Hematology Parameters: Usefulness and Limitations in Monitoring Red Cell Production
Giuseppe d'Onofrio, MD
Immunohematology Services, Catholic University of the Sacred Heart
Rome, Italy
Overview:
A variety of instruments and methods are available for the evaluation of hematological parameters in the context of erythropoiesis. Among the standard parameters and methods used for such evaluation, there exists a strong need for standardization, which should take into account biological, analytical, pre-analytical variabilities, all of which can significantly affect the data being obtained through hematological analysis.
If our goal as clinical laboratory hematologists is to monitor and detect abnormal stimulation of erythropoiesis, we must use valid criteria to assess the value of testing procedures used for this purpose.
This means that we have to select appropriate tests ˆ ones that meet the accepted performance levels in terms of sensitivity, specificity, and predictive value. We also need, most importantly, to standardize the tests used.
Standardize, in this sense, is a very general word, which covers the areas of calibration and quality control, the elimination of imprecision, assuring the accuracy of instruments and methods, and the harmonization between these factors. It also recognizes the importance of biological and analytical variability, and of the performance goals and critical differences between testing methods.
Numerous studies have shown the effects of recombinant EPO on various hematological parameters, including hematocrit, hemoglobin, and reticulocyte counts. EPO has also been shown to affect various properties of red cells themselves, such as shape and size.
Among other things, studies have shown that reticulocyte counts increase much earlier than hematocrit or hemoglobin in response to EPO administration, and that there is a decrease in the reticulocyte count following the cessation of EPO use.
The Australian research group of Robin Parisotto and Mike Ashenden introduced the use of reticulocyte parameters as fine-line indicators of changes in the production of red blood cells.
It should be understood that laboratory data have no value in themselves. They must be subjected to the transformation process known as interpretation. This can be done for our purposes through two methods: transversal evaluation and longitudinal evaluation.
In transversal evaluation, data is compared with that of a reference population or a threshold value, and adjudged to be normal or abnormal. Examples of this type of evaluation include the UCI‚s use of hematocrit cut-off values, and the FIS‚ use of hemoglobin cut-off values.
Longitudinal evaluation involves the comparison of data with earlier data from the same individual and is defined as changed or not changed. This evaluation method is probably much more effective than transversal evaluation, and is the basis for the UCI‚s plans to prepare an effective method of examining athletes periodically.
As an example of the ways in which an athlete is evaluated, consider a hypothetical case study of an individual. This athlete arrives in France today, has his blood taken and analyzed on a Bayer ADVIA 120 analyzer. He then travels to the United States, and after eight days, a new blood sample is taken and analyzed on a Coulter machine. Then, after one week, the athlete flies to Japan and his blood is analyzed there on a Sysmex analyzer. Continuing his travel, blood samples are drawn over the course of several weeks on an ABX analyzer in Jerusalem, and on an unknown analyzer in Moscow. Finally, the athlete returns to France, where blood is drawn once again and analyzed on the original Bayer machine.
We would expect that any change observed over the course of all these analyses is that any change observed in hemoglobin or cell counts were the result of biological variations, and not due to the fact that the analyses were performed on different instruments, at different times, or in different places. However, the lack of testing standardization across all these analyses can be the cause of significant variation, which is the reason standardization has become an area of focus for many international sporting and hematology organizations.
Clinical interpretation of testing data depends on a number of variables, including biological, pre-analytical, and analytical variabilities. Biological variability, including such factors as actual physiological changes and trainings changes, can be within one individual or between different individuals. Pre-analytical variability includes such factors as food and fluid intake, exercise, and posture, prior to or during sampling.
Analytical variability is a combination of analytical imprecision and systematic errors of method. This analytical variability can be lessened through in-laboratory internal quality control methods, and through standard quality assessment schemes and harmonization processes, so that results from different methods and different instruments can be compared.
It is apparent that the issue of instrument calibration is very important. This calibration of blood cell analyzers involves adjustments to correct for many factors, including dilation and signal amplitude in the different analyzer channels. These adjustments can be made by comparing values obtained from an analysis of fresh blood with values obtained by a reference method or through the use of reference materials. This need for calibration can be clearly seen through incidents in which samples obtained from an individual athlete at the same time have been analyzed using the same tools in different laboratories, with significantly different results being obtained.
When we want to quantify increases in erythropoiesis, there are two hematological parameters that are of particular importance: hemoglobin concentration and hematocrit.
Hemoglobin concentration, which is the most precise, accurate, standardized and harmonized hematology parameter, is obtained by photometric measurement after conversion to cyanmethemoglobin, or with new cyanide-free methods. Measurement of this parameter is supported by a unique hemoglobincyanide reference preparation, which was proposed by the International Council for Standardization in Hematology (ICSH), and also by an established reference method, which was proposed and accepted by the ICSH in 1987.
Hematocrit values, on the other hand, can be measured using either manual methods ˆ as a volume ratio of red blood cells to whole blood ˆ or automated methods, in which the basic object of the direct measurement is the electrical impulse generated by the cell when it passes through impedance-based or optical-based analyzers.
There are discrepancies in manual versus automated hematocrit measurements, especially due to the plasma trapping in the corner of red blood cells, which can be as high as 5-6 percent in abnormal samples and up to 20 percent in very ill abnormal cells. This causes a false increase of the mean cellular volume in centrifuge hematocrit. What this means is that when we calculate hematocrit from pulse size and red cell count, an underestimation of the hematocrit can occur. A correction of about three percent on automated hematocrit measurements has been proposed in the past to deal with type of discrepancy, but this would not be valid, as the effect is not linear, since plasma trapping increases with microcytosis and anemia.
Different extraneous factors affect how automated hematocrit measurements are made by different instruments. The fundamental difficulty is that the electrical impulse produced by any cell is only approximately proportional to the volume of the cell (shape effect). Also, aberrant impulses, which do not truly represent cell size, can occur on any counter (coincidence, edge effect, recirculation), occurring more frequently on aperture-impedance counters without sheat flow. Owing to their unusual characteristics, suitable electronic circuits edit these aberrant impulses, resulting in measurement variances.
This information shows that variability is increased in hematocrit measurement, suggesting that hematocrit is not the best parameter for red cell quantitative assessment. This parameter represents red cell production relative to whole blood, and is sensitive to changes in plasma volume, as well as red cell volume. It is also a virtual, or calculated, parameter, which is more difficult to standardize.
Hemoglobin, on the other hand, is the most precise, accurate, direct and standardized blood parameter. It expresses more directly the oxygen-carrying capacity of the blood, which is the target of blood doping.
Another hematological parameter useful to examine is reticulocyte measures. Both absolute reticulocyte and percent reticulocytes are very useful parameters, which, while always standardized, have some measurement problems at very low volumes. Reference values for reticulocyte measurements are very heterogenous, and change from one analyzer to the next.
Different methods of measuring the immature reticulocyte fraction have been shown to have very poor inter-method correlation. However, reticulocyte volume measurements obtained using different methods are well correlated.
In conclusion, hematology laboratories at the start of the new century are provided with a growing number of parameters and methods aimed at assessing the degree and efficiency of erythropoiesis, some of which have been shown to be more accurate or useful than others.
Finally, the importance of how our tests and methods should be selected, standardized, and controlled should be kept in mind as we continue to develop our anti-doping strategies and methods.
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