Hematology analyzers demystified (Proceedings)
The manual PCV and plasma protein are quick, reliable methods for determining blood volume and hydration status when performed correctly. However, more than a bare-bones level of hematologic information has been made available with advances in technology. Wallace H. Coulter revolutionized hematology when he developed an automated means of counting particles that resulted in a patent in the early 1950s. His concept ultimately became known as "The Coulter Principle". Around the same time, others were involved with refining flow cytometry for sizing cells. In the 1980s, rudimentary automated differentials were developed for screening purposes. Continued refinement has resulted in the waning of manual differentials in human medicine and advances in the human health care market have played a significant role in the development of automation for veterinary hematology.
The hematology technology today involves three main principles: centrifugal, impedance (the Coulter principle) and light scatter. Spectrophotometry is shared by impedance and light scatter analyzers for the determination of hemoglobin concentration. Impedance is the most commonly used method in the clinic setting although progress has been made towards a cost-effective light scatter analyzer. A simplified review of the methods is discussed here along with a brief overview of instrument selection, common interferences and quality assurance. The Quality Assurance and Lab Errors sessions provide more in depth discussions of interferences and QA.Centrifugal
The only veterinary centrifugal analyzer available is known as the QBC (Quantitative Buffy Coat) VetAutoread by Idexx. It uses the principle that cells form layers based on cell density when centrifuged in what resembles a giant hematocrit tube. Fluorescent dyes are used to stain lipoproteins, DNA and RNA to help differentiate the cells and the layers are amplified with the use of a float. This system performs reasonably well with normal samples and platelet estimates, and it can identify leukocytosis, the presence of reticulocytes and microfilaria. Since it measures the size of the layers, cell counts are estimated and cell sizes are not directly determined. Eosinophils are reportedly inconsistent in dog samples and there has been poor correlation with absolute reticulocyte counts.
Non-centrifugal sample processing
The sample is handled in a similar fashion by both impedance and at least some flow cytometry-based analyzers prior to the actual counting and sizing of cells: An aliquot of the blood sample is aspirated from the tube and divided into two portions. Analyzers typically use either a shear valve or a syringe displacement mechanism to obtain an aliquot of the sample. The aliquot is diverted into two parts: one to the "white blood cell (WBC) side" with a ~40-fold dilution in a reagent that lyses the erythrocytes for WBC counting and hemoglobin determination, and the second to the "red blood cell (RBC) side" for ~50,000-fold dilution in isotonic solution to allow RBC and platelet (Plt) counting and sizing. After this point, how the cells are counted and sized is unique for each technology.
Impedance analyzers can be obtained from multiple sources including Coulter (AcT), Abaxis (VetScan HM5), Heska (HemaTrue), scil (Vet abc), Drew Scientific (HemaVet 950) and others. These use electronic methods to count and size cells (the Coulter Principle). Known volume of diluted blood is pulled through a small aperture flanked on each side by electrodes. Each cell displaces an equivalent volume of ionic solution as it passes through the aperture and impedes the electrical current passing between the electrodes proportionately, creating an electronic "blip". Because a specific sample volume is analyzed and a detector tracks the number and size of each blip, cell concentrations and their sizes (volume) can be accurately determined. This data is converted to a plot of particle size vs. numbers in the form of a histogram.
On the "RBC" side, both platelets and RBCs are counted and sized. When histograms representing the volume in femtoliters (fl) vs. number of RBCs from several species are compared, human erythrocytes are relatively large (~ 80-98 fl), those of dogs are smaller (62-73 fl), RBCs of cats are smaller yet (37-50 fl) and goat RBCs are tiny (~12-20 fl). The Mean Platelet Volume (MPV) of most species typically falls in the goat erythrocyte size range. Impedance analyzers distinguish types of particles based on size only. In humans, because of the wide size difference between erythrocytes and platelets, these two cell types are easily distinguished. This is not always true in our animal species.