Paroxysmal nocturnal hemoglobinuria – Disease Markiafavy-Micheli

Paroxysmal nocturnal hemoglobinuria - Rather a rare acquired form of hemolytic anemia, related to the change in the structure of red blood cells, neutrophilic granulocytes and platelets. Proceeds from the symptoms of intravascular hemolysis, in which there is hemoglobinuria, gemosiderinuriya, raising the level of plasma free hemoglobin. The disease is often complicated by thrombosis of peripheral veins and blood vessels of the internal organs.

The etiology and pathogenesis of paroxysmal nocturnal hemoglobinuria

The first description of the disease was given in 1882 g. In more detail, it has been described in 1928 g.

Common name of the disease - paroxysmal nocturnal hemoglobinuria (PNG), but it is small corresponds to the essence of the disease, since there is no real paroxysms and not always observed hemoglobinuria.

Intravascular hemolysis with gemosiderinuriey except APG observed in many forms of autoimmune hemolytic anemia with both thermal, and with cold antibodies, especially in forms with thermal hemolysin, as well as in some forms of hereditary hemolytic anemia, related to the disruption of the structure of erythrocyte membranes.

Disease Marchiafava Michele observed at different ages. The reason for the increased destruction of red blood cells is defective red blood cells themselves.

Red blood cells of patients with APG, as indicated above, It is easily destroyed by complement in creating optimal conditions for his actions, or by acidification of the serum, or by increasing the concentration of the erythrocyte complement around by creating a low concentration of ions in the sucrose medium. Red blood cells of patients with APG have increased sensitivity to the effects of antibodies as the agglutinin, and, especially, gemolizinov.

When APG affects not only the red blood cells, but also leukocytes and thrombocytes. Platelets and white blood cells of patients with APG have increased sensitivity to the effects of complement as in the acidification whey, and in a medium with a low ionic strength sucrose. They are many times more sensitive to the action of idioagglutinin, than donor platelets and leukocytes. Thus, platelets and white blood cells have the same defect membrane, as erythrocytes.

Discover immunoglobulins on the surface of red blood cells, leukocytes and platelets, and thus to prove ownership of the APG to the group can not be auto-aggressive diseases most sensitive methods.

There is strong evidence of APG at two separate populations of red blood cells. The most persistent in healthy human cells - reticulocytes are the most fragile in the APG. This indicates that, abnormal erythrocyte population that simply do not survive to maturity, while older mature red blood cells are healthy or almost healthy population.

IN 1970 the study of erythrocyte mulattos, stradaûŝej PNG, found, they contain, as in healthy mulatto, two normal electrophoretic variants of G-6-PD; Option A, widespread among the people of Africa, and option B, peculiar to the population of Europe. After the hemolysis appeared, pathological erythrocytes contain only one embodiment of G-6-PD, and normal erythrocytes - Both electrophoretic variants of G-6-PD. Hence, hemolyzed red blood cells are descended from a single cell.

This fact with high confidence indicates the presence of two populations of red blood cells, differing not only functional, as, eg, when microspherocytosis, but origin: in other words, it confirms the presence of abnormal cell clone, t. it is. somatic mutation. The identity of the destruction of the membrane of red blood cells, neutrophils and platelets suggests, that pathological information likely received cell, following the stem, t. it is. precursor cell myelopoiesis. Neoplastic growth is not typical for PNG. We describe only a few cases of leukemia in the background of the rapid APG. Nonetheless, We can assume, that APG - a variant of a benign tumor of the blood system, long time not giving tumor proliferation, characteristic for other forms of leukemia.

The mechanism of komplementchuvstvitelnosti at APG is unclear.

Substantial changes in the activity of enzymes of glycolysis and pentose-phosphate cycle are absent.

When APG reduced acetylcholinesterase activity of erythrocyte, most likely due to the disruption of the structure of the membrane of red blood cells, since the enzyme is on the surface of the membrane and is easily broken during its defeat. Known, that acetylcholinesterase inhibitors had no effect on survival of erythrocytes. However, the reduction of acetylcholinesterase activity observed with other forms of hemolytic anemia, so can not explain the phenomenon of increased komplementchuvstvi- tions the.

There is evidence, reflecting an increase in the number of unsaturated fatty acids, members of the phospholipids, erythrocyte membrane. It is possible, that changes in lipids - it is only the reflection of any other changes in the erythrocyte membrane structure. In the study patients erythrocyte membrane protein APG by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate found, that a number of protein fractions significantly different from control.

The main role in the pathogenesis of thrombotic complications of intravascular APG assigned erythrocytolysis and stimulate clotting factors, is released from the red blood cells during their decay. When APG destroyed mainly reticulocytes, may be, and this frequency should be explained thrombotic complications of this disease, in contrast to other forms of hemolytic anemia, accompanied by severe intravascular hemolysis.

Clinical manifestations of paroxysmal nocturnal hemoglobinuria

The disease often begins gradually. Patients complain of weakness, malaise, dizziness. Sometimes there subikterichnost sclera. Often, the first complaints are headache, abdominal pain of various localization. The tendency to increased thrombosis causes the patient to see a doctor. Hemoglobinuria rarely the first symptom of the disease, and some patients may be absent at APG. In some cases, it appears the first 2-3 years, and even after 10 years after onset.

One of the characteristic features of the APG - bouts of abdominal pain. Locating it may be very different. Outside the period of crisis abdominal pain, usually, not observed. Often it joins vomiting. Most likely, abdominal pain in patients with APG associated with thrombosis of mesenteric vessels.

Peripheral vascular thrombosis (most of all - the veins of the upper and lower extremities, rarely - renal vessels) as a characteristic symptom of paroxysmal nocturnal hemoglobinuria. In 12 % APG patients observed thrombophlebitis. Thrombotic complications are the most frequent cause of death in this disease.

An objective study of the patient often attracts attention pallor slightly jaundiced. Often there is puffiness of face, sometimes excessive fullness. A slight increase in spleen and liver, although it is not typical for PNG.

For paroxysmal nocturnal hemoglobinuria characteristic symptoms of intravascular hemolysis, the most important of which - an increase of plasma free hemoglobin. This feature periodically observed in almost all patients APG. However, the amount of increase in free hemoglobin plasma varies and depends on the, in any period of the disease surveyed. In the period of crisis, this figure increases significantly, There has also been an increase in the number of plasma metalbumina.

The level of plasma free hemoglobin is dependent on the degree of haemolysis in reality, the content of haptoglobin, filtration degree of hemoglobin in the urine, and the rate of destruction of complex hemoglobin - haptoglobin. In the case of a small degree of hemolysis of the plasma level of free hemoglobin would be insufficient for its filtration through the kidney filter. Thus hemoglobinuria is not obligatory symptom. When passing through the tubules of nephrons allocated hemoglobin partially destroyed and deposited in tubular epithelium. It is the cause of urinary excretion of hemosiderin.

Hemosiderin excreted in the urine in the vast majority of patients with paroxysmal nocturnal hemoglobinuria. Sometimes it does not appear immediately gemosiderinuriya. This is an important, but not specific to the APG signs of disease.

Laboratory findings in paroxysmal nocturnal hemoglobinuria

The content of hemoglobin in patients APG ranges from 1,86- 3,1 mmol / l (30-50 G / l) and even lower to normal in remission.

The number of red blood cells in patients with APG decreases according to the level of hemoglobin decrease. The color indicator of a long period remains close to unity. Where, if the patient loses a significant amount of iron in the urine as hemosiderin and hemoglobin, the iron level is gradually reduced. Low color index observed in about half of patients. Some of them have elevated levels of hemoglobin P, especially in the period of exacerbation.

A significant proportion of patients high content of reticulocytes, but comparatively low (2- 4 %). The number of leukocytes in the APG in most cases reduced. In many patients, it is 1.5-3 F in 1 l, but sometimes reduced to 0.7-0.8 F in 1 l. Leukopenia, usually, there by reducing the number of neutrophilic granulocytes. Sometimes white blood cell count at normal or high APG - up to 10-11 T 1 l.

When APG reduced phagocytic activity of neutrophils.

Thrombocytopenia also often observed at APG, however, the functional state of platelets with the normal. Probably, This explains the rarity of hemorrhagic complications in this disease, although the number of platelets is sometimes lowered to a considerable extent - 10-20 F in 1 l. Typically most patients platelets in T 50-100 1 l. Normal platelet count does not exclude the diagnosis of paroxysmal nocturnal hemoglobinuria.

Bone marrow examination asymptomatic, generally characteristic of hemolytic anemia - a boring red germ hematopoiesis in normal amounts myelokaryocytes. A number of patients there is a slight decrease in the number of megakaryocytes.

The level of serum iron at APG It depends on the stage of the disease, intravascular hemolysis and degree of activity of hematopoiesis. Where, If patients have persistent or frequent hemoglobinuria, as well as the constant gemosiderinuriya, the content of iron in the body, and hence, and decreases in serum, sometimes very significantly. Sometimes APG begins with signs of hypoplasia. Stocks of iron in the body of the patient depends on the APG, one side, of iron loss in the urine, and on the other the intensity of erythropoiesis. Therefore, it should not be considered diagnostic of iron deficiency paroxysmal nocturnal hemoglobinuria.

The level of serum bilirubin at APG in most cases, increased or normal mild, mainly due to indirect. On average, bilirubin is 22-28 mmol / l. It should be taken into account, that the decay of intravascular hemoglobin formed hemoglobin-gaptoglobinovy ​​complex, and which also eventually decomposes with the formation of bilirubin.

In some patients the disease begins with PNG picture aplastic anemia.

Diagnosis of paroxysmal nocturnal hemoglobinuria

When paroxysmal nocturnal hemoglobinuria disrupted the structure of erythrocyte membranes, which is manifested in increased sensitivity to the effects of its complement. This increased sensitivity is detected using a probe Hema, sucrose or more sensitive tests. However, in some cases, especially in the form of hemolysin autoimmune hemolytic anemia, sucrose sample may give a false positive result. Hemolysin are fixed on the surface of their own red blood cells and in the presence of complement in the sucrose medium destroy them, whereas typical APG complement destruction of cells without exposure to antibody.

To eliminate mold hemolysin autoimmune hemolytic anemia hemolysins study recommended by the above method and using cross-sucrose with serum samples of the patient and the donor red blood cells odnogruppnoy, and not vice versa, as is customary. When APG direct test is positive, and cross - negative. When hemolysin form AIHA can be observed as a positive, and a negative cross-sample. In these cases it is expedient to use a third embodiment of the sucrose tests - with using both serum, and red blood cells of the patient. The degree of hemolysis in this embodiment, the minimum and maximum at APG in the form of AIHA hemolysin. To explain such a result can only be highly specific serum antibodies patient hemolysin form of autoimmune hemolytic anemia for the antigen, abounding in erythrocytes of a patient, than donor red blood cells. Direct sucrose sample is positive in these patients is associated, probably, with the presence of antibodies fixed on erythrocytes.

Identify hemolysins by agregatgemagglyutinatsii and others help make the correct diagnosis. It can not be used as a donor serum of patients, as in the case of the contents therein hemolysin result may be false positive. A negative result can occur, if used as a source of complement serum of the patient APG or serum of patients with cirrhosis, rheumatoid arthritis, systemic lupus erythematosus and other diseases, in which the reduced level of complement. Sucrose sample and the sample Hema become less pronounced, If the capture of the patient's blood is used as an anticoagulant, heparin, instead of sodium citrate. After the intensity of the sample hemolytic crisis Hema and sucrose reduced sample, but they do not become negative.

Determination of plasma free hemoglobin by Bing modification Derviz Byalko and in paroxysmal nocturnal hemoglobinuria

The method is based on the ability of hemoglobin to react with benzidine in an acidic medium in the presence of hydrogen peroxide to form a green color, passing first into the blue, and then reddish purple. The color intensity is proportional to the amount of hemoglobin.

Reagents.

1. Acetate buffer 0,1 M, pH 4,6: 27,22 g of sodium acetate dissolved in 1 l of water (0,2 M solution of); in another flask is prepared 1 l 0,2 M solution of acetic acid; mixed 480 ml number 1 and 520 ml number 2.
2. Hydrogen peroxide 0,3 % solution (prepared before use by diluting in perhydrol 100 time).
3. Benzidine hydrochloride 0,1 % solution: 50 mg hydrochloric benzidine dissolved in 35-40 ml of acetate buffer, the solution is heated to 80 ° C water bath and after cooling is adjusted to acetate buffer 50 ml.

Filter the dark bottle and stored at room temperature for no longer 7 days. Pozheltevshiy reagent unsuited, with rapid darkening it should be recrystallized from alcohol benzidine.

Method.

Blood samples taken from a vein in the dry needle siliconized tube, containing any anticoagulant. The blood is centrifuged anticoagulated no more 10 minutes at 1500 / min. After separating the plasma it again centrifuged 10 minutes at 8000 rev / min for deposition of leukocytes.

The pour tube 4 ml acetate buffer, 2 ml hydrogen peroxide, 2 ml and benzidine 0,04 ml of test serum was mixed and allowed to stand 3 m, then poured into a cuvette pathlength 1 cm and photometric against the compensation of the solution at a green light filter. The intensity of the blue color increases during 4-5 min, so the measurement is repeated 3-5 times in a row and record the maximum reading. After 5-6 minutes, serum coloration becomes purplish-brown hue, and the optical density starts to decrease. Of the two identified the best results takes an average optical density.

Compensation solution, consisting of 6 ml acetate buffer, 8 ml hydrogen peroxide, 3 ml benzidine and 0,06 ml of isotonic sodium chloride solution, prepared simultaneously with the sample.

A calibration curve for the output of hemoglobin solution. In blood, taken to prepare a solution of hemoglobin, measured by its content (colorimeter for medical or medical refractometer). A solution of hemoglobin with a concentration of 1 g / l, A solution of a lower concentration: 0,005,0,025, 0,05, 0,1 and 0,2 g / l. From each of the resulting solution is added 0,04 ml of a mixture of 4 ml acetate buffer, 2 ml benzidine and 2 ml hydrogen peroxide.

The basic solution hemolysate (1 g / l) for the calibration curve must be stored in the cold, and working solutions of lower concentration should be prepared immediately before the test.

Normally, in the 1 l blood plasma contains 0,0005 mmol / l (0,01-0.04 G) Hemoglobin.

Determination of urine hemosiderin in paroxysmal nocturnal hemoglobinuria

In the decay of hemoglobin iron it adsorbs the epithelium of the renal tubules and is part of hemosiderin. Exfoliated epithelial cells of the renal tubules in these cases contain a grain of hemosiderin.

A careful study of urine sediment after centrifugation of grain hemosiderin seen without special stains. However, they stand out more clearly in the color of Perls method.

Method.

After centrifugation of urine its upper layer was aspirated, to the residue was added a freshly prepared solution of 1 % ferrocyanide and 1 % hydrochloric acid (mix equal volumes 2 % hydrochloric acid and ferrocyanide). After 10 minute after stirring, the mixture was centrifuged and the precipitate browsing. Grains hemosiderin stained blue.

Determination methemalbumin plasma spectrophotometry

Reagents.

1. Phosphate buffer Isosmotic (pH 7,4): mixed 18 ml 0.15 M solution of sodium dihydrogen phosphate (24,4 Mr. YaN2R04 on 1 l of water) and 82 ml 0,15 M solution of disodium hydrogenphosphate (21,3 г NaH₂AFTER₄ on 1 l of water).
2. Sodium hydrosulfite.
3. Sodium hydroxide 1 M.
4. A solution of human serum albumin (40 g / l).
5. The solution of Hemin (5 mg / 100ml); is prepared immediately by dissolving in a small amount of hemin sodium hydroxide, and then: bringing it to volume with a solution of serum albumin.

Method.

K 2 ml of serum or plasma added 1 ml solution zabuferernogo isoosmotic, pH 7,4. The mixture was centrifuged 30 at 1200-1500 m / min. Determine the optical density at 569 nm. Then, the diluted plasma was placed in a vial and added to approximately 5 mg of dry sodium hydrogensulfate. Through 5 min re-measure the absorbance at the same wavelength. The difference between the two values ​​of optical density corresponds to the optical density methemalbumin. Methemalbumin concentration is determined by the calibration graph.

The calibration curve is derived as follows. Hemin solution with albumin was transferred to a test tube in an amount of 0,01 to 0,1 g / l (from 0,2 to 2 ml). In those tubes, where the amount of the solution less 2 ml, added to 2 ml albumin solution. Then added 1 ml of phosphate buffer isoosmotic. The mixture was centrifuged at 1500 / min, and then optical density is measured. The difference between measurements is plotted according to the number of Hemin.

Normally gemalbumin plasma offline. He appears in the case of intravascular hemolysis, eg, in paroxysmal nocturnal hemoglobinuria, or form hemolysin autoimmune hemolytic anemia, and in the absence of haptoglobin, when heme is bound to albumin.

Probe Hema in paroxysmal night gemoglobinurii

Hema sample based on erythrocytes of patients with lowered resistance APG decrease pH of the medium in the presence of fresh serum complement.

Reagents.

1. A solution of hydrochloric acid 0,2 n.
2. Ammonia solution 0,04 %.

Method.

From Vienna take 5- 6 ml of blood. As an anticoagulant used oxalate or sodium citrate. Simultaneously, 15-20 ml of blood was taken from a donor of the same group. Separate the plasma from the red blood cells in the blood of the patient and the donor.

Serum donor poured 0,5 Six ml tubes, two of them were placed on 20 minutes in a water bath at 56 ° C to inactivate serum complement, four remaining tubes left at room temperature. Red blood cells of the patient are washed twice with isotonic sodium chloride solution, then suspended in the same solution at a ratio of 1:1.

The study design is presented in Table.

The contents of each test tube mix, put on your 1 h in the thermostat at a temperature 37 ° C, and then centrifuged.

For idle sampled 0,05 m red blood cells of the patient and the donor is added separately to the 0,55 ml of isotonic sodium chloride solution.

After centrifugation the degree of hemolysis can be estimated by eye. To quantify the extent of hemolysis in each tube to 4,7 ml 0,04 % Add ammonia solution 0,3 ml surface layer, but in the test tube with idle breakout — 0,3 ml of the reference serum.

To identify 100 % hemolysis to 4,7 ml ammonia solution add 0,3 mL suspension of erythrocytes (0,05 ml erythrocytes patient in 0,55 ml of isotonic sodium chloride solution).

Optical density of the solution is measured at the medical kolorimetre and compare the readings with optical density ammonia solution without red blood cells:

% hemolysis =((IS₁-IS₂)/IS₃)*100;

where E₁ -optical density of analyzing probe; IS₂-optical density blank sample; IS₃-optical density 100 % gemolizata.

Normally, not more than gemoliziruetsja 5 % erythrocyte.

The sample is considered positive the Hema in case, If gemoliziruetsja more 6 % erythrocyte.

In paroxizmalna night gemoglobinurii and gemolizinovoj form of autoimmune hemolytic anemia gemoliziruetsja sometimes up to 50-80 % erythrocyte. For these diseases is characterized by hemolysis only in the presence of serum neinaktivirovannoj (-No. 2). If inactivation of serum does not prevent hemolysis (-No. 3), We can assume the existence of other forms of hemolytic anemia or a mistake in setting research.

For the destruction of the red blood cells of patients with paroxysmal nocturnal hemoglobinuria Wednesday at pH 6,5 requires mandatory presence of complement, contained in the fresh serum.

Saharoznaja tryout with paroxysmal nocturnal gemoglobinurii

Saharoznaja sample is based on the, that red blood cells of patients with paroxysmal nocturnal hemoglobinuria and gemolizinovoj form of autoimmune hemolytic anemia are destroyed in solutions of low ionic strength in the presence of complement.

Reagents.

1. Solution of sucrose: 9,42 sucrose dissolved in 0,005 M fosfatnom buffer; pH 6,2 (91 ml 0,005 M NaH₂AFTER₄ and 9 ml 0,005 M Na₂HPO₄).
2. Reagent for hemoglobin concentration determination: 200 mg red blood salt dissolved in 1 l distilled water, There add 0,5 ml atsetontsiangidrina.

Method.

Blood in a patient taking from Vienna to two tubes is dry (8-10 ml) and with anticoagulant (2- 3 ml). As anticoagulant use sodium citrate. Erythrocytes of blood collection tubes with anticoagulant twice launder izotoniceski solution of sodium chloride. After laundering and supernatant extraction to 0,4 ml erythrocytes add 0,25 ml of isotonic sodium chloride solution.

At the same time taking blood from a donor of the same group of blood in two tubes — with anticoagulant (2-3 ml), It is treated in the same way, as the blood of the patient, and dry (8- 10 ml) to obtain serum. After the separation of the donor's serum 0,5 ml it is placed in a water bath at a temperature 56 ° C 30 min for inactivation of complement. The donor's serum should take daily experience and keep in the cold. Scheme of study is given in table.

Tubes are placed in the thermostat on 30 minutes at 37 ° C, and then centrifuge for 10 min speeds 2000 rpm and assess the degree of hemolysis.

To do this, in the test tube is poured on 3,4 ml reagent for hemoglobin concentration determination and 0,6 ml on- sediments from each test tubes. Through 10 min measure the optical density of the solution in tubes with 540 NM on health refraktometre (spectrophotometer) or medical kolorimetre (DEC fotokolorimetre-m) When a green visor, by comparing the data obtained with optical density solution for hemoglobin determination. Takes the average of the two readings each experience. The calculation is made according to the formula:

% hemolysis in vitro =((IS₁-IS₅)/(IS₈-IS₅))*100

where E₁ -optical density of the solution in a test tube no. 1; IS₅ -optical density of the solution in a test tube no. 5, necessary to exclude the coloring serum; IS₈ -optical density of the solution in a test tube no. 8, where 100% hemolysis.

For cross-saharoznoj samples (-No. 2) 100%-th hemolysis in vitro is investigated No. 7.

Evaluation of results.

Normal in-vitro No. 1, contains red cells probed the patient, hemolysis does not exceed 2-3 %.

In paroxizmalna night gemoglobinurii and gemolizinovoj form of autoimmune hemolytic anemia (AIGA) in this vitro hemolysis exceeds 6 % In test tubes-No. 4, containing the blood of the patient and the donor's serum inaktivirovannuju, test tube # 5, containing erythrocytes and serum donor, and number 6, containing serum instead of isotonic sodium chloride solution — hemolysis should not exceed 1-2 %.

Cross-saharoznaja trial (-No. 2) When gemolizinovoj the form of AIGA usually positive (in the presence of hemolysins in the serum), but it can be negative. In vitro No. 3 (red blood cells and patient's serum) hemolysis as observed at APG, and in gemolizinovoj the form of AIGA. In paroxizmalna night gemoglobinurii maximum hemolysis is detected in normal saharoznoj sample (-No. 1), less pronounced — with its own sample cell patient (-No. 3); No hemolysis in cross-saharoznoj sample (-No. 2).

When gemolizinovoj the form of AIGA the most pronounced hemolysis is detected in a sample with your own blood serum and erythrocytes of sick (-No. 3), somewhat less pronounced — in vitro No. 1. In vitro No. 2 haemolysis is observed frequently, but the lack of it does not exclude form gemolizinovuju AIGA.