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Cytotoxicity

Cytotoxicity testing generally refers to Complement-Dependent Cytotoxicity (CDC). This technique has historically been the gold standard for Human Leukocyte Antigen (HLA) tissue typing, antibody screening, and crossmatching. While molecular methods (PCR) and solid-phase assays (Luminex) have largely replaced CDC for routine typing, understanding the physiology and pathophysiology of cytotoxicity remains vital for interpreting crossmatches and managing platelet refractoriness

The fundamental physiological basis of this test relies on the classical pathway of the complement cascade. When a specific antibody binds to a corresponding antigen on a cell membrane, the Fc portion of the immunoglobulin undergoes a conformational change that allows the binding of the C1 complement complex. This initiates a cascade resulting in the formation of the Membrane Attack Complex (MAC), which punctures the cell membrane, leading to osmotic lysis and cell death

Complement-Dependent Cytotoxicity (CDC) Test Principle

The CDC test, often referred to as the Terasaki microlymphocytotoxicity test, visualizes antigen-antibody reactions by assessing cell viability. The test utilizes lymphocytes because they are rich in HLA antigens. If a specific antibody is present in the patient’s serum that targets an antigen on the test lymphocytes, the antibody will bind. The subsequent addition of an exogenous source of complement (typically rabbit serum) causes lysis of the antibody-coated cells

To visualize this reaction, a supravital dye is added to the reaction well. The principle of dye exclusion is applied here:

  • Live cells (Negative Reaction): Have intact membranes that exclude the dye. Under a phase-contrast microscope, these cells appear small, bright, and refractile
  • Dead cells (Positive Reaction): Have membranes damaged by the complement MAC, allowing the dye to penetrate the cytoplasm. These cells appear large, dark, and non-refractile

Reagents & Test Components

Target Cells (Lymphocytes)

  • T-Lymphocytes: These cells primarily express HLA Class I antigens (A, B, C). They are used to detect antibodies against Class I antigens
  • B-Lymphocytes: These cells express both HLA Class I and HLA Class II antigens (DR, DQ, DP). Testing B-cells increases sensitivity and allows for the detection of Class II antibodies
  • Isolation: Peripheral blood is typically separated using density gradient centrifugation (e.g., Ficoll-Hypaque) to isolate the buffy coat mononuclear cells

Complement Source

  • Rabbit Complement: Rabbit serum is the standard source of complement for CDC testing because it is more effective at facilitating in vitro lysis of human cells than human complement
  • Storage: Complement is heat-labile and must be stored frozen (usually at -70°C or below) until use to preserve activity

Dyes * Eosin Y: A commonly used dye; dead cells appear dark red * Trypan Blue: Dead cells appear blue * Fluorescent dyes: Methods utilizing fluorescein diacetate (stains live cells green) and ethidium bromide (stains dead cells red) are used in some fluorochromatic variations to improve readability

Test Applications & Clinical Significance

HLA Antibody Screening (Panel Reactive Antibody - PRA)

The CDC method determines the percentage of the general population against which a patient has pre-formed antibodies. The patient’s serum is tested against a panel of lymphocytes with known HLA phenotypes

  • Calculation: If a patient reacts with 10 out of 50 panel cells, they have a PRA of 20%
  • Significance: A high PRA indicates the patient is “sensitized,” making it difficult to find compatible platelets or solid organs. High PRA is often caused by prior pregnancies, blood transfusions, or transplants

HLA Crossmatching This is the most critical application of cytotoxicity in transplantation. It mixes the recipient’s serum with the donor’s lymphocytes

  • Positive Crossmatch: Indicates the recipient has pre-existing antibodies against the donor’s HLA antigens
  • Pathophysiology: In solid organ transplant, this leads to Hyperacute Rejection, where the graft is destroyed within minutes to hours via thrombosis and necrosis. In platelet transfusion, this leads to immediate destruction of the transfused platelets

Platelet Refractoriness While lymphocytes are the targets in the test well, the results are directly applicable to platelet transfusions. Platelets express HLA Class I antigens (but not Class II)

  • The Clinical Issue: Patients receiving multiple platelet transfusions may develop HLA antibodies. Subsequent transfusions fail to raise the platelet count (refractoriness)
  • The Solution: An HLA-matched platelet product is required. The CDC test helps identify the specificity of the antibody so that donors lacking that antigen can be selected
  • Correction Calculation: To confirm refractoriness is immune-mediated (and not due to fever or splenomegaly), a Corrected Count Increment (CCI) is calculated. A low CCI after two consecutive transfusions suggests alloimmunization

Modifications & Enhancements

Because standard CDC may not detect low-titer antibodies, enhancements are frequently used:

  • AHG-CDC (Anti-Human Globulin): After the initial incubation of serum and cells, a wash step is performed, and AHG is added before the complement. AHG acts as a bridge, cross-linking antibodies that are bound to the cell but too sparse to activate complement on their own. This significantly increases the sensitivity of the assay
  • DTT Treatment: Dithiothreitol (DTT) can be used to treat serum to distinguish between IgM and IgG antibodies. DTT breaks the J-chain of IgM, inactivating it. If a positive reaction turns negative after DTT treatment, the antibody was IgM. This is clinically relevant because IgM HLA antibodies are generally considered less significant in graft survival than IgG

Interpretation & Scoring

The reaction is scored based on the estimated percentage of dead cells in the well. The standard scoring system typically follows the guidelines set by the American Society for Histocompatibility and Immunogenetics (ASHI):

  • Score 1 (Negative): Less than 10-20% dead cells. This is the background level of cell death
  • Score 2 (Doubtful Negative): 20-30% dead cells
  • Score 4 (Weak Positive): 40-60% dead cells
  • Score 6 (Positive): 60-80% dead cells
  • Score 8 (Strong Positive): 80-100% dead cells

Limitations & Interferences

CYNAP (Cytotoxicity Negative, Absorption Positive) This phenomenon occurs when an antibody binds to the cell surface (proven by absorption studies) but fails to activate the complement cascade to cause lysis. This results in a false-negative CDC result. This may occur if the antigen density on the cell surface is too low to allow the critical spacing required for complement activation

Granulocyte Contamination Granulocytes have Fc receptors that can bind antibodies non-specifically, leading to high background staining or false positives. Proper isolation of lymphocytes is crucial

Autoantibodies If a patient has autoantibodies (antibodies against their own tissues), the crossmatch will be positive against almost all donors. An autocontrol (patient serum + patient cells) must be run to rule this out

Non-HLA Antibodies The CDC test is not perfectly specific to HLA. Antibodies against other lymphocyte antigens (such as Lewis antigens or viral receptors) can cause positive cytotoxic reactions, potentially confusing the interpretation of HLA compatibility