Leukocyte and Platelet Antigen Testing

Platelets carry HLA class I antigens. Antibody to HLA class I is the most common immune cause of platelet transfusion refractoriness. HLA typing and antibody identification can therefore support platelet selection even when the patient is not being evaluated for a transplant.^{1,21 Bloch EM, et al. Technical Manual. 2026.2 American Society for Histocompatibility and… 2025 ASHI Standards for Accredited Laboratories. 2026.}

Complement-dependent cytotoxicity

Complement-dependent cytotoxicity (CDC), also called microlymphocytotoxicity, demonstrates complement-mediated injury to viable lymphocytes. Test lymphocytes are incubated with antibody of known specificity for typing, or with patient serum for a crossmatch. Complement is added, and a vital dye distinguishes cells with damaged membranes from cells that exclude the dye. Positive and negative controls establish complement activity, cell viability, and the valid scoring range. The laboratory assigns the HLA type or crossmatch result from the complete reaction pattern across all wells.^{1,21 Bloch EM, et al. Technical Manual. 2026.2 American Society for Histocompatibility and… 2025 ASHI Standards for Accredited Laboratories. 2026.}

CDC depends on viable cells and complement-fixing antibody. A wash step in an Amos-modified method can reduce interference from anticomplementary serum factors, and an AHG-augmented method can increase detection of weak cell-bound antibody. Reagent choice and scoring follow the laboratory's validated procedure. DNA-based methods now provide the primary HLA type in most settings. CDC remains useful for selected antibody and crossmatch questions.^{1,21 Bloch EM, et al. Technical Manual. 2026.2 American Society for Histocompatibility and… 2025 ASHI Standards for Accredited Laboratories. 2026.}

Molecular typing and antibody assays

PCR with sequence-specific primers, sequence-specific oligonucleotide probes, Sanger sequencing, and next-generation sequencing identify HLA alleles at different levels of resolution. The report must state the loci, resolution, ambiguity, and assay coverage. A genotyping result predicts the encoded HLA type only. Antibody detection requires a separate assay.^{2,32 American Society for Histocompatibility and… 2025 ASHI Standards for Accredited Laboratories. 2026.3 Joint United Kingdom (UK) Blood Transfusion… Chapter 16: HLA typing and HLA serology.}

Cell-based flow cytometry and solid-phase assays detect antibody without requiring complement-mediated cell death. In a single-antigen bead assay, each coded bead carries one HLA antigen, and a fluorescent anti-human immunoglobulin detects bound antibody. Mean fluorescence intensity (MFI) is an assay signal and does not measure antibody concentration. Antigen density, denatured antigen, interference, lot, instrument settings, and analysis rules can change that signal. No universal MFI cutoff defines a clinically important antibody.^{2,32 American Society for Histocompatibility and… 2025 ASHI Standards for Accredited Laboratories. 2026.3 Joint United Kingdom (UK) Blood Transfusion… Chapter 16: HLA typing and HLA serology.}

For platelet support, the HLA laboratory may combine the patient's HLA-A and HLA-B type with the HLA antibody profile. An exact HLA match is one option. Antibody-specific prediction expands the search by excluding donors who carry antigens targeted by the patient's antibodies. A platelet crossmatch tests patient serum against donor platelets and can detect incompatibility caused by HLA or HPA antibody. The crossmatch does not identify the antibody specificity.^{4,54 Association for the Advancement of Blood &…, et al. Circular of Information for the Use of Human Blood and Blood… 2024.5 Cohn CS. Platelet transfusion refractoriness. 2020.}

The transplantation immunology lesson covers detailed transplant typing, donor-specific antibody assessment, and organ crossmatch policy.

At the bench, refractoriness appears as repeated posttransfusion platelet counts that rise little or not at all. Platelet refractoriness is a repeated lower-than-expected response to platelet transfusion. A single low posttransfusion count does not establish it. Active bleeding, fever or sepsis, disseminated intravascular coagulation, splenic sequestration, medications, and other nonimmune factors cause most cases. ABO incompatibility, HLA class I antibody, HPA antibody, and platelet-reactive autoantibody can also reduce the increment.^{4,54 Association for the Advancement of Blood &…, et al. Circular of Information for the Use of Human Blood and Blood… 2024.5 Cohn CS. Platelet transfusion refractoriness. 2020.}

The corrected count increment (CCI) adjusts the platelet-count change for patient size and the platelet dose:

Obtain a posttransfusion count 10 to 60 minutes after each of two sequential transfusions when evaluating a suspected refractory state. In an afebrile, nonbleeding patient receiving conventional platelets that are not pathogen reduced, a 10- to 60-minute CCI is typically greater than 7,500. A CCI below 5,000 with supportive serologic results may indicate immune refractoriness. Published and local criteria differ, and immune and nonimmune patterns overlap, so interpret a low CCI from the trend, the component, and the clinical setting together.^{4,54 Association for the Advancement of Blood &…, et al. Circular of Information for the Use of Human Blood and Blood… 2024.5 Cohn CS. Platelet transfusion refractoriness. 2020.}

Investigation and component selection

1. Record the pretransfusion count, platelet dose, completion time, posttransfusion count time, and clinical factors that can consume or sequester platelets.
2. Calculate the 10- to 60-minute CCI for two sequential transfusions under comparable conditions.
3. If poor increments persist after nonimmune factors are assessed, test for HLA class I and HPA antibodies according to the laboratory algorithm. Obtain the patient's HLA type when HLA-selected support may be needed.
4. Use a limited trial of crossmatch-compatible platelets or select HLA-matched or antibody-compatible platelets. Select HPA antigen-negative platelets when an HPA specificity is present, most platelet crossmatches are incompatible, or HLA-selected products fail.
5. Measure another 10- to 60-minute posttransfusion count. If the increment does not improve despite a compatible laboratory result, reassess nonimmune causes and the selection strategy.

In many services, platelet crossmatching is faster than a donor search and tests HLA and HPA compatibility together. Highly alloimmunized patients may have too few compatible crossmatched donors. HLA matching and antibody-specific avoidance require an HLA-typed donor inventory and an interpretable antibody profile. Name the strategy used in the laboratory report, and use the word "matched" only for HLA-matched units.^{5,65 Cohn CS. Platelet transfusion refractoriness. 2020.6 American Red Cross. National Reference Laboratory for Specialized Testing.}

Human neutrophil antigen (HNA) nomenclature names antigen systems defined through neutrophil alloantibodies. Some carrier proteins occur on other cells, so an HNA designation does not mean that expression is limited to neutrophils.^{77 Joint United Kingdom (UK) Blood Transfusion… Chapter 17: granulocyte immunology.}

Most HPA system antigens arise from a nucleotide change that produces one amino-acid difference on a platelet glycoprotein. The glycoprotein assignment matters because antigen-capture assays target a specific protein complex.^{1010 Versiti. Human Platelet Antigen (HPA) Database.}

Panel composition follows the population served and the reference laboratory's validation. Low-frequency HPA variants, private antigens, and CD36 deficiency can require specialized testing.

Whole-platelet solid-phase or immunofluorescence assays detect platelet-reactive antibody. A positive reaction can reflect HLA, HPA, ABO, autoantibody, or another platelet-reactive antibody. Glycoprotein-specific assays narrow that result. In the monoclonal antibody immobilization of platelet antigens (MAIPA) method, a monoclonal antibody captures a selected platelet glycoprotein after exposure to test serum, and an anti-human reagent detects human antibody bound to the same complex. Other validated antigen-capture and bead-based assays use related principles. No single method detects every HPA antibody.^{6,106 American Red Cross. National Reference Laboratory for Specialized Testing.10 Versiti. Human Platelet Antigen (HPA) Database.}

HPA genotyping identifies the alleles covered by the assay and predicts the antigen profile. It can confirm an antigen incompatibility, select an antigen-negative donor, and resolve serology when platelets are unavailable or recently transfused. The report must distinguish a genotype-predicted antigen from a serologically demonstrated phenotype and state any alleles the assay does not cover.^{1,101 Bloch EM, et al. Technical Manual. 2026.10 Versiti. Human Platelet Antigen (HPA) Database.}

Fetal and neonatal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) results when maternal antibody targets a paternally inherited fetal platelet antigen that the mother lacks. It can occur during a first pregnancy. Laboratory confirmation evaluates the incompatibility and the antibody together:

1. Test maternal serum for platelet alloantibody with two validated serologic methods when available. Use serum when it is available, because EDTA plasma can substantially reduce anti-HPA-1a detection by MAIPA.
2. Genotype the mother and affected neonate when neonatal DNA is available. The father's genotype can define the incompatibility and inheritance risk when the neonate is unavailable.
3. Crossmatch maternal serum with paternal platelets. This can detect antibody against a private or low-frequency paternal antigen that is absent from a routine panel.
4. Compare serology, genotype, platelet counts, and the clinical findings. A negative routine panel does not exclude FNAIT when the antibody is weak, develops late, or targets an antigen outside the panel. When initial serology is negative but clinical suspicion remains high, arrange reference-laboratory reassessment 2 to 8 weeks after delivery.^{11,1311 Petermann R, et al. Investigations for fetal and neonatal alloimmune… 2018.13 Sachs UJ, et al. Diagnosis and management of fetal and neonatal alloimmune… 2025.}

When the clinical team orders platelets for severe neonatal thrombocytopenia or bleeding, the transfusion service issues them without waiting for test results. Issue the immediately available platelet component under the emergency or local protocol, including random-donor platelets when necessary. Supply HPA antigen-negative or otherwise compatible platelets when they can be provided without delay, and continue the laboratory investigation. State the implicated specificity and family genotypes in the laboratory report, because those findings also affect future pregnancies.^{1111 Petermann R, et al. Investigations for fetal and neonatal alloimmune… 2018.}

Posttransfusion purpura

Posttransfusion purpura typically presents as abrupt, profound thrombocytopenia about 5 to 10 days after transfusion in a previously sensitized patient, often after pregnancy. Anti-HPA-1a is the classic specificity, although other HPA antibodies can occur. Both transfused and autologous platelets are cleared. The exact mechanism of autologous platelet destruction remains incompletely defined.^{1212 Hawkins J, et al. Post-transfusion purpura. 2019.}

The laboratory tests patient serum for HPA antibody and compares the specificity with the patient's HPA genotype. The antibody should target an antigen absent from the patient's predicted profile. The transfusion history, timing, platelet trend, and exclusion of other causes remain necessary because antibody alone does not establish the clinical diagnosis. Documented specificity informs future antigen-negative support.^{1212 Hawkins J, et al. Post-transfusion purpura. 2019.}

The final interpretation should name the specimen, method, antibody class or specificity, antigen panel, genotype coverage, crossmatch result, and component-selection consequence. It should also state the limitation that matters for the case.

Platelet autoantibody testing cannot establish immune thrombocytopenia, and its limited sensitivity means that a negative result cannot exclude the condition. State the method and its limit in every report so the reader knows what a negative result covers.^{11 Bloch EM, et al. Technical Manual. 2026.}