1. Neutrophil antigens include P, I, and HLA class I antigens but not ABO antigens. Important human neutrophil antigens (HNA) systems include HNA-1 to HNA-5 which are defined by alleles of genes encoding neutrophil surface glycoproteins. 2. HNA typing is performed using PCR or sequencing based techniques while HNA antibody detection uses assays like GIFT-FC, GCLT, and MAIGA. 3. HNA incompatibility can cause conditions like neonatal alloimmune neutropenia and transfusion related acute lung injury. HNA typing and antibody screening are important for investigating and managing these conditions.

1. NEUTROPHIL ANTIGENS
DR. G.D. ARJUNA SAMARANAYAKA

2. COMMON ANTIGENS
• P and I antigens
• HLA class 1 antigens
• No ABO antigens

6. System Glycoprotein/Gene Allele Epitope(s)
HNA‐1
(NA1, NA2, SH)
CD16b/FcɣRIIIb
FCGR3B*01 HNA‐1a
FCGR3B*02 HNA‐1b, HNA‐1d
FCGR3B*03 HNA‐1b, HNA‐1c
FCGR3B*04 HNA‐1a
FCGR3B*05 HNA‐1b variant
No Glycoprotein FCGR3B*null HNA‐1 null
HNA‐2
(NB1)
CD177 CD177a HNA‐2
No Glycoprotein No allele HNA‐2 null
HNA‐3
(5b, 5a)b CLT2/SLC44A2
SLC44A2*01 HNA‐3a
SLC44A2*02 HNA‐3b
SLC44A2*03 HNA‐3a variant
HNA‐4
(Mart)
CD11b.CD18/ITGAM
ITGAM*01 HNA‐4a
ITGAM*02 HNA‐4b
HNA‐5
(Ond)
CD11a.CD18/ITGAL
ITGAL*01 HNA‐5a
ITGAL*02 (HNA‐5bw)c

7. Investigation
Associated HNA antibody
specificity
Type Class
NAIN
1a, 1b, 1ca, 1da, CD16ba,
2, 3aa, 3ba, 4aa, 4ba, 5aa allo, iso IgG, IgM
ANI (primary)
1a, 1b, CD16ba,
unidentified
auto, iso IgG, IgM
AIN (secondary)
1a, 1b, 1ca, 1da, CD16ba 2,
3a, 4a, 4ba, unidentified
auto, iso IgG, IgM
TRALI 2, 3a allo IgG
Graft failure 2, 3a allo IgG
Kidney rejection 3a allo IgG
HNA TESTING—CLINICAL CONDITIONS AND
ASSOCIATED ANTIBODY SPECIFICITIES

9. ANTIGEN TESTING
• Like HPA, HNA typing is performed by PCR‐SSP or sequence‐based
typing techniques
• With the exception of HNA‐2, which currently requires typing fresh
neutrophils with CD177 monoclonal antibodies by GIFT‐FC.

10. PROS AND CONS OF CURRENTLY EMPLOYED HNA
TYPING METHODS
Method Pros Cons
Serology
Quick Need for defined anti‐sera
Cheap Time sensitive (live cells)
Clinically relevant (phenotype) Not suited to high throughput
Molecular (PCR‐SSP)
Cheap Only targets known polymorphisms
Quick Novel/variant not identified
Molecular (RT‐qPCR)
Sample multiplexing Only targets known polymorphisms
Suitable for high throughput
Novel/variant not identified
High initial costs (equipment and
probe/assay design)
Molecular (PCR‐SBT)
Full exon/gene sequence Large number of individual
sequencing reactions set‐up
Identify novel mutations
Protein sequence can be elucidated
Co‐amplification of homologous
genes
Not suited to high throughput

11. NEUTROPHIL ANTIBODIES - TESTING METHODS
• The granulocyte immunofluorescence test by flow cytometry (GIFT‐FC)
and the granulocyte chemiluminescence tests
• good sensitivity but are not specific
• cannot readily distinguish between granulocyte‐specific and HLA class I
antibodies
• For some HNA systems (CD16, CD177 and CD11/18) monoclonal
antibody immobilisation of granulocyte antigens (MAIGA) - determine
HNA specificity
• Bothe above tests – similar to platelet counterparts

12. PROS AND CONS OF CURRENTLY EMPLOYED
HNA ANTIBODY DETECTION METHODS
Method Pros Cons
GAT Simulates ‘in vivo’ agglutination Time sensitive (live cells)
GIFT/LIFT
Detection of both auto‐ and
allo‐membrane‐bound antibody
Time sensitive
GCLT Simulates ‘in vivo’ phagocytosis
Nonspecific
Time and temperature sensitive
rHNA‐3a/‐3b Elucidate HNA‐3 antibody
Cost of creation and maintenance
of cell lines
MAIGA Define HNA antibody specificity Not suitable for high throughput
Fluorescent beads
Quick
No need for fresh cells
Unsuitable for AIN/ANI
Assay cut‐offs are subjective
False positives
Poor correlation with known
anti‐sera (especially HNA‐3a)

13. NEONATAL ALLOIMMUNE NEUTROPENIA
• Maternal antibodies against paternal neutrophil antigens
• Incidence – 0.1-0.2% live births – rare
• HNA- 1 and 2 - commonest
• Clinical presentation
• Bacterial infection with isolated neutropenia
• Neutropenia may be severe – but reversible
• May last up to 32 weeks
• Mx – antibiotics and G-CSF

14. AUTOIMMUNE NEUTROPENIA
• Rare self limiting autoimmunity in young
• Chronic form in adults
• Targets – FcYRIIIb (CD16), CD 177, HNA 1a
• Detection
• Direct immunofluorescence
• Screening of a Patient’s serum with a panel of typed neutrophils in the
indirect granulocyte immunofluorescence and granulocyte
chemiluminescence or granulocyte agglutination test

15. PERSISTENT POST-BONE MARROW TRANSPLANT
NEUTROPENIA
• Autoimmune or alloimmune
• May be a serious complication
• Require serological studies

16. DRUG INDUCED NEUTROPENIA
• Drug induced immune neutropenia (DIIN)
• Drug dependent antibodies form against neutrophil membrane
glycoproteins and cause neutrophil destruction
• Presentation - fever, chills and infections (due to neutropenia)
• Severe neutropenia or agranulocytosis with exposure to
nonchemotherapy drugs - 1.6 to 15.4 cases per million per year
• Possible mechanisms and utility of laboratory testing for antibodies is not
well understood.
• Discontinuation of the suspected drug(s) should be considered.

17. Human neutrophil antigens: Nature, clinical significance and detection
Tom Browne
Rebecca J. Dearman
Anthony Poles
First published: 24 September 2020
REFERENCES

18. THANK YOU