Clinical Profile and Severity of Hemolysis in Adult Patients of Primary Autoimmune Hemolytic Anemia and Their Response to Steroid: A Prospective Cohort Study from Single Institution

Abstract

Autoimmune hemolytic anaemia (AIHA) has traditionally been classified based on the temperature sensitivity of the autoagglutinins as warm (WAIHA), cold (CAIHA) and mixed type. Autoagglutinin may be of IgG or IgM type. The present prospective study was conducted to evaluate the profile of clinical picture, severity of haemolysis, treatment response of steroid. This study on patients of adult primary AIHA was conducted by taking complete history followed by detail physical examination. Laboratory investigations were performed to establish haemolytic anaemia and to assess severity of haemolysis. Immunehematological work up including blood grouping, direct antiglobulin test (DAT), IAT, antibody screening, adsorption elution was performed to diagnose type of AIHA. All cases were followed up to assess the response to prednisolone. All the data were collected and analysed by SPSS 19. Out of 62 primary AIHA cases, female were affected more than male (41:21). WAIHA is most common type (42, 67.8%) followed by mixed (20.9%) and cold AIHA (11.3%). Severity of haemolysis showed significant correlation with the DAT strength and not with type of AIHA. (P < 0.05) On oral prednisolone, 22 cases attended complete remission, while relapse, drug dependency and partial remission was achieved in 13, 9, 3 cases respectively. Severity of haemolysis in AIHA is directly related with DAT strength. WAIHA is most common type and can be managed with oral prednisolone (cr 45.2%), without red cell transfusion in most of cases. Mixed type AIHA cases were presented mostly with severe haemolysis, with minimum therapeutic response to prednisolone and maximum relapse/drug dependency.

Introduction

Immune haemolytic anaemia (IHA) is a clinical condition of reduced red cell survival due to the destruction of red blood cells (RBC) via complement and reticuloendothelial system by binding of antibodies (IgG/IgM) to surface antigen. IHA is classified as either autoimmune, alloimmune, or drug-induced based on the antigenic stimulus responsible for the immune response [1]. Autoimmune haemolytic anaemia (AIHA) is due to the development of antibodies against self-red cells. Autoantibodies are produced because of the alteration in the immune system concerning recognizing self-antigen that is the loss of tolerance [2]. AIHA has traditionally been classified based on the temperature sensitivity of the autoagglutinins as warm (WAIHA), cold (CAIHA), and mixed type. When the red cells are coated with IgG or IgG plus C3d, the antibody is usually reactive at 37 °C, which is termed as warm antibody while only C3d coating red cells will have reactivity at 4 °C termed as cold antibody [3]. Mixed AIHA has both warm and cold autoantibodies. AIHA can be idiopathic (50%) or secondary. Secondary causes are mainly lymphoproliferative syndrome (20%), autoimmune diseases (20%), infections, and tumours [4]. There is not much difference in the presentation of both warm and cold AIHA. These patients may present with hepatosplenomegaly, acrocyanosis of the ears, nose tip, fingers [5].

Surprisingly treatment of AIHA is still not evidence-based but essentially experience-based as there is only one randomized study on WAIHA where comparison of high dose prednisone alone vs prednisolone plus rituximab was analysed [6, 7]. A thorough scan of the literature found few Indian studies with a limited number of patients, which have corroborated the strength of direct antiglobulin test (DAT) with the severity of haemolysis (laboratory parameters) but, without detail correlation with the clinical presentation and therapeutic response in both types of AIHA. Immune suppression with steroid is considered as the first-line therapy in WAIHA, while rituximab therapy in CAIHA is the standard of treatment. Rituximab therapy is expensive and not affordable in most of the developing countries, including India. Various studies have reportedthelow effectiveness (14–35%) of steroids in CAIHA and a high incidence of steroid dependency/relapse [6]. There is not a single study among the patients of CAIHA from entire India regarding the effectiveness of steroids. The natural history of AIHA varies significantly among age groups like pediatric, adults, and elderly (> 60 years), etc. Few Indian studies related to AIHA have not considered this variation in the analysis, thereby compromising the research outcome in specific age groups. Thus the present prospective study was conducted to evaluate the profile of clinical picture, the severity of haemolysis, treatment response of steroid, and their correlation with the type and level of antibodies in adult patients (> 18 years) of primary WAIHA, CAIHA, and mixed-AIHA.

Materials and Methods

The present prospective cohort study was conductedon patients admitted to the Department of Clinical Hematology and Transfusion Medicine for anaemia evaluation with hemolytic picture between September 2015 and May 2018.

Patient Selection

The study population included the patients of primary autoimmune-haemolytic anaemia, in the age group of more than 18 years, and those giving consent for the enrolment in the study. The patients of DAT negative haemolytic anaemia, secondary AIHA and the patients who did not permit to be included in the study population were excluded from the study. A detailed history including the information regarding onset (acute or insidious), infections, recent blood transfusions, intake of drugs, vaccination, and joint pain, etc. were recorded in all cases. General and specific system evaluations, including lymphadenopathy/organomegaly/arthritis were also done. Routine workup relevant for diagnosis of secondary causes and treatment decisions also included like abdominal examination by computed tomographic scan (to search for splenomegaly, abdominal lymphomas, ovarian dermoid cysts, renal cell carcinoma), anti-nuclear antibody (ANA) profile and other antibodies (dsDNA, Ro, etc.), a search for a lupus anticoagulant in case of warm antibodies, or a bone marrow examination and immune fixation (to rule out lymphoproliferative diseases).

Laboratory Evaluation of Haemolysis

All the laboratory details (Haemoglobin, Indirect bilirubin, Reticulocyte count, Serum LDH) ofhaemolysis were collected from the clinical case sheet. Complete blood count (CBC) and reticulocyte count were performed by a five-part automated cell counter (Sysmex 1000i), Liver function test (LFT) by auto-analyzer (Toshiba TBA120FR, Japan) and serum LDH by a fully automated analyzer (Cobas Integra 400 plus, Roche Diagnostics Ltd, Switzerland).

Immunohematological Workup

Blood grouping, DAT, antibody screening and Identification, elution, and adsorption were performed in the department of Transfusion Medicine, as per the American Association of blood banks (AABB) methods. An algorithm was developed for immunohaematological work up to classify AIHA serologically and to give appropriate management (Fig. 1).

Fig. 1.

Flow-chart for immunohematological work up of AIHA case

Blood Grouping

Both forward and reverse grouping was performed by conventional tube technique (CTT) using commercially available antisera anti-A, anti-B, anti-D, (anti-AB, and anti-H as and when required) [Tulip diagnostic Pvt Ltd India] and pooled A, B and O cell prepared from the inventory. If there was any blood group discrepancy, then it was resolute by different techniques, as described in various literature. Basically, cold antibodies showed blood group discrepancy, which was resolved by a prewarmingprocedure (warm saline washing) [8].

Direct Antiglobulin Test

Ten microliters of 4% red cell suspension were taken in a well of the Coomb's cassette (Ortho BioVue, USA). It was centrifuged, and the result was interpreted according to the manufacturers' instruction. If a positive result came, polyspecific and monospecific DAT were performed by conventional tube technique (CTT) using polyspecific and monospecific AHG (IgG + C3d, IgG, C3d) [polyclone/eryclone Tulip Diagnostic Pvt. Ltd, India]. Validation of the negative result was done by adding Coomb's control cell [Agtrol, Tulip Diagnostic Pvt. Ltd, India].

Temperature Sensitivity Test

Six test tubes were taken and appropriately labeled as auto-control and pooled O-cell at 4 °C, 22 °C, 37 °C. For auto-control patient's serum and 5% patient RBC suspension, in the other three test tubes, patient serum and 5% pooled O-cell suspension were taken in 2:1 ratio and kept at three different temperatures for 1hour of incubation. Then the test tubes were centrifuged (1000 RPM for one minute) and observed for agglutination. Autoagglutinin was defined as warm or cold as per the reactivity of the antibody either at 37 °C or 4°/RT respectively. Mixed autoantibody was defined as the antibody having activity at all three temperatures, and both IgG and C3d were detected on monospecific DAT.

Antibody Screening and Identification

Antibody screening and Identification was made with commercially available three cell panel (Reagent Red cell, Surgiscreen, OrthoClinic Diagnostic, USA) and Coomb's glass bead cassette(Ortho BioVue, USA) as per the manufacturers' instruction and the result was interpreted using the antegram.

Polyethylene Glycol (PEG) Adsorption and Elution Test (Cold-Acid Elution)

Elution and adsorption were performed as per AABB methods. If the panpositive result was observed during antibody screening, the serum was adsorbed with PEG (Himedia) by alloadsorption. The adsorbed serum was screened for the presence of any underlying alloantibody.

DAT positive red cells were washed four to six times with large volumes of normal saline. The Glycine–HCl (0.1 M, PH 3.0, Himedia), Phosphate buffer saline (Himedia), and normal saline were used for this procedure. Elute was transferred into a clean test tube and tested in parallel with the last wash's supernatant. Antibody specificity was analyzed as per the result.

Severity of haemolysis was defined as severe or moderate on the basis of presence of all four parameters or less respectively and the parameters were as follows: (1) Hb < 9 g/dl, (2) indirect billirubin > 2 mg/dl, (3) Reticulocyte count > 2%, (4) LDH > 500 IU/L [9, 10].

Diagnosis and classification of AIHA were made to integrate all the clinical parameters with the laboratory data and serological data. Cold agglutinin titer, thermal amplitude test, and Ii specificity were performed in diagnosed cases of cold autoantibody.

AIHA patients were transfused with compatible blood units as like that of other patients. As expected, incompatibility was an issue for transfusion support in AIHA patients. Those patients were transfused with best match blood, which was decided by comparing the strength of agglutination of the cross-match unit (least strength) with auto-control [11]. If underlying alloantibody was present in these AIHA patients, then the antigen-negative blood unit was issued, which was a tedious procedure [12].

All the patients were treated with prednisone (PDN) with a dose of 1 mg/kg/day for two weeks then tapered over 8–12 weeks. Patients were followed up monthlyfor one year. Treatment efficacy was assessed at the end of 3 months, 6 months, and 12 months based on the Hb level achieved following treatment. Complete response (CR) was defined as Hb level ≥ 12 g/dl without a blood transfusion recently and without haemolysis features. A partial response (PR) was established when the Hb level was raised to ≥ 10 g/dl with a minimum rise of 2 g/dl from baseline, but persistent haemolysis is there. At the end of the follow-up, complete remission (cr) was defined as a lasting CR without any treatment and partial remission (pr) as the need for a daily dose of PDN less than 10 mg to maintain at least a long-term remission.

Corticosteroid dependency (DD) was defined as the need for long-term PDN to maintain at least a response or as early relapse (within three months) after the withdrawal of PDN. In every other situation, patients were considered nonresponders and/or had active disease [13]. All the primary AIHA cases were managed with PDN, and the response to PDN was evaluated, as mentioned above. Those patients not responding to PDN were managed with rituximab and non-corticosteroid immunosuppressive agents like azathioprine, cyclophosphamide, cyclosporine, and danazol. However, these nonresponders were excluded from the present study. At the end of one year, CR, PR, DD, and relapse were evaluated.

Statistics

All the data were collected through an Excel sheet and analyzed by SPSS version 19. All the descriptive data were analyzed and mentioned by mean with standard deviation. Chai-squire test was applied to compare the severity of haemolysis w.r.t gender, DAT strength, type of antibody (IgG/C3d), and type of AIHA.

Result

During this study period, 62 primary AIHA cases were registered. Female patients were affected more than males (41:21). The mean age was 35.4 years. All the patients presented with pallor whereas icterus, splenomegaly, and hepatomegaly were present in 62.9%, 32.2%, 11.2% cases, respectively. The laboratory parameters of red cell haemolysis, i.e., reduced haemoglobin, increased reticulocyte count, indirect bilirubin, serum LDH along with the distribution of DAT strength among these patients have been described in Table 1. There were 42 cases (67.8%) of Warm-AIHA, 7 (11.3%) cold and 13 (20.9%) mixed AIHA cases.

Table 1.

Correlation of DAT strength with laboratory findings of red cell hemolysis

DAT Strength	Number of cases (%)	Hb gm/dl (Median/IQR)	Reticulocyte count % (Median/IQR)	Indirect bilirubin mg/dl (Median/IQR)	LDH IU (Median/IQR)
4+	30 (48.4%)	5.8/2.475	4.2/2.125	3.1/1.7	979/164
3+	21 (33.9%)	5.5/2.55	2.7/1.95	2.1/0.95	846/228
2+	8 (12.9%)	6.4/2	2/0.97	1.7/1.1	797/195.2
1+	3 (4.8%)	9.2(Mode)	1.9(Mode)	1.7(Mode)	876(Mode)

The severity of haemolysis was compared with DAT strength, AIHA type, and type of antibody (IgG/C3d), the gender of the patient in Table 2. DAT strength was significantly correlated with the severity of haemolysis. (P < 0.05) Almost all mixed AIHA cases presented with severe haemolysis but warm and cold AIHA cases were of equal proportion of severity. All (7) cases of cold-AIHA and 12 cases of mixed-AIHA showed blood group discrepancies. Only in one case of W-AIHA, autoantibody was detected with underlying anti-E alloantibody. Anti-e antibody specificity was found in one case, whereas the autoantibody specificity could not be determined in other cases. Response to steroid at different time phrase was mentioned in Table 3. At the end of 3 month, CR was attended in 34, 2, 6 cases of warm, cold, and mixed AIHA respectively. But cr was achieved only in 45.2% (19) cases of W-AIHA and one case cold and two cases of mixed AIHA.

Table 2.

Comparison of severity of hemolysis with DAT strength, type of AIHA, gender

N = 62		Severe hemolysis	Moderate hemolysis	P value
DAT Strength	4+	24 (38.7%)	6 (9.6%)	0.001
	3+	14 (22.6%)	7 (11.3%)
	2+	2 (3.2%)	6 (9.6%)
	1+	0	3 (4.8%)
AIHA Type	Warm IgG	24 (38.7%)	16 (25.8%)	0.08
	IgG + C3d	0	1
	C3d	1	0
	Mixed (IgG + C3d)	11 (17.7%)	2 (3.2%)
	Cold	4 (6.5%)	3 (4.8%)
Gender	Male	14 (22.6%)	7 (11.3%)	0.528
	Female	26 (41.9%)	15 (24.2%)

Table 3.

Response to steroid in different types of AIHA patients at different time phrase

Response	CR 3 month	CR 6 month	CR 12 month	PR 3 month	PR 6 month	PR 12 month	DD 12 month	Relapse after12month	Loss to follow up
WAIHA (n = 42) CAIHA (N = 7) MIXED (n = 13)	34 80.9%	32 76.1%	19 45.2%	3 7.1%	4 9.5%	3 7.1%	0	11 26.2%	9 21.4%
	2 28.6%	1 14.3%	1 14.3%	2 28.6%	1	0	5 71.4%	1 14.3%	0
	6 46.1%	4 30.8%	2 15.4%	2 15.4%	2 15.4%	0	4 30.8%	1 7.7%	6 46.1%

CR Complete response, PR Partial response, DD Drug dependency

Discussion

In this prospective study, we have included only the primary AIHA cases. Out of the total 62 cases, 42 cases (67.8%) were W-AIHA. We have detected 7 cases of C-AIHA (11.2%) and 13 cases mixed AIHA (20.9%) in contrast to the Western part of the world where CAIHA is more common than mixed AIHA [5, 14, 15]. The mean age of presentation was 35.7 years, and maximum W-AIHA cases were below 40 years of age, similar to Das et al. In contrast, two studies from Western countries reported the median age between 60 and 70 years and maximum cases were above 40 years of age [14–16]. Females were affected more than males both in warm (29, 69%) and mixed AIHA (9,69.2%), similar to previous studies (58–64%), while cold-AIHA had similar sex distribution [14–17].

Anaemia, icterus, mild splenomegaly and hepatomegaly were common clinical features. The mean Hb, Indirect bilirubin, reticulocyte count, and LDH were 5.73 g/dl, 2.69 mg/dl, 3.6%, and 926U/L, respectively which depicted that most of the cases (68.75%) were presented to us with severe haemolysis. This could be explained by the fact that these patients were referred to this tertiary care hospital late.

Subset analysis has revealed that severe haemolysis was most commonly in mixed AIHA(84.6%), followed by warm (59.5%) and cold AIHA (57.1%), and severity was directly related to the strength of DAT (P < 0.05) [15]. This might be due to the synergistic effect of IgG with C3d in comparison to a single antibody (IgG/C3d) and an increased amount of antibody-coated over RBC [18]. Among the WAIHA, 40 cases (95.2%) were having IgG type autoantibody. Most of the patients were of B RhD positive (35) and O RhD positive (18) blood group similar to the blood group distribution in our population [19]. A cold autoantibody is mostly the common cause of blood group discrepancy in our patient population apart from a clerical error, as described by Mishra et al. [20]. In only one case of AIHA, we were able to detect anti-e autoantibody specificity, which is the commonest antibody, as described in the literature. Das et al. found anti-C, anti-E alloantibodies, but in this study, only one patient of warm AIHA had underlying anti-E alloantibody.

All of our patients received uniform protocol of immunosuppression by oral prednisolone at the dose of 1 mg/kg/day for four weeks, followed by gradual tapering across all types of AIHA. Though 80.9% (34) cases of warm AIHA had attended CR, only 19 (45.23%) cases achieved cr, similar to Sudulagunta et al. [13]. Birgens et al. demonstrated that 50% of the patients in the prednisone alone group achieved either a complete or partial response (CR/PR) at three months. However, nearly half of the responders relapsed within a year. The rituximab group demonstrated a higher CR rate (75% vs. 36%) and higher relapse-free survival at 36 months (70% vs. 45%) [7]. The response to steroids among cold and mixed AIHA was minimal, as most of these patients were drug-dependent. Relapse was observed in eleven cases of WAIHA and one case each of cold and mixed AIHA. The inadequate responses in cold and mixed AIHA have not been explained precisely. Some literature describes that clearance of IgG is earlier than IgM in response to steroids [21]. However, the preferred therapy in this type of AIHA is rituximab, which depletes B cells, which is the main culprit. But the drug is expensive and may not be affordable by many patients of developing countries. Our study has demonstrated that even with a cheaper drug like oral prednisolone, a reasonable clinical improvement can be achieved in both cold and mixed AIHA. However, this may be considered if standard therapy is neither affordable nor tolerable. Another important revelation of the present study is that most of the patients (56 cases; 90.3%) were managed cautiously without red cell support. Transfusion support in these patients is challenging and expensive in most places due to either blood group discrepancy, alloimmunization, or incompatibility with almost every donor unit. Red cell transfusion should be considered in extremely demanding situations after critical evaluation of the patient's profile of antibodies, along with searching the best match blood unit to avoid flaring of the haemolysis.

Study limitations: DAT negative AIHA cases were not included in the study. The severity of haemolysis w.r.t IgG subtype (IgG1/IgG3) was also not analyzed. We did not study response in CAIHA patients to rituximab.

Conclusion: Warm AIHA is the most common type of AIHA followed by mixed AIHA and cold AIHA in decreasing frequency in our population. The severity of haemolysis is more in mixed AIHA, which needs further evaluation by including a large study population. The strength of DAT is directly correlated with the severity of haemolysis. Most of the WAIHA cases can be managed satisfactorily with oral prednisolone and other supportive therapies but without red cell transfusion. Such an approach can be considered as an alternative in CAIHA and mixed AIHA when standard treatment of rituximab is either unaffordable or intolerable.

Funding

None.

Compliance with ethical standards

Conflict of interest

There is no conflict of interest.

Ethical Approval

Approved by the institutional ethical committee.

Informed Consent

Informed consent was obtained from all patients.