Hemodialysis: Chapter 1, Physiological Principles of Hemodialysis - Dr.Gawad
Granulocyte antigens & antibodies and their role in transfusion medicinepptx
1. Granulocyte antigens & antibodies-
Their role in Transfusion Medicine
Presented By- Dr. Shiny
Moderator- Dr. Yadwinder Kaur
2. Granulocytes
•Granulocyte transfusion is required for neutropenic patients with infections not responding
to antibiotics.
•prepared either by pooling multiple units of buffy coat or by apheresis technology.
•Collection by apheresis technology involves donor stimulation with steroids or with G-CSF.
•The preparation of the final product requires a sedimentation agent such as HES to facilitate
the separation between red blood cells and granulocytes.
•Granulocytes must be irradiated and can be stored at 20-24ºC without agitation and used
within a maximum period of 24 hours.
3. •The separation between granulocyte and red blood cell layers is quite difficult, and
granulocyte collection is often contaminated with red blood cells. Hence the unit should
be ABO compatible with the recipient’s plasma.
•Microaggregate filters or leucocyte reduction filters are contraindicated while transfusing
granulocytes as they remove the collected.
•AABB Standards requires that 75% of granulocyte components contain at least 1 × 1010
granulocytes, although the optimal therapeutic dose in adult patients is unknown.
•For infants and children, a dose of 10 to 15 mL/kg may provide an adequate number of
granulocytes per dose.
4. Granulocyte Antigens
•Antibodies against granulocyte (neutrophil) antigens are implicated in the following
clinical syndromes:
neonatal alloimmune neutropenia (NAN)
Transfusion-related acute lung injury (TRALI)
immune neutropenia after HPC transplantation
refractoriness to granulocyte transfusion
chronic benign autoimmune neutropenia of infancy (AIN)
5. •To date, nine neutrophil antigens carried on five different glycoproteins have been
characterized and given human neutrophil alloantigen (HNA) designations by the
Granulocyte Antigen Working Party of the ISBT.
•This nomenclature system follows a similar convention to that used for HPA
nomenclature.
•Several of the antigens on granulocytes are shared with other cells and are not
granulocyte specific.
6. Antigens on FcyRIIIb (CD16b)
•FcyRIIIb (CD16b) is a GP linked protein receptor for the Fc of IgG and is present only
on the surfaces of neutrophils.
•Neutrophils express 100,000 to 200,000 molecules of CD16b.
•The first granulocyte-specific antigen detected was NA1, later named “HNA-1a.”
•3 alleles of HNA-1 have now been identified—HNA-1a, HNA-1b, and HNA-1c
•There are rare individuals (aprx 0.1%) whose neutrophils express no FcyRIIIb (CD16
null) and who can produce antibodies that are reactive with FcyRIIIb when they are
exposed to it through transfusion or pregnancy.
• Antibodies to HNA-1a and -1b have been implicated inTRALI, NAN, and AIN.
7. Antigens on CD177
•Neutrophils from approximately 3% to 5% of people lack expression of CD177 on
neutrophils.
•Lack of CD177 is thought to be caused by a Mrna splicing defect.
•CD177 is expressed only on neutrophils and belongs to the Ly-6/uPAR/snake-toxin
family of proteins.
•A recent report documented cation-dependent heterophilic interactions between
CD177 and the endothelial cell membrane protein PECAM-1(CD31), suggesting a
role for CD177 in neutrophil transendothelial migration to sites of infection.
•Antibodies against HNA-2 have been implicated in NAN, TRALI, and neutropenia
in marrow transplant recipients.
8. Antigens on CTL2
•HNA-3a and HNA-3b are carried on the choline transporter-like protein 2 (CTL2), and a
SNP in the gene (SLC44A2) accounts for the polymorphism.
•CTL2 is also expressed on both T and B lymphocytes, and small amounts are present on
platelets. .
•HNA- 3a antibodies occasionally develop in women after pregnancy, and they are the
most frequent cause of fatal TRALI.
•HNA-3b antibodies are rarely detected, but several have been found during screening of
the serum of multiparous blood donors.
9. Antigens on CD11a and CD11b
•HNA-4a and HNA-5a, both high-prevalence antigens, are present on monocytes and
lymphocytes as well as granulocytes.
•HNA-4a is carried on the CD11b.
•CD11b/18 plays a role in neutrophil adhesion to endothelial cells and phagocytosis of
C3bi opsonized microbes.
•There is some evidence showing that alloantibodies against HNA-4a interfere with
CD11b/ 18-dependent neutrophil adhesion and enhance neutrophil respiratory burst.
• Antibodies against HNA-4a have been implicated in NAN, and autoantibodies against
CD11b/18 have also been described.
10. •HNA-5a is carried on CD11a/18 glycoprotein.
•CD11a/18, like CD11b/18, plays a role in neutrophil adhesion to endothelial cells.
•Antibodies that are reactive with HNA-5a have been found in a chronically transfused
patient with aplastic anemia and have also been reported to be associated with NAN.
•The patient who made the original HNA-5a antibody experienced prolonged survival of
an HLA nonidentical skin graft that was attributed to the HNA-5a antibody.
11. Other Neutrophil Antigens
Neutrophils do not express ABH or other red cell group antigens, but they do express
modest amounts of Class I and II HLA only upon activation.
15. Neonatal Alloimmune Neutropenia
•NAN is caused by maternal antibodies against antigens on fetal neutrophils; the most
frequent specificities are against HNA-1a, HNA- 1b, and HNA-1c antigens.
•NAN may also occur in the children of women who lack the FcyRIIIb protein.
•Neutropenia in NAN can occasionally be life-threatening because of increased
susceptibility to infection.
•Management with antibiotics, IVIG, granulocyte colony-stimulating growth factor,
and/or plasma exchange may be helpful.
16. TRALI
•In TRALI, the causative antibodies are most often found in the plasma of the blood
donor. When these antibodies are transfused, they cause activation of primed neutrophils
that are sequestered in the lungs of certain patients.
•The activated neutrophils undergo oxidative burst releasing toxic substances that damage
pulmonary endothelium and resulting in capillary leak and pulmonary edema.
•Class I and II HLA and HNA antibodies have all been implicated in TRALI.
17. Autoimmune Neutropenia
•Autoimmune neutropenia may occur in adults or in infants. When present in adults, it
may be idiopathic or secondary to such diseases as rheumatoid arthritis or systemic
lupus erythematosus or to bacterial infections.
•In autoimmune neutropenia of infancy, usually occurring in children between the ages of
6 months and 2 years, the autoantibody has neutrophil antigen specificity (usually
HNA-1a or -1b) in about 30% of the patients.
•The condition is generally self-limiting, with recovery usually occurring in 7 to 24
months, and is relatively benign and manageable with antibiotics.
18. Testing for Granulocyte Antibodies and
antigens
•Granulocyte antibody testing is technically complex and labor intensive.
•The inability to maintain the integrity of granulocytes for testing that are stored at room
temperature, in refrigerated conditions, or by cryopreservation requires that cells should
be isolated from fresh blood on each day of testing.
•Class I HLA antibodies that are often present in patient sera complicate detection and
identification of granulocyte antibodies. For these reasons, it is critical that granulocyte
antibody and antigen testing be performed by an experience laboratory using appropriate
controls.
19. GRANULOCYTE AGGLUTINATION TEST
•This was one of the first tests developed for the detection of granulocyte antibodies.
•It is typically performed by overnight incubation of small volumes of isolated fresh
neutrophils with the patient’s serum in a microplate.
•The wells are viewed under an inverted phase microscope for neutrophil agglutination or
aggregation.
20.
21. GRANULOCYTE IMMUNOFLUORESCENCE
test
• This test also requires fresh target cells that are incubated, usually at room temperature
temperature for 30 minutes, and washed in EDTA and phosphate-buffered saline.
•Neutrophil-bound antibodies are then detected with fluorescein isothiocyanate-labeled
antihuman IgG or IgM with either a fluorescence microscope or a flow cytometer.
•A combination of agglutination and immunofluorescence tests is beneficial.
•Other methods include chemiluminescence, SPRCA, and the monoclonal antibody
immobilization of granulocyte antigens (MAIGA) assay, etc.
•The MAIGA assay is used to differentiate between HLA- and HNA-specific antibodies.
22.
23. HNA TYPING
•As with HPA, typing for HNA is largely performed using molecular methods to detect
the allelic variants that determine the antigens.
•Any methods used in HPA typing can be applied to HNA typing with simple
modifications to the primer and probe sequences.
•Readers are referred to several publications on this subject. Because the splicing defect
that results in CD177 deficiency is not known, typing for HPA-2/CD177 requires testing
for CD177 on freshly isolated neutrophils using specific monoclonal antibodies and the
granulocyte immuno fluorescence test.
24. Mixed passive agglutination:
Sera to be tested are incubated with neutrophil extract in wells of U-bottom Terasaki plates.
Antibody binding is detected using sheep erythrocytes coated with antihuman IgG.
The assay has been shown to detect antibodies specific for HNA-1a, -1b, -2a and -3a.
25. Monoclonal-antibody capture
assays:
•The monoclonal-antibody capture, or monoclonal-antibody immobilization of
granulocyte antigens (MAIGA) assay, allows the detection of antibodies to specific
neutrophil membrane glycoproteins.
•It can be used to detect antibodies to FcγRIIIb (CD16), NB1 gp (CD177), leucocyte
function antigen-1 (LFA-1 or CD11a) and complement component C3bi receptor (CR3 or
CD11b).
•In addition, this assay detects antibodies to HNA- 1, -2, -4 and -5. The assay allows
recognition of antibodies to specific neutrophil glycoproteins even when antibodies to
HLA antigens are present.
