Hematology analyzers are mainly used for cell counts and differential leukocyte analysis, but these analyzers can also report several other parameters and provide much more information.
FREMONT, CA: Since its initial development in the 1960s, cell counting and characterization automation have revolutionized the results of hematology analysis. Although many detection techniques are still in use, since its implementation, optical technology has been a critical innovation in automated hematology assessment.
Light, scattered and identified at particular angles, captures a spectrum of cell size, composition, internal complexity, nuclear segmentation, and cytoplasmic granulation data. Innovative expansions of optical flow cytometry in specific have allowed laboratories to achieve outcomes of higher quality for more precise and quick clinical decision-making.
Electrical impedance, radio frequency (RF), optical light dispersion (optical flow cytometry), cytochemistry, and fluorescence are the most prevalent technologies. Optimum combinations of these detection techniques in a brief turnaround moment provide a precise automated complete blood count (CBC) differential, including white blood cell (WBC).
The Development of WBC Differentials
The automatic WBC differential gives the absolute and relative levels of the five distinct kinds of WBCs present in ordinary blood (neutrophil, eosinophil and basophil granulocytes, lymphocytes and monocytes). This offers data for diagnosing and evaluating infections, disorders of the immune system or bone marrow, and hemato-oncological illnesses. Traditionally, a stained blood smear has been used to determine the WBC difference by manually counting and classifying 100 or 200 WBCs. Although extremely imprecise, this technique is still regarded as the WBC differential reference method, and after an automated CBC, assessment can be conducted as a reflex test.
On automated hematology analyzers, WBC differentials are generally conducted today. Although the automated WBC differential is highly accurate and reliable in the absence of pathological cell types, the presence of immature or reactive cells, blasts, and other types of diseased cells is often required to confirm a manual differential.
Impedance and Cytochemistry
Electrical impedance is a method of cell counting and sizing based on measuring changes in the electrical impedance (strength) generated by a particle (i.e., a blood cell). Counting cells and measuring the volume of WBCs, red blood cells (RBCs) and platelets was the first automated technology. As cells pass through an opening of known size, the electrical conductance changes proportionally to the particle size.
Impedance technology can provide a three-part WBC differential in which cells are divided into three dimensions: lymphocytes, mid-range cells, and granulocytes, but it does not allow the granulocyte subtype to be differentiated. The restriction of this technology is that it is based on solely measuring cell size, so it is not possible to separate abnormal cells such as nucleated red blood cells (NRBCs), PLT clumps, gigantic PLTs or unlinked red cells and can interfere with ordinary cell populations.
The cytometry of optical flow offers several benefits over traditional techniques of impedance. A laser light beam is passed through a diluted blood specimen stream during optical light scatter measurement, which is projected through hydrodynamic focusing into the flow cell. As each cell moves, the concentrated light is dispersed in different directions and identified by photodetectors converting the signal into an electrical pulse. For storage and analysis, the electronic signals are transmitted to a computer.
The signals provide data on cellular features such as size, inner complexity, atomic lobularity/segmentation, and cytoplasmic granularity used for cell identification. Using sophisticated software algorithms, cells with comparable light scatter characteristics form a cluster in the scattergram. Some analyzers use only two light angles, while others use optical disperse assessment of multi-angle perspectives.
Multi-Angle Polarized Scatter Separation (MAPSS) technology utilizes four-light scatter detectors to identify different cellular characteristics. A depolarized light detector implementation is a distinctive feature of this technique and enables specific identification of eosinophil granulocytes. The four sensors generate the following signals:
• 0° or Axial Light Loss (ALL): related to size
• 0° to 10° Intermediate Angle Scatter (IAS): about cellular complexity
• 90° Polarized Side Scatter (PSS): about nuclear lobularity/segmentation
• 90° Depolarized Side Scatter (DSS): related to eosinophil granules.
These signals correlate with features of morphology that can be visually determined from a stained slide under the microscope. To classify the WBC subpopulations and provide morphological flagging, various combinations of these four measurements are used.
Hematology in the Future
There have been numerous promising directions that could positively impact the future of the automated WBC differential. The International Council for Standardization in Hematology (ICSH) has suggested a new leukocyte differential reference technique based on the use of monoclonal antibodies (mAbs) and multicolor flow cytometry. This would provide producers with precise and reproducible WBC classification for automated technology development and enhancement.
Another chance for enhancement is to incorporate extra light scatter measurements into the characterization of blood cells, to classify WBC subpopulations more accurately and identify potential immature or pathological cell types.