Comparison of microscopic and instrumental results of individual blood cell types with a focus on platelets

The aim of my thesis is to determine the number of platelets with different methods; then to determine the cause of the measured differences in the number of platelets, followed by determination of algorithm for reliable counting of platelets. In specific situations, the instrument determination of the levels of platelets may be significantly different from the real situation. The actual number of platelets can be obtained by using alternative methods. Microscopic assessment and determination of the number of platelets in sodium citrate are considered as alternative methods. Hypothesis I. When using alternative methods, different results are obtained in part of the population. Hypothesis II. These alternative methods help us to distinguish between true and false thrombocytopenia. The theoretical part deals with the formation, structure, physiology, function, metabolism and characteristics of platelets. The term of thrombocytopenia is defined. Quality control of laboratory analyzes is mentioned. Different anticoagulant products used in hematology are described. There are also described techniques by which platelets can be determined and interference associated with the measurement of blood cells. The practical part includes the processing of biological material in a test-tube with the purple cap containing anticoagulant EDTA (from this test-tube a complete blood count is provided), while a test-tube with the blue cap contains anticoagulant sodium citrate (this test-tube is only for re-determination of the platelets count). Analysis of biological material was performed by the analyzer Coulter LH 750, Beckman Coulter Inc. The Coulter method measures the size of blood cells due to changes in electrical resistance. Manual processing of biological material was performed using a light microscope Carl Zeiss Inc., the total magnification for observation of platelets is 400 times. Number of platelets is evaluated in Bürker chamber in 20 rectangles. Solution of procaine is used to stabilize and highlight the platelets. In the period of 23rd April - 22nd May 2012 at the department of Clinical Hematology, Nemocnice České Budějovice a.s., I examined the platelets count in two different anticoagulants by using different measurement techniques. This comparison is important because if the difference in the number of platelets found in EDTA anticoagulant compared with sodium citrate is higher than 20 %, the samples could be suspected of pseudothrombocytopenia. The measurement included fifty samples which were selected already with some suspicion according to following two criteria: the platelets count had to fall below 100 x 10^9/l and at the same time we had received analyzer report of the presence of clumps. The measured values of platelets were statistically processed and they are shown in charts and graphs in my thesis. For a better idea there are also pictures - especially histograms showing some changes in the number and volume of blood cells. This research has demonstrated the occurrence of the phenomenon PTCP in 0,052% cases. The difference between the techniques was clearly evident. Microscopy does not reach such accurate results as analyzers. Quantity of samples and speed of implementation can not be compared. Nowadays microscopy is used as an auxiliary technique in those cases where the analyzer is too affected by imperfections (for example formation of clumps) that it gives false platelets count. The disadvantage is the time delay from the sampling to the possibility of counting. By alternative methods we reach eventual verification or modification of first results (for example repeated sampling to another anticoagulant)