Modern clinical blood tests in doping control initiatives

Dr.Med., Associate Professor V.V. Dorofeykov1, 2
PhD N.Y. Chernysh2
Dr.Med. A.V. Kalinin1
Dr.Hab., Professor S.E. Bakulev1
Dr.Hab., Professor V.A. Taymazov1
Dr.Hab., Professor S.M. Ashkinazi1
1Lesgaft National State University of Physical Education, Sport and Health, St. Petersburg
2Almazov National Medical Research Centre, St. Petersburg

 

Keywords: blood test, erythrocyte, reticulocyte, doping, doping control.

 

Introduction. Clinical or general blood test is the most common laboratory test in the world. At the same time, it is one of the most important diagnostic techniques that reflects the reaction of the blood-forming organs to various physiological and pathological factors. The test helps evaluate the functional state of the body, for example, its reactivity – based on the leukocyte response rate to infection and other pathological processes, as well as assess the state of erythropoiesis – based on the number of reticulocytes during blood loss or upon returning from highlands, diagnose blood diseases, evaluate the dynamics of changes during inflammatory processes or purulent-septic complications.

Objective of the study was to scientifically substantiate the importance of clinical blood tests for the athlete’s biological passport as part of anti-doping control.

Results and discussion. Today, clinical blood tests are a high-tech computerized study that includes assessment of all three hematopoietic lineages; on the other hand, it is a routine analysis any certified clinical diagnostic laboratory can perform. If 10-15 years ago doctors used conventional microscopic tests and a keen eye to carry out anti-doping control, in recent years it has been done with the use of a high-tech automated clinical analyzer that enables to perform up to 70 blood tests per hour. Modern analyzers determine from 18 to more than 70 different blood indices depending on the purpose, cost of the device itself and reagents. Such devices are produced in the USA, EU countries, Japan and China, but not in Russia, unfortunately. The peculiarities and possibilities of interpretation of the anti-doping control results by a doctor depend on the scope of study. While operating automatic systems, doctors have to strictly follow the rules, as reliable results are only guaranteed if all pre-analytical and analytical requirements are fulfilled. To illustrate the progress in this area it should be noted that, in accordance with the professional standard, a doctor can measure the level of blood cells under the microscope accurate to 10% of the true value, while the margin of sampling error using analyzers does not exceed 1-2% for most indicators. A laboratory doctor can view up to 60-100 blood samples during the working day...

It should be pointed out that, in recent years, the clinical blood test has been used to compile the athletes’ biological passport, and here it is necessary for the athletes to be at rest before the blood sampling for at least 2 hours. In terms of anti-doping control, there is a number of strict requirements for blood sampling. It is to be done in the morning before active physical activities or other medical manipulations, with the written consent of the patients or their parents. Blood is taken from the vein on an empty stomach, when seated. A certified doping control inspector with a medical background can make 3 attempts to take blood from the vein. The importance water schedule must be remembered. There is no doubt, in case of danger to the patient’s health and life, part of these requirements may be neglected (and blood sample can be taken at any time of the day or after meals).

Despite the general norms in the results of analysis of the capillary (from the finger, from the infant’s heel) and venous blood, automatic analyzers involve a preferred venous blood test. This is primarily due to the higher accuracy of this kind of testing. Blood is taken in vacuum tubes containing the minimum allowable amount of anticoagulant for the sample volume indicated on them.       

Freshly obtained blood samples should be gently mixed and placed in a container and transported to the laboratory. The blood cells (erythrocytes, leukocytes and platelets), while in contact with the anticoagulant, change gradually. Platelets are the most sensitive: their volume increases gradually within 2-4 hours and in some patients can reach the size of erythrocytes, which can lead to both a false decrease in platelet count and a false increase of the red blood cell count. In 6-8 hours at room temperature, a slow lysis (destruction) of red blood cells may start. This process is strictly individual, but red blood cells don’t increase during transportation. Leukocytes are deemed the most stable fraction, but they may begin to collapse by the end of the first day. Given these peculiarities, the blood to be transported to an anti-doping or other laboratory, upon reaching room temperature, is placed in a refrigerator and stored at a temperature of 4-8 °C. Blood cannot be frozen, it will destroy the cell structure. Carefully packed samples should be transported in thermostable containers, and prior to the test, the blood must be removed from the refrigerator so that the test material reaches room temperature. Immediately before the test, the sample must be mixed again [1].

In vitro, anti-doping control is carried out using devices of various classes - simple (3-DIFF systems) and high-tech analyzers (5-DIFF). The more automated the system, the less likely it is to influence the results of the “human factor” analysis and possibility of errors. The main and most significant differences between the devices are in their technological characteristics and the number of research parameters. 5DIFF analyzers give a detailed analysis of leukocytes, but in difficult cases, especially oncological diseases of the blood system, there is a need to view the material under the microscope [6].

Red blood cell count also depends on the type of device. The cutting-edge test system being used in the WADA (World Anti-Doping Agency) laboratories generates up to 28 erythrocyte growth indices with the output data presented in a wide variety of presentation charts and diagrams. Doctors who are expected to interpret these data need special trainings for the service.

Lately a growing priority has been given to the tests of erythrocyte precursors (reticulocytes, RET#) in the sport test systems rated by up to 10 test indices. Young erythrocytes are characterized by the presence of aggregated cell organelles - ribosomes and mitochondria - in their cytoplasm after the nucleus loss, and differ by the degree of maturity. This reflects the regenerative capacity of the bone marrow, in particular under the influence of such doping drug as erythropoietin, or after transfusion. Persistent reticulocytosis may indicate continued bleeding. The lifetime of reticulocytes in the bone marrow is about 40 hours, in peripheral blood - 24–29 hours. Normally, reticulocyte count in adults ranges from 0.2 to 1.2% of the total number of erythrocytes [2].

MCVr is the mean cell volume reticulocytes and MSRV is the mean sphered reticulocyte volume. These two volume indicators can be used for diagnosing anemia, monitoring the response to iron supplements, folic acid, and vitamin B12. Changes in MSRV testify to the abuse of drugs stimulating erythropoiesis. Immature reticulocyte fraction is indicative of erythropoiesis activity. An increase in this fraction testifies to an accelerated release of immature cells from the bone marrow. This value increases well before the reticulocyte count and is the most sensitive marker of the state of erythropoiesis activity and treatment efficacy. Ret-Hb / CHr (reticulocyte hemoglobin) is another interesting parameter that characterizes hemopoiesis (synthesis of blood corpuscles) for the last 7 days, and shows the hemoglobin concentration rate in the newly synthesized cells, while MSN and % of hypochromic red blood cells reflect the entire picture of the last 8 -12 weeks [5].

To prove the importance of reticulocyte count in elite sports, we present the biological passport of the world-class female athlete, disqualified in 2018. WADA (see Table 1, pay attention to the number of reticulocytes in Test 4, blood from the vein was taken consistently into the vacuum systems I and II).

 

Table 1. Blood hemoglobin concentration and reticulocyte count in athlete's biological passport (track-and-field athletics)

Vacuum systems

Indicators (haemoglobin and reticulocytes)

1st test

4th test

5th test

I

Hb (g/dl)

Ret %

14.8

0.27

14.7

1.7

15.4

0.89

II

Hb

Ret %

14.8

0.23

14.7

1.75

15.3

0.78

PLT is a platelet count. PLT (platelet) - platelet count. Norm - 150-400 (х109/l). Unlike manual platelet counting, when the red blood cell (RBC) pre-lysis is carried out, automatic blood analyzers provide information of the platelet and RBC count without pre-treatment, sports performance does not matter much.

WBC is a white blood cell count. Norm - 4.5-9.0 (x109/l). The number of leukocytes and leukocyte fractions are determined using various types of hematological analyzers and are measured in relative values (%) and absolute values (#), and since the device analyzes thousands of cells, the absolute value is the best reference value. Leukograms are often analyzed with the microscope in case of systemic inflammations in the body, for instance, after injuries [4].

Erythrocyte sedimentation rate is a nonspecific test most foreign laboratories do not use at all. The results are evaluated by the height of the blood plasma layer (in mm) formed within 1 hour. Any change in the Erythrocyte sedimentation rate may be an indirect sign of an inflammatory process.

Conclusions. To generate reliable clinical analysis results, the athletes must properly prepare for the upcoming procedure, be sure to rest before the blood sampling after training or competition, strictly follow all recommendations of the doping control inspector, and remember to indicate in the protocol that their training cycle runs in the highlands, and they take vitamins and other approved drugs (iron supplements, dietary supplements, etc.).

References

  1. Antonov V.S., Volkov A.S. Avtomatizatsiya gematologicheskogo analiza. Interpretatsiya pokazateley gemogrammy. Ch. 2 [Automatization of hematological analysis. Interpretation of hemogram indicators. Part 2]. Laboratornaya sluzhba, 2013, no. 4, pp. 10-21.

  2. Zenina M.N., Kozlov A.V., Bessmeltsev S.S., Chernysh N.Y. Dopolnitelnye laboratornye markery monitoringa zhelezodefitsitnykh sostoyaniy [Additional laboratory markers for monitoring of iron deficiency states]. Vestnik Severo-Zapadnogo gosudarstvennogo meditsinskogo universiteta im. I.I. Mechnikova. 2014, vol. 6, pp.  34- 42.

  3. Chernysh N.Y., Zenina M.N. Laboratornyie kriterii diagnostiki anemicheskogo sindroma u lits starshey vozrastnoy gruppyi [Laboratory criteria for diagnostics of anemia syndrome in senior people]. Laboratornaya sluzhba, 2018, vol. 2, no. 2, pp. 44-49.

  4. Barbara J. Bain MBBS, FRACP, FRCPath Performing a Blood Count. Blood Cells: A Practical Guide, Fifth Edition https://doi.org/10.1002/9781118817322.ch2

  5. Buttarello M. Laboratory diagnosis of anemia: are the old and new red cell parameters useful in classification and treatment, how? Special Issue: International Society for Laboratory Hematology 2016 Education Issue, vol. 38, pp.123-132.

  6. ICSH guidelines for evaluating blood cell analyzers, including for differential leukocyte and reticulocyte counts International Council for Standardization in Hematology, Writing Group: C. Briggs N. Culp B. Davis G. d'Onofrio G. Zini SJ Machin International Council for Hematology Standardization International Journal of Laboratory Hematology, 2014, vol. 36, pp. 613-627.

Corresponding author: vdorofeykov@ya.ru

 

Abstract

The study analyzes the key provisions for the modern clinical blood tests to estimate athlete’s health and demonstrates benefits of the high-tech computerized clinical blood tests versus conventional microscopic tests, applicable by the doping control projects in elite sports. The cutting-edge test system being used in the WADA laboratories generates up to 28 erythrocyte growth indices with the output data presented in a wide variety of presentation charts and diagrams. Doctors who are expected to interpret these data arrays and graphical presentations need special trainings for the service. It should be mentioned that lately a growing priority has been given to the tests of erythrocyte precursors (reticulocytes, RET#) in the sport test systems rated by up to 10 test indices. The article gives a special attention to the pre-test preparations, including the actions, precautions and potential errors in preparations for the blood tests for biological passport, plus the basics of the blood test data analyses.