Standardized flow cytometric method for the accurate determination of platelet counts in patients with severe thrombocytopenia

Abstract

Background: The therapeutic option of prophylactic platelet (PLT) transfusion in cases of severe thrombocytopenia critically depends on the availability of accurate and precise counts because clinical decisions are widely based on decision or trigger points. Although often applied in current practice at a level of 20 Gpt/L, there is increasing evidence that the trigger points could safely be reduced to10 or even 5 Gpt/L. In order to facilitate this downward revision, it is necessary to have PLT counting methods that are able to provide reliable results in the appropriate decision range. Methods: Postchemotherapy‐induced pancytopenia PLT counting was performed in patients with hematological malignant disorders. This study describes a novel flow cytometric method that utilizes a PLT‐specific monoclonal antibody (CD41a) in conjunction with fluorescent reference beads in order to derive absolute platelet numbers. Results: Applying a mathematical model, this flow cytometric method was shown to have a detection limit of 0.24 Gpt/L and a lower limit of quantification (coefficient of variation [CV] = 10%) of 1.1 Gpt/L. These values are a substantial improvement on previously reported results for the Technicon H1 automated instrument or manual hemocytometry. Moreover, although the flow cytometry and Technicon H3 methods were found by supplementary analyses to show a reasonably good correlation, the hematology instrument showed a distinct tendency to overestimate PLT counts at low levels. Conclusion: It is proposed that this standardized immunoplatelet method offers the best approach in evaluating, at the clinical level, the possibility of lower PLT transfusion triggers. It can be used to evaluate the performance limitations of automated hematology analyzers that are widely used at the present time. Cytometry (Comm. Clin. Cytometry) 42:284–289, 2000. © 2000 Wiley‐Liss, Inc.

Evaluation of the platelet counting by Abbott CELL‐DYN® SAPPHIRE™ haematology analyser compared with flow cytometry

“…In this study, as expected, the higher degree of correlation was observed between the CD61‐immunoplatelet and reference method, particularly in thrombocytopenic samples. These results therefore confirm the agreement between the flow cytometry and CD Sapphire immunoplatelet procedures, and according to other studies (Kunz et al. , 2000, 2001) provide evidence that the immunological method appear to be superior to the optical and impedance method in CD Sapphire.…”

“…Another limitation is platelet count underestimation resulting from exclusion of larger platelet forms from conventional counting methods. Optical methods using two dimensions of light scatter, are less prone to these problems, but there are still some circumstances when optical analysis may also be erroneous (Kunz et al. , 2000).…”

Impact of immunological platelet counting (by the platelet/RBC ratio) on haematological practice

“…The main conclusion of the study was that the CD61 ImmunoPLT method was more accurate than any other technology. This finding agrees with other studies comparing the CD61 method with the flow cytometric reference method but is not really unexpected because both immunological methods are essentially measuring the same parameter, albeit with a number of methodological differences (see above) (15)(16)(17)(18). Interestingly, the three optical methods were not shown to be superior to the impedance methods, agreeing with the recent study of Sandhaus et al (10).…”

Immunoplatelet counting: potential for reducing the use of platelet transfusions through more accurate platelet counting

“…Immunoplatelet counting uses a platelet‐specific monoclonal antibody, most commonly CD61 or CD41, to identify the platelets in a whole blood sample for analysis with a flow cytometer. The immunoplatelet count has been used in studies, evaluating it against the other platelet counting methods, and the results from various groups have suggested that its use could potentially reduce the number of platelet transfusions used in supportive therapy (Dickerhoff & Von Ruecker, 1995; Harrison et al, 2000; Kunz et al, 2000). The immunoplatelet count has recently been shown to be suitable for use as the new reference method and has been recommended for use as such by the International Council for Standardization in Haematology (ICSH) (Harrison et al, 2001; ICSH Expert Panel on Cytometry and International Society of Laboratory Hematology Task Force on Platelet Counting, 2001).…”