European Journal of Cardiovascular Medicine

The term "primary immunodeficiency diseases" (PID) refers to a set of genetically and clinically diverse illnesses that impact various innate and adaptive immune system cells, including neutrophils, macrophages, dendritic cells, complement proteins, natural killer cells, and T and B lymphocytes (1–3). Currently a total of 485 primary immunodeficiency diseases have been identified (4,5). Recent studies have reported an association of inborn errors of immunity with severe COVID-19 (6).

The European Society for Immunodeficiencies registry contains information on the clinical details of over 25000 patients with primary immunodeficiency disorders. The registry is helpful to support treatment decisions for patients with no genetic diagnosis in addition to the registry purposes(7). The International Union of Immunological Societies (IUIS) expert committee (EC) classifies 485 primary immunodeficiencies into ten groups (8).

The present study attempts at reporting and classifying various primary immunodeficiency diseases diagnosed in children at a tertiary care hospital, describing the clinical, immunological and haematological profile in these patients.

OBJECTIVES

PRIMARY OBJECTIVE:

To describe the clinical profile, immunological profile and flow cytometry findings in different types of Primary immunodeficiency diseases.

 SECONDARY OBJECTIVE:

1.To study the pattern of complete blood count values in various Primary immunodeficiency diseases.

2.To describe the bone marrow findings in whichever case possible especially in Bone marrow failure syndromes.

This is a descriptive cross-sectional study conducted in the Department of Pathology and Department of Paediatrics, Government Medical College, Kozhikode from June 2021 to July 2022. The study population included children within 14 years of age presenting in the Paediatric out-patient department with the warning signs of Primary immunodeficiency diseases during the study period. All patients with secondary immunodeficiency conditions such as HIV, malignancy and immunosuppressive drugs were excluded. 70 cases were selected.

Clinical details were collected from OP records, IP records and from patients according to proforma. Samples were studied for complete blood count, peripheral smear and bone marrow findings, NBT and Flow cytometry studies. Clinical features and laboratory findings were described. Clinical diagnosis of PID is made based on the ESID registry. Classification is done according to the International Union of Immunological Societies expert committee guidelines. Descriptive statistics were used because the sample includes a variety of primary immunodeficiency diseases.

Institutional Ethics Committee clearance was obtained prior to the start of the study. Confidentiality was maintained.

Demographics:

The study included 70 cases with the age of the patients ranging from 6 days to 13 years. Males predominated with a frequency of n = 46 (66%). Age of onset of the disease is the age at which symptoms started developing. The most frequent age group of disease onset was found to be 1 – 5 months of age with n = 29 (41.4%). The next commonest age group for the onset of PID was <1 month of age with n = 15 (21.4%). All the cases had an age of onset under 4 years.

All the cases of Severe Combined Immunodeficiency (SCID) showed an early onset of symptoms which was <5 months of age. Majority of the DiGeorge syndrome and Leukocyte Adhesion Deficiency (LAD) cases also showed an early age of onset of <1 month.

Distribution of cases:

70 cases of primary immunodeficiency diseases were studied.

Table 1 - Distribution of cases according to IUIS category

According to the IUIS 2022 categorization, the distribution of primary immunodeficiencies in our study is shown in Table 1. Combined immunodeficiency with associated or syndromic features is the IUIS category with the greatest number of cases in the study (31%). No cases were obtained under the category of Complement deficiencies and Phenocopies of PID.

Table 2 - Distribution of PID cases according to subtypes

The most common case encountered was Hyper IgE syndrome (HIES) followed by X-Linked Agammaglobulinemia (XLA) and DiGeorge syndrome. 1 case of Wiskott Aldrich Syndrome (WAS) and 1 case of MCM4 deficiency were also studied among Combined immunodeficiency with associated or syndromic features. MCM4 deficiency or Immunodeficiency 54 is an autosomal recessive PID with decreased numbers of NK cells(5,9). The category of Predominantly antibody deficiencies included 4 cases of Hyper IGM and 2 cases of Common Variable Immunodeficiency (CVID) along with XLA. Congenital defects of phagocytes included 5 cases of LAD, 5 cases of Chronic Granulomatous Disease (CGD), 1 case of cyclic and congenital neutropenia and 1 case of Schwachman-Diamond syndrome. 7 cases of Auto-inflammatory disorders were obtained out of which 6 cases where Hyper IgD syndrome and 1 case was Familial Cold Autoinflammatory syndrome (FCAS). Immunodeficiencies affecting cellular and humoral immunity constituted 7 cases, out of which 5 were SCID and 2 were CID. One of the CID cases was that of Immunodeficiency 76 (FCHO1 deficiency) in which there is low T cell proliferation, normal number of B cells and normal immunoglobulin profile. It is associated with increased activation-induced T-cell death(5). There were 3 cases of Diseases of immune dysregulation, out of which 2 were Hemophagocytic Lymphohistiocytosis (HLH) and 1 was Autoimmune lymphoproliferative syndrome (ALPS). 2 cases of Bone marrow failure - Aplastic anaemia were studied. Defects in intrinsic and innate immunity constituted 2.9%, with 1 case of Chronic mucocutaneous candidiasis (CMC) and 1 case of Mendelian susceptibility to mycobacterial diseases (MSMD).

Family history:

Consanguinity in parents:

13% cases showed a history of third-degree consanguineous marriage in the parents. Consanguinity appears to be a contributing factor for the development of PID(10).

A history of pregnancy loss in mother was present in 30% cases and 20%  of the patients had a sibling with similar complaints.

Clinical presentations:

Recurrent pneumonia, fever and other infections were the most common presentations. Recurrent fever was present in 71% of the cases.

The most common clinical signs were crepitations and pallor followed by lymphadenopathy. Facial dysmorphism was also present in about 18.5% cases, majority of which is constituted by DiGeorge syndrome.

Figure 1 - Mild prognathism, microtia, atretic external auditory canal and mild prognathism in a case of LAD

Respiratory system was the most commonly affected system (73%), followed by Skin (51%) and GIT (36%). Recurrent pneumonia was the most common respiratory system presentation followed by upper respiratory tract infections.

 Table 3 – System-wise presentation of PID cases

Skin manifestations were the most common in HIES. Skin abscess and pruritic rashes were frequent.

Figure 2 - Eczematous scalp lesion in CGD

Cardiovascular system was the most commonly affected system in DiGeorge syndrome with the commonest presentation being congenital heart defects such as VSD, PDA, ASD and TOF. A history of poor feeding and cyanotic episodes were present during the neonatal period in 2 cases. Other associated features were absent thymus and hypocalcaemia.

Pneumonia was the single most common presentation in LAD. 3 out of 5 cases of LAD had a history of delayed fall of umbilical cord and umbilical sepsis.

Figure 3 - Ulcer without pus in a case of LAD

Laboratory findings:

Hemoglobin values: Aplastic anaemia showed the least mean haemoglobin value (6.6 g/dL) followed by HLH (8.4g/ dL).

Total leukocyte count:

Total leukocyte count was markedly increased in case of LAD. It was found to be low in cases of HLH, SCID and CID.

The absolute lymphocyte count (ALC) was found to be low in cases of SCID and CID.

The absolute neutrophil count was high in the cases of LAD and CGD. It was low in case of cyclic and severe congenital neutropenia.

Platelet count: Thrombocytopenia was observed in cases of WAS, HLH and aplastic anaemia.

Peripheral smear:

Peripheral smear examination showed neutrophilia in all cases of LAD. Neutropenia was observed in the case of cyclic and severe congenital neutropenia. Pancytopenia was observed in all cases of HLH and aplastic anaemia.

Figure 4 - Peripheral smear under 100X, showing neutropenia in a case of cyclic and congenital neutropenia

Figure 5 - Peripheral smear under 100X, showing neutrophilia with shift to left in a case of LAD

Figure 6 - Peripheral smear showing neutrophilia in a case of LAD

Figure 7 - Peripheral smear under 100X showing thrombocytopenia in WAS

Bone marrow examination:

Bone marrow findings were available in 3 cases which includes 2 cases of aplastic anaemia and 1 case of HLH. The bone marrow biopsy of aplastic anaemia showed markedly reduced cellularity with marrow spaces replaced by fat. Bone marrow aspirate of HLH case showed haemophagocytosis.

Figure 8 - Bone marrow aspirate showing erythrophagocytosis in a case of HLH

Figure 9 - Bone marrow trephine biopsy showing markedly hypocellular marrow with marrow spaces replaced by haemorrhage and fat in a case of aplastic anaemia

NBT test:

NBT test was negative in the cases of CGD.

Figure 10 - Abnormal NBT assay – Stimulated. No bluish granules in neutrophils

Immunoglobulin Profile:

XLA, SCID and CVID cases showed decreased levels of 2 or more immunoglobulins.

Table 3 - Immunoglobulin profile of XLA cases.

9 out of 10 HIES cases studied showed elevated IgE levels.

Table 4 - IgE levels in HIES cases

Flow cytometry:

DHR Test: Abnormal DHR test was seen in cases of CGD, where there is no shift in fluorescence with stimulation.

Figure 11 - DHR Control - shows the rightward shift of plot with stimulation

Figure 12 - DHR Test - shows absence of rightward shift with stimulation

Flow cytometry was done for 2 LAD cases which showed absent CD18 and CD11c on neutrophils.

Figure 13 - Absent CD18 and CD11c on neutrophils in LAD 1

All SCID cases showed deficiency of absolute T lymphocyte (CD3, CD4 and CD8) and B lymphocyte (CD19) count. CD56 counts were within normal limits. Absence of naïve T helper cells and naïve T cytotoxic cells were also seen.

Table 5 - Lymphocyte subset analysis in SCID cases

Figure 14 - Lymphocyte subset analysis in a case of SCID.

Clinical profile:

Combined immunodeficiency with associated or syndromic features is the IUIS category with the greatest number of cases in the study followed by Predominantly antibody deficiencies. The most common individual case was HIGE followed by XLA and DiGeorge syndrome. However, the most prevalent disease identified varies in different studies as it is greatly influenced by the location of study, demographics and diagnostic facilities available. CGD was found to be the most common PID in a study conducted by Verma et al at a tertiary care hospital in north India (11). In a study conducted by Chinnabandar et al, disorders of immune dysregulation constituted the majority (12). Antibody deficiencies were the major category in most of the studies conducted outside India, especially with a sample size more than 50 (13).

Males were the predominantly affected gender in this study as in many similar studies(12). The commonest age of onset of the disease in this study was 1-5 months.

The commonest presentations were recurrent pneumonia, fever and skin infections. Recurrent pneumonia was the most common presentation in the study conducted by Verma et al (11). The clinical heterogeneity of PIDs explains the varying clinical profile in different studies.

Family history consanguineous marriage in parents appears to be a genetic factor for the development of PID in children. Siblings with similar complaints or pregnancy loss in mother were identified in significant number of cases.

Haematological findings:

Haemoglobin value was found to be reduced in many of the PIDs studied but it was significantly low in cases of HLH and aplastic anaemia. Total leukocyte count was markedly high in case of LAD and it was significantly low in case of HLH, SCID and CID. Congenital neutropenia showed reduction in the ANC. Also, thrombocytopenia was observed in cases of WAS, aplastic anaemia and HLH. Haemophagocytosis was observed in the bone marrow aspirate of the HLH patients. Bone marrow biopsy of the patients with aplastic anaemia showed hypocellular marrow with marrow spaces replaced by fat.  The haematological parameters obtained in the study were similar to those described in the literature (14,15). The pattern of CBC values is helpful but not specific for diagnosis of any PID.

Immunological profile:

XLA and SCID cases showed decreased levels of 2 or more immunoglobulins whereas, HIES cases studied showed elevated IgE levels. CVID case showed reduced levels of IgA and IgM.

Flow cytometry:

Flow cytometry provided strong evidence for the diagnosis of CGD, LAD and SCID. CGD was diagnosed based on abnormal DHR test results when there is no shift in fluorescence after stimulation. LAD cases showed absent CD18 and CD11c on neutrophils. Deficiency of absolute T lymphocyte (CD3, CD4 and CD8) and B lymphocyte (CD19) count was observed in SCID cases. Absence of naïve T helper cells and naïve T cytotoxic cells were also seen in SCID cases. According to Kanegane et al, while genetic analysis provides a conclusive diagnosis, flow cytometry plays an crucial role in the cost-effective evaluation of PID suspects (16–18,18–20).  Flow cytometry testing properly diagnosed and categorized 73% of the cases in another study conducted by Meshaal et al in Egypt, in which the sample size was 1510. Phagocytic defects were the most common in this study followed by SCID and CID(18).

 Management of PID:

To significantly reduce disease-related morbidity and enhance patient outcomes, early identification and treatment are essential. Treatment for PID is complicated and typically combines supportive and curative methods. Immunoglobulin replacement therapy, antibiotic prophylaxis, cytokine replacement etc constitute the supportive treatment. Hematopoietic stem cell transplantation is a curative therapy for PIDs like SCID, CID, WAS, CGD and HLH(21,22).

 A delay in diagnosis may make the curative treatment impossible and can cause morbidity and mortality of the patient. Hence, this study aims at increasing awareness on the clinical and laboratory features of PIDs to prevent the delay in diagnosis and ensure a quality life to the patient. The importance of flow cytometry in the diagnosis of various primary immunodeficiencies is also demonstrated in the study since it is a very cost-effective method for evaluation of PID suspects.

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