Classical hematology

For biochemical investigations, peripheral blood collected in EDTA (0.5–4 mL, depending on the test) is required. Most analyses (complete blood count, osmotic fragility testing of erythrocytes, enzyme activity measurements) should be performed within 48 hours after sampling; serum is required for assessment of iron status.

For human genetic testing, peripheral EDTA blood is used.

As classical hematology mainly deals with inherited disorders, written informed consent in accordance with applicable genetic testing regulations is required before performing human genetic analyses.

For all classical hematology requests, a complete blood count including red blood cell indices and reticulocyte count is performed, which facilitates the evaluation of anemia and the interpretation of the subsequent test results.

Hemoglobin electrophoresis separates and quantifies the different hemoglobin fractions to detect abnormal hemoglobins (structural variants such as HbS in sickle cell disease) and to provide evidence for an underlying thalassemia.

In suspected hereditary spherocytosis, binding of the fluorescent dye eosin‑5‑maleimide (EMA) to erythrocytes is measured by flow cytometry; fluorescence is typically reduced in patients with hereditary spherocytosis, but may show different patterns in other erythrocyte membrane disorders.

In routine practice, the PINK test is equivalent to the acidified glycerol lysis test (AGLT) and reflects the osmotic fragility of erythrocytes as a functional parameter of membrane defects, particularly in hereditary spherocytosis.

Enzyme activities can be measured in washed erythrocytes to detect enzyme deficiencies; however, these assays are only partially standardized and are susceptible to preanalytical influences. Therefore, molecular genetic testing is often used to identify sequence variants causing an enzyme defect, whereas enzyme activity measurements are reserved for the evaluation of sequence variants of uncertain clinical significance.

To identify sequence variants that cause hereditary anemia, different human genetic methods are applied. Next‑generation sequencing (NGS) is used to detect single‑nucleotide variants and small insertions/deletions (indels), while larger copy‑number variants (CNVs), such as deletions, can be identified using techniques like MLPA.

Depending on the patient’s phenotype and biochemical findings, candidate genes associated with the suspected disease group (hemoglobinopathy, membranopathy, or enzymopathy) are analyzed; if these targeted approaches do not reveal a causative variant, whole‑genome sequencing (WGS) can be used to search the entire genome for relevant alterations.

Human genetic methods are also used to clarify additional disorders within classical hematology, such as cyclic neutropenia, ACKR1/DARC‑associated neutropenia (ADAN), or familial erythrocytosis.