of pyruvate kinase deficiency

Clinical manifestations of deficiency pyruvate kinase activity depend on the degree of haemolysis. In some cases, increased hemolysis is manifested at birth and is accompanied by the occurrence of severe hemolytic crisis, causing the need for blood transfusions to maintain hemoglobin in the sub-normal levels. In other cases, the disease can occur almost asymptomatic with the first signs of increased hemolysis (subikterichnost sclera and skin) aged 17-30.

Generally; compared with microspherocytosis, pyruvate kinase deficiency has a more severe.

Almost all patients with palpable spleen. The slight increase in its disclosed some anomalies heterozygous carriers, although they have anemia, usually, missing. A number of patients possible splenomegaly. A significant increase in liver rarely found. In isolated patients with leg ulcers identified.

Laboratory indicators of activity of pyruvate kinase deficiency

The majority of patients with deficiency of pyruvate kinase activity observed moderate anemia (4,34- 6,21 mmol / l, or 70-100 g / l), However, during the crises hemoglobin may be reduced to 3,1 3,72 mmol / l (50-60 G / l). Content eritrotsitov- 2.5-3.0 T 1 l. Hemolytic crisis can be caused by infection. There are cases of hemolysis gain during menstruation.

Red blood cells of patients with pyruvate kinase deficiency characterized by macrocytosis with normohromiey, slight anisocytosis and poikilocytosis. There was a fair polychromatophilia. Sometimes found mishenevidnye red blood cells and red blood cells with scalloped edges or in the form of mulberry, often identified basophilic erythrocyte punktatsiya, Jolly corpuscles. Some patients in the peripheral blood revealed a significant number of erythrokaryocytes, during the crisis, it can be increased up to 60-70 %. ESR, white blood cell count, leukogram, platelet counts within the normal range. As with other forms of hemolytic anemia, in the bone marrow revealed a sharp irritation red germ hematopoiesis.

The level of bilirubin, usually, increased due to indirect.

The free plasma hemoglobin normal, but it may be reduced levels of haptoglobin. Hemosiderin in urine is absent. Contents of fetal hemoglobin and hemoglobin A₂ within the normal range.

Osmotic resistance of erythrocytes pyruvate kinase deficiency in most cases normal, sometimes reduced after day incubation. Autogemoliz, usually, upgraded to varying degrees. There are cases of pyruvate kinase deficiency with normal autogemolizom. In the first reported cases of pyruvate kinase deficiency has been registered type II autogemoliza, corrects the addition of ATP, and not correcting or slightly correcting the addition of glucose. Subsequently, many cases have been described with enzyme deficiency type I autogemoliza or no. Therefore, it is not recommended to apply the method autogemoliza for the differential diagnosis of hemolytic anemias. The lifespan of red blood cells with deficiency of pyruvate kinase activity is always shortened.

Type inheritance pyruvate kinase activity autosomal recessive. Heterozygotes practically healthy, According to various researchers, the severity of the deficit have ranged from 50 to 70 % norms.

Diagnostic activity of pyruvate kinase deficiency

The diagnosis of pyruvate kinase deficiency of red blood cells can be expected to deliver in the case of hemolytic anemia, flowing over the years with the increase in spleen, lack of peripheral blood smears signs microspherocytosis, elliptotsitoza and stomatotsitoza.

Qualitative tests for determining the activity of pyruvate kinase. For this purpose, the method is widely used fluorescent stain, based on, that in the course of the reaction, kataliziruemoy piruvatkinazoy, nikotinamiddinukleotid oxidized and the fluorescence disappears.

The activity of pyruvate kinase in healthy individuals ranged from 4,0 to 7,7 mmol (min / 1010 erythrocytes) or 4,0 to 7,7 Power. The majority of homozygous disease enzyme activity less 1 Power. However, there may be cases slight decline in activity of the enzyme. Diagnosis of a rare form of hemolytic anemia is very difficult, and a diagnosis can be made only in the study of enzyme kinetics.

Differential diagnosis of hemolytic anemia, associated with deficiency of pyruvate kinase

The differential diagnosis of hemolytic anemia, associated with deficiency of pyruvate kinase, necessary to carry out other forms of hereditary hemolytic anemia.

Since most patients deficient in pyruvate kinase activity unlike individuals, suffering microspherocytosis, elliptotsitozom, stomatocitozom, hemolytic anemia, conditioned carriage unstable hemoglobin, and thalassemia disease is inherited in an autosomal recessive, that this pathology is found only in the same generation and almost never seen among parents.

When hereditary anemia, conditioned carriage unstable hemoglobin, especially in severe disease, sometimes proband is the first carrier of the mutation, and then the disease is transmitted to the next generation. Therefore, the differential diagnosis must be carried out with hemoglobinopathies, conditioned carriage unstable hemoglobin, as well as recessive inherited hemolytic anemia, caused by deficient activity of other enzymes.

It plays a supporting role hemoglobin stability, yego electrophoresis. To exclude other fermentopathia investigated the activity of other enzymes of erythrocytes, and to exclude autoimmune hemolytic anemia carried Coombs, agregatgemagglyutinatsionnaya.

Fluorescent method for determining the activity by piruvatkiiazy Beutler

Reagents.

1. Fosfoenolpirovinogradnaya acid (tritsiklogeksilammoniynaya salt) 0,15 M solution of, neutralized to pH 7.0-8.0 0,2 M solution of NaOH (0,03 ml).
2. ADF 0,03 M solution of, neutralized (0,1 ml).
3. NAD-N 0,015 M solution of, neutralized (0,1 ml).
4. Magnesium sulfate 0,08 M solution of (0,1 ml).
5. Phosphate buffer solution of potassium 0,25 M, pH 11 7,4 (0,05 ml).
6. Distillirovannaya water (0,62 ml). The mixture can be stored at - 20 ° C is not more than a week.

Method.

For research use blood, taken with heparin or sodium citrate. It is centrifuged, aspirated and prepare plasma 20 % suspension of red blood cells by adding four volumes of isotonic sodium chloride solution.

Erythrocyte suspension in an amount 0,02 ml is mixed with 0,2 ml of reagent and incubated for 30 minutes at 37 ° C. A droplet was placed on the filter paper, and browsing the fluoroscope. Baseline fluorescence was detected immediately after the addition of erythrocytes to the mixture before incubation. Normally after 30 min of incubation it disappears, and the activity of pyruvate kinase deficiency remains. This is, NADH which fluoresces under the influence of UV radiation in the area, close to visible, whereas NAD does not fluoresce. Because the solution above is unstable and easily degraded, requires monitoring. You should carefully separate the layer of white blood cells, since the activity of pyruvate kinase deficiency erythrocyte enzyme activity in leukocytes can be normal.