Ultrastructural and morphometric analysis of enlarged platelets in congenital isolated asplenia

Introduction. Congenital asplenia is an extremely rare condition that can be separate entity due to a specific defect of spleen development or may occur in the context of a malformation syndrome. The patients with asplenia have thrombocytosis and susceptibility to life-threatening infections. Case report. We report a 52-years-old female patient with isolated congenital asplenia with pseudothrombocytopenia and giant platelets. Estimation of platelets life with ra-dioactive indium showed normal lenght of platelets life (9 days). Flow cytometric analysis of platelets showed normal expression of CD41 and CD42b antigens. The mean platelet diameter of asplenic patient measured on the ultrathin sections by the transmission electron microscope was significantly higher than in the healthy individuals (3.81 ± 1.16 µm vs. 2.37 ± 0.61 µm, p < 0.05). There were very few platelets of diameter more than 4 μ m found in healthy individuals (around 1%) in comparison to > 40% of the patient’s platelets. The ultrastructural studies revealed normal morphology of megakaryocytes. The platelets were uniformly spheroid in shape with conspicuous pseudopodia and the centralization of granules. There were no marginal bands of microtubules inside the platelets. Conclusion. The first case of congenital asplenia with the pseudothrombocytopenia and giant platelets is presented. We discussed the pathogenesis of giant platelets and possible relation of observed ultrastructural changes of platelets with the severe three-vessel coronary artery disease in our patient.


INTRODUCTION
Congenital asplenia is very rare condition (1 case in 20.000 live births) that occurs sporadically,but alsomay have familial association [1].Congenital asplenia can be a separate entity due to a specific defect of spleen development or may occur in the context of a malformation syndrome [2].Since the spleen is a major producer of antibodies and splenic macrophages have a major role in bacterial phagocytosis, the patients with asplenia or hyposplenia are susceptible to lifethreatening or fatal septicemia caused by encapsulated pathogens [2,3,4].Life-threatening infections usually occur in childhood, but have been described also in adult patients [2,3,4].All cases with isolated congenital aspleniapublished so far had thrombocytosis [5,6,7] together with a common finding of small platelets [2].
Platelet production represents the final stage of megakaryocyte development [2].It is commonly accepted that during the final stages of differentiation, megakaryocytes extend cytoplasmic protrusions referred to as proplatelets [8].Proplatelets are branching long processes that extend from mature megakaryocytes into the sinusoidal blood vessels of the bone marrow.Proplatelet formation is dependent on the function of microtubules [9].Microtubular coils similar to those observed in blood platelets can be detected only at the ends of proplatelets and not within the platelet-size beads found along the length of proplatelets [10].Thon at all recently identified a previously unrecognized intermediate cell, which they termed a preplatelet [11].Preplatelets are defined as discoid cells, anucleate platelet progenitors 3-10 µm across that are retainingthe capacity to convert into barbell-shaped proplatelets [11,12].These preplateletsmay be relatedto bothyoung (reticulated) platelets associated with increased RNA content, and large plateletscommonly seen in inherited/acquired macrothrombocytopenias [11,12].Preplatelets reversibly convert into barbell-shaped proplatelets in the blood that divide to form two platelets [11,12].When the number of peripheral microtubules is increased, the spectrin-based membrane skeleton becomes disassembled and preplateletsturn out to be incapable of undergoing further barbell proplatelet conversion, resulting in terminal platelets of a larger size [11,12].Macrothrombocytopenia may therefore represent a failure to convert preplatelets to barbell-proplatelets [11,12].We report a unique case of isolated congenital aspleniapresentedwithpseudothrombocytopeniaand enlarged platelets.The patient also hadsevere coronary heart disease, with no history of life-threatening infections or bleeding.
The particular aim of this case report was ultrastructural and morphometric analysis ofenlarged platelets obtained from patient , speripheral blood and bone marrow.

CASE REPORT
A 52-year old woman referred to Hematology department in 2003 because of pseudothrombocytopenia.She had a 5-years history of arterial hypertension and 2-years history of depressive neurosis.There was no history of abdominal surgery, infections, thrombosis or bleeding.She had no history of prolonged menstrual bleeding.Her two sons had normal number of platelets, normal spleen on abdominal ultrasonography and absence of gigantplatelets in peripheral blood smear.Physical finding was normal.Full blood counts and white cell differential werein normal range, except for platelet number which appeared to be low (54x10 9 /L) and mean platelet volume which was high (14.1fL).However, when platelets wereanalyzed using CD61 (GPIIIa) MoAbs (ImmunoPLT method) or when they were counted in chamber,platelet number was found to be around 100x10 9 /L.On blood smear platelets were unusually large and Howell-Jolly bodies were present in red blood cells.The platelet aggregation in response to ristocetin, ADP, TRAP and adrenalin, as well asprothrombin time, aPTT and bleeding time were normal.Platelet survival analyzed by radioactive indium showed normal life span (9 days) with decreased index of production of platelets in the bone marrow (0.24, normal 1.0).
Flow cytometry analysis of platelets showed normal expression of the CD42b and CD41 antigens.Bone marrow aspirate and trephine biopsy showed mild hypercellularity, normal number of megakaryocytes, mild reactive changes without signs of malignant infiltration.
Routine biochemistry and thyroid hormones were in referent range.Immunological tests (ANA, RF, antibodies against cardiolipin and β 2 -glycoprotein)and lupus anticoagulanswere negative.Cytogenetic analysis showed normal 46,XX karyotype.Radiography of the chest showed normal finding.The spleen was not visible by abdominal ultrasonography and computer tomography of abdomen were unremarkable.Scintigraphy using 99mTc-labeled red cells showed absence of splenic tissue in abdominopelvic cavity.
Ultrastructural analysis of platelets was done by using transmission electron microscope (TEM, FEI Morgagni 268D) and showed abnormal morphology of platelets, both in peripheral blood and in the bone marrow (Fig. 1A-D).Platelets were uniformly spheroid in shape with conspicuous pseudopodia and the centralization of granules (Fig. 1D).There were no marginal bands of microtubules inside the platelets(Fig.1D).Megakaryocytes of normal morphology were found on ultrathin sections of bone marrow aspirates (Fig. 1E).
Platelets in bone marrow were also large, spheroid with numerous pseudopodia and the centralization of granules with no signs of dilatation of open canalicular system in the majority of them (Fig. 1F-H).
In 2009 pacemaker was implanted because of tachy-brady form of atrial fibrillation.In July 2012 she complained onchest pain and fatigue.Coronarography revealed three-vessel coronary artery disease.Urgent surgical revascularization of the heart was successfully done, followed by anticoagulant therapy.On her last follow-up (April 2016) she was in good clinical condition and without complains.

DISCUSSION
Isolated congenital asplenia is a rare condition.Literature review [2] only yielded about 50 cases since the first report of Myerson and Koelle in 1956 [13].This report described 24 sporadic and 26 familial cases of isolated congenital asplenia, majority in children [2].Our patient did not have other somatic anomalies beside aspleniaand this condition was not found in any family member.Therefore, we concluded that our patient had sporadic isolated congenital asplenia.
Majority of the reported adult cases of congenital asplenia presented with thrombocytosis [2].On the contrary, our patient had near normal number of plateletswhich were unusually large.To our knowledge, this is the first reported case ofasplenia with giant platelets.More than 40% of measured asplenicpatient's platelets diameters were larger than 4µm, while the diameters of platelets obtained from healthy individuals ranged above that value very scarce, around 1%.The largest measured diameter in patient sample(6.7 µm) wasalmost two folds higher than the largest value of platelets found in healthy individuals.
The unusually large platelets were noticed before in macrothrombocytopenias [14].
Although the platelets seen in our case have largesize, they differ from the appearance of the macrothrombocytes seen in the macrothrombocytopenias.Ultramicrographs of the previously published cases with macrothrombocytopenias revealed aggregates with large vacuoles and areas mostly devoid of dense bodies and alpha granules [14].In the case presented here there were no large vacuoles seen in platelets while both dense bodies and alpha granules were apparent.
Some reported patients with congenital asplenia had thromboembolic complications.An adult case of isolated congenital spleen agenesis complicated by thrombocytosis and chronic thromboembolic pulmonary hypertension was previously described [15] as well as the patient with congenital asplenia, thrombocytosis and myocardial infarction [5].Our patient also had diffuse coronary artery disease but without thrombocytosis.However, her cardiovascular condition might be related to ultrastructurally altered platelets.It was previously shown that higher-than normal mean platelet volume (MPV) may be considered as risk factor for vascular complications [16].It was previously noticed that platelet size correlates with platelet reactivity and that larger platelets have greater prothrombotic potential.Elevated platelet size (MPV) was found to be associated with increased platelet aggregation, increased expression of adhesion molecules and elevated risk of cerebro-and cardiovascular diseases [16,17].
Number of platelets in our patient determined by automatic counter was significantly lower than determined immunologically by CD61 or by counting in a chamber due to the inability of automatic counter to recognize large platelets.Precise determination of platelets count in patients with giant platelets requires the use of immunological method or counting platelets in chamber.It is particularly important when patient has indication for anticoagulant or antiplatelet therapy, as it was in our case.
It is well known that in resting state platelets are discoidwhereas activated platelets develop pseudopodia or extensions from the cell wall [11].Platelet activation is also consistent with certain morphological features such as dilatation of open canalicular system [18].However, open canalicular system did not show any dilatation in cells of the specimens that we studied.Both platelets seen in the bone marrow and peripheral blood were rounded and there were no prominent extensions from the cell wall and the marginal microtubule coils could not be spotted.Perhaps large platelets seen in our patient with asplenia were not activated cells, but rather cells that represent or resemble proplatelets.Namely, proplatelets have an average diameter of 2-4μm and can be distinguished from platelets by their diameters, (>2μm vs. ≤2 μm, respectively) [11].
The conversion from pre-to proplatelet is driven by microtubule-basedforces, which are governed by two major biophysical properties:microtubule coil diameter and microtubule coil thickness [11].Interestingly, these forces both regulate and predict the size of circulating platelets generated by proplatelets, providing an explanation for the approximately 2µm diameter of platelets [11].According to the Thon et al, circular preplateletsare released into the blood, rapidly convertinto barbell proplatelets, and undergo fast rounds of abscissionthat result in mature platelets, or alternatively, preplatelets may becometrapped in the microcapillaries of the bone marrow, lung, orspleen where intravascular shear forces drive proplatelet toplatelet production [11].In respect to, absence of the spleen can lead to partial absence of transition of proplatelets to platelets.