Monocyte monolayer assay

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The image on the left shows a monocyte actively phagocytizing an antibody-sensitized red blood cell. The image on the right shows multiple RBC that have been phagocytized by a single monocyte. Phagocytosis of RBC by Monocyte.jpg
The image on the left shows a monocyte actively phagocytizing an antibody-sensitized red blood cell. The image on the right shows multiple RBC that have been phagocytized by a single monocyte.

The monocyte monolayer assay (MMA) is used to determine the clinical significance of alloantibodies produced by blood transfusion recipients. [1] The assay is used to assess the potential for intravascular hemolysis when incompatible cellular blood products are transfused to the anemic patient. When donor cells possess substances that are not produced by the recipient, the recipient's immune system produces antibodies against the substance; these are called alloantibodies. Specific white blood cells, called monocytes, are tasked with ingesting foreign material and become activated during certain inflammatory events. [2] [3] These activated monocytes come in contact with antibody-sensitized red blood cells (RBC) and may or may not exhibit phagocytosis (ingestion) and destroy the donor red blood cells. If monocytes destroy the RBC, the antibody attached to those RBC is considered clinically significant. [1]

Contents

Background

Blood banking

Blood banking is a concentration within a clinical laboratory that analyzes specimens from potential transfusion recipients and provides compatible blood products to the healthcare team in charge of that patient's care. Several routine tests are performed including blood typing (determination of ABO/Rh status), antibody screening, serologic cross-matching, direct antiglobulin testing, and antibody identification. Beyond the presence of naturally occurring antibodies (isohemagglutinins) to the ABO and Rh(D) blood group antigens, additional immune-stimulated antibodies are considered unexpected alloantibodies. [4]

The identification of unexpected antibodies is a labor-intensive process, and sometimes requires the addition of special laboratory techniques to aid in the proper identification of the antibody. Among these techniques are elutions, adsorptions, and enzyme treatment. [4] [5] Some patients produce antibodies to high frequency antigens. That is, the red cell antigens are present in a significant portion of the human population. It may be questionable and very difficult to assess if the antibody is considered clinically significant or not. [6] A clinically significant antibody is an antibody that is capable of causing in vitro hemolysis or a decreased survival of transfused donor red blood cells. [7] Antibodies to high frequency antigens can be assessed for clinical significance using the monocyte monolayer assay. [8]

Hemolytic transfusion reaction

There are many different varieties of abnormal reactions to blood transfusion. Among these, a potentially life-threatening reaction is known as a hemolytic transfusion reaction. This is an immune mediated reaction where recipient antibodies attack donor red blood cell antigen(s), causing hemolysis of donor cells. The reaction may occur during, immediately after, or up to 28 days later. An acute reaction is observed within the first 24 hours, whereas a delayed reaction will be observed between 24 hours and 28 days after transfusion. [4]

Alloantibody formation and clinical significance

When talking about the ABO blood group system, Landsteiner's Law states that if an individual possesses the A and/or B antigen, they will not form antibodies to these antigens. [9] However, if an individual does not have either A or B antigens, they will naturally produce anti-A and anti-B antibodies. [10] According to the International Society of Blood Transfusion (ISBT), 43 blood group systems containing hundreds of different red blood cell antigens have been described. [11] With some exceptions, many non-ABO blood group system antigens require a sensitizing event to stimulate antibody production. In other words, the immune system must be exposed to the antigen in order to illicit antibody production. Exposure to antigens can occur through blood transfusion, stem cell/bone marrow transplant, and pregnancy. [4]

The clinical significance of an alloantibody depends on its ability to cause a decrease in donor red blood cell survival. [12] Characteristics of clinically significant alloantibodies include: immunoglobulin G antibody subclass, reactivity at body temperature, and ability to cause red blood cell agglutination in the presence of anti-human globulin (AHG) in an indirect antiglobulin test. [4] Sometimes, clinical significance of an antibody can be difficult to determine. [6] Antibodies to high prevalence red cell antigens can sometimes mask the detection of clinically significant alloantibodies because the corresponding antigen is present on most, if not all, of the screening red blood cells used to detect these antibodies possess the antigen. This is where the monocyte monolayer assay may be useful.

Principle

The MMA is a very labor-intensive, manual laboratory testing method. The following steps are performed in this assay: [13]

  1. Anticoagulated blood is collected from normal, healthy individuals. Acid citrate dextrose is preferred. [6]
  2. Peripheral blood mononuclear cells (PBMC) are harvested from the blood sample using a Ficoll-Paque® density gradient.
  3. The PBMC's are washed using a phosphate-buffered saline (PBS) and then suspended in tissue culture media in order to keep the monocytes viable.
  4. The PBMC-media mixture is then added to a tissue culture chamber slide. Monocytes will adhere to the glass slide forming a monocyte monolayer.
  5. Serum is mixed and incubated at body temperature (37°C) with a 5% group O RBC suspension. (This step sensitizes or coats RBC with antibody)
  6. The sensitized RBC's are washed with PBS to remove any unbound antibody or interfering substances.
  7. The washed, sensitized RBC are then added to the monocyte monolayer tissue culture chamber slide.
  8. After a 60-minute, 37°C incubation, the supernatant is removed from the chamber slides and rinsed with PBS.
  9. Once the slide is completely dry, it is stained with a Wrights-Giemsa stain.
  10. At least 600 (200 if positivity is greater than 20%) monocytes are observed under the microscope for evidence of RBC phagocytosis.
  11. A positive and negative control is also performed in tandem with the patient specimen for quality assurance.

Interpretation

If the positive or negative controls fail, than the entire testing procedure is invalid and must be repeated. Criteria for a positive MMA will vary by laboratory, though the originally established threshold set by Sandra Nance et al., is >20% phagocytic activity observed. [13]

Clinically, a positive MMA would indicate that the patient's serum used in the assay contains clinically significant antibodies that are capable of causing antibody-mediated phagocytosis.

See also

Related Research Articles

<span class="mw-page-title-main">Blood type</span> Classification of blood based on antibodies and antigens on red blood cell surfaces

A blood type is a classification of blood, based on the presence and absence of antibodies and inherited antigenic substances on the surface of red blood cells (RBCs). These antigens may be proteins, carbohydrates, glycoproteins, or glycolipids, depending on the blood group system. Some of these antigens are also present on the surface of other types of cells of various tissues. Several of these red blood cell surface antigens can stem from one allele and collectively form a blood group system.

<span class="mw-page-title-main">Blood transfusion</span> Intravenous transference of blood products

Blood transfusion is the process of transferring blood products into a person's circulation intravenously. Transfusions are used for various medical conditions to replace lost components of the blood. Early transfusions used whole blood, but modern medical practice commonly uses only components of the blood, such as red blood cells, white blood cells, plasma, platelets, and other clotting factors.

<span class="mw-page-title-main">Hemolytic disease of the newborn</span> Fetal and neonatal alloimmune blood condition

Hemolytic disease of the newborn, also known as hemolytic disease of the fetus and newborn, HDN, HDFN, or erythroblastosis fetalis, is an alloimmune condition that develops in a fetus at or around birth, when the IgG molecules produced by the mother pass through the placenta. Among these antibodies are some which attack antigens on the red blood cells in the fetal circulation, breaking down and destroying the cells. The fetus can develop reticulocytosis and anemia. The intensity of this fetal disease ranges from mild to very severe, and fetal death from heart failure can occur. When the disease is moderate or severe, many erythroblasts are present in the fetal blood, earning these forms of the disease the name erythroblastosis fetalis.

The direct and indirect Coombs tests, also known as antiglobulin test (AGT), are blood tests used in immunohematology. The direct Coombs test detects antibodies that are stuck to the surface of the red blood cells. Since these antibodies sometimes destroy red blood cells they can cause anemia; this test can help clarify the condition. The indirect Coombs test detects antibodies that are floating freely in the blood. These antibodies could act against certain red blood cells; the test can be carried out to diagnose reactions to a blood transfusion.

<span class="mw-page-title-main">Cross-matching</span> Testing before a blood transfusion

Cross-matching or crossmatching is a test performed before a blood transfusion as part of blood compatibility testing. Normally, this involves adding the recipient's blood plasma to a sample of the donor's red blood cells. If the blood is incompatible, the antibodies in the recipient's plasma will bind to antigens on the donor red blood cells. This antibody-antigen reaction can be detected through visible clumping or destruction of the red blood cells, or by reaction with anti-human globulin. Along with blood typing of the donor and recipient and screening for unexpected blood group antibodies, cross-matching is one of a series of steps in pre-transfusion testing. In some circumstances, an electronic cross-match can be performed by comparing records of the recipient's ABO and Rh blood type against that of the donor sample. In emergencies, blood may be issued before cross-matching is complete. Cross-matching is also used to determine compatibility between a donor and recipient in organ transplantation.

Autoimmune hemolytic anemia (AIHA) occurs when antibodies directed against the person's own red blood cells (RBCs) cause them to burst (lyse), leading to an insufficient number of oxygen-carrying red blood cells in the circulation. The lifetime of the RBCs is reduced from the normal 100–120 days to just a few days in serious cases. The intracellular components of the RBCs are released into the circulating blood and into tissues, leading to some of the characteristic symptoms of this condition. The antibodies are usually directed against high-incidence antigens, therefore they also commonly act on allogenic RBCs. AIHA is a relatively rare condition, with an incidence of 5–10 cases per 1 million persons per year in the warm-antibody type and 0.45 to 1.9 cases per 1 million persons per year in the cold antibody type. Autoimmune hemolysis might be a precursor of later onset systemic lupus erythematosus.

In ABO hemolytic disease of the newborn maternal IgG antibodies with specificity for the ABO blood group system pass through the placenta to the fetal circulation where they can cause hemolysis of fetal red blood cells which can lead to fetal anemia and HDN. In contrast to Rh disease, about half of the cases of ABO HDN occur in a firstborn baby and ABO HDN does not become more severe after further pregnancies.

Hemolytic disease of the newborn (anti-Kell1) is the second most common cause of severe hemolytic disease of the newborn (HDN) after Rh disease. Anti-Kell1 is becoming relatively more important as prevention of Rh disease is also becoming more effective.

Hemolytic disease of the newborn (anti-Rhc) can range from a mild to a severe disease. It is the third most common cause of severe HDN. Rh disease is the most common and hemolytic disease of the newborn (anti-Kell) is the second most common cause of severe HDN. It occurs more commonly in women who are Rh D negative.

The term human blood group systems is defined by the International Society of Blood Transfusion (ISBT) as systems in the human species where cell-surface antigens—in particular, those on blood cells—are "controlled at a single gene locus or by two or more very closely linked homologous genes with little or no observable recombination between them", and include the common ABO and Rh (Rhesus) antigen systems, as well as many others; 44 human systems are identified as of December 2022.

<span class="mw-page-title-main">Rh blood group system</span> Human blood group system involving 49 blood antigens

The Rh blood group system is a human blood group system. It contains proteins on the surface of red blood cells. After the ABO blood group system, it is the most likely to be involved in transfusion reactions. The Rh blood group system consisted of 49 defined blood group antigens in 2005. As of 2023, there are over 50 antigens among which the five antigens D, C, c, E, and e are the most important. There is no d antigen. Rh(D) status of an individual is normally described with a positive (+) or negative (−) suffix after the ABO type. The terms Rh factor, Rh positive, and Rh negative refer to the Rh(D) antigen only. Antibodies to Rh antigens can be involved in hemolytic transfusion reactions and antibodies to the Rh(D) and Rh antigens confer significant risk of hemolytic disease of the fetus and newborn.

The MNS antigen system is a human blood group system based upon two genes on chromosome 4. There are currently 50 antigens in the system, but the five most important are called M, N, S, s, and U.

<span class="mw-page-title-main">Packed red blood cells</span> Red blood cells separated for blood transfusion

Packed red blood cells, also known as packed cells, are red blood cells that have been separated for blood transfusion. The packed cells are typically used in anemia that is either causing symptoms or when the hemoglobin is less than usually 70–80 g/L. In adults, one unit brings up hemoglobin levels by about 10 g/L. Repeated transfusions may be required in people receiving cancer chemotherapy or who have hemoglobin disorders. Cross-matching is typically required before the blood is given. It is given by injection into a vein.

Hemolytic disease of the newborn (anti-RhE) is caused by the anti-RhE antibody of the Rh blood group system. The anti-RhE antibody can be naturally occurring, or arise following immune sensitization after a blood transfusion or pregnancy.

This page is currently under construction.

The Vel blood group is a human blood group that has been implicated in hemolytic transfusion reactions. The blood group consists of a single antigen, the high-frequency Vel antigen, which is expressed on the surface of red blood cells. Individuals are typed as Vel-positive or Vel-negative depending on the presence of this antigen. The expression of the antigen in Vel-positive individuals is highly variable and can range from strong to weak. Individuals with the rare Vel-negative blood type develop anti-Vel antibodies when exposed to Vel-positive blood, which can cause transfusion reactions on subsequent exposures.

The Junior blood group system is a human blood group defined by the presence or absence of the Jr(a) antigen, a high-frequency antigen that is found on the red blood cells of most individuals. People with the rare Jr(a) negative blood type can develop anti-Jr(a) antibodies, which may cause transfusion reactions and hemolytic disease of the newborn on subsequent exposures. Jr(a) negative blood is most common in people of Japanese heritage.

<span class="mw-page-title-main">Blood compatibility testing</span> Testing to identify incompatibilities between blood types

Blood compatibility testing is conducted in a medical laboratory to identify potential incompatibilities between blood group systems in blood transfusion. It is also used to diagnose and prevent some complications of pregnancy that can occur when the baby has a different blood group from the mother. Blood compatibility testing includes blood typing, which detects the antigens on red blood cells that determine a person's blood type; testing for unexpected antibodies against blood group antigens ; and, in the case of blood transfusions, mixing the recipient's plasma with the donor's red blood cells to detect incompatibilities (crossmatching). Routine blood typing involves determining the ABO and RhD type, and involves both identification of ABO antigens on red blood cells and identification of ABO antibodies in the plasma. Other blood group antigens may be tested for in specific clinical situations.

The Lan blood group system is a human blood group defined by the presence or absence of the Lan antigen on a person's red blood cells. More than 99.9% of people are positive for the Lan antigen. Individuals with the rare Lan-negative blood type, which is a recessive trait, can produce an anti-Lan antibody when exposed to Lan-positive blood. Anti-Lan antibodies may cause transfusion reactions on subsequent exposures to Lan-positive blood, and have also been implicated in mild cases of hemolytic disease of the newborn. However, the clinical significance of the antibody is variable. The antigen was first described in 1961, and Lan was officially designated a blood group in 2012.

<span class="mw-page-title-main">Antibody elution</span> Laboratory procedure

An antibody elution is a clinical laboratory diagnostic procedure which removes sensitized antibodies from red blood cells, in order to determine the blood group system antigen the antibody targets. An antibody elution is deemed necessary when antibodies of the immunoglobulin class G (IgG) are found sensitized (bound) to peripheral red cells collected from a blood product transfusion recipient. IgG antibodies are detected using an assay known as the direct antiglobulin test.

References

  1. 1 2 "Through the Microscope: Monocyte Monolayer Assays".
  2. ↑ "https://www.cancer.gov/publications/dictionaries/cancer-terms/def/monocyte". www.cancer.gov. 2011-02-02. Retrieved 2023-01-09.{{cite web}}: External link in |title= (help)
  3. ↑ Avery's diseases of the newborn. Christine A. Gleason, Sandra E. Juul (Tenth ed.). Philadelphia, PA. 2018. ISBN   978-0-323-40172-2. OCLC   1013816550.{{cite book}}: CS1 maint: location missing publisher (link) CS1 maint: others (link)
  4. 1 2 3 4 5 Modern blood banking & transfusion practices. Denise Harmening (Seventh ed.). Philadelphia, PA. 2019. ISBN   978-0-8036-9462-0. OCLC   1048659017.{{cite book}}: CS1 maint: location missing publisher (link) CS1 maint: others (link)
  5. ↑ Strobel, Erwin (2004). "Use of the enzyme method for antibody identification". Clinical Laboratory. 50 (9–10): 575–580. ISSN   1433-6510. PMID   15481633.
  6. 1 2 3 "Mastering the monocyte monolayer assay | Canadian Blood Services". www.blood.ca. Retrieved 2023-01-09.
  7. ↑ Makroo, Raj Nath; Arora, Bhavna; Bhatia, Aakanksha; Chowdhry, Mohit; Luka, Rosamma Nakamatathil (July 2014). "Clinical significance of antibody specificities to M, N and Lewis blood group system". Asian Journal of Transfusion Science. 8 (2): 96–99. doi: 10.4103/0973-6247.137442 . ISSN   0973-6247. PMC   4140072 . PMID   25161347.
  8. ↑ Arndt, Patricia A.; Garratty, George (September 2004). "A retrospective analysis of the value of monocyte monolayer assay results for predicting the clinical significance of blood group alloantibodies". Transfusion. 44 (9): 1273–1281. doi: 10.1111/j.1537-2995.2004.03427.x . ISSN   0041-1132. PMID   15318848. S2CID   24871062.
  9. ↑ Marshall, William J.; Lapsley, Márta; Day, Andrew; Ayling, Ruth (5 March 2014). Clinical Biochemistry: Metabolic and Clinical Aspects. Elsevier Health Sciences. ISBN   9780702054785.
  10. ↑ Denomme, Gregory A. (2000). Molecular protocols in transfusion medicine. Maria Rios, Marion E. Reid. San Diego, CA: Academic Press. ISBN   978-0-12-209370-8. OCLC   162129287.
  11. ↑ ISBT. "International Society of Blood Transfusion |ISBT". www.isbtweb.org. Retrieved 2022-10-05.
  12. ↑ Moinuddin, Imran; Fletcher, Craig; Millward, Peter (2019). "Prevalence and specificity of clinically significant red cell alloantibodies in pregnant women - a study from a tertiary care hospital in Southeast Michigan". Journal of Blood Medicine. 10: 283–289. doi: 10.2147/JBM.S214118 . ISSN   1179-2736. PMC   6708388 . PMID   31692490.
  13. 1 2 Nance, S. J.; Arndt, P.; Garratty, G. (November 1987). "Predicting the clinical significance of red cell alloantibodies using a monocyte monolayer assay". Transfusion. 27 (6): 449–452. doi:10.1046/j.1537-2995.1987.27688071692.x. ISSN   0041-1132. PMID   3686653. S2CID   21638521.
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