Problems with existing methods of platelet counting:
(1) Debris and spurious noise may be interfere with accurate counting of small platelets.
(2) Erythrocytes are typically 20 times more numerous than platelets and may interfere with an accurate platelet count.
Method:
(1) Perform a red blood cell count in automated analyzer.
(2) Dilute a fresh (< 4 hours old) blood sample 1:20.
(3) Label the platelets with 2 monoclonal antibodies specific to antigens common to all platelets (such as anti-CD41 and anti-CD61).
(4) The sample is then diluted 1:1000.
(5) The sample is then counted in a flow cytometer.
(6) Endpoint for counting: (a) A minimum of 1,000 platelet "events"; (b) A minimum of 50,000 total "events:
RBC to platelet ratio =
= (number of RBCs counted) / (number of platelets counted)
platelet count per µL =
= (RBC count per µL) / (RBC to platelet ratio)
Performance:
• The method was tested in 11 laboratories in 4 countries using stabilized material. The method excellent intra-assay and interlaboratory precision.
Limitations:
• Several conditions could result in interference, as listed in Table 2, page 463 (thrombocytosis, autoantibodies, congenital platelet disorders, platelet aggregates, cold agglutinins, adherence of platelets to WBCs, fragmented RBCs, fragmented WBCs, abnormalities of platelet size).
• These conditions may interfere with the monoclonal antibody binding sites or block the staining reaction.
