Platelet‐Mimicking RBC Microfragments: PLT‐F Interference on Sysmex XN Analyzers in Pediatrics

Dear Editors,

1.

Despite advancements in modern hematology analyzer technology, accurate platelet (PLT) counting remains among the most error‐prone procedures, requiring multiple checks across the laboratory workflow and an understanding of each system's limitations [1]. This challenge is amplified in pediatrics, where blood volume for routine testing is often limited. While new instruments deliver precise complete blood counts (CBCs) in normal samples, analytical performance decreases in thrombocytopenic samples [2] or in the presence of interfering factors such as red blood cell (RBC) and white blood cell (WBC) fragments [3, 4], abnormal proteins, lipids, or microorganisms (e.g., gametocytes). These interferences can falsely increase PLT counts, potentially masking clinically significant thrombocytopenia and increasing hemorrhagic risk [5].

The XN analyzers (Sysmex, Kobe, Japan) offer three platelet counting technologies with decreasing susceptibility to interference [6]. Impedance counts (PLT‐I) are based on particle volume, making them vulnerable to interference from similarly sized particles. Optical counting (PLT‐O) is more specific, using multidimensional measurement. Fluorescent count (PLT‐F) employs a five‐fold larger sample volume and an oxazine dye that preferentially binds to nucleic acid–rich platelet organelles (ribosomes, mitochondria) [7], minimizing interferences and correlating well with CD41/CD61 flow cytometric counts. Specific alarms are triggered when interferences are detected: the “PLT Abn distribution” flag (PLT‐I channel) appears when the mean platelet volume (MPV) cannot be determined, which is often due to abnormal curve that does not return to the baseline or suspected clumping. The “RET Abn Scattergram” (PLT‐O channel) flags in the presence of debris. Finally, PLT‐F rarely flags (“PLT Abn Scattergram”), and only in case of major interference. Only immunological flow cytometry methods, using highly specific antibodies, are considered totally free from interferences and remain the reference for platelet counts. Interferences have long been associated with cytoplasmic fragmentation of blasts, particularly, in acute myeloid leukemia (AML) and in PLT‐I and PLT‐O channels. Hummel et al. recently reported PLT‐F interference from blast fragments in two adults with AML, but to our knowledge, this has never been described with RBC fragments. We report here six pediatric leukemia cases (3 B‐acute lymphoblastic leukemia [B‐ALL], 1T‐ALL, 2 AML) with major PLT‐F discrepancies due to extensive RBC fragmentation (Figure 1, Table A).

FIGURE 1.

(A) Table A: Main patient characteristics and duration of interference episodes (AML: acute myeloid leukemia; ETP: early T precursor; B‐ALL: B acute lymphoblastic leukemia). (B) Table B: Comparison of platelet counts obtained with the XN‐3000 modes (PLT‐I/PLT‐O/PLT‐F) versus a manual platelet count performed using a counting chamber (Malassez chamber) after dilution with the Thrombo‐TIC reagent (Bioanalytic, ref. 004015–0007) and flow cytometry. ND = not done. (C) Normal platelet count profiles obtained using impedance (PLT‐I) and scattergrams from optical (PLT‐O) and fluorescence (PLT‐F) methods. (D) Examples of platelet count interferences observed in five patients: Patient No. 1 (PLT‐I, PLT‐O and PLT‐F) and Patients No. 2/3/4/6 (PLT‐F) during episodes of intense red blood cell fragmentation. Black circles indicate atypical event clusters distinct from the expected platelet cluster. (E) Reference platelet enumeration by immunophenotypic flow cytometry in whole blood using anti‐CD41‐PC7 (clone P2) and BD Trucount tubes (FACSCanto II, BD Biosciences). Platelets were identified as CD41+ events and displayed on an FSC/SSC dot plot (blue), with Trucount beads gated separately. Acquisition was stopped after 5000 bead events, and the absolute platelet count (platelets/μL) was calculated from the platelet‐to‐bead event ratio using the manufacturer‐assigned (lot‐specific) number of beads per tube.

Abnormal PLT‐I distribution curves prompted PLT‐O and PLT‐F measurements, which were compared to manual platelet counts performed using a counting chamber (Malassez chamber) after dilution with the Thrombo‐TIC reagent (Bioanalytic, ref. 004015‐0007) and to CD41 counts (FACSCanto II, BD Biosciences; BD Trucount tubes; anti‐CD41‐PC7, clone P2) (Figure 1, Table B, C–E). Results showed substantial differences between the PLT‐F versus CD41 counts. Blood smears revealed the presence of numerous spherocytes, schizocytes and microspherocytes, characteristic of an ongoing hemolytic process (Figure 2). These usually trigger the “Fragments?” alarm in PLT‐I and occasionally in PLT‐O, but rarely in PLT‐F, which is generally considered less affected by RBC size. Although the exact mechanism(s) underlying this PLT‐F interference remain hypothetical and cannot be demonstrated from our data, a plausible explanation can be proposed. The PLT‐F method relies on fluorescence flow cytometry using an oxazine dye, with event classification based on forward scatter (size) and side fluorescence intensity. In our pediatric cases, blood smears showed abundant platelet‐sized, fully respherized RBC‐derived microfragments during episodes of extensive hemolysis, making partial overlap with the platelet cloud on the PLT‐F scattergram biologically plausible and thereby leading to apparent interference. This hypothesis warrants confirmation in controlled spiking and/or targeted labeling studies. RBC fragmentation occurred at diagnosis (two patients) and after chemotherapy initiation (four patients). In two patients, it appeared much later (on Days 22 and 48) during aplasia and without residual circulating blasts. Interference lasted a mean of 7 days (range 2–12), decreasing as leukemic burden fell. An atypical non‐platelet cluster, not usually observed in the PLT‐F channel, was observed in several patients (Figure 1D). Although its exact nature cannot be confirmed without dedicated controls, its proximity to the platelet cloud and low fluorescence make it the most likely contributor to the observed PLT‐F interference by partial overlap with the platelet region. This interpretation is supported by blood smears showing abundant RBC fragmentation, with little or no leukemic cytoplasmic debris in most cases; apoptotic blasts were documented only in a subset of patients. Overall, these findings therefore suggest that the atypical PLT‐F events predominantly reflect RBC‐derived microfragments rather than blast‐derived cytoplasmic fragments.

FIGURE 2.

Illustration of the formation of “vesicles” from the erythrocyte membrane (represented by black boxes) on blood smears stained with May–Grünwald–Giemsa (MGG). The smears also show the presence of schizocytes (varying among patients) and very small microspherocytes fully respherized microspherocytes (indicated by arrows), comparable in size to platelets.

Only one patient triggered a “PLT Abn Scattergram” in PLT‐F, and in half of the cases, the PLT‐I showed no abnormal curves.

The differences in platelet counts were significant both analytically and clinically. In three cases (Patients 2, 4, and 5), inaccurate counts on all three channels could have altered transfusion management without confirmation by flow cytometry (Figure 1, Table B). Accurate counts are essential for optimizing platelet support in these patients. Despite extensive testing (enzyme assays, RBC disorder screening, metabolic panels, biochemical profiles), the cause of this marked hemolysis with membrane‐loss “droplets” remains unclear. While Sysmex PLT‐F is generally accurate and specific, these cases highlight the importance of visually inspecting scattergrams, even without flags, for abnormal particle clouds overlapping the platelet cluster. The analyzer's population color codes (e.g., green for platelets) can also signal interference. Laboratories using XN‐series analyzers should be aware of potential PLT‐F interference in acute leukemia patients, especially during intensive chemotherapy when complications associated with cellular fragmentation may occur.

Author Contributions

A.M. and E.L. designed the study, wrote the manuscript, and made the figures. O.F. provided photomicrographs of the blood smears. A.G. provided the Scatter plots from the XN‐3000 analyzer and A.V. corrected the manuscript. All authors have reviewed and approved the submitted version.

Funding

The authors have nothing to report.

Disclosure

All sources are from the laboratory of Robert Debré hospital.

Ethics Statement

The authors have nothing to report.

Consent

All participants provided informed consent before blood sampling performed as part of their treatment.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.