Phenotypic analysis of peripheral blood

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Clinical presentation

Introduction

New Zealand has a population of 4.4 million which is ethnically diverse. Approximately 78% classified themselves as European/Other, 15% Maori, 7% Pacific Islanders, 9% Asian and 1% Middle Eastern/Latin American/African with some identifying themselves with more than one ethnicity (http://www.stats.govt.nz/). CVID has a prevalence of 1:25,000 in Caucasians and is less common in Asians. There are approximately 120 CVID patients in New Zealand from data supplied by the New Zealand Blood Service. For this study a total of 125 patient samples were collected, 48 were diagnosed as CVID, 58 had asymptomatic hypogammaglobulinaemia, three patients had low B cell numbers and the diagnosis of 16 patients was unknown at the time of writing. Our first aim was to understand the clinical manifestations of CVID and hypogammaglobulinaemia and will be presented in this chapter. The focus was on the clinical details of the CVID patients as most hypogammaglobulinaemia patients are asymptomatic.
CVID is characterised by low levels of IgG, IgA and/or IgM. The median total serum levels of IgG, IgM and IgA were 2.19 g/L, 0.33 g/L and 0.11 g/L respectively in a study of 34 CVID patients [95]. Normal values for adults in g/L are: IgG 7 to 14, IgA 0.7 to 4.0 and IgM 0.4 to 2.3.
Some studies have suggested a bimodal peak in onset, one in late childhood and the other in early adulthood [62]. Differences in the mean age at the time of diagnosis were observed. In a report from 1976 on 50 patients the mean age was 39 years for males and 46 years for females [279]. In a 1999 study of 248 patients the mean was 29 years for males and 33 years for females [62]. This suggests that time to diagnosis possibly improved over the years.
Complications seen in CVID patients could be related to infections, such as upper and lower respiratory tract infections, or underlying immune dysfunction, such as autoimmune complications [64]. The complications observed show significant differences in prevalence between countries [61]. Recurrent upper respiratory tract infections in CVID patients resulted in chronic sinus and lower respiratory tract infections, causing bronchiectasis in some patients [64]. The prevalence of bronchiectasis in CVID patients ranged from 4 to 43% in various studies. Bronchiectasis was found in 10 out of 248 patients (4%) in a study by Cunningham-Rundles et al. [62], in 16 out of 65 patients (25%) by Aghamohammadi et al. [280] and in 18 out of 42 patients (43%) by Busse et al. [281].
Granulomatous changes are often diagnosed years prior to the recognition of CVID [282, 283]. Granulomatous changes have been associated with autoimmunity. Ardeniz et al. [283] reported 20 out of 37 (54%) of the patients with granulomatous changes were diagnosed with an autoimmune condition, mainly ITP and AIHA. Normally 20 to 23% of CVID patients develop autoimmune disease [284, 285]. CVID patients with granulomas or autoimmunity are also likely to have a significant deficiency of isotype switched memory B cells [96, 101, 286]. In general, granulomas are known to develop in response to microbial or insoluble agents. Since the lungs are commonly affected by CVID, the search for an environmental antigen triggering this reaction is important [283].
In the report by Cunningham-Rundles, 56 out of a group of 248 CVID patients (22%) developed autoimmune conditions [62]. Increased risk of mortality in CVID patients with an autoimmune disease was observed [64]. Splenomegaly was found in approximately 30% of CVID patients [64]. Non-Hodgkin’s lymphoma was observed in 19 out of 248 CVID patients (8%) [62].
Early diagnosis has proven to be important as intensive IVIG replacement may prevent development and progression of pulmonary complications and diagnostic delay could explain the recurrence of respiratory infections and progression of bronchiectasis observed in some patients [287]. Other studies showed that bronchiectasis did not relate to diagnostic delay or age at symptom onset [61]. Reduced survival was observed in CVID patients with bronchiectasis [62, 64, 280, 288] and as IVIG replacement may prevent development of bronchiectasis, early diagnosis and treatment may reduce mortality.

Methods

A total of 125 asymptomatic hypogammaglobulinaemia and CVID patients were recruited for this study, including 48 CVID patients. Medical specialists located throughout New Zealand collected clinical information based on a questionnaire (Appendix B) developed by Dr. R. Ameratunga. Laboratory results were obtained from LabPlus after receiving consent from the patients. The clinical details of 39 patients were available the time of writing and will be analysed in this chapter. Probability testing was performed using GraphPad Prism 5. Comparisons between patients were made by a two-tailed paired t-test.

Results

The CVID cohort consists of 39 patients (18 females and 21 males). Most of the patients were classified as European (32 out of 39, 82%), two were classified as Maori (5%), two as European/Maori (5%), one Chinese (2.5%) and one as Cook Islander (2.5%). The mean age of the 39 CVID patients at the time of this study was 46 years with a mean age for males of 44 years and a mean age for females of 49 years. The mean immunoglobulin levels, in g/L, were: IgG 4.5, IgA <0.07 and IgM 0.71. Whether Ig levels were taken prior to IVIG replacement was not indicated. A total of 25 CVID patients (64%) were located in Auckland, 35 CVID patients (90%) were located on the North Island of New Zealand and 4 (10%) were located on the South Island of New Zealand (Table 4-1).

Age at symptom onset and diagnosis

The age of symptom onset is the age that marks the beginning of a drop in health for the patient. Patients were asked when they were last in good health. The median age at symptom onset was 10 years for males and 14 years for females. A third of the patients presented the first symptoms within the first five years of life, five patients (14%) between 5 and 9 years of age, six patients (16%) between 10 and 14 years of age, two patients (5%) between 15 and 19 years of age, four patients (11%) between 20 and 24 years of age, three patients (8%) between 25 and 29 years of age, two patients (5%) between 30 and 34 years of age, and four patients (11%) from the age of 35 and above (Figure 4-1 – A).
The median age at diagnosis was 25 for males and 35 for females. Two patients (5%) were diagnosed within the first five years of life, two patients between 5 and 9 years of age, three (8%) between 10 and 14 years of age, five (13%) between 15 and 19 years of age, one (3%) between 20 and 24 years of age, four (10%) between 25 and 29 years of age, six (15%) between 30 and 34 years of age, four (10%) between 35 and 39 of age, five (13%) between 40 and 44 years of age, and seven (18%) from the age of 50 and above (Figure 4-1 – B).

Delay in diagnosis

The delay in diagnosis was correlated to the year of symptoms onset (Figure 4-2). A linear regression was found between the year of symptom onset and the delay in diagnosis. In 1940 it took more than 40 years to diagnose CVID, even though CVID was first described in 1953, it took another 27 years before diagnosis was made. In 1990 on the other hand it took on average less than 10 years to diagnose CVID. The correlation was measured with Pearson’s correlation coefficient (r value) of -0.875 (p = 0.01).

Infections in CVID patients

CVID patients showed a varying number of upper respiratory tract infections in the year prior to diagnosis. Infections that resulted in a week or more time away from school or work were classified as severe infections.
Approximately a quarter (26%) of CVID patients had less than five infections per year prior to diagnosis, 23% had five to nine infections per year, 44% had 10 to 14, 3% had 15 to 19 and 5% had 20 to 24. There were no patients with 25 or more infections per year prior to diagnosis (Figure 4-3 – A).No significant difference was observed between the age of symptom onset and the number of infections per year prior to diagnosis. Patients with an age of onset of up to 10 years had an average number of infections of 12.2 ± 10.9 while patients with an age of onset above 10 years had an average of 11.4 ± 11.7 infections per year prior to diagnosis (p = 0.84).
There was no significant difference between the delay in diagnosis and the number of upper respiratory tract infections per year prior to diagnosis. Patients with a delay of less than 10 years had an average number of infections of 7.3 ± 1.6 while patients with a delay in diagnosis of 10 years or more had on average 9.8 ± 1.1 infections per year prior to diagnosis (p = 0.18)
Figure 4-3: Number of (A) infections and (B) severe infections in CVID patients. A – The number of infections per year in CVID patients prior to diagnosis. B – The number of severe infections in CVID patients prior to diagnosis. Severe infections are those infections that resulted in a week or more time off from school or work.
The total number of severe infections prior to diagnosis varied from zero to more than 25 in total. Less than five severe infections prior to diagnosis were observed in 42% of the CVID patients, 11% had 5 to 9 severe infections, 8% had 10 to 14, 3% had 15 to 19, 18% had 20 to 24 and 18% had 25 or more severe infections prior to diagnosis (Figure 4-3 – B).
There was no significant difference between the age of symptom onset and the number of severe infections per year prior to diagnosis. Patients with an age of onset of up to 10 years had an average number of infections of 9.1 ± 4.9 while patients with an age of onset above 10 years had an average of 8.7 ± 4.9 infections per year prior to diagnosis (p = 0.79). Comparing the number of severe infections per year to the delay in diagnosis showed that there was a significant difference between the two groups. The patients with a delay of less than 10 years had on average 3.8 ± 1.1 severe infections per year while patients with a delay of 10 years or more had an average of 0.7 ± 0.2 severe infections per year (p = 0.003; Figure 4-4 – A). Comparing groups by dividing them based on 5 year intervals, the first group had the highest number of severe infections per year and the differences after that were not significant. Up to five was an average of 4.9 ± 1.4 infections per year, five to nine was 1.1 ± 0.8 infections per year and 10 or more years was 0.7 ± 0.2 infections per year (Figure 4-4 – B).

Hospitalisations prior to diagnosis

In terms of hospitalisations, 81% of the patients were hospitalised less than five times before diagnosis, 8% were hospitalised five to nine times, 3% 10 to 14 times, 3% 15 to 19 times, and 6% 25 times or more, before diagnosis was made (Figure 4-5).

Contents
ABSTRACT
ACKNOWLEDGEMENTS
FUNDING
ETHICS
LIST OF TABLES
CHAPTER 1. B AND T LYMPHOCYTES IN THE IMMUNE SYSTEM
1.1. B CELL DEVELOPMENT
1.2. T CELL DEVELOPMENT
CHAPTER 2. COMMON VARIABLE IMMUNODEFICIENCY (CVID)
2.1. CVID IS A DIAGNOSIS OF EXCLUSION
2.2. CLINICAL CHARACTERISTICS COMMON VARIABLE IMMUNE DEFICIENCY
2.3. IMMUNOPATHOGENESIS OF CVID
2.4. GENETICS OF CVID
2.5. AIMS AND GOALS OF THIS STUDY
CHAPTER 3. MATERIALS AND METHODS
3.1. BUFFERS AND MEDIA
3.2. ANTIBODIES AND ISOTYPE CONTROLS
3.3. MITOGENS AND ANTIGENS FOR LYMPHOCYTE PROLIFERATION ASSAY
3.4. SAMPLE PREPARATION
3.5. MOLECULAR BIOLOGY
3.6. CELLULAR IMMUNOLOGY
3.7. STATISTICAL ANALYSIS AND GRAPHS
CHAPTER 4. CLINICAL PRESENTATION
4.1. INTRODUCTION
4.2. METHODS
4.3. RESULTS
4.4. DISCUSSION
4.5. FUTURE DIRECTIONS
CHAPTER 5. ANALYSIS OF CANDIDATE GENES
5.1. INTRODUCTION
5.2. METHODS
5.3. RESULTS
5.4. DISCUSSION
5.5. FUTURE DIRECTIONS
CHAPTER 6. A CVID FAMILY STUDY
6.1. INTRODUCTION
6.2. METHOD
6.3. RESULTS
6.4. DISCUSSION
6.5. FUTURE DIRECTIONS
CHAPTER 7. PHENOTYPIC ANALYSIS OF PERIPHERAL BLOOD
7.1. INTRODUCTION
7.2. METHODS
7.3. RESULTS
7.4. DISCUSSION
7.5. FUTURE DIRECTIONS
CHAPTER 8. TIM-3: AN EXHAUSTION MARKER
8.1. INTRODUCTION
8.2. METHOD
8.3. RESULTS
8.4. DISCUSSION
8.5. FUTURE DIRECTIONS
CHAPTER 9. SUMMARY AND PERSPECTIVES
9.1. CLINICAL DETAILS
9.2. CANDIDATE GENES
9.3. TACI FAMILY STUDY
9.4. PHENOTYPIC MEMORY B AND T CELL ANALYSIS
9.5. TIM-3
9.6. FUTURE DIRECTIONS
Appendix A : Consent form for the study
Appendix B : Questionnaire for the clinical study
Appendix C : Phenotypic analysis results
REFERENCES

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Common Variable Immunodeficiency in New Zealand