Posttransplant neutropenia (PTN) is common after kidney transplantation and occurs mostly in the first year after transplant, with an incidence ranging from 10% to 55.5% in retrospective studies (1–3). The main cause of PTN is drug toxicity: immunosuppressive treatment such as mycophenolic acid (MPA) or azathioprine, sirolimus, and anti-infectious agents, especially valganciclovir (VGCV) and trimethoprim-sulfamethoxazole (TMP/SMX) (4, 5). Cytomegalovirus infection is the leading cause of infectious neutropenia. Large granular lymphocytes (LGL) proliferation, hematological diseases, and recurrence of primary autoimmune diseases may also be responsible for PTN.
PTN increases the infectious risk in transplanted patients and the risk of infection-related death (2, 3). PTN had also been associated with acute rejection (2, 3), and this phenomena has been related to the extended withdrawal of MPA in one study (2). Therefore, the understanding of PTN’s mechanisms is critical.
We report here two documented cases of an underdiagnosed entity of PTN and present compelling evidence for an autoimmune mechanism.
A 36-year-old man, with a history of radiochemotherapy treatment for neuroblastoma during infancy, underwent kidney transplantation in November 2011. On day 0 posttransplant, blood cell count was normal. He received basiliximab induction therapy combined with tacrolimus, MPA, and prednisone and infectious prophylaxis, including TMP/SMX and VGCV. He had a good graft recovery with a stable creatinine at 1.3 mg/dL. On day 50 posttransplant, he developed isolated severe neutropenia (absolute neutrophil count [ANC]: <0.5 G/L) and subsequently a Pseudomonas aeruginosa pulmonary infection. ANC was unaffected by MPA dose modifications, which was tapered on day 85, was discontinued on day 92, and was replaced by azathioprine on day 106. A bone marrow aspirate was performed on day 106 and showed active granulopoiesis with maturation arrest at the promyelocytic stage. Hemophagocytosis was not observed. VGCV and TMP/SMX were discontinued on day 116 and switched for valaciclovir and atovaquone, respectively, without affecting the ANC. The patient received granulocyte-colony stimulating factor (G-CSF) twice weekly from day 129 to day 179, which allowed a normal blood granulocyte count. Withdrawal of G-CSF was followed by prompt relapse of severe neutropenia.
A 31-year-old woman with end-stage kidney disease secondary to nephronophtisis associated with a mutation in NPHS1 underwent kidney transplantation. On day 0 posttransplant, blood cell counts were normal. She received anti-thymocyte globulin combined with tacrolimus, MPA, and prednisone, and TMP/SMX and VGCV. She was highly sensitized (poly-immunization with anti-HLA antibodies) and was also treated with plasmapheresis, rituximab (375 mg/m2, one infusion), and three courses of IVIg (2 g/kg). She received high-dose steroids for two episodes of acute cellular rejection (day 22 and day 150) and creatinine stabilized at 2.2 mg/dL. On day 120, she developed an isolated severe neutropenia (ANC 0.4 G/L), which was unaffected by MPA, VGCV, and TMP/SMX withdrawal. Bone marrow smear showed active granulopoiesis with maturation arrest at the promyelocytic stage. She was treated with G-CSF discontinuously from day 139 to day 213, which resulted in an increase of ANC (Fig. 1B).
For both patients, tests for anti-nuclear antibodies, anti-DNA antibodies, rheumatoid factor, cryoglobulinemia, and anti-neutrophil cytoplasmic antibodies were all negative. C3, C4, and CH50 serum levels were normal. Blood and bone marrow PCR assays for CMV, EBV, and parvovirus B19 were negative. LGL were excluded by blood smear, multiparameter flow cytometry, and TCRγ clonality analysis.
Taking into account the bone marrow smear with maturation arrest and the lack of granulocytes recovery after withdrawal of drug potentially associated with toxic and immunoallergic reaction, we hypothesized that the mechanism of the neutropenia may be autoimmune in nature. Granulocyte specific antibodies were negative by granulocyte agglutination test and granulocyte immunofluorescence test for both patients. Cultures of granulocytic progenitors from bone marrow aspirate were performed in the presence of patient and control sera. After 10 days of culture, patients’ sera inhibited autologous granulocytic differentiation, whereas with control serum patients granulocytic differentiation was normal. Similar inhibitions of differentiation of granulocytic progenitors from a control marrow were observed with the patients sera compared with the control serum (Table 1). Therefore, we concluded the mechanism of neutropenia in both patients was immunologic in nature and we hypothesized that it was mediated by an acquired autoantibody present in the patients’ sera.
Patient 1 was treated by intravenous immunoglobulin (IVIg) (2 g/kg) on day 205 and GCSF was discontinued concomitantly. Two weeks after the IVIg infusion, the ANC rose to 11 G/L with a platelet count at 738 G/L. ANC then stabilized around 4 G/L. No additional IVIg infusion was required. A new culture of control granulocytic progenitors was performed (Fig. 1A) with the post-IVIg patient’s serum (3 months later) and the control serum (Table 1). Inhibition of granulocyte differentiation by the patient’s post-treatment serum was no longer observed as compared with control serum.
Patient 2 received IVIg (2 g/kg) on day 221. On day 236, the ANC increased to 1.6 G/L and stabilized around 3 G/L. The post-IVIg serum (2 months later) inhibited 31% of the differentiation of heterologous granulocytic progenitors (Table 1). On day 241, MPA was reintroduced. On day 294, ANC fell to 0 and she developed pyelonephritis. The patient received a second course of IVIg (2 g/kg) and ANC normalized 7 days later while MPA was maintained. Four months later, ANC remains normal (Fig. 1B). Unfortunately, because of bacterial contamination, the differentiation of heterologous granulocyte progenitors with patient sera before and after the second course of IVIg could not be evaluated.
Posttransplant neutropenia is common after renal transplantation, and the mechanism is dominated by drug-induced bone marrow toxicity and viral infections. We describe here two cases of autoimmune neutropenia observed in a 1-year period in our transplant unit.
In our two patients, the lack of idiosyncratic or toxic mechanisms (demonstrated by MPA, TMP/SMX, and GCV withdrawal) and of other usual etiologies of acquired neutropenia suggest an immune mechanism. Bone marrow maturation arrest on the bone marrow smear showing an early block in differentiation led us to perform granulocyte progenitor proliferation tests. The in vitro inhibition of patient and control granulocytic progenitors growth by the patient’s serum but not by the control serum, in conjunction with the clinical response to IVIg reinforced the diagnosis. Of note, to exclude false positivity, the culture of granulocytic progenitors was performed after verifying the absence of anti-HLA antibodies against the control progenitor. The prompt efficiency of IVIg in these two cases strongly suggests an immune mechanism. Interestingly, in the second case, post-treatment serum had a reduced but persistent inhibition activity: this was associated with clinical relapse and the need for a second IVIg course.
PTN is associated with an increased incidence of infectious complications with 24% of bacterial infections during neutropenia (2). G-CSF appears to be safe and effective in severe posttransplant neutropenia, but its effect is only transient (2, 3). Moreover, PTN has been indirectly associated with significantly higher acute rejection rate when MPA was stopped for a period longer than 6 days (2).
The diagnosis of immune neutropenia can be a challenge as granulocyte specific antibodies are rarely detected in etiologies other than allo- and autoimmune neonatal neutropenia. In primary and secondary adult autoimmune neutropenias, detection of granulocyte specific antibodies is highly variable and depends on studies, contexts, and methods (6–8). As mentioned by Capsoni et al., regardless of the method used, the low antibodies titers and their low avidity for the target antigen often make them difficult to detect (9). Non-humoral mechanisms of autoimmune neutropenia have also been reported (10). Granulocyte specific antibodies tests should be performed in posttransplant neutropenia when an immune-mediated neutropenia is suspected but a negative result cannot exclude the diagnosis.
In vitro granulocytes progenitors’ culture has been demonstrated to be an efficient and reliable diagnostic test to demonstrate immune mechanisms in patients with idiopathic neutropenia without identifiable granulocytes’ specific antibodies (10, 11). If the bone marrow smear demonstrates maturation arrest or absence of granulocytes progenitors, ex vivo assessment of granulocyte progenitor growth in the presence of patient and control sera may be helpful. The potential of growth inhibition of the patient’s serum can also be tested on a control marrow, reinforcing the test value. This test usually shows a reduction rather than a total inhibition of the granulocytic differentiation. When positive, this test is considered to be indicative of a humoral mechanism of immune neutropenia. This test is analogous to in vitro erythroid progenitors culture that is commonly used to make the diagnosis of acquired pure red cell aplasia (12).
So far, only one other case of posttransplant AIN has been reported in the literature (13). PTN was supposed to be caused by anti-NA1 antibodies and was successfully treated by high-dose IVIg. However, culture of granulocytic progenitors was not performed.
Two main mechanisms of immune PTN can be hypothesized: alloimmune or autoimmune neutropenia. The former could be induced by an alloantigen expressed by the graft that could be associated with cross-reactivity with an antigen expressed on the patient’s granulocytes, as is observed in post-transfusion purpura or the lymphocyte passenger syndrome (14). Lymphocyte passenger syndrome is, however, usually observed earlier in the posttransplant period (in the first weeks after transplant). Alloimmune antibodies produced by the recipient’s B cells, and which target a persistent graft alloantigen, are predicted to be harder to eradicate; this has been observed in other alloimmune reactions. The ability of our patient’s sera to block control marrow granulocyte progenitor growth suggests an antibody-mediated mechanism that targets a shared or common antigen expressed on cells of the granulocyte lineage.
Autoimmunity in the setting of immunosuppressive therapy is not unusual: other autoimmune cytopenias and asymptomatic autoantibodies (15) or other organ autoimmune damage (16, 17) have been described ranging from 3% to 10% after solid organ transplantation (18). Autoimmune hemolytic anemia and immunological thrombocytopenic purpura have been commonly described in solid organ transplantation, and are sometimes associated with viral infections or posttransplant lymphoproliferative disorders (19–22). Their diagnoses are usually easier than AIN, using direct antiglobulin test and bone marrow aspiration, respectively, which is a possible explanation for the higher reported incidence. Three cases of pure red cell aplasia after kidney transplantation in patients receiving azathioprine therapy that were cured by cyclosporine therapy have also been previously described (23). Immune dysregulation is considered to be the leading cause of autoimmune phenomena occurring during immunosuppressive therapy, ranging from asymptomatic autoantibodies to autoimmune disease (cytopenia, hepatitis, or alopecia aerata) (16, 17). Immunosuppressive drugs, especially tacrolimus, have been suggested to block CD4 regulatory T cells and to favor the emergence of autoreactive B and T cells (24, 25). Tacrolimus has been more frequently associated with autoimmune phenomena than cyclosporine after transplantation. It is unknown whether this difference is a result of its more frequent use or to its more potent T-cell inhibition. A switch from tacrolimus to cyclosporine or rapamycine has been reported to be effective in other posttransplant autoimmune cytopenias (26). Tacrolimus and MPA combination have been found to be significantly associated with more PTN compared to cyclosporine and MPA combination. In one of these studies, the mechanism was presumed to be related to pharmacokinetic interaction between MPA and tacrolimus, but MPA levels were not available to support this hypothesis (2). Recently, De Rycke reported three patients with PTN while receiving tacrolimus and MPA therapy (27). Neutropenia persisted despite withdrawal of MPA and resolved only after tacrolimus was replaced by cyclosporine. As in our two patients, a pharmacokinetic interaction between MPA and tacrolimus seems unlikely because neutropenia persisted after withdrawal of MPA (respectively 73 and 97 days for patients 1 and 2).
Rituximab have also been associated with neutropenia in various settings, the mechanism of which is not fully understood. In our patient number 2, we cannot completely exclude a role of rituximab in the development of neutropenia.
Among 395 patients transplanted over a 2-year period in our center, we identified 15 patients with unexplained PTN lasting for more than 21 days. However, at that time, we did not perform granulocyte progenitor cultures, and the exact mechanism of these neutropenia cannot be formerly hypothesized.
In conclusion, AIN is a possible etiology of PTN that should be suspected in case of persistent unexplained neutropenia. IVIg is safe and may be effective, thereby avoiding extended immunosuppressive withdrawal that could precipitate acute rejection. If autoimmune neutropenia is suspected, granulocyte specific autoantibodies should be assessed, in combination with granulocytic progenitors growth study with patient and control sera when bone marrow aspiration shows maturation arrest or absence of granulocyte progenitors. A prospective study of all the kidney transplant patients presenting with unexplained persistent severe neutropenia will allow us to better evaluate the true prevalence of AIN in this population and to better explore the mechanism and the target antigen.
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