Chapter 4 Leukocyte Differential in Undiluted Whole Blood
4.1 Introduction
4.1.1 Methods of leukocyte differential
Leukocyte differential currently can be performed either manually or by conventional automated blood analyzers. Manual leukocyte differential analysis began over 100 years ago, when Paul Ehrlich stained cell nucleus and cytoplasm granules with aniline dyes developed for the textile industry and found that leukocytes show different colors [3]. Manual blood analysis requires making a blood smear or the use of hemacytometers and then counting by a trained professional. One of the Romanowsky stain (i.e., Giemsa, Jenner, Wright, or Leishman stain) procedures, which are based on cell affinity for neutral, basic, or acidic dyes, is normally used in the procedure for a 5- part differential. It is time consuming, labor intensive, and error prone.
Between the manual blood smear count and automated flow-cytometer-based conventional blood counters, there were also attempts to use a computer-controlled microscope to differentiate leukocytes based on structure and coloration of nucleated blood cells which had been fixed and stained in a blood smear [4−9]. They were largely unsuccessful in practical diagnostic settings due to their inability to improve precision under the requirement of substantial throughput increase, and the inability of algorithmic structural identification of leukocyte subtypes to replace the professional morphologist.
Automated blood counters are usually based on flow cytometry and employ one or a combination of electrical impedance sensing, light scattering measurement, and chemical or immuno staining followed by optical sensing. In electrical impedance sensing, RBCs are removed by lysing reagent saponin, or some more sophisticated lysing reagent including surfactants. During the lysing process, the leukocyte cell volume changes depending on cell type, due to the leakage of cytoplasm contents and cell
nucleus shrinkage in varying amounts [10]. Thus normally 2-part (lymphocytes versus granulocytes) or even 3-part (lymphocytes, neutrophils, and other leukocytes) leukocyte differential can be achieved by simply electrical impedance measurement of particle volume [11−15]. Combining DC and AC impedance, special acidic hemolysis in basophil channel and alkali hemolysis in eosinophil channel, a 5-part leukocyte differential can be achieved [16]. Due to the dynamics of the lysis procedure, dilution, mixing, temperature, and time need to be perfectly controlled, so fully automated instruments are required for reliable 3-part and 5-part differential. Later optical methods based on light scattering and fluorescence staining of organelles, granules, and nuclei becomes an alternative method. Generally, low-angle scattered light contains information on cell size and high-angle scattered light can be used to probe internal composition of the cell. To achieve 5-part differential, eosinophils requires some special stain to change its scattering characteristics from other granulocytes, and basophils need to be counted separately after differential lysis of other leukocytes [1, 10]. The first flow- cytometer-based automated optical leukocyte differential instrument, Hemolog-D by Technicon, uses three different channels: lymphocytes, neutrophils, and eosinophils classified by myeloperoxidase staining; monocytes identified by staining intracellular nonspecific esterase; and basophils identified by Alcian blue staining [17]. Later, Coulter Electronics, Inc., combined electrical impedance and light scattering and developed their VCS technology, which uses DC resistance sensing to measure cell volume; AC impedance sensing to collect information on cell size and internal structure (including chemical composition and nuclear volume); and light scattering for cellular granularity, nuclear lobularity, and cell surface structure [4]. Generally speaking, conventional
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automated blood analyzers are bulky, expensive, and mechanically complex. They require larger sample volumes and generate more waste than the systems developed using microdevices. They are usually found only in hospitals or central laboratories, and it normally takes from hours to even days for a patient to get the results.
Micro blood counters promise to provide a point-of-care solution. They cost less and can be more accurate than a manual count. Particle (bead, erythrocyte, and cultured cell) counting has been demonstrated, for example, by electrical impedance sensing [18−21], light scattering detection [22, 23] and fluorescent sensing [21, 24−27] in microsystems. All of these previous studies use diluted samples. Here human leukocyte counting and differentiation with microfabricated devices from undiluted human blood is reported for the first time. Dilution is normally required as one of the sample preparation steps in blood cell counting and differentiation. There are several reasons for this. First dilution prevents the coincidence effect in which multiple cells appear in the detection zone simultaneously. Because of the ratio of erythrocytes to leukocytes is on the order of a thousand to one, for electrical impedance or light scattering detection of leukocytes, a blood sample dilution factor from one hundred to several tens of thousand is typically required to avoid erythrocyte interference. Even for counting leukocytes in samples where leukocytes are specifically fluorescent labeled, a dilution of at least ten times is performed. Secondly, dilution reduces the risk of sample clogging in the flow chamber.
This is a major concern for macroscopic blood counters, since the flow chamber is used in continuous mode and removing the clog or replacing the flow chamber can be difficult.
The third reason is that the whole blood is diluted during the chemical hemolysis process to remove erythrocytes, especially when electrical impedance or light-scattering detection
is employed. Hemolysis can produce inaccurately high leukocyte counts due to lysis- resistant erythrocytes, and inaccurately low counts because some leukocytes are lysed.
Finally in some protocols an additional fixation buffer is required to conserve the properties of leukocytes.