Getting PLT counts right: A solution to pseudothrombocytopenia
Author: Dr. Bao Yejiang Deputy Manager of Laboratory, Zhejiang Cancer Hospital, China
First, a little science time: a review of "blood basics".
Blood is made up of plasma and several types of cells — red blood cells, white blood cells, and blood platelets. Blood platelets (PLT), also called thrombocytes, are produced in your bone marrow and help to stop bleeding.
Platelets have important roles in physiological and pathological processes such as hemostasis, wound healing, thrombogenesis and most inflammatory reactions. If for any reason your blood platelet count falls below normal, the condition is called thrombocytopenia. Thrombocytopenia often occurs as a result of complications of other diseases, such as leukemia or aplasitc anemia. However, falsely low platelet counts, or pseudothrombocytopenia (PTCP), though easily unrecognized, are found in clinical cases. It is an in vitro phenomenon caused by platelet clumping that results in reporting of a spuriously low platelet count by automatic hematology analyzers.
Causes of platelet clumping include EDTA-dependent agglutination, blood-collection factor, cold agglutination, and platelet satellitism.
Pseudothrombocytopenia may lead to misdiagnosis, unnecessary examinations and inappropriate treatment. Therefore, it is very important to establish a proper method to prevent inaccurate platelet counting. The most common approaches to identifying pseudothrombocytopenia include drawing and testing additional blood specimens and using different anticoagulants such as citrate and heparin, but these methods may cause discomfort on the patient and are time-consuming.
Is there a painless, hassle-free solution? Let's look at a clinical case.
We conducted a routine CBC test for one of our patients on Mindray CAL 8000 New Generation Cellular Analysis Line, which reports routine platelet counts using the conventional impedance method (PLT-I). The result showed as below:
As is shown in above figure, the platelet count using PLT-I method was reported to be 20, much lower than the normal number between 100~300. A peripheral blood smear revealed clumped platelets which resulted in pseudothrombocytopenia.
The reticulocytes (RET) analysis channel on Mindray CAL 8000 and BC-6000 series can be activated to report platelet count as well, but it uses optical method (PLT-O) instead. In comparison, the same blood specimen was analyzed again on CAL 8000, utilizing both methods for PLT counting.
The report showed two different numbers. The platelet count using PLT-I method was 24, closer to the former count, while the RET channel with optical method (PLT-O) produced a normal count of 106, which conformed with the results obtained from blood film validation. For further confirmation, we repeated a CBC test using a new blood draw from the patient.
The result indicated that the pseudothrombocytopenia observed in the patient's first blood draw was completely verified. Could this be a mere coincidence? We conducted extensive case studies for validation, with some of the results shown as below.
It is observed that the RET/PLT-O channel on Mindray's high-end hematology analyzers could prevent the incidence of pseudothrombocytopenia substantially.
Since 2016, our laboratory has been using Mindray's high-end hematology analyzer for automatic correction of pseudothrombocytopenia instead of the conventional approach of blood redraw and retesting. We have preset the validation rules. Once PLT clumps are flagged and the reported PLT count is less than 100, the RET/PLT-O channel will be activated automatically to analyze and report a more accurate PLT-O count. Of the 400 cases of pseudothrombocytopenia caused by PLT clumping related to blood draw factor or EDTA-dependent agglutination, over 90% have been corrected via the RET/PLT-O channel on Mindray CAL 8000.
What are the technologies behind Mindray's solution to PLT clumping?
Here's a short video produced by Mindray to demonstrate how it works.
Mindray's R&D team explained the principles of the technologies. In the RET/PLT-O channel of Mindray's hematology analyzers, the pre-heating pool will heat up the reagent according to the temperature of the environment so that the fluorescent dye (which accounts for the majority of the reaction volume) can reach the reaction temperature rapidly.
After the platelet clump samples pass through the sample rotary valve and mix with the pre-heated fluorescent dye, they will subsequently be injected into a thermostat (42°C) titanium alloy reaction chamber to form a high-speed swirl so that the fluorescent dye will be completely mixed with the samples.
At the same time, the stirrer bar in the reaction chamber will mix up the reaction solution at a high speed of 1400 r/min. During this intense concussion, the physical structures of the platelet clump samples will be mechanically destroyed.
In the meantime, the "depolymerization component" in the reagent also acts on the clumped platelets, destroying the molecular structure of the platelet clumps, and combines with individual platelets to prevent re-aggregation.
After depolymerization, the stained platelets will pass through the laser one by one under the sheath flow for 3D analysis using the SF cube technology.
This is how pseudothrombocytopenia samples are corrected and tested on the RET/PLT-O channel of Mindray's BC-6000 series Auto Hematology Analyzers.
About the author:
Research and Development Department, and In-vitro Diagnostics Department, SHENZHEN MINDRAY BIO-MEDICAL ELECTRONICS CO., LTD.