This open label, prospective two dose comparison study was conducted in the Department of Clinical Haematology in collaboration with Dept of Pharmacology of S.C.B Medical College & hospital, Cuttack, Odisha.

Newly diagnosed patients of ITP were enrolled from July 2012 to June 2014. Patients were defined as newly diagnosed ITP as per recent ITP international working group consensus report, that has divided the disorder into three phases: newly diagnosed ITP (≤ 3 months), persistent ITP (3 months-1 year) and chronic ITP (> 1 year) [7]. Only typical cases of thrombocytopenia were included for the study for which no special investigation was done. Recommendation of American Society of Haematology evidence base practice guideline (ASH- 2011) was followed for the diagnosis of patients for inclusion in this study [8]. This includes a careful history taking, physical examination, review of the complete blood count, peripheral blood smear examination and exclusion of other secondary causes of thrombocytopenia like Anti- Phospholipid syndrome, autoimmune thrombocytopenia, common variable immunodeficiency, drug induced thrombocytopenia, infection with cytomegalovirus, HIV, H. Pylori, SLE and other vaccine side effects. The principles of indication of Anti D treatment were strictly followed as per the ASH -2011 guidelines. The protocol was approved by institutional ethics committee before starting the study and informed consent was obtained in all cases.

Newly diagnosed case of ITP with:

A total 150 patients meeting the inclusion criteria were enrolled in the present study. The patients were divided into two groups randomly considering male female proportion, age range and pretreatment platelet count (<10x10⁹/Litre). Eighty-four patients were treated with Anti-D at the dose of 75μg/kg while 64 patients received the conventional dose of Anti-D (50μg/kg). Physical examination, assessment of severity of bleeding and complete blood count was evaluated in all cases every day during first week and in every alternate day next week. Response was evaluated at the end of 7^th day and was categorized into Complete Response(CR), Response (R) and No Response (NR) as per the criteria of international working group [8].

CR: TPC ≥100x10⁹/Litre measured in two occasions > seven days apart and absence of bleeding.

R: TPC > 30x10⁹/Litre and greater than two fold increase from base line measured in two occasions > seven days apart and absence of bleeding.

NR: TPC <30x10⁹/Litre or less than two fold increase in TPC from base line seven days apart or the presence of bleeding.

Time to response was calculated as time taken from starting anti-D to achievement of CR or R.

Primary End Point: The quality of response (CR, R, and NR) at the end of one week of administration of Anti D was considered as primary end point of the study.

Secondary End Point: Incidence of haemolysis and other side effects during the study period.

Patients were divided into two groups with compared demographic and other parameters. Anti-D (INDKDIV001, Synergy Diagnostics Pvt. Ltd. Thane India) was provided to all enrolled participants of this study. These hospitalised patients were included randomly to each of the two groups who received either 50μg/Kg or 75μg/kg of Anti–D as a slow IV push over 3–5 min [9]. All patients were pre-medicated with paracetamol and diphenhydramine. Steroids were not routinely given prior to treatment. Two patients in each group were lost to long-term follow-up and were not included in the analysis of long-term treatment response.

Any haematological or other adverse events were actively searched in the study participants. The observed adverse events were noted in a standard CDSEO form. Special importance was given to search for incidence of haemolysis that is manifested in the form of decrease in haemoglobin concentration or any other side effects.

All subjects who fulfilled the criteria were included in the analysis as long as adequate data were obtained to measure the rise in platelet count and fall in haemoglobin within 14 days of the infusion. The highest count was chosen, reflecting the best response. Dose of 75μg/kg body weight as single bolus intravenous dose was given to all subjects while the second group received the usual dose of 50μg/kg body weight. Data on rise in platelet count were analysed after logarithmic transformation in order to meet distributional assumptions of the analysis. All analyses were performed using SPSS software version 2014 for analysis. All the patients were enrolled randomly to both groups from indoor.

Male patients predominated in both the groups (45/39 and 38/26 in two groups). The age range of the admitted patients was found out to be between 4-14 years with median age of presentation between 6-7 years [Table/Fig-1]. Median haemoglobin (Hb) was 10.8 g/dl (8.2-12.2) Although haemolysis was seen in nearly all infusions, with a median haemoglobin fall of 1.2 g/dl (range, 0.2 to 2.4 g/dl), the decrease in haemoglobin was greater than 1.5 for only three infusions and maximum fall in haemoglobin (2.4) was noted in a child with an underlying anaemia. When safety of Anti-D was evaluated in terms of decrease in haemoglobin level (due to haemolysis) higher dose (75μg/kg) dose produced 1.2gm decrease in haemoglobin level (range: 0.2-2.4). Standard dose of Anti-D (50μg/kg) produced one gm decrease (0.2-2) in haemoglobin concentration due to haemolysis. Relapse rate was slightly less (in 18 patients) with higher dose Anti-D while 22 number of patients had relapse with 50 μg/kg dose of Anti-D. However, the difference was not statistically significant. No mortality was observed in both the groups till the end of study period [Table/Fig-1].

Complete Response (CR) was observed in 85% of patients treated with higher dose of Anti-D while usual dose of Anti-D produced positive response in 70% of patients. Partial Response (R) was found in 16% and 14% of patients treated with high dose and usual dose of Anti-D, respectively. Importantly, incidence of NR was totally absent in patients treated with 75μg/kg anti D. Nine patients were landed to No Response (NR) category treated with 50μg/kg Anti-D therapy. Thus, overall response (OR) was significantly high (99%) in patients treated with 75μg/kg dose of Anti-D compared to 86% with 50μg/kg dose [Table /Fig-2].

Although ITP in children is usually of the acute, self-limiting type, the chronic form can occur even in young children; some cases continue for longer than 6 months, yet still remit entirely. In children, in whom the risk of post-splenectomy sepsis is higher, a goal of therapy is to delay or avoid splenectomy. Our data demonstrate that the effect of Anti-D on platelet count in children treated for acute ITP is dose-related. Haemolysis is not dose-limiting, at least in the dose used in this study; majority of patients showed a fall in haemoglobin of less than 2.4 g/dl.

Our hypothesis for dose dependence of increase in platelet count without a similar effect on haemolysis can be described as: with administered amounts of Anti-D, red blood cells are bound, but can be removed from the circulation only at a rate determined by the number of Fc receptors in the spleen. As red blood cells are removed, Fc receptors become available, allowing additional cells to be bound and destroyed. However, the rate of haemolysis will reach maximum constant rate, easily compensated by a modest increase in the reticulocyte count. Low doses of Anti-D may manifest a dose effect on haemolysis because these doses may be insufficient to saturate Fc receptors, as the fall in haemoglobin using lower doses is less than that seen in our study. For platelets, in contrast, marrow production is already accelerated in ITP due to feedback from the thrombocytopenia. Thus, as soon as Fc receptors are blocked by antibody-coated red blood cells, the platelet count is poised for a rapid increase. Furthermore, we hypothesize that the platelet count should remain relatively high as long as Fc receptors are blocked. Therefore, a higher dose of Anti-D will elevate the reticulocyte count and platelet count for a longer period of time without creating a greater degree of anaemia.

Although acute ITP is usually more responsive to a given therapy than is chronic ITP, our data show in addition that the acute form has a steeper dose response.

Acute ITP is a dynamic disease, with resolution in a few months, so subsequent therapy will always seem more effective than initial therapy. Chronic ITP is a more static disease, so the effect of the order of therapies (or dose levels) will be more clearly manifest.

Historical data using only low doses of IV Anti-D (25 to 60μg/kg/d) found no evidence of increase in efficacy with increasing doses of Anti-D; however, it did suggest that haemolysis increases with dose. To determine if a dose response was indeed present and its effect on either platelets or haemoglobin level, we analysed data using a larger dose range in 84 children treated with intravenous Anti-D immune globulin for acute ITP at single institution [9]. Multiple clinical trials in both adults and children have shown that anti-D effectively raises the platelet count in majority of the treated patients and is associated with an acceptable safety profile. Duration of effect appears to be comparable to IVIG, with some reports of longer duration of effect in anti-D treated patients [10].

Salama et al., in 1983 reported an increase in platelet counts in ITP patients who were also positive for rhesus D antigen after the infusion of IV Anti-D [11]. Current wisdom favours the notion that IV anti-D coats the RBCs that are positive for D antigen and these opsonized RBCs in turn compete with opsonized platelets in the spleen for sequestration [12,13]. Reports suggest that a dose of 75 μg/kg over 3-5 minutes is more efficacious than a 50μg/kg dose, although the side effects are more common with the higher dose. Some of the commonly encountered infusion-related side effects are fever, chills, nausea, and headache. Another important adverse effect is a fall in haemoglobin secondary to haemolysis. Usually the fall in haemoglobin is not more than 2g/dl [14,15], but in rare cases, haemolysis can be severe and can lead to renal failure and disseminated intravascular coagulation [16,17].

One randomized study found no significant difference in efficacy between IVIG, Anti-D and oral prednisone, but the dose of Anti-D used (25ug/kg/d X 2) was suboptimal. Our study was not designed to compare the efficacy of Anti-D with that of IVIG or corticosteroids. However, the overall efficacy of Anti-D at the doses used in this study is comparable to historical data for these therapies for acute ITP in this population. This supports differing mechanisms of treatment effect. We have demonstrated that Anti-D is safe and effective for acute ITP in children, even at the relatively high doses used in our patients. Because of its low cost (compared with IVIG), favorable safety profile and ease of administration, the presence of a dose effect suggests that higher doses may offer a therapeutic advantage over IVIG or corticosteroids.

Our study showed that, haemolytic anaemia thought previously to be dose-limiting, was not clinically significant at these higher doses in the subjects, although caution should be taken in those with pre-existing anaemia or haemolysis. Our study indicates that the optimum dose of Anti-D in the treatment of acute ITP has not been found, demonstrating the need for a prospective study to determine the speed, duration and degree of platelet increase and haemolysis for different doses of Anti-D. Although, a dose of 75μg/kg body weight demonstrated positive results in majority of our subjects, rapidity and duration of response are clinically at least as important as the absolute or relative increase in the platelet count. Since rapidity, duration and degree of rise in the platelet count may not directly correlate, they should be assessed specifically in future studies. It is believed that intravenous Anti-D immune globulin functions by binding to Rh-positive red blood cells in the circulation. The antibody is then bound to Fc receptor in the reticuloendothelial system, mostly in the spleen, thus blocking the system’s effect on platelets and increasing their survivals. This mechanism requires the patient to be both Rh-positive and to have a functioning spleen.

Accurate measurement of rapidity and duration of response are not readily available from this protocol. Platelet counts after study duration (14days) were not observed on a regular basis and many patients were followed for only 3months with monthly platelet counts, making duration of response difficult to measure accurately and to analyse objectively. Another study limitation was the fact that patients did not have a 12-hour platelet count. One of the goals of a planned study will be to obtain a 12-hour count on all patients so the rate of rise can be quantified.

Intravenous Anti-D immunoglobulin at the dose of 75μg/kg produced higher rate of complete response and overall response compared to 50μg/ kg dose in newly diagnosed patients of Immune Thrombocytopenic Purpura. However, there was no significant difference in median time of response. Incidence of haemolysis was not significantly higher in higher dose group.