The Coulter principle
The Coulter principle states that particles pulled through an orifice together with an electric current, produce a change in electrical impedance that is proportional to the size of the particle traversing the orifice. This is based on the principle that cells are relatively poor conductors of electricity in relation to the diluent fluid. Initially, the original Coulter Counter counted and measured only red blood cells. Later, as the technology and instrumentation evolved, it enabled clinicians to also count and measure white blood cells. In the 1970s, further refinements enabled technologists to also separate the platelets.
Evolution of automated cell counters
Traditionally, the blood counts were performed manually using the hemocytometers and the differential leukocyte counts by studying the peripheral blood smears (also referred to as the 100-cell slide differential, eyecount leukocyte differential or manual counts). The Coulter principle led to the availability of the Coulter counters and thereafter, the development of sophisticated automated blood-cell analyzers. The level of sophistication has been rising ever since. Technological advancements have made it possible to incorporate increasingly more analysis parameters as possible into single instrument platforms, in order to minimize the need to run a single sample on multiple instruments. The modern versions of analyzers are capable of measuring white blood cells (WBC), WBC differentials (5 part differentials), red blood cells (RBC), hemoglobin (HGB), platelets, mean corpuscular volume (MCV), and mean platelet volume, and automatically calculating hematocrit (HCT), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), RBC distribution width, plateletcrit, and platelet (PLT) distribution width. The other crucial considerations in automatic analyzers are the speed with which they perform and the number of specimens they can process per batch (reduction in turnaround time due to high throughput).
Bone marrow cell counts and extended differential counts
Automated analyzers are being developed for analysis of bone marrow aspirates and peripheral blood to aid in the preliminary classification of peripheral blood and bone marrow disorders . Newer parameters are being made available as components of the extended differential count (hematopoietic progenitor cells, immature granulocytes, and erythroblasts), the immature reticulocyte fraction, the reticulocyte indices, the fragmented RBCs, and the immature platelet fraction .
Point-of-care testing (POCT) is becoming an important adjunct to hematologic laboratory practice. Analyzers with capability to perform 5 part WBC differential leukocyte counting are becoming available for use in the areas such as intensive care units .
Manual versus automated cell counting
Although peripheral blood smear examination provides information that cannot be obtained from automated cell counting, it has certain limitations and special considerations.
Limitations of manual cell counting
- Experience is needed to make technically adequate smears consistently.
- Non-uniform distribution of WBCs over the smear, with larger leukocytes concentrated near the edges and lymphocytes scattered throughout.
- There is a non-uniform distribution of red blood cells as well, with small crowded red blood cells at the thick edge and large flat red blood cells without central pallor at the feathered edge of the smear.
- It is subjective, labor-intensive, and statistically unreliable (only 100-200 cells are counted),
- It is imprecise with reported Coefficients of Variation (CV) ranging from 30 – 110 % .
- Cell identification errors in manual counting: This is mostly associated with distinguishing lymphocytes from monocytes, bands from segmented forms and abnormal cells (variant lymphocytes from blasts). The monocytes tend to be underestimated and the Lymphocytes tend to be overestimated.
Advantages of the automated analyzers
Cell counting with these instruments is rapid, objective, statistically significant (8000 or more cells are counted), and not subject to the distributional bias of the manual count. They are also more efficient and cost effective than the manual method. Some of these cell counters can process 120-150 samples per hour. In addition, the precision of the automated differential makes the absolute leukocyte counts reliable and reproducible.
Disadvantages of the automated analyzers
The automated hematology analyzers also may produce cell counts which are falsely increased or decreased. Some analyzers, particularly the impedance based counters check only the volume and number of particles and may not be able to correctly distinguish tiny clumps of platelets and nucleated red blood cells. Platelet clumps may be misclassified as leukocytes or erythrocytes, and nucleated red blood cells can be misclassified as leukocytes or, specifically, lymphocytes. Furthermore, large or unidentifiable atypical cells, toxic immature neutrophils, and markedly reactive lymphocytes can also be misclassified. Moreover, large or unidentifiable atypical cells, toxic immature neutrophils, and markedly reactive lymphocytes can also be misclassified.
Software-generated WBC suspect flags
These ‘flags’ are warnings generated and displayed by the machine to alert the laboratory personnel that the machine has detected some abnormality in cell population or distribution that needs attention. The machines are preprogrammed by the manufacturers to generate certain flags according to an internal algorithm. Some examples of these flags include, blast flag, atypical flag, flag for discrepancy of the two WBC counts, nucleated RBC (NRBC) flag, flag for immature granulocytes as well as others.
In the current state of the art of cell analysis, there are two technologies used for counting and classifying cells. These are generally known as "flow cytometry" and "image cytometry." The flow cytometry technology (based on the original Coulter principle), essentially consists of passing cells one at a time through a sensing zone of a flow cell, and cell counting and sizing is performed by calculating the changes in the electrical impedance or by the “optical” technique, in which scattering of a laser beam (‘light scatter’, ‘optical scatter’) by each individual cell is measured. Both methods use cell by cell analysis. Therefore, due to the high concentration of cells in whole blood, it is necessary to dilute the blood samples prior to analysis so that individual cells can be isolated for sensing within a flowcell. Modern cell analyzers are multi-channeled and perform automated hematology measurements simultaneously using different techniques. For example, the Cell-Dyn.RTM. 3000 instrument, uses "impedance" measurements to count and size RBCs and PLTs, "absorption" measurements to determine the concentration of hemoglobin in RBCs (MCH), and "optical scatter" measurements to count and classify WBCs and the five part differential. Newer, specialized type of flow cytometry techniques such as the Fluorescence-activated cell sorting, are now available and these are based upon incorporating certain substances known as “fluorophores” as labels into the cells. Immunological analysis for cell antigens (such as on the lymphocytes) is now possible by fluorescence flow cytometry using monoclonal antibodies.
For the RBC count and MCV measurements, a diluent is added that makes the red cells absorb fluid by osmosis until they become spherical in shape (without altering their volume). They are then analyzed. Because RBCs outnumber WBCs by about 700 to 1, for leukocyte analyses, it is desirable that all of the RBCs are removed from the solution. One way of doing this is to lyse them by adding a special solution which does not affect the WBCs and platelets. This ensures that it is no longer necessary to process over ten thousand RBCs for every single member of the less numerous leukocyte subpopulations. After the lyse reaction is completed, the machine washes the sample to remove any debris left over from lysed RBCs, which may significantly distort white cell counts. Now, the WBCs and PLTs can be analyzed. Other methods of isolating WBCs from a sample of diluted whole blood, by using magnetic particles that specifically bind to WBCs, agitating the sample at specified frequencies, and placing the sample in close proximity to a source of magnetic field have been introduced. The new methods provide for isolation of WBC preparations with high yield, purity, and viability. These methods have been designed for compatibility with automation protocols for rapid processing of multiple samples.
Three-part and five-part differentials
A basic understanding of this concept may be useful especially for clinicians, as 3-part differential instruments may still be in circulation and terminologies such as “Mids %” may still be prevalent in laboratory reports and continue to baffle the unwary. A 3-part differential instrument differentiates the leukocytes into 3 subpopulations namely, lymphocytes, granulocytes, and the mid-cell fraction (eosinophils, basophils, monocytes, and precursors of WBCs) by electronic sizing. Specially formulated reagents cause the WBC membrane to shrink around the nucleus while keeping the cell intact, allowing separation of WBCs according to their volume. In the leukocyte histogram, the lymphocytes fall within the small-cell region, neutrophils within the large-cell region, and the remaining cells into the mid-size cell region. The 5-part differential screen reports on all the five subpopulations namely, neutrophils, eosinophils, basophils, monocytes and lymphocytes.
Automation of some of the most commonly requested laboratory tests (the CBC and differential counts) has reduced the number of technologists needed for performance of these tests . As a result, there has also been a dramatic reduction of the numbers of medical technologists and technicians in medical laboratories . While automated blood analyzers count significantly more cells to create a differential count than technicians , they are perhaps still less efficient in detecting abnormal WBCs. Therefore, whenever a hematological malignancy or rare hereditary disorder is suspected in a patient, a microscopic differential count must be performed independently of whether the automated hematology analyzer gives a suspect flag message or not. At present, the automated analyzers are not error-free. With further technological refinements, this situation may well change.
“A just cause is not ruined by a few mistakes.”
Fyodor Dostoyevsky (Russian Novelist and Writer, 1821-1881).
“Everything that can be counted does not necessarily count; everything that counts cannot necessarily be counted.”
CITE THIS ARTICLE:
M. Aroon Kamath, M.D.. Automated blood-cell analyzers. Can you count on them to count well?. Doctors Lounge Website. Available at: http://www.doctorslounge.com/index.php/blogs/page/17172. Accessed March 05 2015.
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