Indications for evaluation of neutrophil function

• Severe systemic infections in children. Ex: sepsis, osteomyelitis, meningitis
• Infections at unusual sites. Ex: hepatic or brain abscess
• Recurrent bacterial infections- Ex: pneumonia, sinusitis, lymphadenitis, otitis media
• Infections with unusual pathogens- Ex: Aspergillus pneumonia, disseminated candidiasis, Serratia marcescens, nocardia species, burkholderia cepacia
• Chronic gingivitis/ recurrent aphthous ulcers

Defective adhesion 

• Leucocyte adhesion deficiency
  • Autosomal recessive inheritance
  • Subtypes
    • Type 1- Deficiency of beta 2 integrins and CD11/CD18 complex. 
    • Type 2- Lack of fucosylation of ligands for selectins
    • Type 3- Mutation of FERMT3 which encodes kindlin-3 on hematopoietic cells. Normally this protein binds to beta integrin
  • Defects are seen in adhesion related functions such as
    • Adhesion to endothelial cells
    • C3b/ Opsonin mediated phagocytosis
    • Particle triggered respiratory burst activation
    • Degranulation
  •  Clinical features
    • Patient has increased risk of infections with marked leucocytosis, without pus formation at infection site.
    • Severe and progressive generalized periodontitis
  • Investigations
    • Flow cytometry- To look for expression of CD11b and CD15 (Selectin) on neutrophils
    • Hemogram- Neutrophilia
      • Baseline—15,000- 60,000/cmm
      • During infections- >1,00,000/cmm
    • Bone marrow aspiration- Granulocytic hyperplasia
  • Treatment
    • Prophylactic antibiotics and cotrimoxazole
    • Maintain oral hygiene
    •  Bone marrow transplantation in severe cases
• Drug induced

Defective locomotion and chemotaxis

• Neonatal neutrophils
• Actin polymerization abnormalities
  • Recurrent pyogenic infections
  • Autosomal recessive
  • Generally lethal
  • Treatment- HSCT
• IL-2 administration
• Cardiopulmonary bypass
• Drugs- Ethanol, glucocorticoids

Defective microbial killing

• Chronic granulomatous disease
  • X Linked and autosomal recessive inheritance
  • 2/3^rd of cases have mutation of CYBB gene, which encodes gp91phox component of the membrane cytochrome b protein complex
  • Defect – Lack of respiratory burst and super oxide production due to defect in NADPH oxidase
  • Neutrophils ingest but do not kill catalase positive organism
  • Morphology of neutrophils is normal
  • Clinical features
    • Recurrent infection with low grade pathogens such as Serratia marcescens, enterobacteraceae, staphylococci
    • Recurrent pneumonia, osteomyelitis, lymphadenitis, hepatic abscess 
  • Morphology - Granulomas are seen in affected tissues.
  • Investigations  
    • Nitro blue tetrazolium dye test. 
      • NBT (Yellow dye) + Neutrophils + Micro organism 
      • Normal –Increased leukocytic production of H₂O₂ and O₂  which reduce Yellow NBT to formazan, a blue compound
    • Flow cytometry using dihydrorhodamine-123 fluorescence
      • If H2O2 is present, there is oxidation of dihydrorhodamine-123 to Rhodamine 123 which has fluorescence.
      • Gives absolute number of cells affected and also degree of deficiency
    • PCR to identify the genetic defect. This can be used for prenatal diagnosis.
  • Treatment - 
    • Prophylactic antibiotics, cotrimoxazole, itraconazole
    • All routine vaccinations including live viral vaccines can be given.
    • Rec. INF- Gamma- 50mcg/m2- 3 times a week- Enhances phagocytic function and killing by non-oxidative mechanisms.
    • Surgical drainage if needed
    • HSCT- 
      • Only option of cure
      • Should be done if there are recurrent infections despite of aggressive medical treatment and there is a matched sibling donor
    • Gene therapy- It is being tried on experimental basis
• RAC-2 deficiency
  • Autosomal dominant
  • Dominant negative inhibition by mutant protein of Rac-2 mediated function. Failure of membrane receptor mediated O2 generation and chemotaxis.
  • Present with recurrent infections and neutrophilia
• Myeloperoxidase deficiency
  • Autosomal recessive  
  • MPO gene is located on chromosome 17.
  • 4 mutations have been identified.
  • It is a benign disorder in which infection is not a frequent complication
  • MPO stain is negative
  • Acquired forms- Acute myeloid leukemia, MDS, CML, Batten disease, lead intoxication, ceroid lipofuscinosis
• Hyper IgE syndrome- Discussed in primary immunodeficiency section.
• Neutrophil G6PD deficiency
• Glycogen storage disease Ib
• Extensive burns
• Ethanol toxicity
• End stage renal disease
• Diabetes mellitus

Abnormal structure of nucleus or organelle

• Hereditarymacropolycytes
• Specific granule deficiency
  • Autosomal recessive
  • Functional loss of myeloid transcription factor (Gff-1 or C/EBPE) which regulates specific granule formation.
  • There is absence of intracellular pool of leucocyte adhesion molecules
  • Reduced defensins, gelatinase, collagenase, vitamin B12 binding protein, and lactoferrin
  • Eosinophils also lack granules
  • There is impaired chemotaxis and bacterial activity
  • Peripheral smear- No specific granules but contain azurophilic granules in neutrophils. Bilobed nuclei in neutrophils.
  • Presents with deep seated abscesses
  • Common pathogens include S. Aureus, Ps. Aerogenosa
  • Treatment- Antibiotics
• Alder Reilly anomaly
  • Autosomal recessive
  • Defect – Incomplete break down of mucopolysaccharides due to various enzyme deficiencies.
  • Peripheral smear   
    • Leukocytes
    • Large purplish organelles in the cytoplasm
    • They stain metachromatically with toluidine blue
    • Gasser’s cells – Inclusions in lymphocytes tend to occur in clusters in the shape of dots or commas and are surrounded by vacuoles. (They are also seen in Hurler’s & Hunter’s disease)
• May Hegglin anomaly
  • Autosomal dominant
  • Peripheral smear
    • Granulocytes have large basophilic and pyroninophilic inclusions. They consist of RNA.
    • Giant platelets containing few granules.
• Chediak Higashi disease
  • Autosomal recessive
  • Defect in CHSI/LYST gene- located on chromosome 1. It encodes a protein involved in regulation of granule fusion.
    • Abnormal fusion of cytoplasmic membranes
    • Impaired locomotion.
    • Decreased cell activity in tissues.
    • Defective chemotaxis due to defect at membrane receptor level.
  • Clinical features  
    • Recurrent pyogenic infections invloving mucus membrane, skin and respiratory tract          
    • Skin hypopigmentation and Silvery hair - Due to failure to disperse melanocytes
    • Lymphadenopathy 
    • Hepatosplenomegaly- Usually due to associated HLH
    • Development of HLH is known as accelerated phase
    • Bleeding time- Prolonged due to platelet storage pool defect
    • Neurological symptoms- Peripheral and cranial neuropathies, autonomic dysfunction, weakness, sensory defect, ataxia
  • Investigations:
    • Peripheral smear: Leukocytes (Neutrophil, lymphocytes and monocytes)
      • Giant gray green peroxides positive bodies in cytoplasm.
      • Formed by aggregation & fusion of primary azurophilic & specific granules
    • Hemogram- Neutropenia- Due to death of myeloid precursors within the bone marrow
    • Bone marrow examination- Normal to hypercellular
    • Flow cytometry- Reduced expression of CD11b/CD18 due to altered membrane fluidity
    • Microscopy of hair shaft- Large speckled pigment clumps as opposed to normal pattern of finally divided pigment of melanin spread along the length of the shaft.
  • Differential diagnosis (Partial albinism with exagerrated bleeding and recurrent infections)
    • Griscelli syndrome
    • Hermansky- Pudlak syndrome
  • Treatment
    • High dose ascorbic acid
      • Infants- 200mg/day
      • Adults- 6gm/day
    • Prophylactic antibiotics
    • HSCT- Potential curative therapy

Familial Mediterranean fever 

• It is a type of autoinflammatory disease (Periodic fever syndrome)
• First presentation is seen in childhood
• Presents with sporadic paroxysmal attacks of fever, serosal inflammation (such as peritonitis) and neutrophilia
• Spontaneous resolution is seen after 1-3 days
• Autosomal recessive disorder
• Mutation of MEFV gene, which codes for pyrin protein
• There is neutrophil overactivity and tissue infiltration
• Because of chronic inflammation, these patient often have amyloid A amyloidosis.
• Treatment-
  • Colchicine
  • Severe refractory disease- HSCT
• Other autoinflammatory diseases include
  • Hyper-IgD syndrome- Mevalonate kinase deficiency
  • TNF receptor associated periodic syndrome
  • Cryopyrin associated periodic syndrome
  • PAPA syndrome- Pyeogenic arthritis, pyederma gangrenosum, and acne
  • PAMI syndrome- PSTPIP1 associated myeloid related proteinemia inflammatory syndrome
  • Majeed syndrome- Sterile, chronic recurrent multifocal osteomyelitis with pain and swelling around the joints, recurrent febrile episodes and congenital dyserythropoietic anemia