Common features of hemolytic anemias are as follows:

• Increased rate of red blood cell destruction

• Variable degree of intensity of hemolysis

 • Transient marrow response, increasing up to six- to eight-fold on baseline erythropoiesis

• Progressive decrease in the marrow’s ability to respond to demands

• Reduction in erythrocyte survival up to 15–20 days without anemia (compensated state, limited in time and variable in magnitude)

The wide variety of hemolytic conditions makes it necessary to proceed systematically, subdividing hereditary and acquired disorders on an etiopathogenetic basis (Table 1). Membrane defects represent the most numerous groups of hereditary hemolytic conditions, and among them, hereditary spherocytosis is relatively more frequent. The main clinical features to be detected and evaluated in the hereditary forms are listed below.

Table1. Etiopathogenetic classification of hemolytic anemias

• Degree of anemia:

– Absent, sometimes in crisis

– Variable, moderate to mild (compensation)

– Severe with cardiovascular syndrome

 • Presence of jaundice:

– Neonatal– Persistent, episodic, absent

– Acholuric, no itching

• Episodes of aplastic crisis:

– From human parvovirus type B19 (HPV) → inhibition CFU-Es

– Hb drop of 20–60 g/L

– Erythroid hypo/aplasia (folate deficiency)

• Splenomegaly

• Cholelithiasis ➔ it is often the first symptom

 • Lower limb ulcers, especially in cases of spherocytosis and sickle cell trait

 • Skeletal changes due to erythroid expansion in the marrow

Hemolytic crises can be characterized by acute onset (e.g., after a noncompatible transfusion) similar to those of acute febrile illness with pain in the back, abdomen, headache, vomiting, oligo/anuria, pallor, tachycardia, or subacute onset with gradual and insidious progression, with few symptoms, good compensation at least initially, jaundice, pallor, then crises with worsening. From the point of view of the clinical laboratory, the diagnostic indicators should be sought in different directions, as reported in Tables 2, 3, and 4.

Table2. Hemolytic disorders: signs of increased red blood cell destruction

Table3. Hemolytic disorders: signs of intravascular hemolysis

Table4. Hemolytic disorders: signs of accelerated erythropoiesis

In hemolytic conditions, direct and indirect antiglobulin tests (the Coombs test) should always be performed. However, it should be considered that 2–5% of patients with anemia of immunohemolytic origin are falsely negative due to the reduced sensitivity of the test.

The evaluation of erythrocyte osmotic fragility is based on tests that are parallel to those described for the screening of thalassemias; however, the mechanism and meaning are completely different, which makes these fragility tests useful for screening and therefore sensitive but little specific. In these tests, the principle is always that of hemolysis for osmotic stress, obtained either with progressively hypoosmotic solutions of NaCl or with solutions of glycerol in the acid buffer. Hemolysis begins when the erythrocyte surface/ volume ratio reaches a critical volume, which in a normal subject is ~165%, and occurs at a NaCl concentration of 0.45–0.50% to reach a 50% hemolysis value at 0.40–0.45% NaCl. If hemolysis is observed at higher NaCl concentrations, this is referred to as increased fragility due to the shift of the hemolysis curve to the left (Fig. 1). A normal erythrocyte swells when placed in 0.6% NaCl; hemolysis begins at 0.42% NaCl when the erythrocyte volume rises to ~145% of normal; in 0.35% NaCl, hemolysis is complete. In borderline cases, the test can be made more sensitive by per forming it on a blood sample preincubated at 37 °C for 24 h under sterile conditions. The test is positive for conditions associated with hereditary spherocytosis. Evaluation of erythrocyte osmotic fragility is also performed with variants of the glycerol test, previously described for thalassemias, using an acidified glycerol solution. Additional commercial tests are the Pink Test and the Osmored B.

Fig1.  Osmotic fragility test in progressively hyposmotic NaCl solutions of a sample from a normal subject and one from a subject with hereditary spherocytosis before and after incubation. (Copyright EDISES 2021. Reproduced with permission)

The self-hemolysis test, once widely used for the evaluation of erythrocyte fragility but now less practiced, is based on the evidence of spontaneous hemolysis after incubation of sterile defibrinated blood at 37 °C for 48 h in the presence of two parallel tubes containing glucose and ATP. In spherocytosis and various enzyme deficiency conditions, hemolysis is corrected by both additions; however, the test is not very sensitive and specific.

Various hemolytic conditions show the presence of Heinz bodies. These are bodies of denatured hemoglobin adherent to the erythrocyte membrane. Causal conditions include glucose- 6-phosphate dehydrogenase (G6PD) deficiency, the presence of unstable hemoglobins, some hemolytic thalassemias, and some chemical agents. Heinz bodies are visible microscopically after staining with supravital staining (e.g., methyl violet). In enzymatic deficiency conditions, their presence is accentuated by incubation with in vitro oxidizing agents (e.g., acetylphenyl hydrazine).

The glutathione stability test evaluates the residual reducing capacity of the erythrocyte under conditions of oxidative stress. Glutathione is mainly present in the erythrocyte in its reduced form (GSH), at concentrations in the reference range of 5.3–6.8 μmol/g Hb. Under normal conditions, these values vary little, even under oxidative stress (e.g., with acetylphenyl hydrazine). In enzyme deficiencies, particularly G6PD deficiency, GSH falls to values near zero.

Other laboratory tests generally used in hemolytic conditions include measurement of intrinsic enzyme activities of erythrocytes, assessment of hemoglobin balance when unstable hemoglobins are suspected, and Ham’s test for paroxysmal nocturnal hemoglobinuria (PNH), a membrane structure disorder, performed to reveal increased sensitivity of cells to complement lysis.

The diagnostic approach to hemolytic anemias involves the establishment of two conditions, the first of which is pre liminary: (1) demonstration of the presence of the hemolytic state and (2) determination of the specific cause of hemolysis. In the differential diagnosis of hemolytic conditions, it is important to consider conditions that may mimic the presence of hemolysis (Table 5).

Table5. Conditions mistaken for hemolytic state

Once the presence of hemolysis has been demonstrated, the process of identifying the specific cause must begin with an accurate medical history, a microscopic examination of the blood smear, and antiglobulin testing. Based on the data obtained, patients can be distinguished into five groups:

 1. Patients with exposure to hemolytic agents.

 2. Patients with positive antiglobulin tests.

 3. Patients with a negative antiglobulin test are likely to have spherocytosis.

 4. Patients with morphological abnormalities (ellipsocytes, schistocytes, ovalocytes, etc.).

5. Patients with a negative antiglobulin test and normal morphology, on which to proceed with investigations on erythrocyte enzymes, hemoglobin balance, PNH tests, etc.

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مواضيع ذات صلة

liquid biopsy (Fluid biopsy, Fluid phase biopsy)

lipoproteins (High-density lipoproteins [HDLs, HDL-C], Low density lipoproteins [LDLs, LDL-C], Very-low-density lipoproteins [VLDLs], Lipoprotein electrophoresis, Lipoprotein phenotyping, Lipid fractionation, Non-HDL cholesterol, Lipid profile)

lipoprotein-associated phospholipase A2 (Lp-PLA2 PLAC test)

Sickle Cell Anemia (Hemoglobinopathy S, Sickle Cell Disease)

Laboratory Diagnosis (Phenotypic) of Thalassemias

Pathophysiological and General Clinical Aspects of β-thalassemias

lipase (Pancreatic lipase, Lipoprotein lipase)

leucine aminopeptidase (LAP)

Legionnaires disease antibody test

Sideropenic and Assimilated Anemia

Automated Differential Count

Differential Leukocyte Count (Leukocyte Formula)