Keywords inherited platelet disorders - guideline - diagnosis
Introduction
Inherited platelet disorders (IPD) are a heterogeneous group of diseases causing bleeding
of variable severity that can either be associated with complex systemic diseases
or occur as isolated entities.[1 ] The diagnosis of these disorders is comprehensive and often challenging. Many IPD,
particularly those without a reduction in platelet count below 100,000/µL, often remain
undetected until bleeding symptoms manifest.[2 ] The clinical consequences of IPD typically result in a mild to moderate bleeding
tendency. However, co-factors such as medications, surgeries, or other hemostatic
challenges can lead to clinically significant bleeding. Typical symptoms of IPD include
mucocutaneous bleeding such as epistaxis, menorrhagia, hematomas, petechiae, and bleeding
during invasive procedures and surgeries. Additionally, bleeding can occur suddenly
and unpredictably.[3 ]
[4 ]
To assess platelet function, a diagnostic algorithm is recommended. Despite the availability
of numerous testing methods, only a few are suitable for clinical practice.[3 ] The application of different laboratory diagnostic tests is dependent on the patient's
history, clinical presentation, and local resources, which frequently require close
collaboration with specialized hemostasis centers and molecular genetics laboratories
for the following:
To facilitate the diagnosis of IPD as close to the patient's residence as possible.
To limit the diagnostic process to the essential steps.
To ensure that the diagnosis provides tangible benefits including adequate treatment
to the patient.
To support the counseling of affected individuals and their families.
The ThromKidplus study group of the Permanent Pediatric Commission of the Society for Thrombosis and
Haemostasis Research (GTH) has updated the AWMF Guideline for the “Diagnosis of Platelet
Disorders” (AWMF Registry Number 086–003), which is summarized here in this manuscript.
ThromKid, initiated in 2004 as a project of the Permanent Pediatric Commission of
the GTH, aims to improve the diagnosis and treatment of patients with IPD. These efforts
have led to the development of two guidelines for Germany, Austria, and Switzerland,
providing information on standardized diagnostic procedures and consensus-based treatment
recommendations.[5 ]
[6 ] The ThromKidplus group, which now also includes recommendations for adult patients, further focuses
on refining existing guidelines, establishing criteria for quality control in specialized
hemostasis laboratories, and designating competence or reference centers to support
other centers in diagnosis and therapy.[7 ]
[8 ]
[9 ]
[10 ]
[11 ] Another important aspect of this initiative is the establishment of a patient registry
for IPD,[12 ] contributing to the overall effort to enhance patient care and outcomes.
Methods
Literature Basis of the Guideline
As an S2k guideline, the recommendations are derived from expert consensus and current
literature. The references cited in this manuscript were selected based on their relevance,
but no systematic literature search was performed. Original studies and systematic
reviews supporting the recommendations and aligning with current clinical and laboratory
standards were prioritized.
Determination of Consensus Strength for Recommendations
In the context of the AWMF guideline development, the consensus strength for the recommendations
was determined through a structured voting process. The recommendations were presented
to eligible participants, followed by an opportunity for questions and suggestions
for modifications. Voting was conducted either on the original recommendations or
proposed amendments. If necessary, discussions led to alternative proposals, followed
by a final vote. Voting options included “agree,” “disagree,” or “abstain.” The strength
of the consensus was classified based on the percentage of agreement among participants:
Strong consensus : >95% of participants agreed.
Consensus : >75% agreement.
Majority agreement : >50 to 75% agreement.
No majority agreement : <50% agreement.
The consensus process involved experts from the ThromKidplus study group as well as representatives from various medical societies. The consensus
conferences and voting were moderated neutrally by a member of the AWMF, ensuring
an unbiased discussion and adherence to formal consensus methodology. This approach
ensured that the final recommendations reflected a broad consensus among the expert
panel, with all relevant expert opinions considered during the guideline development
process.
The guideline includes a total of 42 recommendations, covering various diagnostic
aspects. In this manuscript, only the key recommendations are highlighted, which received
a strong directive ('should' recommendations), to focus on the most relevant and actionable
guidance.
Key Updates in the Guideline
Key Updates in the Guideline
Diagnostic Algorithm
The updated guideline introduces a structured diagnostic algorithm for IPD ([Fig. 1 ]), outlining a stepwise approach that integrates clinical assessment and laboratory
testing to ensure accurate diagnosis. It emphasizes the importance of a thorough patient
history and physical examination, alongside essential tests. The algorithm prioritizes
clinical history and exclusion of plasmatic coagulation disorders. Blood counts and
morphological platelet assessment can provide insights into size, granulation, and
shape abnormalities. Advanced diagnostics, including aggregation assays, luminometry,
flow cytometry, immunofluorescence microscopy, and genetic testing, may require referral
to specialized centers. The workflow minimizes unnecessary visits by allowing and
supporting blood or DNA samples to be sent to specialized laboratories for timely
evaluation.
Fig. 1
Algorithm to support a rational approach to diagnose inherited platelet disorders. A structured approach consists of assessment of family history, clinical investigations,
and laboratory tests. A standardized bleeding assessment tool (BAT), such as the ISTH-BAT
for adults or pedISTH-BAT for children, should be used to evaluate bleeding symptoms.
In girls and women with menorrhagia, the Pictorial Blood Assessment Chart (PBAC) may
be more appropriate. Mean platelet volume (MPV) and immature platelet fraction (IPF)
values are supportive for further investigations. Notably, MPV and IPF are device-dependent
and require internal reference ranges. Blood smear examination provides essential
morphological insights beyond platelet size and granularity, including abnormalities
in erythrocytes and leukocytes. The term “unlikely” indicates that conditions such
as immune thrombocytopenia (ITP), thrombotic thrombocytopenic purpura (TTP), hemolytic
uremic syndrome (HUS), and pseudothrombocytopenia should be excluded based on clinical
and laboratory findings before proceeding with specialized diagnostics. NGS, next-generation
sequencing.
Key Recommendations for Diagnostic Algorithm
Key Recommendations for Diagnostic Algorithm
A standardized questionnaire[13 ]
[14 ] should be used to assess the patient's bleeding tendency (strong consensus, 96%).
The pedISTH-BAT should be used for children and the ISTH-BAT for adults (consensus,
86%).
In suspected IPD cases, a complete differential blood count, including platelet size
distribution, mean platelet volume (MPV),[15 ] and quantification of the immature platelet fraction (IPF),[16 ] if available, should be conducted (strong consensus, 100%). As measurement methods
may influence values, MPV and IPF results should be interpreted accordingly.
A microscopic morphological cell assessment on routinely stained blood smears should
be performed (strong consensus, 100%).
In cases of repeated, clinically relevant, unexplained bleeding tendency, extended
hemostasis diagnostics, including platelet tests listed in the guideline, should be
performed (consensus, 91%).
Pre-analytics
Pre-analytics involves the initial steps of handling, preparing, and processing blood
samples, with standardized procedures to ensure accuracy and minimize variability.
The updated guideline provides recommendations on medication and dietary restrictions,
blood collection, sample storage, and transportation to maintain sample integrity
and ensure reliable results, particularly in platelet function testing.
Key Recommendations for Pre-analytics
Key Recommendations for Pre-analytics
Medications and nutritional supplements that impair platelet function should, where
medically acceptable, be discontinued or paused in a timely manner before platelet
function testing (strong consensus, 95%).
Blood collection for platelet function diagnostics should not be performed immediately
after venipuncture (consensus, 90%). Ideally, the first 3 to 4 mL of blood drawn should
be discarded.
During blood collection for platelet function analysis, bubble formation and overly
rapid withdrawal should be avoided (strong consensus, 100%).
Underfilled or overfilled blood collection tubes with citrate as an anticoagulant
should not be processed further (strong consensus, 100%). According to the guideline,
3.2 or 3.8% citrate tubes should be used, maintaining a 9:1 blood-to-citrate ratio.
Plastic (polypropylene) or siliconized glass tubes are recommended. Vacutainer® tubes
should be avoided for flow cytometry due to the potential risk of platelet activation
caused by the suction associated with this system.
Blood samples should be transported at room temperature without shaking and mechanical
stress (strong consensus, 100%).
The shipment of blood samples for flow cytometric analysis should be restricted and
only conducted after consultation with the analyzing laboratory (strong consensus,
100%).
Analysis of platelet aggregation should be completed within 4 hours after blood collection
(strong consensus, 100%).
Aggregometry
Aggregometry assesses platelet function by measuring light transmission (LT) changes
in response to specific reagents (agonists/inducers). It is a key diagnostic tool
for evaluating platelet function and identifying specific platelet disorders.[17 ]
[18 ]
[19 ]
[20 ] Light transmission aggregometry (LTA) and impedance aggregometry (IA) are commonly
used methods that provide quantitative data on platelet function.[21 ]
[22 ]
LTA remains the recommended method for diagnosing IPD.[23 ] It indirectly measures platelet aggregation by detecting changes in LT as platelets
form aggregates in response to agonists/inducers. IA is mentioned in the guideline,
but due to its lower sensitivity compared with LTA, it is not recommended as a primary
diagnostic tool for IPD.
The updated guidelines provide detailed instructions on using specific agonists to
evaluate platelet aggregation responses. The recommended agonists/inducers and their
standard final concentrations are as follows:
Adenosine diphosphate (ADP): 2.0 to 3.0 µM (doubling of concentration if needed).
Collagen: 2 µg/mL (higher concentration if needed and depending on the type of collagen, e.g.,
5 µg/mL).
Epinephrine: 5.0 µM (higher concentration if needed, e.g., 10 µM).
Ristocetin: 0.5/0.6 mg/mL (to induce vWF binding to platelet GPIb for detection of von Willebrand
disease—VWD of type 2B and platelet type VWD), and 1.2 to 1.5 mg/mL.
Arachidonic acid: 1.0 to 1.5 mM (to assess cyclooxygenase pathway function).
Thrombin receptor-activating peptide (TRAP): 10 µM (for PAR-1 activation).
The chapter also discusses the interpretation of results, potential pitfalls, and
troubleshooting tips to address common challenges encountered during aggregometry
testing.
Key Recommendations for Aggregometry
Key Recommendations for Aggregometry
LTA should be used as the primary method for diagnosing IPD (consensus, 90%).
In LTA, not only the maximum aggregation (%) but also the curve shape (lag time, aggregation
rate, disaggregation) should be evaluated (strong consensus, 100%).
Pathological aggregometry findings should be confirmed in a separate follow-up examination
(consensus, 94%).
Flow Cytometry
Flow cytometry is a key diagnostic tool for analyzing membrane protein expression
and post-exocytotic markers of platelets.[24 ]
[25 ] It uses fluorescently labeled antibodies to detect specific surface markers, enabling
the characterization of platelet receptor expression and activation status.
Flow cytometry is essential for diagnosing receptor defects, specifically Glanzmann
thrombasthenia, Bernard-Soulier syndrome, and storage pool disorders (SPD; α-/δ-granules)
as well as other rare IPD.[26 ]
While flow cytometry primarily assesses surface markers, the mepacrine assay enables
evaluation of dense (δ)-granules.[27 ]
[28 ] Mepacrine is a fluorescent dye that accumulates in δ-granules and is released upon
platelet activation. The resulting decrease in intracellular fluorescence serves as
a surrogate marker for granule content and secretion. The assay is robust and can
be performed up to 24 hours after blood collection.[28 ]
Another important functional application of flow cytometry is annexin-V staining,
which assess phosphatidylserine (PS) exposure, a key marker for procoagulant platelet
activity.[29 ] Defects in PS exposure are characteristic of specific IPD, including Scott syndrome
and Stormorken syndrome. In Scott syndrome, platelets exhibit defective PS exposure,
which can be identified by reduced annexin-V binding upon activation. In contrast,
Stormorken syndrome is characterized by increased basal annexin-V and CD62P staining,
reflecting heightened platelet activation. These assays can be complemented by platelet-dependent
thrombin generation tests to validate procoagulant defects.[30 ]
[31 ]
Although IPD typically affect the entire platelet population, variability in subpopulations
may occur due to residual protein expression, differential activation states, or compensatory
mechanisms. Flow cytometric analysis of platelet subpopulations may therefore provide
additional diagnostic insights.
The updated guideline includes protocols for using flow cytometry to assess platelet
surface markers, granule release, and platelet–leukocyte interactions. The guideline
also covers the interpretation of flow cytometry data and the standardization of reporting
results. Additionally, it highlights advanced techniques such as the use of specific
fluorescently labeled antibodies and multi-parameter analysis for comprehensive characterization
of platelet function.
Key Recommendations for Flow Cytometry
Key Recommendations for Flow Cytometry
Flow cytometry should be used for the diagnosis of Glanzmann thrombasthenia, Bernard-Soulier
syndrome, and SPD (strong consensus, 100%).
For validation, flow cytometry should be combined with other specialized tests for
platelet function analysis (consensus, 81%).
Device- and method-specific, internal laboratory reference ranges should be established,
and measurements should be conducted with regular control samples (consensus, 91%).
Age should be considered in the evaluation of results (especially in young children)
(strong consensus, 100%).
Immunofluorescence Microscopy
Immunofluorescence microscopy is considered a valuable supplementary tool for the
detailed morphological analysis of platelet components and diagnosis of certain IPD.
This technique involves the use of fluorescently labeled antibodies to detect specific
receptor-, granule-, and cytoskeleton-related proteins within platelets, allowing
for a detailed examination of platelet structure and potential activation in vivo.[32 ]
[33 ]
[34 ] The updated guideline provides expanded guidance on the use of immunofluorescence
microscopy for diagnosing IPD. In contrast to other methods, both stained and unstained
blood smears can be mailed to specialized laboratories for further evaluation.
Key Recommendations for Immunofluorescence Microscopy
Key Recommendations for Immunofluorescence Microscopy
Blood smears should only be prepared from fresh EDTA blood (at room temperature) (strong
consensus, 100%).
When performing staining, a control should always be included in the assessment (consensus,
82%).
Molecular Genetic Methods
Molecular genetic methods, including next-generation sequencing (NGS), provide a comprehensive
analysis of multiple genes associated with IPD, including both platelet function defects
(IPFD) and disorders affecting platelet number and structure.[35 ]
[36 ] It is essential for confirming a suspected diagnosis and for characterizing the
specific genetic background of an IPD. It is used for identifying heterozygous carriers
and enabling precise sub-classification of specific disorders.
Genetic testing may be especially useful in patients with bleeding symptoms where
an IPD is highly suspected, but standard platelet testing fails to provide clarity,
e.g., Quebec platelet disorder.[37 ]
[38 ] In complex diseases, where platelets are not the primary concern (“syndromes”),
genetic testing may be prioritized. Furthermore, in cases of suspected inherited thrombocytopenia,
e.g., ANKRD26-related thrombocytopenia, functional platelet assays may be of limited
diagnostic value, and early genetic testing can facilitate diagnosis.[39 ] Conversely, when no clear diagnostic suspicion exists, functional platelet testing
should be prioritized.
Extracted DNA is extremely stable and can be sent to specialized laboratories for
further evaluation.
The updated guidelines incorporate NGS into the diagnostic workflow, offering recommendations
for gene panel selection, data interpretation, and reporting in accordance with the
American College of Medical Genetics and Genomics (ACMG) guidelines.[40 ]
[41 ]
[42 ]
[43 ]
Key Recommendations for Molecular Genetic Methods
Key Recommendations for Molecular Genetic Methods
Molecular genetics should be used for the diagnosis of IPD (strong consensus, 95%).
Molecular genetics should be used to identify heterozygous carriers of IPD (consensus,
78%).
Future Developments
The updated guideline includes a new chapter on future developments in the diagnosis
of IPD. This chapter highlights emerging technologies and research fields that have
the potential to further advance this topic. Topics covered include the use of artificial
intelligence in diagnostics and novel biomarkers for platelet function. The goal is
to provide a perspective on how the diagnosis of IPD may evolve in the upcoming years.
The chapter also discusses the potential impact of personalized medicine and precision
health approaches on the treatment and management of patients with IPD.
Erratum Please note: in the ThromKidplus study group the name Carlo Zaninetti has online been inserted.
