Neutrophil Testing

The Red Cross National Neutrophil laboratory areas of expertise include granulocyte antibody screening, extended antibody identification (MAINA), HLA antibody screening and drug antibody testing. The detection of antibodies to human neutrophil alloantigens (HNA) remains very technically difficult due to the labile nature of granulocytes and the frequent presence of HLA Class I antibodies which also react with neutrophils. Consequently, expertise in this area has been confined to a small number of laboratories. One of the founding members of the International Society of Blood Transfusion (ISBT) Working Party on Granulocyte Immunobiology, our laboratory has participated in its quality assurance exercises, the International Granulocyte Immunology Workshop (IGIW), since its inception in 1989.

Contact Information & Hours

American Red Cross Neutrophil Immunology Laboratory

North Central Blood Services

100 South Robert Street

St. Paul, MN 55107-1489

Monday-Friday: 8:00am – 4:30pm Central

Email: BRSTPaulMNNeutrophil@redcross.org

Phone: (855) 216-9202

Benefits of selecting the ARC National Neutrophil Laboratory

We are the only laboratory in the United States that routinely utilizes a combination of the granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) for detecting HNA antibodies.
1. The ISBT Working Party on Granulocyte Immunobiology has recommended that a combination of GIFT and GAT remains the best means of HNA antibody detection, and has been the standard approach in most laboratories participating in the IGIW^1,2,3,4.
The Red Cross laboratory has achieved a 100% score when detecting and identifying HNA antibodies in the 2008-2017 IGIW surveys.
The laboratory is one of four that are responsible for organizing, preparing and distributing IGIW survey samples to participating laboratories.
The only neutrophil laboratory in the United States that routinely offers a monoclonal antibody immobilization of neutrophil antigen (MAINA) assay. This test is used to distinguish neutrophil antibodies from HLA Class I antibodies when both are present in the same specimen. MAINA is highly sensitive and is suggested when testing for neutrophil antibodies in children with a presumptive autoimmune neutropenia diagnosis.
The laboratory includes extremely rare donor cells in our neutrophil panel that assists in the detection of HNA-2, HNA-3a, HNA-3b, HNA-4a, HNA-4b, HNA-5a and FcγRIIIb (CD16) antibodies.
The laboratory provides phenotyping for HNA-2. Other neutrophil laboratories that provide molecular typing for HNA alleles do not provide HNA-2 typing results.

Laboratory References

Bux J, Chapman J. Report on the Second International Granulocyte Workshop. Transfusion 1997;37:977-83.
Lucas G, Rogers S, et al. Report on the Fourth International Granulocyte Immunology Workshop: progress toward quality assessment. Transfusion 2002;42:462-68.
Bierling P, Bux J, Fung YL, Schuller R, et al. Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leucocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung injury. Vox Sang 2009;96:266-69.
Lucas G, Porcelijin L, Fung YL, Schuller R, et al. External quality assessment of human neutrophil antigen (HNA)-specific antibody detection and HNA genotyping from 2000 to 2012. Vox Sang 2013;105:259-69.

Clinical Indications

The presence of antibodies directed toward neutrophils results in their destruction and leads to a clinical condition known as neutropenia. Neutropenia is defined as an abnormally low concentration of neutrophils. Neutrophils are the most abundant white blood cells found in your blood stream and are essential in fighting off bacterial and fungal infections. Neutrophil antibodies can be found in the blood of women following pregnancy, multi-transfused individuals, patients with autoimmune disease and donors causing transfusion reactions. Our tests can aid in the clinical diagnosis of the following disorders:

Alloimmune neonatal neutropenia (ANN)

ANN is the result of antibodies directed against human neutrophil antigens (HNA) transplacentally transferred to the fetus from the maternal circulation. The mother is alloimmunized by fetal neutrophils crossing the placental barrier during pregnancy. Alloimmunization can occur anytime following the first trimester of her first pregnancy. Once the mother’s antibodies are present in fetal circulation they will bind to fetal neutrophils expressing the corresponding neutrophil alloantigens and will be removed from circulation. Neonates affected by ANN are almost always neutropenic at birth, although cases have been reported where neutropenia has been delayed by 1 to 3 days.

ANN is often asymptomatic and goes undiscovered unless the absolute neutrophil count (ANC) is less than 1.5 x 10⁹/L. At this point the newborn may become susceptible to bacterial or fungal pathogens. Fatalities have been reported in 5 percent of cases, although most cases are mild presenting with fever, malaise, skin infections such as cellulitis and omphalitis, mucosal or respiratory infections (stomatitis, otitis media and pneumonia), and urinary infections. The incidence of ANN has been estimated to be 0.1 to 0.2 percent.

Autoimmune neutropenia (AIN)

These antibody coated neutrophils are removed from circulation by the spleen where they are phagocytized by macrophages. AIN is classified as primary or secondary cases. Primary AIN is not associated with other immunological or hematological abnormalities. Primary autoimmune cases are observed most frequently in newborns with an incidence of 1/100,000. Primary AIN is usually diagnosed during the first few months (5-15 months) with severe neutropenia seen at presentation with Absolute Neutrophil Counts (ANC) between 500-1500 neutrophils/µL. The clinical course is usually benign or at any rate self-limiting, with a tendency to resolve spontaneously by 2 or 3 years of age. Severe infection such as pneumonia, sepsis, and meningitis are observed in approximately 12% of patients.

Chronic idiopathic neutropenia in the adult differs from the neonatal autoimmune form in that it appears much later life with the incidence higher among females than males (70% to 30% respectively). The adult form exhibits little tendency toward spontaneous remission. It remains clinically benign with only 35% of cases positive for neutrophil autoantibodies.

Secondary AIN is associated with other autoimmune disorders such as rheumatoid arthritis (RA), Felty’s syndrome or systemic lupus erythematosus (SLE). Secondary AIN is most commonly observed in individuals between 40 to 80 years of age. Severe or life-threatening infections are not common in patients with secondary AIN, however a pattern of recurrent cutaneous and upper and lower respiratory tract infections was noted.

Transfusion-related acute lung injury (TRALI)

TRALI is defined as an acute lung injury (ALI) that usually develops within 6 hours after the transfusion of any blood component. Recently, the incidence of TRALI was determined to be approximately 1:4000 transfusions. Clinical features seen in TRALI are characterized by the acute onset of hypoxemia, tachypnea, cyanosis, fever, hypotension and radiographic evidence of bilateral pulmonary infiltrates in the absence of circulatory overload. The mechanism of TRALI is associated with the activation of neutrophils located in the pulmonary capillary vasculature. Activation of neutrophils is primarily related to the passive transfer of leukocyte antibodies found in transfused blood components, or on rare occasions leukocyte antibodies in the recipient. This is referred to as “immune TRALI”. A secondary mechanism known as “non-immune TRALI” is the result of the transfusion of cytokines and soluble biologically active lipids found in cellular blood components. Therefore, testing plasma from implicated donors and recipients is critical when evaluating suspected TRALI cases.

Laboratory investigation of TRALI

The National Neutrophil Immunology Reference Laboratory has been performing leukocyte antibody testing in suspected transfusion reactions since TRALI was first described by Popovsky and Moore in 1985. As recommended by the International Granulocyte Immunology Workshops (IGIW), we use a combination of the granulocyte immunofluorescence test (GIFT) and the granulocyte agglutination test (GAT) when detecting and identifying neutrophil antibodies. Results obtained from the IGIW have confirmed that GAT is an essential technique when detecting agglutinating antibodies such as HNA-3a which has have been associated with severe and multiple fatal cases of TRALI. Two IGIW assessments have included HNA-3a antibodies where laboratories using GIFT alone failed to detect antibody to HNA-3a in samples involving TRALI fatalities. Because both techniques are rarely offered in reference laboratories, it is essential to identify laboratories that routinely offer a combination of GIFT and GAT when detecting neutrophil antibodies. Our laboratory also offers state of the art bead based flow cytometry assays to detect and identify HLA Class I and Class II antibodies.

Drug-induced neutropenia

Drug-induced agranulocytosis (DIAG) results in a profound decrease or even complete disappearance of granulocytes from peripheral blood. Absolute neutrophil counts (ANC) less than 100 neutrophils/µL are observed in a majority of cases. Individuals diagnosed with DIAG are at a very high risk of experiencing life-threatening and even fatal infections. The annual incidence of DIAG is approximately 2.4-15.4 cases per million. Many more cases present with mild or moderate neutropenia (ANC of 500-1000/ µL) that are often referred to as drug-induced neutropenia (DINP). DINP appears to evolve slower and may resolve spontaneously. Drugs which are most commonly involved in immune-mediated neutropenia include antibiotics, analgesics, antiarrhythmias, antimalarials and antithyroids.

Specialized Methods

The International Society of Blood Transfusion (ISBT) - Granulocyte Immunobiology Working Party (GIWP) has strongly recommended the use of both the granulocyte immunofluorescence test (GIFT) AND the granulocyte agglutination test (GAT) techniques in the detection of granulocyte (neutrophil) antibodies. It is of vital importance to use both the GAT and GIFT methods when detecting HNA antibodies. International Granulocyte Immunology Workshop (IGIW) external quality assessments have demonstrated that HNA antibodies detected by GAT have NOT BEEN DECTECTED by GIFT. Two IGIW assessments have included HNA-3a antibodies where laboratories using GIFT alone failed to detect antibody to HNA-3a samples involved in TRALI fatalities.

Both of these laboratory assays require viable intact granulocytes for detection and identification of human neutrophil antigen (HNA) antibodies. Granulocytes are intrinsically designed to respond to physiologic priming signals, so they must be handled very carefully. Once activated it is impossible to interpret any serologic test result involving these cells, as false positive results abound. Granulocytes are also extremely labile and must be used within 24 hours following collection. This necessitates ready access to panel cell donors with known HNA types. Finally, the presence of HLA antibodies in the test sera can make identifying HNA antibodies difficult as granulocytes also express HLA class I antigens on their cellular membrane. This obstacle can be overcome with the use of the monoclonal antibody immobilization of neutrophil antigens (MAINA) technique.

The isolation of a pure granulocyte suspension from peripheral blood is accomplished using the Ficoll-Hypaque discontinuous gradient technique. The double-density gradient centrifugation of leukocyte rich plasma yields a granulocyte suspension with a purity of 90 - 99%. Preparing a panel of donors to include all known HNA currently defined by ISBT using a suspension of pure granulocytes will aid in the detection and identification of HNA antibodies.

Granulocyte Aggulation Test (GAT)

This test is biphasic and is based upon the instrinsic response of granulocytes to aggregate when stimulated by antibodies reacting to cognate cell surface antigens. The resulting aggregation is the consequence of the granulocyte activation that occurs during the sensitization phase, which induces the granulocytes to form pseudopods and slowly migrate toward one another during the aggregation phase, until membrane contact is established. GAT provides the best observation as to how the antibodies affect PMNs in vivo, as the observed aggregation is the result of viable PMNs becoming sensitized by antibodies that lead to chemotaxis and producing homotypic PMN:PMN agglutinates that are distinct from passive cross-linking via IgM. Serum or plasma from the specimen being analyzed is incubated with a granulocyte suspension. The reactions are evaluated using an inverted-phase microscope. These reactions are graded based upon the percentage of granulocytes involved in the aggregates. Both IgG and IgM antibodies are detected by this method.

Granulocyte Immunofluorescence Test (GIFT)

Normally, immunoglobulins are present on neutrophil surfaces but these proteins are nonspecifically attached via the granulocyte Fc receptor and can be removed by the use of paraformaldehyde. Paraformaldehyde is also used to stabilize the cell membrane. Serum or plasma is incubated with an optimized concentration of granulocytes. During this incubation HNA reactive antibodies will bind to their cognate antigen epitopes. After a wash step that removes unbound antibodies, the granulocytes are then incubated with F(ab')² fragments of a fluorescent conjugated anti-human antibody. The assay's performance is optimized with the use of a fluorescent secondary probe that can detect IgG, IgM and IgA antibody isotypes (to ensure the detection of both primary and secondary immune responses) and the use of F(ab')² lg fragments to prevent the probe from binding high concentrations of Fc receptors that may remain on the granulocyte surface membrane following paraformaldehyde treatment. Granulocytes go through another wash cycle and are analyzed for HNA antibodies using a flow cytometer.

MAINA

The monoclonal antibody immobilization of neutrophil antigens (MAINA) assay allows the detection of antibodies to specific neutrophil membrane glycoproteins. MAINA relies on the capture of neutrophil-specific antigen-antibody (ag-ab) complexes by a murine monoclonal antibody onto a solid-phase surface. The benefits of this test are twofold; first, this is currently the most sensitive assay available for the detection of granulocyte antibodies, and second, the assay is designed to detect only HNA antibodies even when HLA antibodies are present in the test specimen. The disadvantage of this procedure is its complexity.

Granulocytes are incubated with test serum. This granulocyte suspension is then washed to remove any unbound immunoglobulins and incubated with a murine monoclonal antibody conjugated to a specific neutrophil glycoprotein. After another wash step, the granulocyte membranes are disrupted in a mild detergent and centrifuged. The resulting lysate is then transferred to polystyrene microwells coated with anti-mouse immunoglobulins and incubated. The subsequent tri-molecular complex of neutrophil antigen-patient HNA antibody-murine monoclonal antibody (ag-ab-ab) present in the lysate is captured onto the solid phase, whereas any HLA antigen-antibody (ag-ab) complexes (if present) are removed with a subsequent wash step. Remaining complexes are then detected by the addition of anti-human IgG conjugated to horseradish peroxidase followed by incubation with substrate resulting in a color change for specimens' positive for HNA antibody. The reaction is then analyzed with a spectrophotometer.

HLA Antibody Screen, Class I, IgG

The presence of HLA antibodies in a patient's specimen can make identifying HNA antibodies difficult as granulocytes also express HLA class I antigens on their surface membrane. If no HLA Class I antibodies are detected by this method it suggests that the antibody identified in GAT and/or GIFT is granulocyte specific. If HLA Class I antibody is detected, utilizing the MAINA technique will verify if HNA antibody is also present.

This test is a qualitative solid phase enzyme linked immunosorbent assay (ELISA) that uses microwells coated with affinity purified HLA Class I glycoproteins. During the incubation of the patient's sample HLA Class I antibody, if present, will bind with its cognate antigen epitope. A wash step will remove unbound antibodies from the microwells and anti-human IgG conjugated to an enzyme is added followed by another incubation step. This anti-human conjugate reagent will attach to the solid phase matrix if antibody to HLA Class I was bound during the initial incubation and will be detected with the addition of a substrate reagent. The reaction is then analyzed with a spectrophotometer.

These assays are used for the detection and identification of HLA Class I and HLA Class II antibodies associated with transfusion-related acute lung injury (TRALI). These assays are performed by the American Red Cross National HLA laboratory using the LABScreen® Mixed and LABScreen® Single Antigen test systems.

HLA Antibody Screen for Transplantation, Class I & II
HLA Antibody Identification, Class I, lgG
HLA Antibody Identification, Class II, lgG

Neutrophil Typing

Typing HNA antigens or alleles can be ascertained by using serologic techniques (GAT and GIFT) for the former or molecular methods for the later. Neutrophil typing can be valuable when confirming HNA antibody specificity previously detected in patient samples, in cases of alloimmune neonatal neutropenia (ANN) where parental HNA types provide information on the probable risk of affected neonates, and in identifying blood donors that are at high risk of producing HNA-3 antibodies which have been implicated in severe or fatal cases of TRALI.

DNA based genotyping is performed using sequence specific primer- polymerase chain reaction (SSP-PCR) techniques. These methods can determine the genotype of patients for the following neutrophil antigen systems; HNA-1, HNA-3, HNA-4, and HNA-5. Advantages of using genotyping methods over serologic methods are related to the specimen tested, as sample age, handling, storage and shipping are less stringent. Genotyping is also widely used and is considered the gold standard technique in neutrophil typing.

HNA Nomenclature

The Granulocyte Immunobiology Working Party (GIWP) of the International Society of Blood Transfusion (ISBT) established nomenclature of granulocyte alloantigens.

Allele nomenclature

In general the official gene name is followed by an asterisk and by a two digit allele number. Genetic variations of standard alleles are numbered consecutively and are not extended by a third or fourth digit.
New alleles are numbered consecutively according to the first publication.
Only missense mutations within exons leading to an amino acid exchange are included.

Antigen nomenclature

Granulocyte antigens will be called HNA (human neutrophil alloantigen).
The antigen nomenclature is based upon the serologically defined epitope(s) on glycoproteins.
The glycoprotein (antigen system) is coded by a number: HNA-1, HNA-2, etc.
The phenotypic variation of the antigen is assigned given in lower case letters: HNA-1a, HNA-1b, etc.
HNA-2 is an isoantigen without allelic variation and therefore should be named HNA-2.
Glycoproteins, which are encoded by one allele can exhibit more than one epitope. For example, HNA-1b and HNA-1c are encoded by FCGR3B*03 on glycoprotein FcγRIIIb (CD16).
One epitope can be encoded by different alleles. For example, HNA-1a is encoded by FCGR3B*01 and FCGR3B*04.
Only slightly altered epitopes can be identified by adding the subscript “var” in cases which antibodies specific for the original epitope bind with reduced affinity of altered reactivity pattern. For example, HNA-3a_var for HNA-3a with an additional Leu151Phe exchange.
The cellular expression of HNA is not necessarily restricted to neutrophil granulocytes. For example, HNA-3 is expressed on neutrophils, lymphocytes, platelets and tissues.
Antigens originally identified on other cell types and whose main clinical impact depends on cells other than neutrophils keep their original nomenclature and are not included in HNA nomenclature. For example, Naka (GPIV, CD36) located on platelets and monocytes.

HNA alleles and antigens

Allele	Description	Glyco-protein	Epitopes		HNA frequencies North Americans (%)	HNA frequencies Africans (%)	HNA frequencies Asian (%)
FCGR3B*		FcgRIIIb, CD16
FCGR3B*01			HNA-1a		56 -62	46 - 66	90
FCGR3B*02			HNA-1b	HNA-1d##	89	78 - 84	52
FCGR3B*03			HNA-1c	HNA-1b	5	23 - 31	0
FCGR3B*04	FCGR3B*01 316G>A		HNA-1a		NT	NT	NT
FCGR3B*05	FCGR3B*02 244A>G		HNA-1bvar.		NT	NT	NT
FCGR3B*null	FCGR3B gene deletion		HNA-1 null, no epitope		NT	4	0 - 0.2

CD177		CD177	HNA-2		97	98	99

SLC44A2*01		CTL2	HNA-3a		89 - 99	NT	NT
SLC44A2*02			HNA-3b		NT	NT	NT
SLC44A2*03	SLC44A2*01 451C>T		HNA-3a var.		NT	NT	NT

ITGAM*01		CD11b	HNA-4a		96	NT	NT
ITGAM*02		CD11b	HNA-4b**		NT	NT	NT

ITGAL*01		CD11a	HNA-5a		96	88	65
ITGAL*02		CD11a	--		NT	NT	NT

## HNA-1d is antithetical to HNA-1c

**HNA-4b is actually under investigation

NT = Not Tested

How to Order Testing

Tests and CPT Codes

HLA Antigen Typing HLA-A, B and C HLA-DRβ1 & DQ β1	CPT code(s) 81372 81376 x 2
HLA Antibody
HLA Antibody Screen for Transplantation, Class I & II	86828
HLA Antibody Identification, Class I, lgG	86830
HLA Antibody Identification, Class II, lgG	86831
HLA Antibody Screen, Class I, lgG	86829
Neutrophil (HNA) Antibody
Neutrophil Antibody Identification (includes) Granulocyte Agglutination (GAT) & Granulocyte lmmunofluorescence (GIFT)	86021x2
Monoclonal Antibody Immobilization of Neutrophil Antigens (MAINA)	86021x3
Drug Dependent Neutrophil Antibody	86021x3
Transfusion-Related Acute Lung Injury (TRALI) Investigation (Donor and Recipient)	86021x2, 86828, 86830, 86831
Neutrophil Crossmatch	88102
Neutrophil Typing
Molecular Typing (HNA-la, -lb, -lc, -3a, -3b, -4a, -4b, -5a, -5bw)	81479x9
Serological Typing (HNA-la, -lb, -lc, -2, -3a, -4a)	86021x4
Platelet (HPA) Antibody
Platelet Antibody Screen (Serum)	86022
Platelet Associated lmmunoglobulin	86023x2
Heparin Antibody	86022
Platelet Antibody, Crossmatch	86022x2
Platelet Typing
Serological Typing (HPA-1a)	86903x2

Order Form

Specimen Requirements

Requirements for all our tests are available on our Request for Neutrophil Services form.
Specimens without sufficient information for unique identification will be rejected.
We will send a report for every specimen that we receive in our laboratory. If the specimen was rejected we will provide detailed information why we could not test it.
The integrity of the specimen is essential for accurate results. Integrity of the specimen is considered to be compromised when a sample is contaminated in shipment or leaks during transit. Incorrect sample types, such as a segment from a donor component or a specimen that was collected in a gel separator tube, will also be rejected. In some cases we can perform testing on suboptimal specimens, such as when a specimen exhibits hemolysis or if lipemia is present.

Shipping Requirements

Ship according to federal and local requirements for Shipping Biological Substances Category B. (Contact your overnight carrier for specific requirements.)
Serum and plasma specimens should be separated from the red cells and frozen immediately. Frozen specimens should be insulated and packed with dry ice. Dry ice is considered a hazardous material and must be packaged following Department of Transportation (DOT), International Air Transport Association (IATA) and your carrier requirements.
Refrigerated specimens should be packed with frozen or cold gel (ice) packs. Insulate the specimen tubes to prevent freezing.
Specimens shipped at ambient temperature should be insulated to prevent damage from extreme shipping temperatures.
Specimens should be sent by overnight carrier Monday through Thursday to ensure weekday delivery. The Neutrophil Immunology Laboratory is not staffed on weekends or the following holidays: New Year’s Day, Memorial Day, the Fourth of July, Labor Day, Thanksgiving and Christmas Day.