Pathology, Genetics, and Immunology

Workshop Overview

  • Specialization: Pathology, Genetics, and Immunology

  • Year: 2025

  • Instructors: Dr. Wail Hassan, Dr. Paula Monaghan-Nichols, Dr. Mark Nichols

Learning Objectives

  1. **Chronic Granulomatous Disease (CGD)

    • Identify major clinical diagnostic features.

  2. Physiological Role and Defects

    • NADPH Oxidase, O-, H2O2, HOCl in CGD.

  3. Affected Immune Cells

  4. Granuloma Formation

    • Involved cells and triggers.

  5. Genetic Basis of CGD

    • XLR and AR gene roles.

    • Note: No pedigree drawing required.

  6. Test Interpretation

    • Dihydrorhodamine test and Nitroblue Tetrazolium Test.

  7. Treatment Options

    • Long-term care in CGD patients.

Case 1: Initial Presentation

  • Patient: Zaki, 2-month-old male.

  • Symptoms: Poor feeding, lethargy for two days, high fever (rectal 39.5˚C).

  • Immediate actions needed:

    • Physical examination

    • Blood tests

    • Review past medical history

Past Medical History

  • Birth: 38 weeks, 2 days, 9 pounds, uncomplicated.

  • History of Infections:

    • Sepsis

    • Skin infections

    • Gastroenteritis

    • Pneumonia

  • Physical Examination:

    • Height and weight within 25th to 50th percentile

    • Vital signs: Heart rate 160, SpO2 89%

    • Chest exam: Rales noted, mild retraction

    • Skin: 3 cm inguinal abscess, right axillary abscess (2.5 cm), scattered indurated hyperpigmented plaques (C).

Concerning Features in Case 1

  • Main Concerns:

    • Recurrent/persistent infections

    • Growth restriction

    • Suspected pneumonia (low oxygen)

    • Suspected sepsis

    • Multiple abscesses

Tests to Consider

  • Ordered Tests:

    • Complete Blood Count (CBC)

    • B-cell, T-cell, CD4+/CD8+ T-cell counts

    • Blood culture

    • Abscess exudate culture & Gram stain

    • C-reactive protein

    • Erythrocyte sedimentation rate (ESR)

    • Chest X-ray

Laboratory Results from CBC

  • Results:

    • WBC: 22.0 (Normal Range: 8.4-14.4 1000/mm³)

    • RBC: 3.00 (Normal Range: 3.32-4.80 million/mm³)

    • Hemoglobin: 10.5 (Normal Range: 10.8-14.6 g/dL)

    • Hematocrit: 29.5 (Normal Range: 32.0-44.5 %)

    • Neutrophils: 66% (Normal Range: 26-54%)

    • Lymphocytes: 21.5% (Normal Range: 43-53%)

    • Monocytes: 9% (Normal Range: 4.7-12.5%)

    • Eosinophil: 3% (Normal Range: 0-4%)

    • Basophils: 0.5% (Normal Range: 0-1%)

  • Conclusion: (Evaluation pending)

Immune-Cell Counts

  • Results:

    • B cells: 12.0% (Normal Range: 11.1–29.3%)

    • T cells: 51% (Normal Range: 49-97%)

    • CD4+ T cells: 41% (Normal Range: 37-69%)

    • CD8+ T cells: 25% (Normal Range: 6-41%)

  • Conclusion: (Evaluation pending)

Microbiology Results

  • Gram Stain:

    • Gram-positive cocci in clusters, suggests staphylococci.

  • Cultures:

    • Blood culture and abscess exudates grew Staphylococcus aureus after 24 hours.

  • C-reactive Protein: 107 mg/dL (Reference: up to 0.3 mg/dL)

  • ESR: 45 mm/hour (Reference: 3-13 mm/hour)

  • Conclusion: (Evaluation pending)

Chest X-Ray and Findings

  • Assessment:

    • Plain X-ray (A)

    • Computed tomography (B & C)

  • Conclusion: (Evaluation pending)

Major Laboratory Findings

  • Abnormal Findings:

    • Leukocytosis

    • Neutrophilia

    • Lymphocytopenia??

    • Mild anemia

    • Elevated C Reactive Protein

    • Elevated ESR

    • Staphylococcus aureus infection

    • Pneumonia

Family History

  • Patient Profile:

    • Zaki’s sisters (9, 7)

    • Father (42), paternal aunt (38, 3 children)

    • Mother (38), maternal siblings (36, 34, etc.)

    • Grandparents: Paternal (67, 69) and Maternal (deceased)

  • Conclusion: (Determining inheritance pattern X-linked or AR)

Major Laboratory and Clinical Findings

  • Identified Abnormal vs Normal Lab Results

    • Recurrent/persistent infections

    • Growth restriction

    • Pneumonia and skin infections present

    • Suspected sepsis and multiple abscesses

Differential Diagnosis

  • Considerations:

    • Severe Combined Immunodeficiency (SCID)

    • MIRAGE Syndrome

    • ELANE-Related Neutropenia

    • Chronic Granulomatous Disease

  • Tests Needed:

    • Confirmatory laboratory tests for differential diagnosis

Dihydrorhodamine (DHR) Test

  • Results for Zaki:

    • Q: What is the principle behind the DHT test?

    • A: Reactive Oxygen Species (ROS) reduce non-fluorescent DHR123 to fluorescent rhodamine 123.

Dihydrorhodamine (DHR) Family Test Results

  • Result: Analysis for a 5-year-old sister to inform inheritance patterns.

Nitroblue Tetrazolium (NBT) Test

  • Results:

    • Patient vs Healthy Control outcomes.

  • Q: What is the principle behind the NBT test?

  • A: ROS reduce colorless NBT to deep blue formazan.

Genetic Testing for CGD

  • Possibilities for Diagnosis:

    • Autosomal Recessive CGD (e.g., CYBCA, CYBC1, NCF1, NCF2, NCF4)

    • X-Linked Recessive (e.g., CYBB)

  • Pathogenic variants frequency: Detailing the occurrence of mutations

Further Family History Insight

  • Inheritance Questions:

    • Establishing risks for cousins in the absence of new mutations.

  • Carrier frequency: Inherited patterns in family tree analysis.

Summary of Patient Test Results for CGD

  • Genomic Testing Outcomes:

    • Family members and mutation implications for confirmatory diagnosis.

Protein Functions in CGD

  • Normal Function:

    • Phagocytes synthesize ROS through NADPH oxidase.

    • Superoxide generation is crucial for microbial destruction.

  • NADPH Oxidase Composition:

    • 5 subunits (3 cytosolic: p40phox, p47phox, p67phox; 2 membranous: gp91phox, p22phox).

    • Activation requires Rac2 and Rap1 proteins.

    • Activity dependent on coalescing NADPH oxidase subunits.

Glucose 6 Phosphate Dehydrogenase's Role

  1. Essential for NADPH production, required by the oxidase system.

  2. NADPH transfers electrons to produce superoxide (2e-).

  3. Superoxide is converted to hydrogen peroxide via superoxide dismutase.

  4. Hydrogen peroxide can convert to HOCl for microbial killing or be broken down by catalase to maintain cellular integrity.

  5. Alternative pathways for hydrogen peroxide exist, highlighting significance in cell physiology.

Case 2: Initial Presentation

  • Patient: Kylan, 3.2-year-old male.

  • Symptoms: Fever for 15 days, bilateral neck swelling for the last 3 days.

  • Clinical History: Previous health events concerning failure to thrive and recurrent abscesses noted.

Concerns in Case 2

  • Major Features:

    • Bilateral neck swellings

    • Persistent fever

    • History of failure to thrive

    • Recurrent infections and skin abscesses

Physical Examination Findings

  • Biopsy Results: Evaluation of cervical lymph nodes required for granuloma indication.

Why Granulomas Form

  • Discussion of immune responses involved in granuloma formation by multiple cell types (T, B, and macrophages).

CBC Results for Kylan

  • Measurement Data:

    • WBC: 28 (Normal Range: 8.4-14.4 1000/mm³)

    • RBC: 2.9 (Normal Range: 3.32-4.80 million/mm³)

    • Hemoglobin: 9.9 (Normal Range: 10.8-14.6 g/dL)

    • Hematocrit: 28 (Normal Range: 32.0-44.5%)

    • Neutrophils: 70% (Normal Range: 26-54%)

    • Lymphocytes: 19.5% (Normal Range: 43-53%)

Summary of Major Laboratory Findings Kylan

  • Findings:

    • Highlighted abnormalities vs clinical findings like skin lesions and recurrent infections.

Pedigree Analysis (Kylan's Family)

  • Probabilities:

    • Inferred genotypes added to pedigree.

    • Estimated risks of carrier status discussed against family history.

Confirmatory Laboratory Tests

  • Considerations for further confirming diagnosis within the context of Kylan's history and current findings.

Patterns from Case 2

  • Cell test results from DHR, showing responses compared to healthy controls and family members.

Genetic Confirmation and Diagnosis

  • Testing Implications:

    • Mutations indicating X-linked or autosomal recessive conditions in CGD confirmation.

Conclusion of Major Clinical Manifestations in CGD

  1. Pneumonia: 69% incidence.

  2. Abscess Formation: Skin and liver abscesses.

  3. Lymphadenopathy and osteomyelitis are less common.

Diagnosis Criteria for CGD

  • Key Indicators:

    • History of frequent infections

    • Absence of respiratory burst

    • Neutrophil assay results (failure to reduce NBT and DHR).

    • Characteristics of abnormal wound healing and granuloma formation.

Treatment Strategies

  • Management Approaches:

    • Infection prophylaxis

    • Vaccination schedules

    • Colitis management strategies

    • Hematopoietic stem cell transplant and its benefits shown in patients.

  • Consideration of chemotherapy and gene therapy through advancements since early trials.

Follow-Up and Care Recommendations

  • Screening Routines:

    • Regular lab checks every 3-4 months.

  • Avoiding Environmental Risks:

    • Strategies to mitigate exposure to hazards (organic matter, inappropriate vaccines, etc.).

Survival Rates and Prognosis

  • Mortality and Morbidity:

    • 90% reaching adulthood; primary cause of morbidity linked to infections.

    • Median survival data indicates significant follow-up duration.

Questions Section

  • Open for further discussions or inquiries regarding CGD and related case studies aiding clinical understanding.