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Keywords:

  • cyclic thrombocytopenia;
  • T-cell receptor;
  • thrombopoietin;
  • cyclosporine

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Case report
  5. Discussion
  6. References

Summary. Cyclic thrombocytopenia is a rare disorder characterized by periodic platelet count fluctuations of unknown aetiology. We report on a female patient with cyclic changes of platelet counts ranging from 6 × 109/l to 753 × 109/l in 4-week intervals. Platelet counts were inversely correlated to thrombopoietin levels suggesting production failure. Reticulocyte counts and neutrophil counts showed similar, but less prominent, fluctuations. Clonal T-cell receptor rearrangement was detected in bone marrow samples as well as in peripheral blood. Cell typing of blood lymphocytes revealed a relative increase in CD3+ T cells. Treatment with cyclosporine A resulted in a substantial improvement of platelet counts. Taken together, we provide evidence for clonal T-cell mediated bone marrow failure with cyclic impairment of thrombopoiesis responsive to cyclosporine therapy.

Cyclic thrombocytopenia (CTP) is a rare disorder characterized by periodic oscillations of the platelet count. The aetiology of CTP is poorly understood and various pathogenetic mechanisms have been proposed. In some patients, immune-mediated platelet destruction seemed to be responsible for thrombocytopenia with an inverse relationship between platelet-associated antibodies and platelet counts (Yanabu et al, 1993). In another patient, antibodies against megakaryocyte progenitor cells have been described to inhibit megakaryocyte growth (Zent et al, 1999). Megakaryocyte progenitor cell count fluctuations have been reported to precede cyclic changes of platelet counts (Nagasawa et al, 1995) and, in one patient, the patient's peripheral mononuclear cells suppressed megakaryopoiesis (Dan et al, 1991). However, no data on the underlying molecular mechanisms have been published to date. We report on a CTP patient in whom clonal T-cell receptor rearrangement was demonstrated.

Detection of clonal T-cell receptor (TCR) gamma gene rearrangements by polymerase chain reaction (PCR) analysis of genomic DNA. DNA was prepared from bone marrow and peripheral blood using cell lysis, proteinase K digestion (Boehringer Mannheim, Mannheim, Germany), phenol extraction and ethanol precipitation, according to standard protocols. Analysis of TCR gene rearrangements was performed using a multiplex PCR system, containing eight V primers complementary to the various V(ariable) segments and two consensus J primers complementary to the J(oining) segments of the TCR gene, in a single PCR reaction as previously reported (Födinger et al, 1999).

Thrombopoietin (TPO) measurement. Serum TPO was measured using a commercially available sandwich enzyme immunoassay (R & D Systems, Minneapolis, USA), according to the manufacturer's guidelines. The normal range of serum TPO in our laboratory was 50–200 pg/ml.

Detection of antiplatelet antibodies. Platelet immunoglobulin (Ig)G antibodies against glycoprotein (GP) IIb/IIIa and GP Ib/IX were analysed as described previously (Panzer et al, 1997).

Progenitor cells. Colony-forming unit granulocyte macrophage (CFU-GM, normal range: 50–936/ml), burst-forming unit erythroid (BFU-E, normal range: 120–1862/ml) and colony-forming unit megakaryocyte (CFU-Meg, normal range: 24–616/ml) were cultured from peripheral blood obtained on 1, 8 and 12 March 2001 as described (Geissler et al, 1990).

Case report

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Case report
  5. Discussion
  6. References

A 56-year-old woman presented in June 1999 with recurrent haematomas and severe thrombocytopenia. Platelet counts were found to oscillate between 6 × 109/l and 753 × 109/l in 4-week intervals (Fig 1). Physical examination was unremarkable. Splenomegaly was excluded by ultrasonic examination. Platelet antibodies were not detectable, neither in the patient's serum nor on the autologous platelets. Differential blood counts, coagulation tests and values of blood chemistry were within the normal range. Platelet counts were inversely correlated to TPO levels (Fig 2A). Platelet counts, neutrophil counts and reticulocyte counts were monitored closely during a cycle in March 2001 (1–13 March, Fig 2A and B). Progenitor cells were cultured on 1 March (platelet count 49 × 109/l), 8 March (platelet count 38 × 109/l), and 12 March (platelet count 124 × 109/l). CFU-GM amounted to 52, 980, 1234/ml blood and BFU-E to 167, 980, 1118/ml blood on 1, 8 and 12 March respectively. CFU-Meg were available on 8 March (893/ml blood) and 12 March (1275/ml blood). A bone marrow biopsy obtained at the time of platelet nadir (6 March, platelet count 8 × 109/l) showed a hypercellular marrow with increased myelopoiesis and a normal megakaryocyte count. To avoid bleeding, the patient received a platelet concentrate from a single donor after the biopsy. The platelet count was 46 × 109/l on the following day and the corrected count increment 14 × 109/l [cut-off for a satisfactory corrected count increment: > 5 × 109/l (Kurz et al, 2001)]. After complete recovery of the platelet count, the marrow biopsy revealed a normocellular marrow with a slight increase in morphologically normal megakaryocytes.

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Figure 1. Platelet oscillations were documented between June 1999 and December 1999, August 2000 and November 2000, and February 2001 and January 2002. CSA treatment was initialized on 27 March 2001 ([UPWARDS ARROW]). Platelet counts ranged between 6 × 109/l and 753 × 109/l.

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image

Figure 2. Platelet counts (PLT), thrombopoietin levels (TPO), absolute neutrophil counts (ANC) and reticulocyte counts before (March 2001; A and B) and after (October 2001; C and D) initiation of cyclosporine treatment. Cyclosporine led to a substantial improvement of platelet counts, although cyclic oscillations persisted (A versus C). Neutrophil and reticulocyte counts were also subjected to cyclic changes (B and D).

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Immunophenotypic analysis of peripheral blood lymphocytes revealed a relative increase in CD3+ T cells. Clonal T-cell receptor rearrangement was detected in all bone marrow and peripheral blood samples tested. Immunglobulin rearrangement was polyclonal. Cyclosporin A treatment (2 × 200 mg/d) was initiated on 27 March 2001. Because of gingival hyperplasia and hypertension, the dosage was reduced to 2 × 100 mg/d (corresponding to serum levels between 100 ng/ml and 200 ng/ml). Thereafter, therapy was well tolerated and led to a substantial clinical improvement with platelet counts increasing sufficiently to prevent further bleeding tendency. Cyclic changes of platelets counts persisted, however (Figs 1 and 2C).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Case report
  5. Discussion
  6. References

Cyclic thrombocytopenia is a rare disorder characterized by periodic oscillations of the platelet count. The pathogenesis of CTP is poorly understood and various mechanisms have been proposed (Dan et al, 1991; Yanabu et al, 1993; Nagasawa et al, 1995; Zent et al, 1999).

To date, clonal T-cell receptor rearrangement, as it was detected in our patient, has not been reported. On the basis of this finding, we surmise that a clonal T cell-mediated process is responsible for the disease in our patient. Clonal T-cell receptor rearrangement is usually found in neoplastic disorders involving the T-cell lineage. In particular, clonal CD3+ T cells have been implicated in the pathogenesis of T-cell large granular lymphocyte (LGL) leukaemia, a disorder associated with severe and sometimes cyclic cytopenias (Lamy & Loughran, 1999). In this regard, it is noteworthy that in parallel with the cyclic changes of platelet counts, oscillations of neutrophil counts and reticulocyte counts were also detectable in our patient (Fig 2B and D). Moreover, during one closely monitored cycle, the increase in CFU-Meg, CFU-GM and BFU-E ran in parallel with platelet counts, granulocyte counts and reticulocyte counts. Thus, not only thrombopoiesis, but also – although to a lesser extent – erythropoiesis and granulopoiesis were affected in our patient. In contrast to the findings in patients with T-cell LGL leukaemia, LGL were absent from the peripheral blood or bone marrow in our patient and neutropenia was not apparent.

Immune-mediated platelet destruction has also been implicated in the pathogenesis of CTP (Yanabu et al, 1993). However, increased destruction of platelets obviously did not play a major role in our patient. In particular, antiplatelet antibodies were not detectable and the transfusion of a platelet concentrate resulted in a satisfactory platelet count increment. Furthermore, in immune thrombocytopenia, TPO levels are normal or only slightly elevated, whereas in our patient TPO levels were elevated to an extent that was incompatible with immune thrombocytopenia.

A wide variety of therapeutic regimens, including administration of thrombopoietin, high-dose immunoglobulin, immunosuppressive drugs, splenectomy or hormones, have been given to CTP patients (Telek et al, 1989; Dan et al, 1991; Helleberg et al, 1995; Rice et al, 2001). As a clonal T-cell receptor rearrangement was detected in our patient, indicating a T cell-mediated process, cyclosporine therapy was initiated. Cyclosporine led to a marked improvement, albeit not normalization, of the platelet count. Nevertheless, the major improvement in the patient's clinical presentation, as well as the significant increase in the platelet count during cyclosporine therapy, further supports our concept of a T cell-mediated pathogenesis of CTP in this patient.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Case report
  5. Discussion
  6. References
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