Review

National recommendations of the Croatian Chamber of Medical Biochemists

and Working group for Laboratory hematology of the Croatian Society of
Medical Biochemistry and Laboratory Medicine: Management of samples with
suspected EDTA-induced pseudothrombocytopenia
Lara Milevoj Kopčinović*1,2, Gordana Juričić3, Dragana Antončić4, Fran Smaić5, Brankica Šimac6, Ivana Lapić7,8, Vanja Radišić Biljak9,10
1Department of Clinical Chemistry, Sestre milosrdnice University Hospital Center, Zagreb, Croatia
2School of Medicine, Catholic University of Croatia, Zagreb, Croatia
3Department of Laboratory Diagnostics, General Hospital Pula, Pula, Croatia
4Clinical Department of Laboratory Diagnostics, Rijeka Clinical Hospital Centre, Rijeka, Croatia
5Department of Laboratory Diagnostics, General Hospital Dr. Josip Benčević, Slavonski Brod, Croatia
6Clinical Department of Laboratory Diagnostics, University Hospital Dubrava, Zagreb, Croatia
7Department of Laboratory Diagnostics, University Hospital Centre Zagreb, Zagreb, Croatia
8University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
9Department of Medical Laboratory Diagnostics, University Hospital Sveti Duh, Zagreb, Croatia
10Department of Sport and Exercise Medicine, University of Zagreb, Faculty of Kinesiology, Zagreb, Croatia

*Corresponding author: [email protected]

Highlights
• National recommendations aim to harmonize the management of EDTA-induced pseudothrombocytopenia
• The influence of preanalytical errors should be excluded
• The presence of platelet clumps should be confirmed by peripheral blood smear inspection
• Platelet counts should be determined from the 3.2% sodium citrate sample
• If EDTA-induced pseudothrombocytopenia is confirmed, platelet counts are reported with appropriate comments

Abstract
Pseudothrombocytopenia (PTCP) is defined by the occurence of spouriously low platelet count as a consequence of in vitro platelet aggregation. It
is a rare and benign artifact, not associated with any specific disorder or therapy, that becomes clinically relevant when it is not timely and reliably
recognized. Thus, it may result in inappropriate clinical decisions (i.e. unnecessary further testing, misdiagnoses and potential patients’ mismanage-
ment) unavoidably compromising patient safety. The most common form of PTCP is caused by ethylenediaminetetraacetic acid (EDTA).
Several approaches for the management of samples with EDTA-induced PTCP have been described in the literature. However, expert recommen-
dations are scarce. The scope of these recommendations is to assist in achieving national harmonisation in laboratory management (i.e. detecting
and reporting platelet counts) of samples with EDTA-induced PTCP. These minimal recommendations were prepared by the members of the joint
working group of the Croatian Chamber of Medical Biochemists and Working group for Laboratory Hematology of the Croatian Society of Medical
Biochemistry and Laboratory Medicine, and might be customized according to specific conditions (i.e. personnel and equipment) of each individual
laboratory. These recommendations are primarily intended to all laboratory professionals involved in the management of samples with EDTA-indu-
ced PTCP, but also to other healthcare professionals involved in collecting samples and interpreting complete blood count results.
Keywords: thrombocytopenia; pseudothrombocytopenia; hematology analyzers; procedures; harmonisation

Submitted: March 26, 2024 Accepted: September 9, 2024

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

Introduction
Thrombocytopenia is a common condition char- management (i.e. detecting and reporting platelet
acterized by low blood platelet count (< 150 counts) of samples with suspected EDTA-induced
x109/L) and associated with various disorders (1). PTCP. The proposed minimal recommendations
Unlike thrombocytopenia, pseudothrombocyto- were prepared by professional consensus of the
penia (PTCP) is defined by the occurrence of falsely members of the joint working group of the Croa-
low platelet count as a consequence of in vitro tian Chamber of Medical Biochemists and Working
platelet aggregation (2). This benign and rare phe- group for Laboratory Hematology of the Croatian
nomenon, presents in 0.03-0.27% of the general Society of Medical Biochemistry and Laboratory
population and in up to 15.3% of patients with Medicine, and might be customized according to
thrombocytopenia, and is not associated with any specific conditions (i.e. personnel and equipment)
specific disorder or therapy (2). If not timely and of each individual laboratory. These recommenda-
reliably recognized, PTCP might result in unwant- tions are primarily intended to all laboratory pro-
ed clinical implications. The omitted recognition fessionals involved in the management of samples
of this spurious cause of thrombocytopenia inevi- with EDTA-induced PTCP. Furthermore, they are
tably leads to inappropriate clinical decisions (i.e. also intended to other healthcare professionals in-
unnecessary further testing, misdiagnoses and po- volved in collecting samples and interpreting
tential patients’ mismanagement), unavoidably complete blood count results.
compromising patient safety (2,3).
The most common form of PTCP is caused by the Recommended criteria for raising
anticoagulant ethylenediaminetetraacetic acid suspicion on PTCP
(EDTA). Several approaches for the management
of samples with EDTA-induced PTCP have been
described in the literature. The addition of differ- If at least one criterion is met, the presence of
ent compounds (e.g. amikacin or kanamycin) to PTCP should be suspected and the recommend-
the EDTA-sample with confirmed PTCP in order to ed laboratory procedure for the detection and
prevent the formation of platelet aggregates or management of PTCP should be undertaken.
disaggregate platelets within already existing ag- 1. Low platelet count, i.e. < 100 x109/L, deter-
gregates, rapid EDTA-sample analysis, collection mined on the hematology analyzer in a pa-
and analysis of the EDTA-sample at 37 oC, and use tient without any clinical signs or history of
of alternative anticoagulants (e.g. acid citrate dex- thrombocytopenia or platelet disorders, and
trose, magnesium sulfate or sodium citrate) have without previous platelet count result; and/or
been suggested (2-4). Additionally, the potential
2. A significant decrease in platelet count (delta
usefulness of EDTA-sample vortexing for the pur-
check ≥ 40%) compared to the previous pa-
pose of disaggregating platelet clumps has also
tient result; and/or
been investigated, but there is lack of objective
evidence supporting the claim of its effectiveness 3. The presence of platelet and/or leukocyte-
for the complete disaggregation of platelet clumps associated flags on the hematology analyzer
and consequently accurate platelet counts deter- in conjunction with an altered platelet histo-
mination in samples with PTCP (5-7). However, ex- gram (2,3,8,9) (Figure 1).
pert recommendations dedicated to the manage-
ment of EDTA-induced PTCP are lacking.
Considering the heterogeneity of approaches Pseudothrombocytopenia is influenced by the
available for the management of EDTA-induced presence of immune (i.e. platelet autoantibodies),
PTCP, the scope of this document is to assist in chemical (i.e. anticoagulants) and physical (i.e.
achieving national harmonisation in laboratory time, temperature) factors which altogether con-

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

A B
PLT flag(s):
PLT Clumps?

Figure 1. Comparison of platelet histograms obtained by the impedance method on the Sysmex XN-1000 hematology analyzer (Sys-
mex, Kobe, Japan): in (A) blood sample without and (B) blood sample with suspected PTCP. Adapted from (11). PTCP - pseudothrom-
bocytopenia. PLT - platelets.

tribute to the occurrence of this in vitro phenome- (CBC) analysis: the presence of platelet clumps, gi-
non. In the literature, PTCP is most commonly as- ant platelets or any other flag related to abnormal-
sociated with the presence of autoantibodies ities in platelet distribution and/or size (9,10).
cross-reacting with the glycoprotein IIb/IIIa com- Available automated methods can provide reliable
plex (i.e. fibrinogen receptor). Its hidden epitopes platelet counts in the presence of large platelets
become exposed on the platelet membrane after and fragments of other blood cells; however none
reacting with calcium-chelating agents commonly can accurately determine the platelet count when
used as anticoagulants in whole blood sampling aggregates are present in the sample. Importantly,
tubes. the generation of flags related to platelet aggrega-
Pseudothrombocytopenia is most frequently tion depends on the analyzer settings and analyti-
caused by EDTA, but it can be associated with the cal technique used (Table 1). Notably, the imped-
presence of sodium citrate and lithium heparin, as ance method is the most sensitive to the presence
well as other anticoagulants (2,3,5). Once these an- of platelet aggregates (2). Therefore, reviewing the
ticoagulants come in contact with the whole platelet histogram obtained by impedance meth-
blood in which anticoagulant-dependent antibod- od is considered an important step in raising sus-
ies with optimal activity at temperatures below picion of PTCP, regardless of platelet count ob-
normal body temperature are present, platelet ag- tained or flag presence. Atypical platelet histo-
gregation will occur, eventually resulting in PTCP grams, displaying sawtooth features and serrated
(8). curve tails without baseline approaching at 20 fL,
According to the recommendations issued by the might indicate platelet aggregation (Figure 1).
International Society for Laboratory Hematology White blood cell fragments, red blood cell frag-
(ISLH), the peripheral blood smear should be re- ments and microcytes might also interfere with
viewed: a) if the platelet count determined by au- automated platelet counting (2,3).
tomated hematology analyzers is below 100
x109/L (at first patient measurement), and b) if the Recommended laboratory procedure
delta check of the platelet count (i.e. the difference for the detection and management of
between the current and previous patient’s result)
EDTA-induced PCTP
exceeds the criteria of 40% (10). Besides these
quantitative criteria, the ISLH recommends review The recommended procedure for detection and
of the peripheral blood smear regardless of the reporting platelet count when PCTP is suspected
platelet count if any of the following flags are gen- is described below. The procedure is summarized
erated during automated complete blood count in Figure 2.

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

Table 1. Overview of the most common hematology analyzers in Croatia with corresponding flags for suspecting PTCP

Manufacturer Analyzers Instrument flags

PLT clumps
Sysmex
XS, XE, XN, XT, XP, KX series, pocH 100i Plt Abn Distribution
(Sysmex, Kobe, Japan)
Giant Platelets
Platelet Clumps
Beckman Coulter Giant Platelet
ACT, UniCel DxH, LH750, HmX
(Beckman Coulter, Miami, USA) PLT Inter: Debris
RBC-PLT Overlap
PLT-CLM
Siemens
Advia 360, 120, 560, 2120i Plt Clumps
(Siemens, Marburg, Germany)
Large PLT
Mindray
BC 5150, BC 5380,
(Shenzhen Mindray Bio-Medical Plt Clump?
BC 5390
Electronics CO., Ltd., Shenzhen, China)
PLT Clump
Abbott Alinity hq, No MPV result
(Abbott, Santa Clara, USA) Cell-Dyn 1800, 3700, Ruby, Emerald NRBC
NWBC
PTCP - pseudothrombocytopenia. PLT - platelet count. RBC - red blood cell count. MPV - mean platelet volume. WBC - white blood
cell count.

• Platelet count < 100 x 109/L, and/or

• Decreased platelet count (delta
check ≥ 40%), and/or PTCP is suspected
• Platelet flags on the hematology
analyzer

Preanalytical errors
(visible clot?; inappropriate venipuncture site?)

No Yes

Microscopically confirm the presence of PTCP Coagulated sample – reject and repeat
(platelet aggregates? platelet satellitism?) collection
No
Yes
PTCP excluded, report platelet count and
Perform platelet count analysis using a 3.2% sodium platelet-related indices
citrate tube

Significantly higher platelet count observed Inspect the smear from the citrated sample for PTCP
in comparison to the EDTA-sample (platelet aggregates?)
No
Yes Yes

EDTA-induced No EDTA-and citrate-induced

PTCP confirmed PTCP confirmed

Report platelet count and platelet-related indices Report platelet count and platelet-related indices
obtained from the 3.2% sodium citrate tube using the Fonio method

Figure 2. Steps in the management of suspected EDTA-induced pseudothrombocytopenia. PTCP - pseudothrombocytopenia.

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

1. Exclusion of the influence of preanalytical for this purpose, as recommended by the Inter-
errors national Council for Standardization in Hematol-
ogy (15).
Inadequate preanalytical sample handling (i.e.
tube filling or blood mixing after collection)
might lead to falsely decreased platelet count. If After confirmation of platelet aggregates in the
PTCP is suspected according to the previous cri- sample, platelet count and associated platelet pa-
teria, the following should be excluded: rameters should not be reported. The laboratory
1. in vitro activation of the coagulation process, report should clearly indicate the suspected pres-
either by detecting a visible clot or a fibrin ence of platelet aggregates confirmed by micro-
strand in the EDTA-sample. Such samples scopic examination (Figure 4).
should be rejected and sample collection
should be repeated. 3. Measuring platelet count in samples with
2. Selection of inappropriate venipuncture site suspected EDTA-induced PTCP
– sample collection near the infusion line can
lead to contamination of the collected sam-
In samples with microscopically confirmed PTCP,
ple with the fluids being infused and, con-
platelet count and platelet-related indices
sequently falsely lowering all cell counts, in-
(mean platelet volume (MPV), plateletcrit (PCT),
cluding the platelet count. Whenever possi-
and others) should not be reported.
ble, an alternative blood collection site, pref-
erably from the opposite arm, should be se- The responsible medical biochemist/specialist
lected to avoid contamination (12). in medical biochemistry and laboratory medi-
cine should directly contact the patient’s health-
care provider, comment the findings and re-
After exclusion of the influence of preanalytical quest a repeated sample collection using a 3.2%
factors on the platelet count, a reflex testing for sodium citrate tube for platelet count determi-
platelet count analysis might be performed using nation. If the patient’s healthcare provider can-
an alternative analytical method (optical or fluo- not be reached, the patient should be directly
rescence platelet counting), if available. The result invited for repeated blood sampling.
obtained for platelet count analysis using the al- In cases when a corresponding 3.2% sodium cit-
ternative method should be regarded as informa- rate tube is already available in the laboratory
tive, not definitive (13,14). (e.g. when coagulation testing has been re-
quested), it should be used for platelet count
2. Microscopic confirmation of PTCP determination when EDTA-induced PTCP is sus-
pected, without the need for repeated blood
Once preanalytical factors are excluded, PTCP sampling.
should be confirmed by morphologic assess- Platelet count should be determined on the he-
ment of the peripheral blood smear. Slide prep- matology analyzer from the 3.2% sodium citrate
aration and staining might be performed manu- tube and the result should be multiplied by 1.1
ally (stained with May–Grünwald Giemsa, MGG) to eliminate the dilution effect of the liquid cit-
or using an automated slide maker and stainer. rate anticoagulant.
The presence of platelet aggregates or platelet The platelet count and platelet-related indices
satellitism in EDTA-anticoagulated samples, as are reported from the 3.2% sodium citrate tube
shown in Figure 3, should be confirmed by in- after confirmation that the PTCP is EDTA-in-
spection of the peripheral blood smear using duced, that is in cases when:
light microscopy which is the method of choice

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

A B

Figure 3. (A) Platelet aggregates and (B) platelet satelitism in peripheral blood smear prepared from EDTA-anticoagulated blood (16).

Figure 4. Examples of laboratory reports for the following situations: (A) platelet aggregates are found in the EDTA-sample and
EDTA-induced pseudothrombocytopenia is suspected, (B) reporting of platelet count and platelet-associated parameters from the
sodium citrate tube, (C) reporting of platelet count and platelet-associated parameters from the sodium citrate tube if these param-
eters are available as separate requests from the laboratory information system, (D) platelet aggregates are found both in the EDTA
and citrate sample and (E) reporting of the platelet count using the manual Fonio counting method. Reference intervals are derived
from the Croatian national harmonisation project of reference intervals (18).

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

Figure 4. Continued.

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

1) a significantly higher platelet count is ob-

served in comparison to the EDTA-sample plate- port (Figure 4A): Due to suspected EDTA-induced
let count, or pseudothrombocytopenia in the sample, blood
2) the presence of platelet aggregates in the sampling in a tube with sodium citrate as the anti-
3.2% sodium citrate tube is excluded after mi- coagulant is required.
croscopic evaluation of the peripheral blood If platelet clumps are not encountered in the cit-
smear prepared from the 3.2% sodium citrate rated sample, the platelet count and related
tube. platelet indices should be reported as deter-
If the platelet count measured from the 3.2% so- mined by the hematology analyzer from the
dium citrate tube is not significantly higher or 3.2% sodium citrate tube and this should be
platelet aggregates are present in smears pre- clearly indicated on the report as shown on Fig-
pared from the 3.2% sodium citrated sample ure 4B. Additionally, if analyses from the 3.2%
(the incidence is in about 10-20% of patients), sodium citrate tube are available as separate
both EDTA- and citrate-induced PTCP are con- single requests from the laboratory information
firmed. In this case platelet counts might be de- system, platelet count and related platelet indi-
termined using the Fonio manual counting ces should be reported without the need for
method (see Appendix 1) (17). commenting (Figure 4C).
If PTCP with both types of anticoagulants (EDTA
and 3.2% sodium citrate) has been confirmed,
the platelet count and related platelet indices
A „significantly higher“ platelet count (i.e. the ex- (MPV, PCT, etc.) should not be reported. All other
act rise in platelet count which should be obtained results pertaining to CBC should be reported as
from the citrated sample to confirm EDTA-induced determined from the EDTA-sample on the he-
PTCP) cannot be unequivocally and strictly de- matology analyzer. The platelet count should be
fined because it depends on multiple factors (ED- reported as determined by the Fonio manual
TA-sample transport time and temperature, num- counting method from a capillary sample. A
ber and morphology of platelet aggregates pre- comment should be clearly indicated on the
sent in the EDTA-sample, original platelet count, corresponding laboratory report: Analyses were
patient’s medical history, etc.). Thus, the quantifi- performed from the EDTA and citrate sample.
cation of a „significantly higher“ platelet count de- EDTA- and citrate-induced pseudothrombocytope-
termined from the 3.2% sodium citrate tube is in- nia are confirmed in the samples. For future deter-
dividual and depends on the responsible medical mination of the complete blood count, the tube
biochemist/specialist in medical biochemistry and with EDTA should simultaneously be collected with
laboratory medicine. a capillary sample for the determination of the
platelet count (finger prick directly on the slide)
4. Reporting platelet counts (Figure 4D).
When platelet count is determined in a capillary
If EDTA-induced PTCP is suspected due to the sample using the Fonio manual counting meth-
presence of platelet aggregates in the peripher- od, MPV should not be reported and an appro-
al blood smear, the platelet count and related priate comment should be included within the
platelet indices (MPV, PCT, etc.) should not be re- laboratory report: EDTA- and citrate-induced
ported. All other results pertaining to the com- pseudothrombocytopenia are confirmed in the
plete blood count (CBC), should be reported as samples. The platelet count was determined by
determined from the EDTA-sample on the he- manual counting in the capillary blood smear.
matology analyzer. A comment should be clear- MPV cannot be reported (Figure 4E).
ly indicated on the corresponding laboratory re-

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Milevoj Kopčinović L. et al. Management of suspected EDTA-induced pseudothrombocytopenia

5. Reporting platelet count from samples with – review & editing; G Juričić: Conceptualization,
excluded PTCP Methodology, Writing - original draft, Writing – re-
view & editing; D Antončić: Methodology, Writing
- original draft, Writing – review & editing; F Smaić:
If the presence of EDTA- and/or citrate-induced
Writing - original draft, Writing – review & editing.
PTCP is excluded, the platelet count determined
B Šimac: Methodology, Writing - original draft,
on the hematology analyzer from EDTA-sample
Writing – review & editing. I Lapić: Methodology,
should be reported. A comment should be indi-
Writing - original draft, Writing – review & editing.
cated on the laboratory report: The obtained re-
V Radišić Biljak: Conceptualization, Methodology,
sults exclude the presence of EDTA-induced pseu-
Writing - original draft, Writing – review & editing.
dothrombocytopenia.
Potential conflict of interest
None declared.
Author contributions
L Milevoj Kopčinović: Conceptualization, Method- Data availability statement
ology, Writing - original draft, Visualisation, Writing No data was generated during this study.

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Appendix 1: Platelet counting method according to Fonio

Method principle Procedure

Platelet count according to Fonio is a manual The fingertip is disinfected and pricked with a lan-
counting method for determining the number of cet. The resulting drop of blood is transferred di-
platelets and is used only if platelet count cannot rectly from the fingertip to the microscopic slide
be reliably determined by automated counting by placing the upper surface of the slide in contact
methods on a hematology analyzer (e.g. if platelet with the drop of blood. After that, a smear is pre-
aggregates are still present in sample with 3.2% pared using the MGG (Pappenheim) staining
sodium citrate) or when the unreliability of plate- method. Platelets are counted under the immer-
let counts obtained by automated analyzers is sus- sion lens of a light microscope at 100x magnifica-
pected (e.g. in the presence of macroplatelets, an tion. It is necessary to count platelets alongside
unusual histogram or scatter diagram and other with erythrocytes, until the number of a thousand
individual cases based on the laboratory profes- erythrocytes is achieved (usually 5 fields with 200
sional’s assessment) (17). erythrocytes in each). The platelet count is ob-
If EDTA- and citrate PTCP is confirmed, platelets tained by multiplying the erythrocyte number de-
might be counted according to Fonio by collecting termined on the automated analyzer and the
finger stick capillary blood, preparing a stained number of platelets counted in the smear:
smear using the MGG method, and counting plate- Platelet count (x109/L) = A x Erc / 1000
lets per thousand erythrocytes under a light mi-
croscope. If aggregates are not present, but plate- where A is the number of platelets counted per
lets still need to be counted according to Fonio, a 1000 erythrocytes and Erc is the number of eryth-
smear can be made directly from a test tube with- rocytes determined on the analyzer (x1012/L).
out the need for transferring the blood drop di-
rectly from the finger to the slide.

Biochem Med (Zagreb) 2024;34(3):030504 https://doi.org/10.11613/BM.2024.030504

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