An interlaboratory study of a candidate reference method for platelet counting.
TL;DR: A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio and demonstrated that this method meets the criteria for a reference platelet count.
Abstract: A multinational interlaboratory task force explored the important variables of platelet reference counting and developed a candidate flow cytometric reference method based on the RBC/platelet ratio. A multicenter comparison was performed to determine whether the method met the necessary criteria and was precise enough to be recommended as a new reference method. Each laboratory analyzed serial dilutions of normal specimens, stabilized material, and at least 60 patient specimens with a range of platelet counts from 1 to 400 ·10 3 /µL (1-400 ·10 9 /L). Pooled analysis of the serial
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TL;DR: This review discusses the inherited platelet disorders summarising the current state of the art with respect to investigation and diagnosis and suggests how to manage bleeding manifestations with particular attention to surgical interventions and the management of pregnancy.
Abstract: The inherited platelet disorders are an uncommon cause of symptomatic bleeding. They may be difficult to diagnose (and are likely to be under-diagnosed) and pose problems in management. This review discusses the inherited platelet disorders summarising the current state of the art with respect to investigation and diagnosis and suggests how to manage bleeding manifestations with particular attention to surgical interventions and the management of pregnancy.
311 citations
Cites methods from "An interlaboratory study of a candi..."
...A reference method for platelet number using flow cytometry for platelet counting is available (Harrison et al, 2001)....
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TL;DR: There has been interest in the development of an improved reference procedure to enable optimization of automated platelet counting and the possible implementation of a new reference method to calibrate cell counters, assign values to calibrators, and to obtain a direct platelet count on a variety of pathological samples.
Abstract: Summary
The four main procedures for platelet counting are: manual phase contrast microscopy, impedance, optical light scatter/fluorescence and flow cytometry. Early methods to enumerate platelets were inaccurate and irreproducible. The manual count is still recognized as the gold standard or reference method, and until very recently the calibration of platelet counts by the manufacturers of automated cell counters and quality control material was performed by this method. However, it is time-consuming and results in high levels of imprecision. The introduction of automated full blood counters using impedance technology resulted in a dramatic improvement in precision. However, impedance counts still have limitations as cell size analysis cannot discriminate platelets from other similar-sized particles. More recently, light scatter or fluorescence methods have been introduced for automated platelet counting, but there are still occasional cases where an accurate platelet count remains a challenge. Thus, there has been interest in the development of an improved reference procedure to enable optimization of automated platelet counting. This method utilizes monoclonal antibodies to platelet cell surface antigens conjugated to a suitable fluorophore. This permits the possible implementation of a new reference method to calibrate cell counters, assign values to calibrators, and to obtain a direct platelet count on a variety of pathological samples. In future, analysers may introduce additional platelet parameters; a reliable method to quantify immature or reticulated platelets would be useful.
154 citations
Cites methods from "An interlaboratory study of a candi..."
...These two methods initiated the creation of an international multicentre study under the auspices of the ISLH to explore the possibility of developing a reference measurement procedure for platelet counting using CD41 and CD61 based on a red cell (RBC) to platelet ratio (Harrison et al., 2001)....
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...The flow cytometric method for counting platelets has been recommended as a potential reference method and has been the subject of review by the ICSH Expert panel on cytometry (Harrison et al., 2001; ICSH, 2001)....
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...By performing flow cytometric analysis of the ratio of fluorescent platelet events to nonfluorescent red cell events, a highly accurate and precise technique is now available for counting platelets in whole blood (Harrison et al., 2000,2001)....
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153 citations
Cites methods from "An interlaboratory study of a candi..."
...Platelet Count in Human The International Council for Standardization in Haematology and the International Society of Laboratory Hematology has recommended a flow cytometric reference method for platelet counting that utilizes erythrocyte counts, determined by an automated counter, as an internal reference standard.(154,155)...
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TL;DR: The need for external quality control to improve analyser calibration for samples with low platelet counts is re‐emphasized, and the optimal thresholds for prophylactic platelet transfusions should be re‐evaluated.
Abstract: The optimal threshold platelet count for prophylactic platelet transfusions in patients with haematological malignancy undergoing cytotoxic therapy remains uncertain. Recent studies have suggested that for patients with no additional risk factors the clinical decision threshold may be reduced from a platelet count of 20 · 10 9 /l to 10 · 10 9 /l without Summary Although haematology analysers provide reliable full blood counts, they are known to be inaccurate at enumerating platelets in severe thrombocytopenia. If the thresholds for platelet transfusion, currently set at 10 · 10 9 /l, are to be further reduced, it is vital that the limitations of current analysers are fully understood. The aim of this large multicentre study was to determine the accuracy of haematology analysers in current routine practice for platelet counts below 20 · 10 9 /l. Platelet counts estimated by analysers using optical, impedance and immunological methods were compared with the International Reference Method for platelet counting. The results demonstrated variation in platelet counting between different analysers and even the same type of analyser at different sites. Optical methods for platelet counting on the XE 2100, Advia 120, Cell-Dyn 4000 and H3* were not superior to impedance methods on the XE 2100, LH750 and Pentra analysers. All analysers except one overestimated the platelet count, which would result in under transfusion of platelets. This study highlights the inaccuracies of haematology analysers in platelet counting in severe thrombocytopenia. It re-emphasizes the need for external quality control to improve analyser calibration for samples with low platelet counts, and suggests that the optimal thresholds for prophylactic platelet transfusions should be re-evaluated.
131 citations
Cites background or methods from "An interlaboratory study of a candi..."
...These technological principles are well-described (Harrison et al, 2001b)....
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...This is limited by being time-consuming, highly subjective and imprecise at low counts due to the low numbers of cells being counted (Harrison et al, 2001a)....
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...The IRM utilizes two monoclonal antibodies CD41-FITC (Immunotech, Beckman Coulter, France) and CD61-FITC (Becton Dickinson, Oxford, UK) following the method as fully described elsewhere (Harrison et al, 2001b)....
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...This method, as ratified by the International Society of Laboratory Haematology (ISLH), is now the proposed International Reference Method (IRM) for counting platelets (Harrison et al, 2001a)....
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TL;DR: The International Council for Standardization in Haematology and the International Society of Laboratory Hematology recommend the counting of specifically labeled platelets relative to the RBCs with a fluorescence flow cytometer, together with an accurate RBC count determined with a semiautomated, single-channel aperture-impedance counter as a reference method for the enumeration of platelets.
Abstract: The International Council for Standardization in Haematology (ICSH) and the International Society of Laboratory Hematology (ISLH) recommend the counting of specifically labeled platelets relative to the RBCs with a fluorescence flow cytometer, together with an accurate RBC count determined with a semiautomated, single-channel aperture-impedance counter as a reference method for the enumeration of platelets. Fresh EDTA-anticoagulated venous blood specimens are measured within 4 hours of the draw. The specimen is prediluted (1:20) and the platelets labeled with two monoclonal antibodies specific to a cluster of differentiation common to all platelets. A final 1:1,000 dilution is made and at least 50,000 events with a minimum of 1,000 platelet events are counted with a flow cytometer to determine the RBC/platelet ratio. The platelet count is then calculated from this ratio and the RBC concentration of the original blood specimen.
121 citations
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TL;DR: The risk of major bleeding during induction chemotherapy in adolescents and adults with acute myeloid leukemia (except acute promyelocytic leukemia, which the authors did not study) was similar with platelet-transfusion thresholds of 20,000 per cubic millimeter and 10,000 each.
Abstract: Background Prophylactic platelet transfusions are usually administered to patients receiving myelotoxic chemotherapy when their platelet count falls below 20,000 per cubic millimeter. Some observations suggest that lower platelet counts can be appropriate in patients in stable condition, but the safety of lower thresholds is uncertain. Methods We evaluated 255 adolescents and adults (age, 16 to 70 years) with newly diagnosed acute myeloid leukemia (but not acute promyelocytic leukemia), who were treated in 21 centers. One hundred thirty-five patients were randomly assigned to receive a transfusion when their platelet count fell below 10,000 per cubic millimeter (or 10,000 to 20,000 per cubic millimeter in those with a temperature above 38°C, with active bleeding, or a need for invasive procedures), and 120 patients were assigned to receive a transfusion when their platelet count was less than 20,000 per cubic millimeter. Results Patients in the group with a threshold of 10,000 platelets per cubic millimet...
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"An interlaboratory study of a candi..." refers background or methods in this paper
...(2) The receptor is rarely absent or decreased (eg, rare cases of Glanzmann thrombasthenia that should be easily screened out)....
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...Consensus Materials The following were provided to all participating laboratories to minimize interlaboratory variation: (1) 2 FITClabeled antiplatelet antibodies: anti-CD61 (clone RUU-PL 7F12,21 lot number 13222, Becton Dickinson, San Jose, CA) and anti-CD41 (clone P2,22 lot number 26, Beckman Coulter, Hialeah, FL); (2) bovine serum albumin, fraction V (BSA; Sigma, St Louis, MO); (3) Millex-GV filters, mean pore size 0....
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TL;DR: The findings indicate that the threshold for prophylactic platelet transfusions can safely be set at 5 x 10(9)/l in patients without fever or bleeding manifestations and at 10 x 10 (9)/ l in patients with such signs.
Abstract: Early studies suggested that the risk of haemorrhagic complications become unacceptable when platelet counts drop below 20 x 10(9)/l. Because there are insufficient data to define 20 x 10(9)/l as the threshold for prophylactic platelet transfusions, the practicability of a more restrictive transfusion policy has been assessed prospectively in 102 consecutive patients being treated for acute leukaemia. Besides platelet count, the transfusion protocol took into consideration factors such as presence of bleeding, fever, coagulation disorders, and intention to do therapeutic procedures. 31 major bleeding episodes occurred on 1.9% of the study days when platelet counts were 10 x 10(9)/l or less and on 0.07% of study days when counts were 10-20 x 10(9)/l. The findings indicate that the threshold for prophylactic transfusions can safely be set at 5 x 10(9)/l in patients without fever or bleeding manifestations and at 10 x 10(9)/l in patients with such signs. For patients with coagulation disorders or anatomical lesions, or for those on heparin, the threshold should be at least 20 x 10(9)/l. Such a restrictive platelet transfusion policy, which is applicable not only to thrombocytopenia associated with acute leukaemia but also to other forms of hypoproliferative thrombocytopenia, reduces exposure of such patients to blood donors and results in substantial health-care savings.
309 citations
TL;DR: Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo, and is independent of alpha granule secretion.
Abstract: In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.
179 citations