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Absolute Cell Counting
Ian Storie
Applications
In the rearch and especially the clinical tting, there are situations that require not just the percentage positive of a particular cell population but the actual absolute number positive cells. There are three main areas in the clinical tting that require the preci and accurate determination of absolute cell counts.
CD4+ Lymphocyte Enumeration
法语学习The absolute CD4+ T lymphocyte count has long been recognized as a uful laboratory tool for the staging of HIV infected patients. It can also be ud to asss the likelihood of opportunistic infections, the timing of the administration of preventative treatment and, more recently, for monitoring the effect of new antiviral therapies. Absolute CD4+ T lymphocyte counts of less than 200 cells/μL are generally accepted as being the cut-off point for the laboratory diagnosis of HIV infection. It should be noted that dias other than HIV can result in a reduced CD4+ T lymphocyte count, and that a diagnosis of HIV infection cannot be made on the CD4+ T lymphocyte count alone.CD34+ Hematopoietic Progenitor Cell Enumeration
Mobilization, harvesting and transplantation of CD34+ progenitor cells are now well-recognized tech
niques. By monitoring absolute CD34+ cell levels after growth factor-induced mobilization, it is possible to ensure the maximum number of cells can be collected with the minimum number of time-consuming and expensive harvesting procedures.
Residual White Blood Cell Enumeration
The prence of white blood cells (WBCs) in blood products has been shown to lead to febrile reactions, alloimunization, as well as the transmission of infectious agents (e.g. EBV, CMV and CJD). Many countries have now adopted a policy of filtering all red cell, platelet and fresh plasma products for transfusion to remove the WBCs. Quality control of the procedure involves screening a number of randomly lected filtered units and performing a residual WBC (rWBC) count. In Europe, the upper limit is 1×106 WBCs per unit, which equates to a count of around 3.3 WBCs/μL for an average 300 mL unit.In all the above applications, it is esntial to accurately identify the cell population of interest, and this population may be prent at very low levels. The development of strategies using multiple gating regions, employing both light scatter and fluorescence parameters, have aided in this process. For example, the u of CD45 (pan leucocyte marker) versus side scatter results in a more reproducible and accurate lymphocyte gate.1
Likewi, the u of the ISHAGE gating protocol for CD34 determinations has resulted
in more reproducible results.2
Methods
There are two flow cytometric methods that can be employed to derive absolute
counts.
Dual-Platform Approach
The dual-platform approach employs the u of three parate measurements obtained
彼嘉卸妆油from two different instrumentation platforms. A flow cytometer is ud to determine the
cells of interest as a percentage of a “reference” population (e.g. CD3+/CD4+ cells as
a percentage of the total lymphocyte population). Then a hematology analyzer provides
an absolute white blood cell count (WBC) and the lymphocyte percentage. The absolute
CD4+ T Lymphocyte count is, thus, the product of the absolute WBC, the lymphocyte
percentage and the CD4+ T lymphocyte percentage.
With the dual-platform approach, it is important to ensure that the cell population of
interest, as well as its reference population, can be accurately identified. Different
gating strategies have been employed to aid the identification of lymphocytes for
CD4+ T lymphocyte enumeration. Initially, light scatter gating using FSC vs. SSC was
quarters
ud. The main drawback of this method is the incread likelihood of non-lymphocyte
contamination in the gate, including cell debris, platelets, monocytes and basophils.
This method is now not recommended for CD4+ T lymphocyte determinations3 and has
been superded by the u of light scatter and an immunological marker, for example
SSC vs. CD45. Cell debris and platelets are CD45 negative, monocytes have a slightly
higher side scatter signal then lymphocytes, and basophils have a dimmer expression
of CD45 than normal lymphocytes and, thus, can easily be excluded (Figure 1).
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109Figure 1. The importance of the correct identification of the reference population in dual-platform
determinations. A and
B show the potential contamination in a Forward vs Side Scatter gate drawn around the lymphocyte population. This gate includes cell debris and platelets, (yellow), monocytes (cyan) and basophils (green). Note how the basophils (green) sit in the middle of the lymphocyte population on Forward Side Scatter. In
C and D, CD45 vs Side Scatter is ud to gate the debris, and monocytes and basophils are excluded.
It has been well documented that the dual-platform method has a higher inter-laboratory coefficient of variation (CV) due to the way different hematology analyzers derive the absolute WBC and lymphocyte count, and not to the CD4+ T lymphocyte percentage from the flow cytometer.4 The variance is en to increa dramatically with WBC counts less than 0.1×109/L.5 That means this approach cannot be ud for rWBC determinations, where very low counts in the order of 0.001×109/L may be encountered.
Single-Platform Approach
物流工程考研
In the single-platform approach, the absolute cell count is derived from the flow cytometer itlf, without the need for a hematology analyzer. A very preci, known volume of sample is mixed with relevant antibodies. A gating strategy is employed to accurately identify the cell population of interest and exclude any contaminating cells; identification of a “reference” population is not required. The cell population of interest can then be related back to the original blood volume by a variety of different methods.
A B
C D
Microbeads. The u of microbeads is by far the most common approach to single-platform absolute counting. Microbeads are small fluorescent latex
particles with a diameter of 5-10 µm. There are two basic methodologies for
using microbeads. The first us a tube containing a lyophilized pellet of an exact
2011高考试卷
number of microbeads, to which a known volume of sample is accurately added.
In the cond method, equal volumes of sample and microbeads are accurately
added to a tube. In this approach, the microbeads are in a liquid suspension
medium of known concentration, which is supplied by the manufacturer. Byqvga
counting the number of cell events and the number of bead events and knowing
the initial concentration of beads, it is possible to determine the absolute cell
count using the following formula:
短裙英语怎么说Absolute Cell Count =Number of cell events x Concentration of beads
(cells/μL) Number of bead events (beads/μL)
Using time as an acquisition parameter can give a lot of internal quality control information
about the preparation and acquisition of that sample. A histogram plot of time will show
if the count rate of the sample has been stable over the acquisition period (Figure 2).
Recent studies have demonstrated that the number of beads acquired in a fixed time
period is remarkably constant for most flow cytometers.6 If all the sample preparation
pipetting has been performed accurately, then for each tube acquired, the number of
beads counted in a fixed time should be constant. It is then possible, for each batch of
beads ud, to derive a Mean ± 2 Standard Deviation (SD) range of beads counted in
a fixed time period. Bead counts outside of this range could be indicative of potential
pipetting errors, and the sample should be restained.
Volumetric. With the volumetric method, an exact and reproducible volume of stained sample is pasd through the flow cell, and the number of positive cell
events can be directly related to the known volume of sample acquired. The exact
volume of sample can be measured using either precision syringes or by utilizing
two nsing electrodes that monitor the movement of the sample and, hence,
sample volume. With volumetric systems, all pipetting steps must be made with
great precision. The final dilution of the sample must also be calculated and taken
into account.
Microfluorimetry. With this method, blood stained with the relevant antibody is placed into a capillary tube of preci known dimensions. A lar scans the
capillary, and the number of positive events is counted. This can be related to
the precily known volume of the capillary.
ebeamFlow Rate Calibration method. This is a recently developed method aimed at reducing costs in resource poor countries.9 The method utilizes the fact that with
most bench top flow cytometers, the flow rate of sample through the flow cell is
vision是什么意思remarkably constant. Hence, the volume of sample acquired in a fixed time period
will be constant. An accurately prepared dilution of counting microbeads in lysing
reagent can be ud to calculate the volume of sample acquired in a defined
fixed time period. Accurately prepared stained samples without counting beads
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111can then be acquired over the same time period. The number of events in the cell population of in
terest can be expresd per volume acquired, and if the dilution factor for the blood sample is known (i.e. ratio of blood volume to total volume), then the cell count per microlitre of blood can be calculated. A correction factor for the incread viscosity between the tube containing beads plus lysing reagent and the tube containing sample is required. This factor varies for different lysing reagents ud.
The u of single-platform absolute counting methods are becoming more popular, especially the u of microbeads. With accurate pipetting techniques, the results can
智能化的意思be preci and reliable.
Figure 2. The plots illustrate the value of using time for data acquisition. The first plot shows a normal steady flow rate, indicated by the level appearance of the histogram. The cond plot shows that towards the end of the data acquisition period, the sample flow rate began to decrea due to a partial blockage in the flow cell.
Technical Considerations
Since the u of microbeads is the most common approach to single-platform absolute counting, the technical considerations discussion will focus on that methodology. Accurate and preci pipetting is perhaps the most important factor in obtaining reliable results with this methodology. It is recommended that a “wet tip” rever pipetting technique be employed for the dispensing of sample and liquid pha beads. A description of that technique follows:
