Diagnosis of megaloblastic anemias

When collecting the patient's anamnesis, attention is paid to:

• long-term use of antibiotics and anticonvulsants;
• diet/nutrition type;
• presence and duration of diarrhea;
• surgical interventions on the gastrointestinal tract.

A complete blood count reveals:

• anemia;
• increase in erythrocyte indices (MCV - can be up to 110-140 fl, RDW);
• erythrocyte macrocytosis;
• many macroovalocytes;
• pronounced anisopoikilocytosis of erythrocytes;
• the presence of Jolly bodies and Cabot rings;
• leukopenia (up to 1.5x10 ⁹ /l);
• hypersegmentation of neutrophil nuclei (5 or more segments);
• thrombocytopenia (up to 50x10 ⁹ /l).

When examining the bone marrow, signs of megaloblastic hematopoiesis are found:

• the cells are large;
• nuclei are granular, striated;
• the cytoplasm of the cells is more mature compared to the nucleus;
• dissociation of the nucleus and cytoplasm is more pronounced in more mature cells;
• cells containing nuclei with weakly condensed chromatin are found;
• multiple, sometimes pathological mitoses;
• nuclear remnants, Jolly bodies;
• cells containing 2 or 3 nuclei;
• qualitative disorders of erythropoiesis;
• huge (giant) metamyelocytes with horseshoe-shaped nuclei;
• hypersegmentation of neutrophils;
• multinucleated megakaryocytes.

Urine analysis results reveal persistent proteinuria (a sign of specific impairment of vitamin B12 absorption _in the ileum). Determine:

• _{Vitamin B 12} level in blood serum: normal values - 200-300 pg/ml;
• serum folate level: normal value - more than 5-6 ng/ml (low - less than 3 ng/ml, borderline - 3-5 ng/ml);
• folate level in erythrocytes: normal value - 74-640 ng/ml;
• orotic acid excretion level for the diagnosis of oroturia.

The deoxyuridine test is performed to differentiate between vitamin B12 _and folate deficiency.

The Schilling test is performed to determine IF activity and absorption of vitamin B12 _in the intestine.

The Schilling test using a commercial IF is performed in case of an impaired simple Schilling test to differentiate between IF pathology and a specific malabsorption of vitamin B12 _in the ileum (Imerslund-Gräsbeck syndrome) or transcobalamin II deficiency. In case of superimposed bacterial infection, the test should be repeated after a course of antibacterial therapy (after treatment with tetracycline, the test usually returns to normal).

A study is conducted of gastric acidity (initial and after stimulation with histamine), the content of IF in gastric juice (including after the addition of hydrochloric acid to detect antibodies to IF in gastric juice), and a biopsy of the gastric mucosa.

Antibodies to IF and parietal cells are determined in the blood serum.

In addition, the level of holotranscobalamin II in the blood serum is determined: in case of vitamin B ₁₂ deficiency, the concentration of holotranscobalamin II (cobalamin associated with transcobalamin II) is significantly lower than normal values, which precedes a decrease in the level of total cobalamin in the blood serum.

The concentration of methylmalonic acid and homocysteine is determined in the blood serum and urine: in case of folate deficiency, the content of methylmalonic acid is within the normal range, while homocysteine is elevated.

To diagnose congenital methylmalonic aciduria, it is possible to determine methylmalonate in the amniotic fluid or in the urine of the pregnant woman.

Malabsorption tests are performed.

Alizerin red is used for differential diagnostics between vitamin B ₁₂ deficiency and folic acid deficiency. When staining bone marrow smears, megaloblasts formed due to vitamin B ₁₂ deficiency are stained, not folic acid deficiency.

Absorption of vitamin B ₁₂ is assessed by the Schilling test, which uses a radioactive vitamin. The patient ingests a small amount of vitamin B ₁₂ labeled with ^57Co; in the body, it combines with the intrinsic factor of the gastric secretion and enters the terminal ileum, where it is absorbed. Since the absorbed vitamin becomes bound to proteins in the blood and tissues, it is not normally excreted in the urine. A large dose (1000 μg intramuscularly) of non-radioactive vitamin is then administered parenterally to saturate circulating cobalamin-binding proteins (transcobalamins I and II) and ensure maximum excretion of the radioactive vitamin absorbed from the intestine in the urine. Normally, 10-35% of the previously absorbed vitamin appears in the daily portion of urine; in patients with cobalamin deficiency, less than 3% of the administered dose is excreted. The association of malabsorption of the vitamin with the absence of intrinsic factor can be confirmed by a modified Schilling test: the radioactive vitamin is administered together with 30 mg of intrinsic factor. If the malabsorption of vitamin B ₁₂ is caused by the absence of intrinsic factor, the radioactive vitamin is absorbed in adequate quantities and excreted in the urine. On the other hand, if the malabsorption of the vitamin is due to an abnormality of the receptor zones in the ileum or other causes associated with the intestine, the absorption processes of the vitamin are not normalized after the introduction of the intrinsic factor. If the absorption disorder is not compensated for, the test can be repeated after a course of broad-spectrum antibiotics (suppression of bacterial overgrowth) and subsequent use of pancreatic enzymes (correction of pancreatic insufficiency). The Schilling test is indicative only with careful urine collection. The Schilling test for detecting malabsorption of vitamin B ₁₂ is not used in children, since it involves the introduction of a radioactive drug into the body.

To exclude folate malabsorption, the following test is performed: the patient receives 5 mg of pteroylglutamic acid orally, which leads to an increase in the level of folate to 100 ng/ml within an hour. If the level of folate in the blood serum does not increase, folate malabsorption is considered proven.

Examination plan for a patient with megaloblastic anemia

1. Tests confirming the presence of megaloblastic anemia.
   1. Clinical blood test with determination of the number of reticulocytes and morphological characteristics of erythrocytes.
   2. Biochemical blood test, including determination of bilirubin and its fractions, serum iron.
   3. Myelogram.
2. Tests to clarify variants of megaloblastic anemia.
   1. Morphological examination of bone marrow smears stained with aliserin red.
   2. Special methods:
      1. determination of the concentration of vitamin B ₁₂ in blood serum;
      2. determination of folic acid concentration in blood serum and erythrocytes;
      3. urinary excretion level of methylmalonic acid;
      4. urinary excretion level of formi-glutamic acid.
3. General clinical examination to clarify the cause of anemia: urine and stool tests, endoscopic examination, determination of gastric juice secretion, X-ray and morphological (as indicated) examination of the gastrointestinal tract, specialist examinations, all examinations according to individual indications.

[ 1 ], [ 2 ], [ 3 ], [ 4 ], [ 5 ], [ 6 ], [ 7 ], [ 8 ]