Introduction

As a major lymphoid organ, receiving 5% of cardiac output every minute, the spleen has a fundamental role in the reticuloendothelial system. It removes antigens, aging platelets, and erythrocytes from the circulation and participates in iron recycling, as well as in the production of lymphocytes, immunoglobulins, and opsonins¹. It serves as a storage site for blood components and supplies erythrocytes and platelets in increased demand, such as trauma¹.

The knowledge of splenic functions is mostly based on murine studies. However, morphological and physiological differences between humans and other non-primate species may obscure understanding of splenic functions¹,². Ex-vivo studies are preferred, due to limitations in in-vivo human testing, to analyze the interactions between the functioning spleen and circulating blood components. Ex-vivo studies are performed in splenectomized patients by comparing preoperative conditions with postoperative ones. However, there are some difficulties regarding the evaluation of the post-splenectomy state. Along with surgical stress, any indication itself for splenectomy may interfere with the evaluation process by promoting cellular abnormalities³.

Immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia that is characterized by autoantibody-mediated destruction of platelets and suppression of platelet production⁴. As a primary site of platelet removal, the spleen plays a critical role in the pathogenesis of ITP. It also participates in the production of anti-platelet antibodies⁴. Although splenectomy is associated with a better outcome than any other therapy, it is generally reserved (as second-or third-line therapy) for patients who fail medical therapy, as it poses short and long-term risks⁴. Although several efforts have been made to predict splenectomy responses, they have not been fully successful⁴.

This study aimed to compare the post-splenectomy blood changes of ITP patients with those of trauma patients, 1 month and ≥ 6 months after surgery. The comparison of ITP patients with trauma patients, who are hematologically normal people, may clarify the post-splenectomy changes in patients with ITP.

Material and method

Medical records of patients, who had undergone total splenectomy for ITP and trauma at a tertiary center between January 2009 and December 2019, were retrospectively reviewed. Exclusion criteria included partial splenectomies, patients younger than 18 years of age, patients with congestive heart failure, patients with chronic pulmonary diseases, patients with chronic liver diseases, patients with malignant diseases, and patients with autoimmune diseases other than ITP. The patients, who had not had a blood test in the 1^st month and/or ≥ 6 months after surgery and/or who had died before the blood samplings were also excluded.

The diagnosis of ITP was based on isolated thrombocytopenia in the absence of other causes of thrombocytopenia. In cases where platelet counts were < 30 × 10⁹/L and/or patients had signs and symptoms of bleeding, first-line therapy was administered with 1 mg/kg/day oral methylprednisolone. In addition to steroid therapy, some patients had taken danazol, azathioprine, cyclosporine, cyclophosphamide, vincristine, thrombopoietin receptor agonists, intravenous immunoglobulin. For the management of ITP, medical treatment was applied initially, and splenectomy was indicated when medical treatment was exhausted in long-term follow-up. Appropriate preparation of patients for surgery was performed.

The data included demographics, clinical findings, laboratory findings, and therapeutic interventions. The following characteristics were recorded:

The study was conducted according to the principles set forth by the Helsinki Declaration of 1975. Approval from the Human Ethics Committee of the Institution was obtained.

Results

The current study included 52 patients, who had undergone splenectomy for ITP (57.7%), and trauma (42.3%). The mean age was 37.1 ± 15.4 (range: 18-76) years. Of the 52 patients, 33 (63.5%) had one or more systemic diseases including ITP (n: 30), hypertension (n: 2), diabetes mellitus (n: 2), coronary artery diseases (n: 1), and psychiatric disease (n: 1, depression). All trauma cases were operated on with open technique. Of the ITP cases, 80% were operated with laparoscopic technique. Transfusion of blood components was required in 48.1% of the cases. The mean length of hospitalization was 9.9 ± 15.8 (range: 2-96 days) (Table 1). All ITP patients had a complete response to splenectomy, which means platelet count > 100 × 10⁹/L and the absence of bleeding without any treatment after splenectomy.

Discussion

It was not surprising that hematocrit and hemoglobin values increased within the normal range after therapy in both groups. The post-operative 1^st-month increase in MCVs in trauma patients was thought to be a compensatory mechanism for blood loss. ITP, as a chronic inflammatory disease, had lower hematocrit, hemoglobin, and MCV levels compared to those of trauma patients in the post-operative period even after iron repletion.

Thrombocytosis is defined as a platelet count >500×109/L, and extreme thrombocytosis is defined as a platelet count >1,000×109/L. Thrombocytosis is either a reactive process (secondary to infection, trauma, surgery, and malignancy) or is caused by a clonal bone marrow disorder⁵. It is a normal physiologic response to splenectomy, which may persist in up to 30% of splenectomized patients, and cessation of splenic sequestration is thought to be a major participatory mechanism⁶. In the current study, 1 month after surgery, the increase in platelet count was more prominent in the trauma group but declined slightly overtime. The preoperative and postoperative 1^st-month platelet counts were statistically higher in the trauma group. However, the difference disappeared ≥ 6 months after. All ITP patients had a complete response to splenectomy.

Leukocytes constitute the cellular components of the immune system and participate in host defense. Normal levels of leukocytes are 4.000-11.000 per microliter of blood. Leukocytosis is an expected finding after splenectomy; thus, the diagnosis of postoperative infection in those patients may be a challenge⁷-⁹. Leukocyte count rises, irrespective of the reason for splenectomy⁷. The increase is initially confined to neutrophils. However, neutrophil count returns to within the normal range within weeks or months, and lymphocyte and monocyte counts rise steadily⁷.

Leukocytosis is a well-known response of the body to trauma. It is mainly due to neutrophilia, caused by redistribution of neutrophils from the storage pool to the circulation, and not due to increased production¹⁰. Neutrophils are one of the first defenders of inflammatory cells to migrate toward the site of injury. So, leukocytosis, especially neutrophilia, is an expected finding in trauma patients as in the current study. In trauma patients, the leukocyte count decreased compared to that recorded preoperatively, but it was still above the normal range. In ITP patients, the leukocyte count did not change statistically following splenectomy, though leukocytosis was present already before the surgery. Neutrophil counts decreased permanently in both groups during the period of recovery from surgery. The change in lymphocyte and monocyte counts followed a different pattern between the groups. In trauma patients, lymphocyte and monocyte counts initially did not change but increased ≥ 6 months after surgery. However, in ITP patients, lymphocyte and monocyte counts increased gradually overtime. The postoperative 1^st-month lymphocyte count was significantly higher in the ITP group, but the difference disappeared within 6 months.

Which mechanism, therefore, is responsible for leukocytosis after splenectomy? The spleen participates in the regulation of leukocytes. Along with the preservation of lymphoid tissue, it may also sequester leukocytes produced elsewhere⁷. Thus, the removal of the spleen causes the leukocyte count to rise. Could there be any mechanism other than the cessation of sequestration that could cause leukocytosis? In order to determine this, several questions must be answered. First: why does the leukocyte count not change significantly in ITP patients, while it remains stable above the normal range in trauma patients? Second: why do lymphocyte and monocyte counts in trauma patients increase several months later than those of ITP patients? Third: could the high basophil level in ITP patients be a unique finding?

ITP is an autoimmune disease, and inflammation plays a central role in pathogenesis. It was clear that neutrophils were responsible for the preoperative leukocytosis in ITP patients, and neutrophilia was ameliorated by both splenectomies, and also the withdrawal of steroid treatment in some patients. Thus, despite the increase in lymphocytes, monocytes, eosinophils and basophiles, total leukocyte count, which was already above the normal range, did not change in this group. The NLR and PLR have been found to be potential markers of systemic inflammation and used to predict prognosis and treatment response in various benign and malign diseases¹¹-¹³. The NLR was used in a small number of studies for the prediction of treatment response to medical therapy in ITP patients, and their results were contradictory¹⁴,¹⁵. In a recent study, it was concluded that splenectomy might improve the weakened immune system of patients with cirrhosis, possibly by reducing suppressive cell fractions and amplifying the effector cell population. In that study, splenectomy was found to decrease NLR¹⁶. We supposed that, based on the decrease in the NLR and PLR, splenectomy did not only eliminate sequestration but also ameliorated the inflammation in ITP patients.

In ITP patients, the effect caused by the cessation of sequestration on lymphocytes and monocytes is likely to emerge several weeks to months later as in the trauma patients. It seems there was another mechanism responsible for the early increase in lymphocyte and monocyte counts. This mechanism, causing lymphocyte and monocyte counts to increase without inflammatory effects, must be particularly complex, and its effect must not have lasted longer than a few months as the leukocyte counts were comparable with those of the trauma patients, ≥ 6 months after splenectomy. Furthermore, it must have been negated in the presence of a spleen. Although the mechanism that triggers inflammation in ITP has not yet been determined, direct interaction of monocytes with stimulated T- lymphocytes might be a major pathway for the production of proinflammatory cytokines and their inhibitors in monocytes, which has been postulated as a possible mechanism in the pathogenesis of any chronic inflammation¹⁷,¹⁸. Depending on T-cell type and T-cell stimulus, multiple ligands/counter-ligands are involved in the contact-mediated activation of monocytes, and the outcome of the inflammatory process is determined by the balance between proinflammatory cytokines and their inhibitors, which are produced in the monocytes. We suppose that ITP, as a chronic inflammatory disease, might possess similar inflammatory processes¹⁷,¹⁸. Further research is needed to identify inhibitory and/or stimulatory molecules involved in the inflammatory process.

Basophils are the least common type of leukocytes, constituting about 0.5-1% of circulating leukocytes. In the current study, basophils were the only leukocyte type, which was significantly higher in the ITP group ≥ 6 months after surgery. Basophils participate in the regulation of immune response and allergic reactions by producing histamine and serotonin. They also secrete endogenous heparin for anticoagulation¹⁹. Low basophil percentages or counts are thought to be responsible for the increased risk of thrombosis²⁰. Although patients with ITP have a low platelet count with a tendency to bleeding, they occasionally develop venous and/or arterial thromboembolic events by a mechanism, which has not yet been fully elucidated. It may occur as a result of inflammatory processes acting on thrombosis, or as a result of complications of medical and/or surgical treatment. Comorbid diseases may also increase the risk for thromboemboli in ITP patients²¹. It was not known whether the increase in basophil percentage was a compensatory mechanism in the protection of ITP patients against thromboembolism or whether it was a participant in the inflammatory process itself. Only three patients of the study population, two patients with ITP and the other with trauma, developed thromboembolic complications following splenectomy. Further research is needed for clarification.

Small case numbers and a retrospective design were weaknesses of the current study, while the comparison with a normal population (trauma patients) was a strength.

Conclusions

Splenectomy, irrespective of the indications, resulted in an increase in hemoglobin concentration, hematocrit, and platelet levels. It did not alter leukocyte count in ITP patients but led to leukocytosis in trauma patients. Although lymphocyte, monocyte, and eosinophil counts increased after splenectomy in both groups, total leukocyte count, which was already above the normal range, did not change in ITP patients. Neutrophils were responsible for the preoperative leukocytosis in ITP patients, and neutrophilia was ameliorated by splenectomy and also withdrawal of the steroid therapy in some patients Decreased NLR and PLR supported the finding that splenectomy ameliorated inflammation in ITP patients. Splenectomy resulted in a change in percentages of leukocytes in favor of basophils in ITP patients, but did not result in any change in trauma patients. It is not known whether the increase in basophil percentage was a compensatory mechanism to protect ITP patients against thromboembolism or whether it was a participant in the inflammatory process itself. There are many issues that require clarification with regard to the inhibitory and/or stimulatory molecules involved in the inflammatory process in ITP patients. Our knowledge of the spleen is encompassed by the literature, and everything we don’t yet know falls outside of that repository. Further research is needed to illuminate this mysterious organ.