Additional methods of blood testing: determination of reticulocyte hematocrit. How to count reticulocytes. Causes of low reticulocyte count, treatment

… Determination of the reticulocyte count is an important indicator of bone marrow function.

Reticulocytes- these are young forms of erythrocytes formed from normoblasts after they have lost their nucleus (nucleated, immature erythrocytes), the maturation of which occurs within 24-48 hours after release into the peripheral blood from the bone marrow, and reflecting the regenerative ability of the bone marrow (i.e. erythropoietic bone marrow activity).

MORPHOLOGY AND CLINICAL SIGNIFICANCE OF RETICULOCYTES

Reticulocytes are usually larger than mature red blood cells. The cytoplasm of reticulocytes contains a basophilic network (reticulum) in the form of small grains, individual filaments, glomeruli, etc., which are aggregated ribosomes and mitochondria.

According to the degree of maturity, there are 5 types of reticulocytes:
(1) reticulocytes that have a nucleus (erythronormoblasts), and their granularity is located in the form of a dense rim around the nucleus;
(2) reticulocytes having a granular-mesh substance in the form of a ball or lump;
(3) reticulocytes, granular in the form of a dense network;
(4) reticulocytes, having a granular-mesh substance in the form of individual threads;
(5) reticulocytes containing individual granules.

Remember: normally, according to G. A. Alekseev, almost 80% of reticulocytes belong to groups IV – V.

Normally, peripheral blood contains 0.2 - 1% reticulocytes (“%” is the content of reticulocytes from the total number of red blood cells). This indicator reflects the possibility of reticulocytes entering the peripheral blood and their further transformation into mature erythrocytes (maturation), as a variant of the norm, already in the peripheral blood (within several hours). ( ! ) In normal erythropoiesis, most red blood cells pass through the reticulocyte stage in the bone marrow.

An increase in their reticulocytes in the peripheral blood (reticulocytosis) is noted:
with hemolytic anemia (the number of reticulocytes can reach 60% or more (increasing especially strongly during hemolytic crises);
in case of acute blood loss (a reticulocyte crisis occurs 3–5 days after blood loss), including an increase in reticulocytes, which allows one to suspect hidden bleeding (for example, in patients with peptic ulcer of the gastrointestinal tract, typhoid fever);
for malaria;
with polycythemia;
in the treatment of iron deficiency anemia with iron (several days (3 - 10) after the start of antianemic treatment of pernicious anemia);
with acute lack of oxygen;
with tumor metastases to the bone marrow.

It should be remembered that in the case of increased destruction of erythrocytes, the proportion of reticulocytes may exceed 50% due to an artificial increase in the number of reticulocytes (as noted earlier, the proportion of reticulocytes is calculated as a percentage of all erythrocytes). In such cases, to assess the severity of anemia, the “reticular index” is used, which is calculated by the formula: (% reticulocytes x hematocrit) / 45 x 1.85, where 45 is a normal hematocrit, 1.85 is the number of days required for new reticulocytes to enter the blood. If index< 2 – говорит о гипопролиферативном компоненте анемии, если >2-3, then there is an increase in the formation of red blood cells.

True reticulocytosis: an increase in the number of reticulocytes in the peripheral blood with a simultaneous increase in the number of reticulocytes in the bone marrow.

False reticulocytosis: absence of an increased number of reticulocytes in the bone marrow with an increase in their number in the peripheral blood (which indicates increased leaching of reticulocytes from the bone marrow into the peripheral blood).

A factor leading to an increase in the % value of reticulocytes may be the use of the following drugs: corticotropin, antimalarial drugs, antipyretic drugs, furazolidone (in young children), levodopa.

Remember: reticulocytosis, without a corresponding erythronormoblastic reaction of the bone marrow, is observed when certain areas of it are irritated by cancer metastases or inflammatory foci.

A decrease in the number or absence of reticulocytes (reticulocytopenia) is observed:
for aregenerative aplastic and hypoplastic anemia;
for anemia caused by insufficiency of iron, vitamin B12, folic acid (microcytic-hypochromic and megaloblastic anemia);
with thalassemia;
with sideroblastic anemia;
with cancer metastases to the bone;
for radiation sickness and radiation therapy;
when treated with cytostatics;
for autoimmune diseases of the hematopoietic system;
for kidney diseases;
with alcoholism;
with relapse of Addison-Biermer anemia;
with myxedema.

Factors distorting the result of laboratory testing of the % content of reticulocytes:
incorrect choice of anticoagulant or insufficient mixing of blood with the anticoagulant;
prolonged squeezing of the hand with a tourniquet;
taking sulfonamides (both underestimated and overestimated results are possible);
blood transfusion shortly before the study;
hemolysis of a blood sample.

Indications for prescribing a reticulocyte test:
assessment of erythropoiesis activity in conditions accompanied by hemolysis or blood loss;
assessing the ability of bone marrow to regenerate after cytotoxic therapy and bone marrow transplantation;
the value of restoring erythropoietin synthesis after kidney transplantation;
doping control for athletes (taking erythropoietin);
diagnosis of ineffective hematopoiesis or decreased production of red blood cells;
differential diagnosis of anemia;
detection of impaired regenerative capacity of the bone marrow due to deficiency of iron, vitamins B12, B6, folate, copper and monitoring of appropriate therapy;
assessment of response to therapy with erythropoietin and erythrosuppressors.

METHODS FOR COUNTING THE NUMBER OF RETICULOCYTES

(1) Counting the number of reticulocytes in a smear after staining them with special dyes.

This method is in practice the most used method due to the fact that it is simple, fairly cheap and does not require special expensive equipment, and accordingly can be used in any clinical diagnostic laboratory.

The principle of the method is based on identifying the granular-reticular substance of reticulocytes during supravital staining with alkaline paints (saturated solution of brilliant cresyl blue in absolute alcohol / Azur I solution / Azur II solution) with further counting them in a blood smear. Reticulocyte staining is carried out either on glass or in a test tube.

Counting is carried out using a microscope: smears prepared using one of the above methods are microscopically examined with an immersion lens; in the smear, reticulocytes and erythrocytes are colored yellowish-greenish, the granular-filamentous substance in reticulocytes is blue (when stained with azure II and brilliant cresyl blue) or bluish-violet (when stained with azure I).

(2) Reticulocyte count using fluorescence microscopy.

This method is simple and requires little time, it is more accurate than the conventional method, since fluorescent microscopy reveals the smallest grains of a mesh-filamentous substance, but it is only possible with a fluorescent microscope and special dyes, and therefore is available only to a few laboratories .

The principle of counting the number of reticulocytes using fluorescent microscopy is based on the ability of the reticulocyte substance to fluoresce after treating the blood with acridine orange. Blood is mixed with acridine orange in a test tube or mixer in a ratio of 1 part blood and 10 parts paint (the mixture can be stored for no more than 5 hours). The mixture is stirred for 2 minutes, a drop of the mixture is applied to a glass slide and covered with a coverslip. In this case, the liquid should not go beyond the coverslip.

Microscopy using a ZhS-17 light filter. In the preparation, red blood cells have dark green outlines and do not fluoresce, and in reticulocytes, the granular-mesh substance glows bright red, making reticulocytes easy to count. In blood stabilized with heparin or sodium citrate, reticulocyte fluorescence is not observed.

(3) Automatic reticulocyte count using a hematology analyzer.

In modern hematological analyzers, the technology for counting blood cells is based on the conductometric method proposed by H. Wallace and Joseph R. Culter in 1947. The principle of the method is to count the number and determine the nature of impulses that occur when a cell passes through a small-diameter hole (aperture), on both sides of which there are two electrodes isolated from each other. Each passage of a cell through the aperture is accompanied by the appearance of an electrical impulse, which is recorded by an electronic sensor. The division of cells into categories (erythrocytes, leukocytes, platelets, sediment) is carried out by the device based on an analysis of the amplitude of the received pulses. To determine the concentration of cells, it is enough to pass a certain volume of sample through the channel and count the number of pulses that are generated.

It should be noted that in addition to the classical parameter of reticulocytes - the relative (%) content of reticulocytes (RET%, percent of reticulocytes), determined using methods 1 and 2 of laboratory diagnostics, in connection with the advent of high-tech hematological analyzers (method 3) it has become possible obtain (for example, using a patented fluorescent dye for the Sysmex-XT-2000i analyzer) additional informative reticulocyte parameters:
reticulocytes with low RNA content, the most mature (LFR%, low fluorescence reticulocyte fractions, fraction of reticulocytes with low fluorescence);
reticulocytes with average RNA content(MFR%, medium fluorescence reticulocyte fractions) - fraction of reticulocytes with average fluorescence);
reticulocytes with high RNA content(HFR%, high fluorescence reticulocyte fractions) - fraction of reticulocytes with high fluorescence);
immature reticulocyte fraction(IRF%, Immature Reticulocyte Fraction).

The differentiation of reticulocytes, based on the degree of maturity and, accordingly, the content of nucleic acids, is a reflection of the hematopoietic activity of the bone marrow.

Methodology (Sysmex -XT- 2000i analyzer). In a flow cell, cells cross a semiconductor laser beam, causing high- and low-angle scattering of the beam and excitation of a fluorescent dye. This makes it possible to determine the different stages of reticulocyte maturity based on the RNA content in the cells and the intensity of their luminescence. Automated reticulocyte counting is highly accurate (more than 30,000 red blood cells are counted) and reproducible (coefficient of variation is about 6%). This technology ensures accurate counting of reticulocytes even at extremely low concentrations.

To conduct these studies, the following workplace equipment is required:

1. Slides.
2. Ground glass slides.
3. Wet chamber.
4. Mixers for white and red blood cells.
5. Glass with a hole.
6. Microscope.
7. Diamond cresyl blue.
8. Absolute ethyl alcohol.
9. Methyl alcohol or Nikiforov’s mixture (equal parts of 96° ethyl alcohol and ether).
10. Gayem's reagent or 3% sodium chloride solution.
11. Immersion oil.
12. A set of tools for pricking a finger.

Depending on the structure of the granular-reticular substance, five types of reticulocytes are distinguished: dust-like, corolla-shaped, incompletely reticulated, fully reticulated and ball-shaped (see figure).

Taking blood for the purpose of counting reticulocytes in it is carried out as directed by the doctor. To count the number of reticulocytes, they are stained. For this purpose use:

1. Method of intravital (supravital) staining of reticulocytes.
2. Method of simultaneous supravital staining of platelets and reticulocytes according to Alekseev.

Let's consider these methods in more detail.

Intravital (supravital) staining of reticulocytes

This method requires the following reagents:

1. Absolute ethyl alcohol: place calcined anhydrous (white) copper sulfate into a dry container with a ground-in stopper and add 1/4 of the absolute alcohol. The contents are shaken. Absolute alcohol (alcohol that does not contain water) can be obtained by dehydrating the alcohol with previously calcined copper sulfate. Dehydration is stopped when copper sulfate stops turning blue.
2. A saturated solution of diamondcresyl blue in absolute alcohol: add 1 g of diamondcresyl blue to 80 ml of absolute alcohol.
3. Azur I.

Technique for intravital staining and reticulocyte counting

The slides are pre-coated with a saturated solution of diamond-cresyl blue. A freshly released drop of blood is spread onto a glass slide with paint, as when preparing a blood smear. The specimen is immediately placed in a moist chamber for 6-8 minutes, then air-dried and examined microscopically (7X eyepiece, 90X objective with condenser raised). Reticulocytes are counted in the same way as with a field of view limiter. Reticulocytes, stained with brilliant cresyl blue, are pale green with a blue or dark blue mesh (see figure).

Instead of diamond cresyl blue, for staining reticulocytes, you can use, according to Magina’s recipe, azure I paint (1 g azure I per 40 ml of absolute alcohol; kept at room temperature for 2 days).

Simultaneous supravital staining of platelets and reticulocytes according to Alekseev

For this method, Alekseev’s paint is used. 1 g of Azur II paint is poured into a 100 ml flask and a reagent of the following composition prepared in a separate flask is added: 5 g of sodium citrate, 4 g of sodium chloride and 45 ml of distilled water. The contents of the flask are slowly heated on an asbestos grid with constant stirring, without bringing to a boil. Cool and filter. The filtrate is used as a working paint solution.

Technique for simultaneous staining and counting of platelets and reticulocytes

Alekseev’s paint is added to the leukocyte mixer up to the 1.0 mark, the tip is wiped of paint and placed on the table. A deeper than usual injection is made into the finger. Blood is drawn up to 4/5 of the volume of the expanded part of the mixer, making sure that no air bubbles get into it. The blood is quickly blown out of the capillary part of the mixer onto a cotton swab, and the rest of the contents into the well of the slide. From the hole, the liquid is again drawn into the mixer. By repeating this manipulation two or three times, the blood is mixed with the reagent. Having collected the liquid into the mixer for the last time, it is left in a horizontal position for 15-30 minutes to stain reticulocytes and platelets. Then shake the mixer for a minute and release 1-2 drops. Thin smears are prepared from subsequent drops. The preparations are dried in air, fixed with methyl alcohol and stained with Romanovsky paint at a dilution of 1-2 drops per 1 ml of water for 35-45 minutes. The paint is washed off with water, and the smears are dried and microscopically examined. In a smear stained according to Alekseev, the reticulocytes are pink with a blue mesh (see figure), and the platelets are bluish-lilac.

Reticulocytes and platelets are counted simultaneously per 1000 red blood cells in the same way as described in the topic “Platelets”. Results for reticulocytes are expressed in promille; platelets are calculated per 1 mm3 of blood (for the calculation, the number of red blood cells counted in a given patient is taken into account).

In the blood of healthy individuals there are 5-10% reticulocytes.

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Reticulocytes (clinical significance and laboratory diagnostics)

(1) reticulocytes that have a nucleus (erythronormoblasts), and their granularity is located in the form of a dense rim around the nucleus;

(2) reticulocytes having a granular-mesh substance in the form of a ball or lump;

(3) reticulocytes, granular in the form of a dense network;

(4) reticulocytes, having a granular-mesh substance in the form of individual threads;

(5) reticulocytes containing individual granules.

With hemolytic anemia (the number of reticulocytes can reach 60% or more (increasing especially strongly during hemolytic crises);

In case of acute blood loss (a reticulocyte crisis occurs 3–5 days after blood loss), including an increase in reticulocytes, one can suspect hidden bleeding (for example, in patients with peptic ulcer of the gastrointestinal tract, typhoid fever);

When treating iron deficiency anemia with iron (several days (3 - 10) after the start of antianemic treatment of pernicious anemia);

In case of acute lack of oxygen;

With tumor metastases to the bone marrow.

Abnormal form of hemoglobin;

For aregenerative aplastic and hypoplastic anemia;

For anemia caused by insufficiency of iron, vitamin B12, folic acid (microcytic-hypochromic and megaloblastic anemia);

With sideroblastic anemia;

With cancer metastases to the bone;

For radiation sickness and radiation therapy;

When treated with cytostatics;

For autoimmune diseases of the hematopoietic system;

For kidney diseases;

With relapse of Addison-Biermer anemia;

Incorrect choice of anticoagulant or insufficient mixing of blood with the anticoagulant;

Prolonged squeezing of the hand with a tourniquet;

Taking sulfonamides (both underestimated and overestimated results are possible);

Blood transfusion shortly before the study;

Hemolysis of a blood sample.

Assessment of erythropoiesis activity in conditions accompanied by hemolysis or blood loss;

Assessing the ability of bone marrow to regenerate after cytotoxic therapy and bone marrow transplantation;

Evaluation of restoration of erythropoietin synthesis after kidney transplantation;

Doping control for athletes (taking erythropoietin);

Diagnosis of ineffective hematopoiesis or decreased production of red blood cells;

Differential diagnosis of anemia;

Detection of impaired regenerative capacity of the bone marrow due to deficiency of iron, vitamins B12, B6, folate, copper and monitoring of appropriate therapy;

Assessment of response to therapy with erythropoietin and erythrosuppressors.

Reticulocytes with low RNA content, the most mature (LFR%, low fluorescence reticulocyte fractions);

Reticulocytes with average RNA content (MFR%, medium fluorescence reticulocyte fractions) - fraction of reticulocytes with average fluorescence);

Reticulocytes with high RNA content (HFR%, high fluorescence reticulocyte fractions) - a fraction of reticulocytes with high fluorescence);

Immature Reticulocyte Fraction (IRF%, Immature Reticulocyte Fraction).

Counting reticulocytes in a smear after staining them with special dyes

Counting reticulocytes in a smear after staining them with special dyes is in practice the most used method for counting the number of reticulocytes. This is due to the fact that the method is simple, quite cheap and does not require special expensive equipment, and therefore can be used in any clinical diagnostic laboratory.

Principle of the method

Detection of the granular-reticular substance of reticulocytes during supravital staining with alkaline paints with their further counting in a blood smear.

Reagents

You can use one of the following dyes:

1. A saturated solution of brilliant cresyl blue in absolute alcohol (to prepare absolute alcohol, you need to soak 96% ethanol in several changes of calcined copper sulfate powder). For 100 ml of absolute alcohol take 1.2 g of paint.
2. Solution of azure I: azure I – 1 g, ammonium oxalate – 0.4 g, sodium chloride – 0.8 g, ethyl alcohol 96% – 10 ml, distilled water – 90 ml. The paint solution in a closed bottle is placed in a thermostat at 37° C for 2–3 days and shaken vigorously periodically. Then cool to room temperature and filter through a paper filter. The solution is stored in a dark glass container. If sediment appears, the paint should be filtered again.
3. Azur II solution: Azur II – 1 g, sodium citrate – 5 g, sodium chloride – 0.4 g, distilled water – 45 ml. The solution is left in a thermostat at 37° C for 2 days, stirring occasionally. To speed up dissolution, the paint can be heated over low heat for 15 - 20 minutes, without bringing it to a boil. Cool to room temperature and filter. Store in dark glass containers.

Special equipment

Progress of determination

Reticulocyte staining is carried out either on glass or in a test tube.

Staining of reticulocytes on glass

When staining reticulocytes on glass, a well-washed and degreased glass slide is heated over a burner flame. Using a glass rod, apply a drop of one of the dyes listed above to the glass and prepare a smear of paint using ground glass. A glassograph is used to mark the side of the glass on which a smear of paint is applied. In this form, glass can be prepared for future use and stored in a dry, dark place. Before analysis, prepare a wet chamber. Typically, a Petri dish with rolls of moistened cotton wool or filter paper placed around the edges is used for this. Apply a drop of blood to a smear of paint, prepare a thin smear from it and immediately place it in a humid chamber for 3 to 10 minutes. Then the smears are air dried.

Staining reticulocytes in vitro

The staining of reticulocytes in a test tube differs when different dyes are used.

Method 1 – staining of reticulocytes in a test tube with brilliant cresyl blue.

Before use, prepare a working solution of brilliant cresyl blue in a test tube based on a drop of 1% potassium oxalate solution and 4 drops of brilliant cresyl blue paint solution. Add 0.04 ml of blood to the paint (two pipettes to the 0.02 mark). The mixture is thoroughly but gently mixed and left for 30 minutes. Then mix again and prepare thin smears.

Method 2 - staining of reticulocytes in a test tube with Azur II.

0.05 ml of azure II dye solution and 0.2 ml of blood are placed in a test tube. The mixture is thoroughly mixed and left for 20 - 30 minutes. Mix again and prepare thin smears.

Method 3 - staining of reticulocytes in a test tube with Azure I.

Place 0.3–0.5 ml of azur I dye solution and 5–6 drops of blood into a test tube using a pipette from a Panchenkov apparatus. The test tube is closed with a rubber stopper, the mixture is thoroughly but carefully mixed and left for 1 - 1.5 hours (reticulocytes stain better with an exposure of 1.5 - 3 hours). Mix and prepare thin smears.

Currently, ready-made, factory-made dyes for reticulocytes are commercially available. Reticulocytes are stained using them according to the attached instructions.

Reticulocyte count.

Smears prepared using one of the above methods are examined microscopically using an immersion lens. In the smear, reticulocytes and erythrocytes are colored yellowish-greenish, the granular-filamentous substance in the reticulocytes is blue (when stained with azure II and brilliant cresyl blue) or bluish-violet (when stained with azure I).

Photos of reticulocytes:

Find the fields of view where the red blood cells are located separately. In these fields of view, it is necessary to count at least 1000 red blood cells and note among them the number of red blood cells containing a granular-filamentous substance. Greater accuracy is obtained when counting 2000 - 3000 red blood cells.

Due to the fact that there are a large number of red blood cells in the field of view, which makes counting difficult, it is necessary to limit (reduce) the field of view. To do this, you can use either a special eyepiece, in which you can reduce the field of view to the required size, or use a special “window” (a circle with a diameter slightly smaller than the eyepiece is cut out of paper, a small diamond is cut out in the center of the circle and the resulting window is inserted into the eyepiece).

The number of counted reticulocytes is expressed per 100 (in percent) or per 1000 (in ppm) red blood cells.

Example: When counting 1000 red blood cells in a smear, it was revealed that 15 red blood cells out of 1000 have a granular-mesh substance of varying degrees, that is, they are reticulocytes. Therefore, the number of reticulocytes in this case is 1.5% or 15‰.

Literature:

• Directory "Laboratory research methods in the clinic" edited by Menshikov V.V. - Moscow, "Medicine", 1987
• Lyubina A. Ya., Ilyicheva L. P. and co-authors - “Clinical laboratory studies” - Moscow, “Medicine”, 1984.

Reticulocyte count using fluorescence microscopy

The method of counting the number of reticulocytes using fluorescent microscopy is simple and requires little time, and is more accurate than the conventional method, since fluorescent microscopy reveals the smallest grains of a reticular filamentous substance.

Reticulocytes

Reticulocytes are young red blood cells formed after normoblasts lose their nuclei. A characteristic feature of reticulocytes is the presence in their cytoplasm of a granular-filamentous substance (reticulum), which represents aggregated ribosomes and mitochondria.

Pathological inclusions in red blood cells

Jolly bodies (Howell-Jolly bodies) are small round violet-red inclusions µm in size, found 1 (less often 2 - 3) in one erythrocyte. They represent the remainder of the nucleus after removal of its RES. They are detected with intense hemolysis and “overload” of the RES, after splenectomy, with megaloblastic anemia..

Cytochemical study of lipids

Cytochemical study of lipids is based on the use of fat-soluble dyes (sudan III, sudan IV, black sudan, etc.). To identify neutral fat, Sudan III is used, which colors the fat orange. Lipoids are detected better by Sudan black (black staining).

Urinalysis according to Nechiporenko

The Nechiporenko method in domestic laboratory diagnostics is the most common method for the quantitative determination of formed elements in urine. This method is the simplest, accessible to any laboratory and convenient in outpatient practice, and also has a number of advantages over other known quantitative methods for studying urine sediment. Using the Nechiporenko method, the number of formed elements (erythrocytes, leukocytes and cylinders) in 1 ml of urine is determined.

©18 Laboratory diagnostics

Reticulocyte - what is it in a blood test? Standard indicators and designation

A reticulocyte is a precursor of an erythrocyte, that is, it is an erythrocyte in a young form. In the blood of a healthy person their content is 0.2-1.2%. This ratio reflects the content of reticulocytes in the blood relative to red blood cells. They ripen in 4-5 days.

Basic Concepts

A characteristic feature of young forms of erythrocytes is that the cytoplasm contains a granular-filamentous substance, which is aggregated ribosomes and mitochondria. This substance can be detected using a special method of staining blood smears. This staining of reticulocytes is called supravital, that is, it is performed without prior fixation of the cells.

Reticulocyte groups

In total, experts distinguish five main groups of reticulocytes. They differ in the reticular mesh. Its density and age are in inverse proportion: the thicker the mesh, the younger they are. Let's figure out what the youngest reticulocyte is.

This is a cell that has a mesh in the form of a thick ball. These cells belong to the first group. Reticulocytes of a more mature form appear in the form of a substance that has a clearly visible mesh. In the fourth and fifth groups it is represented by individual threads and grains. Most often, the blood of healthy people contains reticulocytes of the latter groups, that is, more mature ones. As a rule, they make up about 80% of all others. With enhanced regeneration, which is characteristic of some pathologies, the number of reticulocytes of the first three groups increases. Such pathologies include:

Reticulocyte crisis accompanying B12 deficiency anemia.

Functions performed by reticulocytes

A reticulocyte is also an erythrocyte. It has the same function as red blood cells. That is, cells also transfer oxygen molecules to various tissues and organs, but the efficiency of this process is much lower than when oxygen is transferred by mature red blood cells. Reticulocytes can adsorb iron molecules contained in hemoglobin. This is possible thanks to transferrin-sensitive receptors. Why are reticulocytes counted? More on this later.

Blood sampling mechanism

A blood sample is taken to determine this indicator as part of a general analysis. If there is a need to determine the level of reticulocytes, then the doctor who ordered the test indicates the need for additional counting.

This test does not require special preparation, but traditionally it is taken on an empty stomach in the morning. At the same time, if there is a need, it can be taken at any time of the day. Blood for analysis should be taken from a finger prick; its examination is carried out in the laboratory, in the hematology department.

The number of reticulocytes is counted in a supravital stained smear. The microscopic method is used, that is, their number is simply counted by placing a blood sample under a microscope. Today there is a hardware method of counting. It is used in modern laboratories.

The designation for reticulocytes in a blood test is RET.

Carrying out analysis

There are several methods for conducting such a study:

• The glass is washed, dried and heated using a special burner. Next, one of three dyes is applied to it, then a drop of blood is added, after which a thin smear is made. Place the glass in a Petri dish or other chamber for a certain time, then dry it in fresh air.
• If you use a test tube, then a coloring substance is placed in it, and a drop of blood is added there. The mixture must be stirred. Wait time - from 20 minutes to 3 hours, it depends on what reagent is used. Then the liquid in the test tube is mixed again, and smears are taken from it.

It is difficult to say which method provides more accurate results. They are both good in their own way, but the doctor may find it necessary to carry out the analysis in a certain way, then he must indicate this in the direction.

Indicators

When calculating the level of reticulocytes, gender differences in the norm are significant only after 12 years. Until this age, the norm for children of both sexes is the same. At about the age of about one year, girls begin menstruation, which is regular. As a result of monthly blood loss, the range of fluctuations of erythroid cells expands.

1. In newborns, the norm is in the range from 0.15 to 1.5%.

2. In children aged two weeks – from 0.45 to 2.0%.

3. In children aged one to two months - from 0.25 to 0.95%.

4. In children aged six months – from 0.2 to 1.0%.

5. In children aged two to six years - from 0.25 to 0.75%.

6. In children aged six to twelve years - from 0.25 to 1.3%.

7. In men over twelve years of age - from 0.25 to 1.7%.

8. In women over twelve years of age - from 0.12 to 2.1%.

What does it mean when reticulocytes are elevated?

Increased number of reticulocytes in the blood

With anemia of various etiologies, determining the number of reticulocytes plays a special role. If their content is increased in the blood, then this pathology is called reticulocytosis. If the increase is accompanied by an increase in hemoglobin levels, this indicates that the bone marrow has good regenerative ability. This may occur if the following factors are present:

Hemolytic anemia of various etymologies. It is a series of diseases that are characterized by the destruction of red blood cells. This phenomenon is called hemolysis. In this case, the content of reticulocytes in the blood can reach 60%. If a hemolytic crisis occurs, this figure may worsen.

Poisoning with hemotoxins causing hemolysis. Hemotoxins include viper venom and some medications that are used to treat erythremia. Malarial toxins can also cause hemolysis.

Levodopa therapy for Parkinson's disease.

Long-term use of certain anti-inflammatory and antipyretic drugs.

Therapy with the drug "Erythropoietin" for anemic diseases.

Bone marrow metastases.

The recovery period after radiation or chemical therapy.

Reticulocytosis can be false or true.

Why take a reticulocyte test? This question is often asked.

False and true reticulocytosis

True reticulocytosis is characterized by an increase in the number of young reticulocytes in the blood, which is accompanied by an increase in their number in the bone marrow.

False reticulocytosis is characterized by an increase in the number of reticulocytes exclusively in the peripheral blood, while in the bone marrow their level remains normal or slightly reduced.

Reduced performance

The process of reducing the number of reticulocytes is observed when erythropoiesis is suppressed. Occurs as a result of the influence of the following factors:

Long-term use of sulfonamides.

Taking certain medications, such as chloramphenicol or carbamazepine.

Chronic infectious diseases.

Some severe kidney diseases affecting erythropoiesis.

Myxedema or other thyroid dysfunction.

Tumors in the bone marrow.

We looked at what a reticulocyte is. This is a young red blood cell in human blood.

The reasons for the increase in reticulocytes, how to determine the functionality of the hematopoietic system?

Sometimes, when prescribing a clinical examination, a general blood test may separately indicate RTC or RET - abbreviations that indicate the content of reticulocytes. They are anucleate cells that precede erythrocytes (red blood cells) in the process of their formation. Reticulocytes in a blood test, their number and ratio with red blood cells reflect the condition and reproductive functionality of the bone marrow, as well as the entire hematopoietic system.

Medical certificate

You can define in detail what reticulocytes are in this way: these are round or stellate-shaped cells that are formed in the bone marrow and transformed into red blood cells under the influence of the glycoprotein hormone erythropoietin.

The color of reticulocytes is usually pink with a blue tint. Their distinctive feature from the later form is the presence in the structure of residual RNA, mitochondria and other organelles that look like granular and filamentous formations, the loss of which indicates transformation into a mature cell.

The functional responsibilities of reticulocytes coincide with erythrocytes - to carry oxygen, but with much less efficiency due to the low hemoglobin content in them. They are mainly concentrated in the red bone marrow, flat and tubular bones, and only mature red cells enter the blood. But a small fraction of immature reticulocytes is also allowed in the peripheral bloodstream, which indicates activation of the process of red blood cell production (erythropoiesis).

The process of blood formation

Erythropoiesis begins with the transformation of the erythroblast, a cell containing a nucleus but without hemoglobin, into a pronormblast. Next, in the erythroid series, a basophilic erythroblast is formed, which already has the makings of hemoglobinization.

As the progenitors of red blood cells accumulate a substance that retains oxygen (hemoglobin), the cell goes through the stages of polychromatophilic and then oxyphilic normoblast. During this period, the nucleus is still preserved, the involution of which leads to the formation of a directly young erythrocyte - a reticulocyte, which already has the ability to retain and transport oxygen and carbon dioxide.

Young red blood cells remain at the site of their formation for up to 40 hours, after which a small part of them, under the influence of erythropoietin, enters the blood vessels, where they move freely for 1.5 or 2 days. During this time, the cell loses its reticulum (residual thread-like formations) and turns into an erythrocyte.

Stages of reticulocyte development

In the blood, reticulocytes undergo five stages of maturation as the reticulum involutes:

1. stage zero - when the basophilic substance (mesh intracellular substance) has the shape of a nucleus, that is, the accumulation of granularity looks like a kind of corolla;
2. the first stage – the basophilic substance (BS) acquires a mesh structure in the form of a ball;
3. second stage – the granular network is evenly distributed throughout the cell;
4. third stage - BV looks like separate threads;
5. fourth stage – BV is distributed along the periphery of the cell in the form of small grains.

In a healthy body, reticulocytes in the bloodstream should be found only in stages III and IV of maturation (80 percent), and 0 - II in single copies. But excessive death of red blood cells or oxygen starvation stimulates increased production of erythropoietin by the kidneys, which leads to the release of immature cells of all stages and even normoblasts into the vessels.

Blood test for reticulocytes

Counting reticulocytes in a blood smear is not necessary for a hematological examination, but is part of the general clinical analysis. Only if certain suspicions arise does the doctor make a special designation in the analysis.

Reasons for the study

The reasons for testing the amount of RTC in the blood are to check the functioning of the bone marrow and kidneys in producing blood cells and erythropoietin in such cases:

• if you suspect anemia of various origins;
• if necessary, control drug therapy for deficiency of iron, vitamin B12 and folic acid;
• for monitoring when taking erythropoietin-containing drugs, erythrosuppressors;
• in case of severe external bleeding or suspected internal bleeding;
• due to the need to take medications that neutralize toxins resulting from bites of poisonous insects or snakes;
• to control the production of red blood cells during bone marrow or kidney transplantation;
• to determine cancer.

The last point is related to the characteristics of metastasis of some cancer tumors to blood-forming organs.

Method of detection

If the nucleus is lost, staining methods with only acidic dyes are applicable to blood cells. By tinting the blood smear, the elements of the erythroid series are indicated, which are then detected using light microscopy. To isolate reticulocytes from the total mass of red blood cells, it is necessary to use special preparations that change the color of the granular structure of the cells.

The detection process itself consists of mixing blood plasma with brilliant cresyl blue or acridine orange dye in vitro (in vitro) or on glass in the proportions: 1 part blood to 10 parts of the substance.

Methods on glass and in vitro are fundamentally different. In the first case, mixed blood smears with dye are immersed in a humid environment for 10 minutes, after which they are dried and the cells are counted under a microscope. In the second option, the composition mixed in a test tube is left for up to 3 hours, then mixed again, a smear is made and counted.

When the stellate cells of the reticulocytes become distinguishable and the degree of their maturity is determined (by the structure of the tinted BV), information about the state of the bone marrow can be obtained.

Acceptable indicators

The normal level of reticulocytes corresponds to the process of blood formation in a healthy body. To organize the data, a special table is used, in which normal ranges are graded by age and gender of the person.

The norm of reticulocytes in the blood of children

The reticulocyte norm in children varies depending on the specific period of formation of the hematopoietic system. Thus, in newborns up to 14 days, the permissible rate is from 0.15 to 1.5 percent in relation to red blood cells. Normal values ​​for infants up to two months are from 0.45 to 2.1 percent, and up to six months - 0.25 - 0.9 percent. In a child from six months to one year, values ​​from 0.2 to 1 percent are considered healthy, and they stabilize until the age of six. Further, the range narrows: from 7 to 12 years it ranges from 0.2 to 1.3 percent.

Reticulocytes in the blood of adults

The further permissible content of reticulocytes in the blood depends more on the gender of the child than on the age, and corresponds to adult indicators. This is due to the beginning of the menstrual cycle in girls, which implies monthly blood loss and, accordingly, an increase in the production of blood cells during this period. Standard indicators:

• the permissible level of young red blood cells in the blood of women and young girls from 13 years of age will be from 0.12 to 2.05 percent (from 1.2 to 20 ppm, respectively);
• The normal reticulocyte count in men ranges from 0.24 to 1.7 percent of the total erythroid mass.

Thus, in an adult, a constant range of the permissible concentration of young blood cells, which contain a granular-mesh substance, is established. In this case, increased reculocytes or a reticulocyte crisis indicate certain disturbances in the functioning of the hematopoietic system.

Increased reticulocytes

Elevated reticulocytes (or otherwise reticulocytosis) can have both positive and negative implications for the subject. An increased number of immature blood cells occurs in the following cases:

• With positive results from therapy with iron supplements, vitamin B12 and folic acid, which usually occur within a week.
• When replenishing blood volume after significant blood loss.
• With positive results of treatment of oncological diseases with antitumor drugs or radiation therapy.

Important! Reticulocytosis is observed with oxygen deficiency at a significant altitude above sea level.

Negative values ​​of reticulocytosis include:

• The consequences of diseases such as typhoid fever, which provoke internal bleeding.
• Reticulocytes are increased during intoxication with chemical poisons or foreign agents.
• False reticulocytosis shows the presence of immature cells in the blood in the absence of them in the bone marrow, which indicates inflammatory processes or metastases in the organ.
• Reticulocytosis is observed in malaria, hemolytic anemia or thalassemia.

An increased content of immature blood cells in the blood (reticulocytosis) is fraught with thickening, which can subsequently lead to thrombosis, heart attack or stroke.

Reticulocytes in the blood during pregnancy

In pregnant women, the total blood volume increases due to plasma, so its dilution and a decrease in the level of red blood cells are observed. Due to the fact that there is not enough hemoglobin in the blood, anemia develops, which activates the process of erythropoiesis. Therefore, pregnant women have a high percentage of reticulocytes. The clinical picture of this phenomenon corresponds to the norm for the physiological adaptation of the body to bearing a child.

Reticulocytes are low

Reticulocytopenia (when the level of reticulocytes is reduced) often indicates damage to the kidneys and/or bone marrow. Factors that reduce the content of young red blood cells are:

• Anemia caused by a lack of folic acid, vitamin B12 or iron, which leads to the destruction of red blood cells.
• Kidney diseases, but they can lower the level of reticulocytes slightly.
• Alcohol abuse, which will show a decrease in RTC due to suppression of renal function, suppression of bone marrow and direct cell destruction as a result of intoxication.

Important! Malignant tumors can also reduce the level of reticulocytes in the blood. This depends on the area of ​​bone marrow to which the cancer has metastasized.

The level of reticulocytes in the blood itself is not a diagnosis. If there is a significant deviation from the permissible range, it sets the general direction for the diagnosis.

Detection of the granular-reticular substance of reticulocytes when stained with brilliant-cresyl blue without prior fixation.

Reagents:

1% solution of brilliant cresyl blue: 50 mg of dye is dissolved in 5 ml of physiological solution and 20 mg of sodium citrate is added (the solution is stored in a dark glass container, good for several days).

Methodology:

Apply 2 drops of a 1% solution of brilliant cresyl blue paint and 1 drop of blood onto a glass slide with a well. Stir carefully with a glass rod and the mixture is placed in a humid chamber (Petri dish, into which lightly moistened rolls of gauze or cotton wool are placed around the edges) for 30 minutes at room temperature. Then smears are made and dried.

Reticulocyte count:

In smears, red blood cells are stained yellowish-green, and the granular-reticular substance in reticulocytes is blue and violet.

The smears are examined microscopically with an immersion lens. Reticulocytes are counted per 1000 red blood cells (greater accuracy is obtained when counting 2000-3000 red blood cells).

An increase in the number of reticulocytes is observed with:

stimulation of erythropoiesis (blood loss, hemolysis, reticulocyte crisis with successful treatment of B12-deficiency anemia, acute lack of oxygen).

A decrease in the number of reticulocytes is observed with:

inhibition of erythropoiesis (aplastic and hypoplastic anemia, untreated B12-deficiency anemia, cancer metastases to the bones),

kidney diseases, endocrine diseases.

An increase in their reticulocytes in the peripheral blood (reticulocytosis) is noted: with hemolytic anemia (the number of reticulocytes can reach 60% or more (increasing especially strongly during hemolytic crises); with acute blood loss (a reticulocyte crisis occurs 3–5 days after blood loss), in including, an increase in reticulocytes allows one to suspect hidden bleeding (for example, in patients with peptic ulcer of the gastrointestinal tract, typhoid fever); with malaria; with polycythemia; when treating iron deficiency anemia with iron (several days (3 - 10) after the start of antianemic treatment of pernicious anemia); with acute lack of oxygen; with metastases of tumors to the bone marrow. It should be remembered that in the case of increased destruction of red blood cells, the proportion of reticulocytes may exceed 50% due to an artificial increase in the number of reticulocytes (as noted earlier, the proportion of reticulocytes is calculated as % of all red blood cells In such cases, to assess the severity of anemia, the “reticular index” is used, which is calculated using the formula: (% reticulocytes x hematocrit) / 45 x 1.85, where 45 is a normal hematocrit, 1.85 is the number of days required for the receipt of new reticulocytes into the blood. If index

METHODS FOR COUNTING THE NUMBER OF RETICULOCYTES

(1) Counting the number of reticulocytes in a smear after staining them with special dyes. This method is in practice the most used method due to the fact that it is simple, fairly cheap and does not require special expensive equipment, and accordingly can be used in any clinical diagnostic laboratory. The principle of the method is based on identifying the granular-reticular substance of reticulocytes during supravital staining with alkaline paints (saturated solution of brilliant cresyl blue in absolute alcohol / Azur I solution / Azur II solution) with further counting them in a blood smear. Reticulocyte staining is carried out either on glass or in a test tube. Counting is carried out using a microscope: smears prepared using one of the above methods are microscopically examined with an immersion lens; in the smear, reticulocytes and erythrocytes are colored yellowish-greenish, the granular-filamentous substance in reticulocytes is blue (when stained with azure II and brilliant cresyl blue) or bluish-violet (when stained with azure I). (2) Reticulocyte count using fluorescence microscopy. This method is simple and requires little time, it is more accurate than the conventional method, since fluorescent microscopy reveals the smallest grains of a mesh-filamentous substance, but it is only possible with a fluorescent microscope and special dyes, and therefore is available only to a few laboratories . The principle of counting the number of reticulocytes using fluorescent microscopy is based on the ability of the reticulocyte substance to fluoresce after treating the blood with acridine orange. Blood is mixed with acridine orange in a test tube or mixer in a ratio of 1 part blood and 10 parts paint (the mixture can be stored for no more than 5 hours). The mixture is stirred for 2 minutes, a drop of the mixture is applied to a glass slide and covered with a coverslip. In this case, the liquid should not go beyond the coverslip. Microscopy using a ZhS-17 light filter. In the preparation, red blood cells have dark green outlines and do not fluoresce, and in reticulocytes, the granular-mesh substance glows bright red, making reticulocytes easy to count. In blood stabilized with heparin or sodium citrate, reticulocyte fluorescence is not observed. (3) Automatic reticulocyte count using hematology analyzer. In modern hematology analyzers, the technology for counting blood cells is based on the conductometric method proposed by H. Wallace and Joseph R. Culter in 1947. The principle of the method is to count the number and determine the nature of impulses that occur when a cell passes through a small-diameter hole (aperture), on both sides of which there are two electrodes isolated from each other. Each passage of a cell through the aperture is accompanied by the appearance of an electrical impulse, which is recorded by an electronic sensor. The division of cells into categories (erythrocytes, leukocytes, platelets, sediment) is carried out by the device based on an analysis of the amplitude of the received pulses. To determine the concentration of cells, it is enough to pass a certain volume of sample through the channel and count the number of pulses that are generated. It should be noted that in addition to the classical parameter of reticulocytes - the relative (%) content of reticulocytes (RET%, percent of reticulocytes), determined using methods 1 and 2 of laboratory diagnostics, in connection with the advent of high-tech hematological analyzers (method 3) it has become possible obtain (for example, using a patented fluorescent dye for the Sysmex -XT- 2000i analyzer) additional informative reticulocyte parameters: reticulocytes with a low RNA content, the most mature (LFR%, low fluorescence reticulocyte fractions); reticulocytes with average RNA content (MFR%, medium fluorescence reticulocyte fractions) - fraction of reticulocytes with average fluorescence); reticulocytes with high RNA content (HFR%, high fluorescence reticulocyte fractions) - a fraction of reticulocytes with high fluorescence); immature reticulocyte fraction (IRF%, Immature Reticulocyte Fraction). The differentiation of reticulocytes, based on the degree of maturity and, accordingly, the content of nucleic acids, is a reflection of the hematopoietic activity of the bone marrow. Methodology (Sysmex -XT- 2000i analyzer). In a flow cell, cells cross a semiconductor laser beam, causing high- and low-angle scattering of the beam and excitation of a fluorescent dye. This makes it possible to determine the different stages of reticulocyte maturity based on the RNA content in the cells and the intensity of their luminescence. Automated reticulocyte counting is highly accurate (more than 30,000 red blood cells are counted) and reproducible (coefficient of variation is about 6%). This technology ensures accurate counting of reticulocytes even at extremely low concentrations.

Reticulocytopenia and reticulocytosis, causes of development, diagnostic value of their detection.

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Reticulocytes (reticulocytus)

Reticulocytes are young red blood cells formed after normoblasts lose their nuclei.

A characteristic feature of reticulocytes is the presence of a granular-mesh substance, which appears with supravital staining, i.e., without preliminary fixation of the cells.

Electron microscopy showed that granular-mesh structures are remnants of the endoplasmic reticulum, ribosomes and mitochondria containing RNA. In reticulocytes, protein (globin), heme, purines, pyrimidine nucleotides, phosphatides, and lipids are synthesized to a small extent, but RNA is not synthesized in them. For 2 days, the reticulocyte remains in the bloodstream, after which, as the RNA decreases, it becomes a mature erythrocyte.

In smears stained using conventional hematological methods, reticulocytes are grayish-pink in color - polychromatophilic, i.e. painted with different dyes.

Currently, a unified method is used to count the number of reticulocytes after staining them

• brilliant cresyl blue,
• Azure I or
• Azure II directly on glass or in a test tube.

1. Principle of the method

Detection of the granular-reticular substance of erythrocytes when stained with alkaline paints with their further counting in a blood smear.

2. Reagents:

a) a saturated solution of brilliant cresyl blue in absolute alcohol (to prepare absolute alcohol, you need to soak 96% ethanol in several changes of calcined copper sulfate powder): 1.2 g of paint per 100 ml of alcohol;

b) azur I solution: azur I - 1 g, ammonium oxalate - 0.4 g, sodium chloride - 0.8 g, ethyl alcohol 96% - 10 ml, distilled water - 90 ml. The paint solution in a closed bottle is placed in a thermostat at 37 °C for 2–3 days and shaken vigorously periodically. Then cool to room temperature and filter through a paper filter. The solution is stored in a dark glass container. If sediment appears, the paint should be filtered again;

c) Azur II solution: Azur II - 1 g, sodium citrate - 5 g, sodium chloride - 0.4 g, distilled water - 45 ml. The solution is left in a thermostat at 37 °C for 2 days, stirring occasionally. To speed up dissolution, the paint can be heated over low heat for 15–20 minutes, without bringing it to a boil. Cool to room temperature and filter. Store in dark glass containers.

3. Coloring

Coloring on glass:

• a well-washed and degreased glass slide is heated over a burner flame. Using a glass rod, apply a drop of one of the dyes to the glass and prepare a smear of paint using ground glass. Mark the side of the glass on which the paint stroke is applied with a steklograf. In this form, glass can be prepared for future use and stored in a dry, dark place;
• A drop of blood is applied to the glass prepared in this way, a thin smear is made, and the glass is immediately placed in a damp chamber. To do this, use a Petri dish with a lid, into which slightly moistened rolls of gauze or cotton are placed around the edges;
• The smears are kept in a humid chamber for 3–5 minutes and then dried in air. The granular-mesh substance of reticulocytes is painted violet-blue, clearly standing out against the greenish-bluish background of erythrocytes.

In vitro staining:

• method 1: before use, prepare a working solution of brilliant cresyl blue in a test tube based on a drop of 1% potassium oxalate solution 4 drops of paint solution 1. Add 40 μl of blood to the paint (two pipettes to the 0.02 mark). The mixture is thoroughly but gently mixed and left for 30 minutes. Stir and prepare thin smears;
• method 2: 0.05 ml of dye solution 3 and 0.2 ml of blood are placed in a test tube. The mixture is thoroughly mixed and left for 20–30 minutes. Stir and prepare thin smears;
• method 3: 0.3–0.5 ml of dye solution 2 and 5–6 drops of blood are placed into a test tube using a pipette from a Panchenkov apparatus. The test tube is closed with a rubber stopper, the mixture is thoroughly but carefully mixed and left for 1–1½ hours (reticulocytes are better stained with an exposure of 1½–3 hours). Mix and prepare thin smears.

4. Reticulocyte count

In smears, red blood cells are colored yellowish-greenish, the granular-filamentous substance is blue or bluish-violet.

• Smears prepared using one of the above methods are examined microscopically with an immersion lens;
• it is necessary to count at least 1000 red blood cells and note among them the number of red blood cells containing a granular-filamentous substance. For uniform thin smears in which the red blood cells are located in one row, select a field of view in which there are, for example, 50 red blood cells, and then calculate 20 such fields of view;
• In practice, for greater accuracy, they use a special eyepiece, in which the field of view can be reduced to the required size. If you don’t have a ready-made eyepiece, you can easily prepare it by unscrewing the ×7 eyepiece, putting a piece of paper with a small square cut out into it and screwing it on. The number of counted reticulocytes is expressed per 1000 or per 100 red blood cells.

NORMAL: 0.5–1.2% (30–70 × 109/l)

An increase in the number of reticulocytes is observed with:

• blood loss (especially acute);
• hemolytic anemia, especially during a crisis (up to 20–30%);
• against the background of treatment of megaloblastic anemia with vitamin B12 (reticulocyte crisis - an increase in the number of reticulocytes on the 4th–8th day of treatment).

A decrease in the number of reticulocytes is characteristic of:

• aplastic and hypoplastic anemia;
• untreated megaloblastic anemia;
• radiation sickness;
• taking cytostatic drugs.

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Reticulocytes normal and counting their number

Blood cells, reticulocytes, the norm of which varies due to many factors, are unformed forms of red blood cells, and they can be found in the bone marrow and peripheral blood. As a rule, reticulocytes in the blood mature in three to five days, and then these cells turn into mature red blood cells. Moreover, the number of unformed red blood cells in newborn babies is significantly greater than in adults.

Detection of reticulocytes:

When identifying reticulocytes, the norm of which is determined using a special table, it is necessary to take into account their differences from mature red blood cells. This type of blood cell either contains a whole nucleus, or its remains are observed in the composition of a granular-filamentous substance. To determine the number of such cells, it is necessary to study the color of the blood smear in the laboratory. To do this, a blue diamond solution is used, which is applied to degreased and pre-washed glass, and only then a smear is made.

When reticulocytes are detected, the norm of which is different, immediately after the smear, the glass used should be placed in a Petri dish - a special wet chamber, then kept for five minutes, thoroughly dried in fresh air, and then microscoped. Moreover, the substance of the granular-filamentous type inside the reticulocyte is usually painted in a violet-blue hue, and the background of the erythrocytes stands out due to its bluish-green tint.

If the Gehl-Meyer method is used, the color of immature blood cells can be seen much better, but this requires a Widal tube. In this vessel, a couple of drops of blood are mixed with a diamond solution and sodium chloride, then the test tube is closed with a lid, and the smear itself is performed within an hour.

Reticulocyte count:

When calculating the number of immature blood cells, you need to take 1000 red blood cells as a basis, and then consider reticulocytes, the norm of which usually varies at the level of 0.2% -1.2% of the number of adult red blood cells. On average, in most people, the number of immature red blood cells is usually 0.7% of the total number of fully mature blood cells. If the number of reticulocytes is 10% or more higher than the maximum permissible norm, a person develops reticulocytosis - a disease accompanied by regular acute bleeding and hemolytic anemia.

When counting reticulocytes (the norm in children is usually high), it is worth checking that their number does not exceed the limit values ​​typical for adults. The standards also depend on gender, since in women 2.07% of immature red blood cells is still considered an acceptable parameter, and in men this figure should not exceed 1.92%.

In cases where the level of reticulocytes is significantly increased, this indicates the presence of disorders in the body such as malaria and thalassemia, polycythemia and hypoxia, anemia, all kinds of hemolytic syndromes, but such indicators are also possible during treatment with cyanocobalamin. If the level of immature red blood cells is low, a person may experience aplastic and hypoplastic anemia, all kinds of kidney diseases and myxedema, as well as the spread of tumor metastases to the bones.

To diagnose the severity of anemia, it is necessary to calculate the “reticulocyte index,” which is calculated as the ratio of the percentage of reticulocytes and normal hematocrit values, multiplied by the number of days required for immature cells to enter the peripheral blood. If the resulting index does not exceed two, the indicator indicates a hypoproliferative component of anemia, and if it exceeds two, it indicates the likelihood of increased formation of red blood cells.

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Blood test. Blood analysis

Determination of hemoglobin concentration

Among the methods for determining hemoglobin, the most widely used methods are based on colorimetry, i.e., determining the intensity of color. The simplest of them is the determination of hemoglobin using visual colorimetry in a Sali hemometer, which is a wooden stand with a central graduated test tube, on the sides of which there are sealed glass tubes with a color standard (hydrochloric acid hematin in glycerin). A 0.1% solution of hydrochloric acid is first poured into the central test tube to the mark corresponding to 2 or 3 g%, then carefully add (exactly!) 0.02 ml of blood taken from a finger with a special pipette attached to the hemometer. The pipette is washed with the surface layer of acid and, after mixing the blood and acid with a glass rod, it is left for 5 minutes to form hydrochloric acid hematin. Then, by adding distilled water and constantly stirring with a stick, the color of the liquid in the central test tube completely matches the standards. The hemoglobin concentration corresponds to the level of the solution along the lower meniscus. Hemoglobin concentration can be expressed either in g% hemoglobin or in arbitrary units. 16.67 g of hemoglobin is taken as 100 units.

The concentration of hemoglobin in the blood in women ranges from 11.7 to 15.8 g/o or from 117 to 158 g/l, in men - from 13.3 to 18 g% or from 133 to 180 g/l.

Taking blood to count the formed elements

To count formed elements, blood is diluted in mixers (melangers) or test tubes, which has recently become more widely used. To count red blood cells, blood is diluted 200 times with a 3% sodium chloride solution; if we use a hemometer pipette with a volume of 0.02 ml to take blood, then we must take 4 ml of sodium chloride solution. To count leukocytes, the blood is diluted 20 times, therefore 0.02 ml of blood and 0.38 ml of 3% are taken tinted solution of methylene blue acetic acid, which is necessary to cause the destruction of red blood cells. Blood collection must be done with great accuracy, since with such small volumes, the entry of an air bubble or residual blood on the outside of the pipette leads to an increase in the determination error. Before filling the chamber, it is necessary to wipe there is ground glass in the chamber so that rainbow rings appear. Before filling the chamber, the diluted blood is thoroughly mixed, as the cells settle on the walls of the test tube or mixer ampoule, after which a drop of blood is introduced under the ground ground glass of the chamber and left for 1 minute alone for the cells to settle .

Counting of formed elements is carried out at low microscope magnification (8X objective, 15X or 10X eyepiece) in a darkened field of view.

Red blood cells are counted in 5 large squares (16x5 = 80 small squares) located in different parts of the chamber; you can take squares located diagonally. Red blood cells lying inside the square and those on the upper and left sides of it are subject to counting; red blood cells lying on the bottom and right sides are not counted, since they already belong to other squares.

By counting the number of red blood cells (A) in 5 large squares, find the arithmetic mean number of red blood cells in one small square A/80, i.e. in 1/4000 µl. Therefore, to find the number of red blood cells in 1 μl, we must multiply the number obtained from division by 4000 and by 200 (dilution).

Thus we get the following formula:

X=A*4000*200/80,

where X is the number of red blood cells in 1 μl, and A is the number of red blood cells in 5 large squares. If we counted red blood cells in 5 large squares and diluted the blood 200 times, then the total multiplier for the number A will be 10,000.

The normal content of red blood cells in the blood of women is 4-5 * 106, men - 4.5-5.5 * 106.

Leukocytes are counted in 100 large squares, not divided into small ones, which corresponds to 1600 small ones. Thus, the number of leukocytes found in 100 squares is divided by 1600, multiplied by 4000 and multiplied by 20 (dilution). In this case, to obtain the result, it is enough to multiply the number of counted leukocytes by 50. The normal content of leukocytes in the blood is 5 * 103 - 8 * 103 in 1 μl.

Blood color index. The color index of blood is a number that shows the average hemoglobin saturation of an individual erythrocyte of a given blood compared to the saturation of an individual erythrocyte of normal blood. The normal hemoglobin content is taken to be 100 units, and the normal number of erythrocytes is 5,000,000. The value of the color index of healthy people varies from 0.9 to 1.1. Study of a peripheral blood smear. In blood smears, the morphology of red blood cells is studied and the leukocyte formula is calculated, i.e. the percentage ratio between different types of leukocytes. In order for the study to be successful, the preparation, fixation and staining of blood smears must be carried out with great care. To prepare a smear, use a clean, fat-free glass surface at a distance of 0.5 cm from the edge of the slide, touch the drop of blood at the puncture site on the finger, and then place the ground cover glass at an angle of 45° to the slide and bring the first to the drop of blood so that it spreads along the back edge of the cover slip and the lungs movement, without sharp pressure, make a smear. The smear should end in a “broom” on the slide. The smear is dried in air; when dry, a good, thin smear has a yellow color.

With a simple, sharpened pencil, the patient's name and the date of the study are written in the middle of the smear, after which the smears are fixed in methyl alcohol for at least 5 minutes and stained using the Romanovsky-Giemsa method.

The paint consists of a mixture of acidic (eosin) and basic (Azur II) dyes. This staining method makes it possible to differentiate cells. The first stage of working with a smear is to assess the morphology of red blood cells. To do this, choose a thin place where the cells lie separately, and not in the form of coin columns. Normal erythrocytes are anucleated, pink-stained cells, round, approximately the same diameter - 7.5 microns, erythrocytes have the shape of a biconcave disk, therefore, in the smear they have clearing in the center and a more intensely colored periphery.

(module direct4)

Preparing a thick drop

To test blood for Plasmodium malaria, a thick drop is made. Blood is taken in the usual way from the flesh of the finger. A drop of blood protruding from the injection site is touched with the surface of a glass slide. 2-3 drops applied separately are smeared with the corner of another glass. The dry smear is poured (without fixation) with Romanovsky paint for 30-40 minutes, then the painted drop is carefully rinsed with water and the preparation is dried in a vertical position.

Sample analysis data

An increased number of erythrocytes and hemoglobin in the blood can occur with a disease of the red elements of the blood - erythremia, then the number of erythrocytes reaches 9-106 or more. An increased content of erythrocytes in the blood can occur secondarily, that is, when there is no disease of the red leaf of hematopoiesis, and the number of red blood cells increases as a result of diseases of other organs and systems (with decompensated diffuse pneumosclerosis, pulmonary emphysema, some types of congenital heart defects, vascular sclerosis of the pulmonary artery system, right heart defects, circulatory failure of the third degree, etc.). This symptom is called erythrocytosis.

Morphologically altered red blood cells appear in hyperchromic (megaloblastic) anemia. In this case, large red blood cells with a high content of hemoglobin (macrocytes), embryonic red blood cells (megaloblasts), which are usually not found in peripheral blood, are found in the blood. The morphology of erythrocytes also changes in hypochromic anemia: small erythrocytes (microcytes) appear that are changed in shape (poikilocytes) and erythrocytes with a low hemoglobin content (hypochromic erythrocytes).

Leukocyte count

When calculating the leukocyte formula, it is necessary to determine the structural features of the cytoplasm and nuclei of cells. The belonging of a cell to one group or another is determined based on the totality of all the characteristics of the cytoplasm and nucleus. When calculating the formula, one must adhere to the same method of moving the glass under a microscope. The most commonly used method is microscopy in 4 places of the smear. It is known that leukocytes are distributed unevenly in a smear: there are fewer lymphocytes at the edges than in the middle, and there are more monocytes at the end of the smear than at the beginning. Therefore, when calculating the leukocyte formula, it is best to move along a broken line, counting all the cells encountered. Counting 200 cells is the practical minimum for routine clinical studies. Peripheral blood leukocytes, depending on the presence or absence of granularity in the cytoplasm, are divided into granulocytes (neutrophil, eosinophil, basophil) and agranulocytes (monocytes and lymphocytes).

Neutrophils. Cell size is 10-12 microns. The cytoplasm of the cells is pale pink with fine, abundant, purple granularity. Normally, band (2-4%) and segmented (60-65%) neutrophils are found in the blood.

Eosinophils. The cells are the same in size as neutrophils, sometimes a little larger, the cytoplasm is filled with large yellowish-red granules, the nucleus usually has two segments of the same size. Eosinophils are normally 2-4%. Basophils. This is the smallest granulocyte in size. The nucleus is irregular, multi-lobed, occupies almost the entire cell, the pale pink cytoplasm contains large dark purple granules. Basophil granules dissolve in water, and sometimes, as a result of washing off when coloring the preparation, colorless cells remain in place of the granules. Normal basophils are 0.1%. Lymphocytes. The cell size ranges from 7 to 10 microns. The kernel has a compact structure, round or bean-shaped. The cytoplasm of the cells is pale blue with a clearing zone around the nucleus (perinuclear), sometimes individual azurophilic grains of red-violet color are found in the cytoplasm. In peripheral blood, there are normally 20-35% lymphocytes. Monocytes. Cell size ranges from 12 to 20 microns. The core is often horseshoe-shaped, sometimes irregular in shape. The cytoplasm is more extensive than that of lymphocytes, ash-blue in color with fine, delicate, reddish granularity. Monocytes are normally 6-8%.

An increased level of leukocytes in the blood is called leukocytosis, and a decreased level is called leukopenia.

Reticulocyte staining and counting

Reticulocytes are young red blood cells with a thin blue retina or granularity in the cytoplasm. These cells characterize the activity of red hematopoiesis. To determine the count of reticulocytes, the method of supravital (intravital) staining is used. Smears of brilliant cresyl blue paint are made on the slides in absolute alcohol, and then a blood smear is made on the paint-coated glass in the usual way and placed in a humid chamber for 3-5 minutes, after which it is dried and microscoped with an immersion lens. Normally, there are 8-10 reticulocytes per 1000 red blood cells, their number is usually expressed as a percentage (0.8-1%) or in ppm (8-10% o) in relation to red blood cells. Reticulocytes are cells that characterize increased production of red blood cells in bone marrow.

They appear in a large percentage in the peripheral blood with hypochromic anemia (“pernicious anemia”), with hemolytic anemia and other diseases. A reduced content of reticulocytes and their complete disappearance in the peripheral blood are observed during exacerbation of hyperchromic anemia.

To prevent aggregation (sticking together) of blood platelets, a skin puncture is made through a drop of a 14% solution of magnesium sulfate applied to the finger. The blood is mixed with magnesium and thin smears are prepared on glass slides, which are then fixed and stained according to Romanovsky for 2 hours. The number of blood platelets per 1000 red blood cells is determined, and knowing the number of red blood cells in 1 μl, the number of platelets in 1 μl of blood is calculated. Normal platelet counts range from 250,000 to 400,000.

Determination of ESR

The erythrocyte sedimentation rate is determined in blood mixed in a 4:1 ratio with sodium citrate. The reaction is performed in a Panchenkov apparatus. The Panchenkov capillary is washed with sodium citrate, then the citrate is taken up to the 50 mark, where the letter P (reagent) is written, and it is blown into a Vidalev test tube. Using the same capillary, blood is taken twice up to the K mark (blood) and mixed with citrate. The same capillary is filled with blood mixed with citrate to division 0 and placed vertically in a tripod for an hour. After an hour, the size of the plasma column formed above the settled erythrocytes is noted in millimeters, which is a measure of the erythrocyte sedimentation rate. Normally, ESR in men is 10 mm/h, in women - 14 mm/h.

The erythrocyte sedimentation rate increases in inflammatory, acute and chronic diseases, in malignant tumors and other diseases.

Rh factor compatibility test

2-3 ml of the recipient's blood is taken into a test tube without citrate, after blood clotting, the clot is circled with a glass rod and the blood is centrifuged. Two drops of serum from this test tube are applied to a Petri dish, half a drop of donor blood is added to it, mixed and the dish is placed in a water bath (42-45°). After 10 minutes, the cup is removed and examined in the light with gentle rocking. The appearance of agglutination will indicate the inadmissibility of transfusion of this blood.

Determination of reticulocyte count– a clinical blood test that counts young red blood cells produced by the bone marrow. The study complements the general blood test. Reticulocyte counting is performed for the differential diagnosis of various types of anemia, assessing the functioning of the bone marrow and spleen, monitoring the conditions of patients with iron deficiency, vitamin B12 or folic acid deficiency, renal failure, and cancer. Blood is taken for research from a vein or capillaries. The reticulocyte count is determined using the flow cytometry method. Reference values ​​for men are 23-70 thousand/µl, for women – 17-63.8 thousand/µl. The analysis timeframe does not exceed 1 business day.

Reticulocytes are the precursors of red blood cells. They are produced by the bone marrow when stem cells differentiate and divide. Most of the reticulocytes are located in the bone marrow; mature red blood cells mainly enter the bloodstream. The proportion of reticulocytes in the blood is about 0.5-2%. When performing the analysis, the number of cells of this type and their percentage of the total number of red blood cells are determined. The results allow us to evaluate the production of red blood cells in the bone marrow and determine the degree of activity of this process.

The body maintains a stable level of red blood cells in the blood. When their quantity decreases as a result of hemolysis, impaired synthesis, bleeding, compensatory mechanisms are activated to restore normal concentrations. By changing the level and percentage of reticulocytes, it is possible to determine whether the production of red blood cells in the bone marrow is increased or whether this process is inhibited. If the bone marrow is functioning normally, the loss of red blood cells leads to a subsequent increase in their number and the number of reticulocytes. A decrease in the level of these cell types occurs when the red bone marrow is damaged by a tumor, metastases, radiation therapy and chemotherapy. To carry out the analysis for reticulocytes, the biomaterial is capillary or venous blood. The study is performed using a hematology analyzer using flow cytometry. The results are used in general therapeutic practice, hematology, pediatrics, infectious disease, and surgery.

Indications

The reticulocyte test is used to evaluate the production of red blood cells by the bone marrow. The results of the study allow us to differentiate different types of anemia. Based on the level of reticulocytes, they are divided into hyporegenerative and regenerative. In the first case, test values ​​are reduced, red blood cell production is insufficient, and bone marrow function is depressed. This group includes hemolytic anemia in chronic diseases, general intoxication, cachexia, deficiency of proteins, iron, and vitamins. Regenerative anemia is accompanied by a compensatory increase in the level of reticulocytes against the background of increased destruction or loss of red blood cells. This group includes posthemorrhagic and some hemolytic anemias. A reticulocyte test can be used to determine the severity of anemia, then its results are supplemented by indicators of the level of red blood cells, hemoglobin and red blood cell indices.

Determination of the level of reticulocytes is indicated for reduced levels of red blood cells, hemoglobin, and complaints of frequent fatigue, headaches, shortness of breath, blood in the urine or stool. When anemia is diagnosed, an analysis is prescribed to assess the effectiveness of treatment. It is especially often used to monitor anemia caused by deficiency of iron, vitamin B12 or folic acid, as well as developing against the background of renal failure and chronic diseases. To test bone marrow function, a reticulocyte test is indicated when red blood cell counts are elevated to determine the cause of polycythemia.

A blood reticulocyte test is not used to make a diagnosis. Its main purpose in clinical practice is to draw up a plan for further diagnostic procedures and monitor the success of therapy. The number of reticulocytes in the blood is relatively stable, therefore, when the level of red blood cells and hematocrit decreases, the analysis indicators become artificially high. This can be avoided by calculating the reticulocyte index - the absolute number of reticulocytes (the percentage of reticulocytes multiplied by the hematocrit). Another feature of this study is that the results indicate recent bone marrow activity, as immature red blood cells circulate in the blood for 2 days.

Preparation for analysis and collection of material

It is recommended to donate blood for reticulocyte analysis in the morning, on an empty stomach. The minimum interval between food intake and the procedure is 3-4 hours. At least 24 hours in advance, you must stop intense physical activity, drink alcohol, and avoid exposure to stress factors. When prescribing a test, it is worth telling your doctor about the medications you are using, as some drugs affect the level of reticulocytes in the blood. 30 minutes before the collection procedure, you need to stop smoking and eliminate physical and emotional stress.

The level of reticulocytes is determined in capillary or venous blood. Accordingly, the sampling is made from the ring finger or from the ulnar vein. The blood is placed in a tube with an anticoagulant and sent to the laboratory. Currently, hematological analyzers are used almost everywhere to count reticulocytes. The study is performed using flow cytometry - cells in a fluid flow move through a very narrow capillary and are counted when irradiated with a laser. Analysis execution time – 1 day.

Normal values

Reticulocyte test results display the absolute number of cells per unit volume of blood and the percentage of the total number of red blood cells. Normally, in female patients, the reticulocyte level ranges from 17*109 to 63.8*109 cells/l, in male patients – from 23*109 to 70*109 cells/l. The proportion of cells of this type in the total number of red blood cells changes with age:

• from birth to 2 weeks – from 0.15 to 1.5%;
• from 2 weeks to 1 month – from 0.45 to 1.4%;
• from 2 to 6 months – from 0.25 to 0.9%;
• from 6 months to 2 years – from 0.2 to 1%;
• from 2 to 6 years – from 0.2 to 0.7%;
• from 6 to 12 years – from 0.2 to 1.3%;
• from 12 to 18 years old - from 0.12 to 2.05% for girls, from 0.24 to 1.7% for boys;
• from 18 years of age - from 0.59 to 2.07% for women, from 0.67 to 1.92% for men.

A physiological increase in the level of reticulocytes in the blood occurs during oxygen starvation caused by ascent to a high altitude or descent under water, prolonged stay in an unventilated room, smoking, or drinking alcohol. In addition, an increase in the concentration of these cells is sometimes detected in pregnant women and is considered as a normal variant.

Level up

The cause of an increase in the level of reticulocytes in the blood may be bleeding. The concentration of red blood cells decreases; in order to restore it, the bone marrow increases the production of their precursors. In acute bleeding, the number of reticulocytes increases on the 3rd or 4th day; in chronic bleeding, it remains persistently elevated all the time. In diseases accompanied by hemolysis (destruction of red blood cells), the reticulocyte count is significantly higher than normal, sometimes the deviations reach 300%. The most common pathologies of this group are hereditary defects of erythrocytes, their autoimmune destruction, and toxic damage due to malaria. Other reasons for increased reticulocyte levels may be polycythemia, inflammatory processes, bone marrow cancer or the spread of metastases into it, restoration of bone marrow function after radiation and chemotherapy. In the treatment of anemia, normalization of reticulocyte concentration indicates the success of therapeutic measures.

Level reduction

The cause of a decrease in the level of reticulocytes in the blood may be anemia caused by a deficiency of iron, vitamin B12 or folic acid. In such cases, the production of blood cells is reduced due to a lack of “building material”. Another reason for a decrease in the level of reticulocytes is anemia that develops against the background of bone marrow damage or suppression of its functions. Changes of this type are determined by alcohol intoxication, decreased activity of the thyroid gland, chronic infections, autoimmune pathologies, kidney diseases, exposure to toxic substances, including medications, and radiation.

Treatment of abnormalities

In clinical practice, a reticulocyte test is most often prescribed after a general blood test when red blood cells and hemoglobin levels decrease. The reticulocyte level allows you to evaluate bone marrow function, red blood cell synthesis, determine the type of anemia, and monitor the effectiveness of its treatment. If the results of the study deviate from normal, you should seek advice from your attending physician - general practitioner, pediatrician, hematologist, or surgeon. To prevent the influence of physiological factors on the number of reticulocytes, it is necessary to exclude situations that cause oxygen starvation - ascents to heights or descents under water, prolonged stay in a polluted or stuffy room, smoking, drinking alcohol.

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