Postscript

Introduction and bottom line

• Ethylenediaminetetraacetic acid (EDTA):
  • EDTA is routinely used as an anticoagulant for routine hematological analysis.¹
  • EDTA is a polyprotic acid which irreversibly chelates calcium, preventing clotting and stabilizing the fluidity of whole blood samples for electronic particle counting.
  • An advantage of EDTA is that it allows the best preservation of cellular components and blood cell morphology.
  • A rare disadvantage of EDTA as anticoagulant is spuriously low platelet count or pseudothrombocytopenia.
• EDTA-induced pseudothrombocytopenia:
  • A rare in vitro phenomenon (0.07 to 0.20% of the general population and 0.1 to 2% of hospitalized patients).
  • Caused by in vitro clumping, which in turn is mediated by EDTA-dependent platelet membrane modification and antibody binding:
    • The antibody targets a cryptic antigen inside the surface of glycoprotein IIb/IIIa that is exposed only when the glycoprotein experiences a conformational change in the presence of EDTA. Through its calcium chelating effect, EDTA dissociates the GPIIb/IIIa complex leading to exposure of the GPIIb epitope.
    • These EDTA-dependent antibodies:
      • Function optimally at cold temperature (between 0°C and 4°C).
      • Can belong to any immunoglobulin idiotype (most commonly IgG and IgM).
      • Cause clumping platelets in vitro, including on Wright-Giemsa stained peripheral blood smears.
• Suspect pseudothrombocytopenia in a patient with thrombocytopenia when no bleeding is present. 
• Confirm diagnosis by demonstrating platelet clumps on peripheral smear and normalization of platelet count when blood collected in a non-EDTA containing tube, preferably Na+ citrate. 
• Failure to recognize pseudothrombocytopenia may lead to unnecessary diagnostic testing and even result in unnecessary treatment.

 

Frequently asked questions

• Does pseudothrombocytopenia correlate with defect in platelet number or function in vivo?
  • No, it is purely an in vitro artifact.
• Is platelet clumping quantified?
  • No, it is read as absent or present.
• Are all cases of pseudothrombocytopenia caused by EDTA-dependent antibodies?
  • No, preanalytic variables such as poor handling of blood may cause platelet aggregation in the test tube. That being said, when we talk about pseudothrombocytopenia, we are referring to EDTA-induced pseudothrombocytopenia unless otherwise stated. 
• What is the difference between platelet clumping and platelet satellism?
  • Platelet clumps consist of aggregates of platelets, while platelet satellism consists of platelets rosetting around polymorphonuclear neutrophils. Both are mediated by EDTA-dependent antibodies, but the two generally do not occur in the same patient. 
• Can anticoagulants other than EDTA cause platelet clumping and pseudothrombocytopenia?
  • Yes, very rarely but through seemingly different mechanisms than with EDTA.

Definitions

• Pseudothrombocytopenia (spurious pseudothrombocytopenia) is thrombocytopenia resulting from an in vitro artifact.
• EDTA-dependent pseudothrombocytopenia is the phenomenon of a spurious low platelet count due to the presence of agglutinating anti-platelet antibodies that react with platelets in blood anticoagulated with EDTA.
• Platelet clumping is defined by the presence of at least 3–5 clustered platelets on peripheral blood smear. 
• Platelet satellism is defined by the presence of at platelets rosetting around polymorphonuclear neutrophils.

Epidemiology

• Pseudothrombocytopenia can occur in:
  • Otherwise healthy individuals.²
  • Patients with underlying conditions that have the ability to cause cryptoantibodies:
    • Autoimmune diseases (most common)
    • Malignancy 
    • Atherosclerosis-related diagnoses
    • Liver disease 
• EDTA-dependent pseudothrombocytopenia has been shown to appear during hospitalization, particularly after an infection.³
• Prevalence:⁴
  • 0.07 and 0.2% in a general outpatient population 
  • 0.13%-2% in hospitalized patients 
  • 0·013% among 76,498 unselected Polish blood donors
  • 15-17% in outpatients evaluated for isolated thrombocytopenia
  • Does not appear to be  age- or sex-dependent

Differential diagnosis

• Pseudothrombocytopenia may be observed in the setting of:
  • EDTA-dependent antibodies, which in turn may be associated with:
    • Platelet clumping
    • Platelet satellism
      • A rare in vitro phenomenon presenting with platelets rosetting around polymorphonuclear neutrophils.
      • Platelet rosetting is observed mainly around PMN, but occasionally also around eosinophils, basophils, lymphocytes or monocytes, too.
      • Does not typically occur with platelet clumping.
  • Preanalytical errors such as:
    • Poor mixing of sample and anticoagulant (which can cause platelet aggregation).
    • Low anticoagulant concentrations in the test tubes.
    • Transfer of blood accidentally drawn without anticoagulant to a collection tube anticoagulated with EDTA.
  • Giant platelets
• In vivo platelet clumping:
  • Type 2B VWD

History

• In 1969,  Gowland et al described 2 patients with pseudothrombocytopenia. He identified EDTA-dependent platelet clumping as the cause. They wrote: “In this case report we describe the discovery of an antiplatelet antibody in a patient with reticulum-cell sarcoma. The case is remarkable in that a reaction between platelets and antibody was observed only when the anticoagulant employed was EDTA (ethylenediamine tetra-acetate).”

• In 1973, Shreiner and Bell first characterized a novel type of platelet agglutinin in 6 patients:
  • The platelet agglutinin was:
    • Active at 37°C as well as at room temperature.
    • Directly attributed to the presence of EDTA.
  • The number of agglutinated platelets increased gradually for as long as 2 h while most platelets were already agglutinated after 30 min after contact with EDTA.
  • Agglutination activity was also found to be associated with the γ globulin fraction and although  immunoelectrophoretic of one patient sample showed the presence of only immunoglobulin
    G, clumping was inhibited by both anti-IgG and anti-IgM antisera.
  • Platelet agglutination was observed with both EDTA and diethylenetriamine-pentaacetate acide (DTPA), but not when other anticoagulants were used (e.g., sodium citrate and heparin).

Cohort studies

• Study of 18 patients with pseudothrombocytopenia
  • All were inpatients at the time of presentation.
  • (67%) were females.
  • Average age was 56 years.
  • All were severely ill.
  • In 11 patients (61%), presenting platelet counts were over 100 × 10⁹/L.
  • The pseudothrombocytopenia was initially noted a mean of 6 hospital days after admission (range 10-60 days).
• Study of 112 patients with pseudothrombocytopenia:
  • 43 males and 69 females.
  • 76% were outpatients.
  • The average number of platelets in the 112 patients studied was 72 × 10⁹/L.
  • About 30% of the patients had platelet counts below 30 × 10⁹/L.
  • Platelet clumping in citrate-anticoagulated specimens at room temperature was observed in 19 patients, 18 of whom contemporaneously presented EDTA-dependent clumping both at room temperature and at 37°C. 
  • EDTA-dependent antiplatelet antibodies were demonstrated in 93 of the 112 (83%) PTCP subjects:
    • 30 IgG
    • 48 IgM
    • 3 IgA
    • 9 IgG + IgM
  • Citrate-dependent antiplatelet antibodies were detected in 15 cases; 14 IgM, 1 IgA, and no IgG.
  • In four cases of citrate-dependent clumping, no antibodies were detected in the patients sera.
  • The author wrote:

• Study of 192 patients with pseudothrombocytopenia:
  • Mean age was 38.7 years.
  • Adult women comprised the largest group.
  • Platelet counts:
    • Most of the patients had platelet counts < 80 × 10⁹/L.
    • Two patients had automated platelet counts <5 × 10⁹/L, with nearly all the platelets aggregated.
  • 22.9% had one or more associated autoimmune disorders.
  • Platelet cryptantibodies and/or autoantibodies were positive in 30.1%.
  • 8.8% had had previous ITP or the pseudothrombocytopenia evolved into ITP.
  • 34.9% had a positive family history of pseudothrombocytopenia (familial PTCP).
  • None of the patients had bleeding symptoms.
  • Authors conclusion: “Our data suggest that PTCP should be considered a situation of autoimmunity”.

Mechanism

• EDTA anticoagulation is accompanied by time- and temperature-dependent in vitro aggregation of platelets, resulting in falsely low counts. 
• In vitro aggregation of platelets is mediated by:
  • Preformed (perhaps naturally occurring) antiplatelet antibodies interacting with hidden epitopes of platelet GPIIa/GPIII receptor complex made accessible by conformational changes induced by calcium chelating effect of EDTA:⁵
    • Calcium depletion by EDTA induces irreversible conformational changes of the platelet membrane.
    • EDTA-associated alteration of the platelet membrane unmasks hidden platelet receptor epitopes of the fibrinogen receptor GPIIb/IIIa.
    • These epitopes are “neoantigens” which bind ubiquitous non-pathogenic autoantibodies resulting in
      platelet aggregates (clumps).
    • Platelet aggregation leads to a spuriously decreased platelet count.
  • The involvement of the fibrinogen receptor was suggested when platelets from patients with Glanzmann’s thrombasthenia did not bind these antibodies.⁶
• Autoantibodies involved in pseudothrombocytopenia:
  • Most are IgG and react optimally at temperatures below 20°C (cold agglutinins, reacting optimally between 0°C and 4°C).
  • Some autoantibodies, mainly of the IgM class, may aggregate optimally at 37°C. This may thus result in spontaneous aggregation in EDTA and in citrate anticoagulated blood samples.
  • IgA antibodies have also been invoked.
• Hematology analyzers count the resulting platelet clumps as single giant platelets or as small lymphocytes in the white blood cell gate and indicate thrombocytopenia. Platelet clumps may lead to false leukocytosis. 
• Some authors have reported a seasonal fluctuation of pseudothrombocytopenia.
• Note: The modifying effect of EDTA on GPIIb-IIIa, resulting in a neoepitope that binds autoantibodies, is reported to involve the chelating effect of EDTA on calcium ions on the GPIIb-IIIa complex. It is not stated why citrate, which is used to prevent aggregation, does not have this effect despite also being a calcium chelator (though as noted in cohort studies above, citrate may have the same effect as EDTA on platelet clumping). Perhaps the difference relates to EDTA having a much higher calcium ion binding constant. 

Diagnosis

• Diagnostic criteria for pseudothrombocytopenia:
  • Absence of clinical bleeding
  • Automated platelet count <150 × 10⁹/L
  • Normal platelet count using a different anticoagulant (rarely manual counting)
  • Platelet clumps in the blood smear:
    • International Society of Laboratory Hematology consensus guidelines state that a positive finding of platelet clumping is defined as rare/occasional.
    • The Groupe Francophone de’Hematologie Cellulaire (GFHC) defines aggregation as the presence of at least five clustered platelets
    • The Norwegian quality improvement of laboratory investigations (Noklus) defines clumping as the presence of at least 3–5 clustered platelets.
    • Internal procedures at the Laboratory for Medical Biochemistry at Lovisenberg Diaconal Hospital defines a positive finding of platelet aggregates as a minimum of 3 clusters containing a minimum of 3 platelets.
    • Platelet aggregation is mostly referred to as a qualitative phenomenon, being either present or absent.

• Modern hematology instruments:
  • Detect a low platelet count in pseudothrombocytopenia.
  • Flag samples for suspected presence of platelet aggregation.⁷
• Suspect diagnosis if the laboratory finding of thrombocytopenia does not correlate with clinical signs and symptoms. 
• Confirm diagnosis by:
  • Demonstrating platelet clumping on peripheral smear 
  • Finding normal platelet count in blood collected in a different anticoagulant:
    • Sodium Citrate is preferred as alternative anticoagulant; it does not generally induce irreversible conformational changes of the platelet membrane.
    • Not all cases are resolved using sodium citrate as the anticoagulant.⁸. Alternative anticoagulants include:
      • Sodium fluoride
      • Ammonium oxalate 
      • MgSO4
      • Lithium chloride
      • Sodium fluoride
      • CPT (trisodium Citrate, 5’-Phosphate pyridoxal, and Tris)
      • Anti-platelet drugs (acetylsalicylic acid, prostaglandin E1, apyrase, monoclonal antibodies directed
        against GPIIb/IIIa)
      • Potassium azide
      • Kanamycin
      • Amikacin
    • Unfractionated heparin (UFH) not a recommended alternative to EDTA because it makes blood smear
      examination impossible due to cellular halos formation. Heparin can also cause/promote platelet
      activation, and hence cause spurious thrombocytopenia.
    • Other means of reducing platelet aggregation and clumping:
      • Maintain the sample at around 37◦C before testing.
      • Perform rapid analysis of blood specimen, due to time-dependent fall in platelet count from 1 min after blood collection to 4 h afterwards
• The presence of clumps varies over time for a given patient; periods with clumping alternate with periods without clumping with no obvious explanation.

Addendum:

1) Example of CBCs from a patient with pseudothrombocytopenia 

2) Findings from automated analyzers in patients with pseudothrombocytopenia