Antigen Information and Courses from MediaLab, Inc.

A 42-year-old male received 6 units of RBCs during open heart surgery 6 months ago. His antibody screen was negative at that time. He has returned for a follow up surgery and his antibody screen is now positive with both screen cells at the AHG phase.Reactions are occurring at AHG phase, which indicates a possible clinically significant antibody, Jka showing dosage. Refer to Case Study 1 panel below to see reactions of antibody panel.IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not doneCase study 1 conclusion:Patient's previous transfusion 6 months ago exposed him to the Jka antigen, causing the formation of this antibody, which is known for showing dosage.

Most are IgM and not clinically significant May interfere with detection of clinically significant antibodies if they react at AHG phase. Screen cells and panel cells will have positive reactions in IS phase and strength will diminish or antibody will not be detected at AHG phase. Auto control will be positive if the cold antibody is an autoantibody. Binding of antibody to antigen occurs at room or colder temperatures and may start to disassociate from the red cell membrane at warmer temperatures. Reactions will appear weaker or be negative at warmer temperatures. (Example: 4+ at IS phase and W (weak)+ at AHG phase.) PrewarmingIf a non specific cold antibody or cold agglutinin is suspected, warm the sample and testing reagents, including saline, to 37° C. Only do reaction readings at AHG; bypassing the optimum reaction temperature prevents activation and binding of the cold antibody .

Possible antibody is anti-C based on matching reaction pattern of sample at AHG. At least 3 positive reactions are present to rule in this antibody.Pink: negative reactions to use for rule-outsTurquoise: homozygous reactions used for rule-out (exceptions to homozygous rule are Rh group and Kk) Antibodies that can be ruled-out using "3 to rule out" rule: D, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, M, N, S, s, P, LubAntibodies that cannot be ruled out: Cw, Kpa, Jsa, LuaPoints to remember: The pattern of positives and negatives on an antibody panel cell indicates whether that particular antigen is present on the testing cells The phase in which the reactions are occurring will help determine if it is an IgG clinically significant antibody or IgM antibody (usually not considered clinically significant). Stronger reactions seen if antibody exhibiting dosage. Think multiple antibodies if reactions occurring at different reaction phases.

Varying reaction strengths in the same phase could indicate antibody showing dosage, multiple antibodies, or both.Jka and S are the antibodies that are present. Weaker reactions can be seen when either of the target antigens is present alone and/or in the heterozygous state on the cell.4+ reaction in panel cell 1, 4 and 9: Both Jka and S are present4+ reaction in panel cell 7 and 10: S present (homozygous)3+ reaction in panel cell 6: Jka present (homozygous)3+ reaction in panel cells 2 and 8: S present (heterozygous)2+ reaction in panel cell 5: Jka present (heterozygous)

D, E, and Lea did not react with the enzyme panel cells (in green). If they had been present, the reactions would have been enhanced. Fya, Fyb, N, S, and s did not react with the enzyme panel cells (some are shown in green).Looking at the enzyme panel results, we can see the reaction pattern of c (in yellow) and the pattern of non-reaction for Fya (in pink). Suspected multiple antibodies are c and Fya. Fya will not react on the enzyme panel since the Duffy antigens are destroyed by enzymes. Enzymes will enhance the reaction of anti-c.

The selected cells should be antigen-negative for the antibody that you think is present and antigen-positive (homozygous) for what you are trying to rule out. You are designing a panel that addresses your testing needs. Example: JkbIf you suspect that your patient has an anti-Jkb and further rule out cells are needed, then those rule out cells should be negative for Jkb. In the table below, donor cells 1,2, 4, 6, 9 and 10 may be used when creating a select panel to test the patient and help rule out the remaining possible antibodies. The homozygous rule applies when choosing which cells to use for testing (antigens highlighted in light-yellow).Example: Picking cells to rule out CUse panel cell 1 and panel cell 2 (C is in the homozygous state). Explanation: Panel cells 1 and 2 do not contain the antigen Jkb (signified by "0" on cell panel). If these cells are tested with the patient's plasma and the reaction is negative, it can be assumed that the patient does not have an antibody to C. C is now ruled out because there would be a total of 3 negative patient reactions with C positive cells (These two reactions and screen cell I from the antibody screen, shown again below). This should be done for all clinically significant antibodies that you were unable to rule out on the first panel.

Antibodies ruled out with 3 reactions: D, c, k, Kpb, Jsb, Leb, P1, and Lub (panel cells used for rule out are in green). Antibodies still needing selected cells for rule outs: C, Lea, E, M, Jka, Jkb, S, s (need 2 reactions)Fya, N, K (need 3 reactions)e, Fyb (needs 1 reaction) Jsa, Kpa, Cw, and Lua all need three reactions for rule-out but these are all low-frequency antigens. It is difficult to find panel cells with these antigens present to allow testing. They will fall in the "unable to rule out" category.Reactions are occurring in the AHG phase only and there is varying strengths of reactivity, which could indicate dosage and/or multiple antibodies.The pattern of reactivity closely matches Fya (cells 2,5,7,8,9 are positive). Of the remaining antibodies that have no rule-out reactions, anti-K is the possible second antibody (present on cell 2 and 10 and screen cell I). Explanation for the varying strengths in reactions: Panel cell 2: Fya (heterozygous) and K present so stronger reaction of 4+. Panel cell 5 and 8: Fya is heterozygous, so weaker reaction of 2+. Panel cell 7 and 9: Fya is homozygous, so stronger reaction of 3+. Panel cell 10: K is (homozygous, so stronger reaction of 3+.

Purpose: To design a set of panel cells that may help you to rule out additional antibodies and lead to the identification of the antibody that is present in the patient's plasma.Benefit of running selected cell panel: Decreases the use of reagents and specimen. How to choose selected panel cells: If you suspect that a specific antibody is present, the cells you choose for the select panel should be negative for that antigen and positive for the antigen you are trying to rule out (homozygous state).

Suspect dosage if varying strengths in reactivity are seen and reactions are in the same phase. Weaker reactions will be seen if suspected antibody is reacting with antigens in the heterozygous state. Stronger reactions are seen if the antigen is present on the testing cells in the homozygous state. This allows more corresponding antibody to bind with the antigen. Remember the antibodies known for showing dosage are: Rh, Kidd, Duffy, MNSs, and Lutheran. Dosage may be seen if cells are R2R2 (DcE/DcE). These red cells have more D antigen sites so reaction with anti-D may be stronger.Refer to Example 5 on the following page.

No reactions were seen with panel cell 1 and 2. This rules out M,N,and s.Notice that C, E, Fya, and P1 are not present on these cells so as not to interfere with ruling-in or ruling-out of the remaining antibodies.ConclusionThrough the processes of ruling-in and ruling-out and matching of reaction patterns, the antibodies that are identified are C,E, Fya, and P1.If the patient has not been transfused in the past 3 months, antigen typing for Fya may be done to further confirm the presence of Fya.Elution and autoadsorptions may also be necessary to confirm the presence of C,E, Fya and P1.

Immune antibodies occur in the serum of individuals who become sensitized to foreign antigens through pregnancy or transfusion. IgM predominates in the primary response, IgG in the secondary response. Most react at 37°C and are considered clinically significant. Examples include antibodies in the Kell, Rh, Duffy, and Kidd systems. Immune antibodies can be classified as alloantibodies or autoantibodies.Alloantibodies Produced by exposure to foreign red cell antigens which are non-self antigens but are of the same species. They react only with allogenic cells. Exposure occurs through pregnancy or transfusion. Examples include anti-K and anti-E. Autoantibodies Produced in an autoimmune process and directed against one's own red cell antigens. React with patient's own cells and all cells tested. Can possibly mask the presence of other significant antibodies. It is very important to make sure that no underlying significant antibodies are present if an autoantibody is suspected. A positive direct antiglobulin test (DAT) or auto control could indicate the presence of an autoantibody. Examples include cold auto (P or I) or warm auto (Rh specificity).

Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.Enhancement media are sometimes used to further promote agglutination and reduce incubation time. Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases incubation time. Polyethylene glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by removing water molecules from the testing sample. It is the most sensitive of the enhancement media; strengthening almost all clinically significant antibodies. However, it will also enhance some clinically insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings should only be done at the AHG phase. 22% albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination. Albumin does not contribute much to antibody uptake. Longer incubation time is needed with this media than with the previously discussed media. Detection of some IgG antibodies can be enhanced with enzyme test methods. Proteolytic enzymes (papain and ficin) denature some RBC antigens and remove negative charges from the RBC membranes. This reduces the zeta potential, bringing the cells closer together. Enzyme techniques are particularly useful in the identification of Rh antibodies and antibodies in the Kidd, Lewis, P and I systems. However, enzymes destroy some antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s antigens are variable.

Suspect an antibody to a high-incidence antigen if: Reactions with all panel and screen cells are positive (same strength and same phase) Auto control is negative Antibodies to high-incidence antigens include: anti-k, anti-Kpb, anti-Jsb, anti-Lub (highlighted in turquoise)

The most common method of ANA testing is indirect fluorescent assay (IFA) utilizing fluorescein isothiocyanate (FITC) as the marker on the secondary antibody.The fluorescent ANA test uses the indirect fluorescent antibody technique first described by Weller and Coons in 1954. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANAs are present, a stable antigen-antibody complex is formed.After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody conjugated to fluorescein. When results are positive, a stable three-part complex forms, consisting of fluorescent antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized with the aid of a fluorescent microscope. In positive samples, the cell nuclei will show a bright apple-green fluorescence with a staining pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear fluorescence. The cytoplasm may demonstrate weak staining while the non-chromosome region of mitotic cells demonstrates brighter staining.The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)

Slide-based ANA testing using a cell substrate started in the 1950s and continues to be the gold standard method. In the early days of ANA testing, rodent tissue (stomach, liver and/or kidney) was commonly used as the substrate. Rodent tissue however had several drawbacks such as small cell size, a lack of dividing cells (mitotics) and poor antigen expression that made interpretation of ANA patterns difficult. In the 1980s, cultured cell lines were examined for utility as an ANA substrate and the human epithelial- like cell line HEp-2 gained popularity. HEp-2's advantages over rodent tissue are: A large nucleus Better antigen expression Abundant mitotic cells that assist in interpretation of the ANA pattern (if grown properly).More recently a cell line called HEp-2000® has become popular for ANA detection. HEp-2000® is a HEp-2 cell line that has been transfected with the cDNA for overexpression of the SSA/Ro antigen. This results in a substrate with all of the original advantages of HEp-2 plus an added advantage of increased sensitivity for detection of antibodies directed to the SSA/Ro antigen and the ability to identify these clinically significant antibodies during the screening process.(Ref4)It has also been demonstrated that antibodies to SSA/Ro develop early in the disease process.(Ref5) Perhaps most importantly, if a woman has anti-SSA/Ro antibodies and becomes pregnant there is a risk of the antibodies crossing the placenta, resulting in the fetus developing neonatal lupus and congenital heart block in utero.The advantage of using these transfected cells is documented in the current Clinical and Laboratory Standards Institute (CLSI) guidelines for ANA testing. Here they note the "dramatically increased" sensitivity of transfected cells for the detection of SS-A/Ro and the unaltered effect of transfection on other ANA patterns.(Ref6)

This is an example of a Nucleolar ANA pattern.This pattern is characterized by staining in the nucleoli of the interphase cells (a). The nucleolar staining can display subtle variations in staining inside the nucleoli including smooth, speckled and clumpy. All are reported as ANA positive, Nucleolar. In this sample staining is present in the chromosomal area of the metaphase mitotic cells (b) along with some staining in the area outside of the chromosomal area. The staining of the mitotics can be different with different anti-nucleolar antibodies. Remember the ANA pattern is determined by staining in the interphase cells and the mitotics are used to assist in interpretation.Follow-up testing for anti-nucleolar antibodies is very limited and in most cases is not done. There are assays for identification of anti-RNP polymerase antibodies. Anti-nucleolar antibodies are primarily seen in patients with systemic sclerosis.This pattern is reported as ANA positive, Nucleolar; titering is necessary.Nucleolar:Interphase cells Staining only of the nucleoli Clumpy Smooth Speckled Metaphase mitotic cells Can demonstrate variable staining depending on the antigen involved

This is an example of a cell cycle dependent speckled ANA pattern called anti-proliferating cell nuclear antigen (PCNA).With this pattern the antigen that the antibodies are directed to is only expressed during a limited portion of the cell's growth cycle. During other parts of the growth cycle the antigen is not expressed. This creates a pattern where only 30-50% of the cells stain positive. The speckled staining within these positive cells varies between coarse speckled (a) and smooth speckled (b). Cells not expressing the antigen are negative (c).Follow-up testing to confirm antibodies to PCNA is recommended. Anti-PCNA antibodies are specific for SLE.This pattern is reported as ANA positive, Speckled, possible PCNA; titering is necessary.Proliferating Cell Nuclear Antigen (PCNA)Cell cycle dependent pattern:Antigen not expressed in all growth stagesInterphase cells 30-50% of interphase cells stain Coarse to fine speckling in the nucleus Nucleoli do not stain Metaphase mitotic cellsNo staining at this stage of the cell cycle

Antibodies to Scl-70, also known as anti-topoisomerase I antibodies, produce three ANA patterns: homogeneous, speckled and nucleolar. This is because the Scl-70 antigen is present in all three areas. In photographs it is difficult to capture the fine granular speckled staining (a), therefore, in the following photos the patterns are best described as Homogeneous (b) and Nucleolar (c).In cases of mixed homogeneous and speckled ANAs, follow-up testing for anti-dsDNA and anti-ENA antibodies is necessary.When mixed patterns are titered, the endpoint for each pattern is reported.This pattern is reported as ANA positive, Homogeneous, Nucleolar and Speckled; titering is necessary. (It should be noted that anti-Scl-70 cannot be identified simply by the ANA patterns. Follow-up ENA testing is necessary.)Scl-70:Antibodies target topoisomerase IDetection methodsANA: Three patterns present Homogeneous, Specked and Nucleolar Rarely seen with other marker antibodies Confirm by: ID, ELISA, WB, others Clinical significance Poor prognosis May precede onset of symptoms

Transplacental ITP may occur in newborn infants who are born to mothers with ITP. If the mother has had one baby born with thrombocytopenia, it is usually an indication that all subsequent infants will also be born with thrombocytopenia. A very small percentage of babies born with ITP will have severe thrombocytopenia. Neonatal alloimmune thrombocytopenia (NAIT) is caused by platelet destruction that is the result of alloantibodies stimulated by foreign antigens during pregnancy or blood transfusions. Platelet destruction by alloantibodies may occur in neonates if the mother lacks the platelet-specific antigen but the baby has inherited the antigen from its father. When maternal IgG antiplatelet antibodies cross the placenta, immune destruction of the neonate's platelets occurs. The major concern with both of these conditions is intracranial bleeding if the neonate's platelet count is less than 50 X 109/L. NAIT has a high mortality rate due to bleeding into the central nervous system. Prompt diagnosis of the condition and treatment is critical. The thrombocytopenia lasts on average 3 - 4 weeks postnatal until the maternal antibodies have cleared the newborn's system.

There are several immunochemical electrophoresis methods used to investigate protein antigens and antibodies in serum. Two methods will be discussed: Immunofixation electrophoresis (IFE) Electroimmunoassay electrophoresis

To aid in the diagnosis of disease or identification of infectious agents, clinical laboratorians use a variety of methodologies to assist them. Knowing what to look for, or the right question to ask, is vital to obtaining the correct answer. Many diseases and agents have unique causes. The cause of the condition then becomes the "target" to be identified and perhaps even quantified.For example: If Patient A is suspected of having disease X, and disease X requires treatment, it is necessary to prove that disease X exists within patient A. We must know something about what causes disease X; is disease X an antigen, a bacteria, a viral particle, a missequenced piece of DNA?Once the target of interest (in this case disease X) has been identified, the clinical laboratorian can choose the methodology most appropriate to answering the question, "Does disease X exist within Patient A?"

Afenyi-Annan, A., Kail, M., Combs, M.R., Orringer, E.P., Ashley-Kock, A., & Telen, M.J. Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease. Transfusion. 2008;48:917-924. Ataka, K. I. et. al.Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia. Blood: 2008; 11(8) 3991-3997.Ballas, S.K., Sickle Cell Anaemia: Progress in Pathogenesis and Treatment. Drugs 2002: 62(8); 1143-1172.Bianchi, N., Zuccato, C., Lampronti, I., Borgatti, N., and Gambari, R. Fetal Hemoglobin Inducers from the Natural World: a novel approach for the identification of drugs for the treatment of B-thalassemia and Sickle-cell anemia. eCAM: 2009; 6(2)141-151.Centers for Disease Control and Prevention. Sickle cell disease: Symptoms and treatments. Available at: http://www.cdc.gov/ncbddd/sicklecell/symptoms.html. Accessed January 21, 2010.Harmening, Denise M., Clinical Hematology and Fundementals of Hemostatis 4th., F.A. Davis, 2001.Inati, A., Koussa, S. Taher, A., & Perrine, S. Sickle cell disease: New insights into pathophysiology and treatment. Pediatr Ann. May 2008.Kaushansky, K., Lichtman, M.A., Beulter, E., Kipps, T.J., and Prchal, J.T. Williams Hematology 8th Ed. McGraw Hill 2010.Lotspeich-Steininger, Stiene-Martin and Koepke, Clinical Hematology Principles, Procedures, Correlations, Lippincott 1992. McKenzie, Shirlyn B., Textbook of Hematology 2nd ed., Williams and Wilkins 1996. Miale, John B, Laboratory Medicine Hematology 6th ed., Mosby 1982. Niscola, P., Sorrentino, F., Scaramucci, L., de Faritiis, P., & Cianciulli, P. Pain syndromes in Sickle Cell Disease: An update. American Academy of Pain Medicine. 2009:470-480.Rodak, Bernadette, Diagnostic Hematology, W.B.Saunders Co., 1995.Yoon, S.L. & black, S. Comprehensive, integrative management of pain for patients with Sickle-Cell Disease. Journal of Alternative and Complementary Medicine. 2006: 12; 995-1001.

Since anti-D produces the most severe HDFN and was once relatively common, let's begin by reviewing how anti-D is produced.Immunization to D may occur when Rh-negative individuals are exposed to the D antigen, but developing anti-D varies greatly from person to person. Some individuals produce anti-D after being exposed to a small volume of D-positive red cells (e.g., 0.1 mL). For others, a relatively large volume of D-positive cells is required. Yet other persons will never produce anti-D, regardless of exposure.

To understand the history of HDFN due to anti-D, it is useful to review the immune response. A primary (1°) immune response is the response that occurs following the first exposure to a foreign antigen. A secondary (2°)/anamnestic immune response occurs following subsequent exposures. The main differentiating features as related to producing anti-D during pregnancy are shown in the table and figure. 1o immune response 2o immune response 1. Following the first exposure to the D antigen, a lag phase occurs in which no anti-D is produced, but activated B cells differentiate into plasma cells. The lag phase can be as short as several days, but often is longer. 1. When exposure to D occurs in subsequent pregnancies, the lag phase is short (3–7 days) due to the presence of memory B cells that quickly differentiate into antibody-secreting plasma cells. 2. Depending on the antibody detection method, it often takes 5–15 weeks before anti-D is detectable in serologic tests. 2. An increase in anti-D is usually detectable within days. 3. The amount of anti-D produced is relatively low. 3. The amount of anti-D rises to a higher level. 4. Anti-D titers decline fairly rapidly and may become undetectable. 4. Anti-D titers tend to remain higher for longer but eventually decline. 5. The first anti-D produced is mainly IgM (although small amounts of IgG are usually also produced). 5. The main type of anti-D produced is IgG (although small amounts of IgM may be produced).

Exposure to D+ red cells: Anti-D is red cell immune. The usual route of exposure to the D antigen is during pregnancy. Fetal bleeds into the mother occur more commonly at delivery but some may occur antenatally due to small lesions in the placenta or due to placenta previa, amniocentesis, abdominal trauma, abortion, ectopic pregnancy, etc. Transfusion is a relatively rare route of exposure since Rh-negative individuals normally receive only Rh-negative donor red cells. However, Rh-negative transfusion recipients may be exposed to small volumes of D-positive red cells in Rh-positive platelet concentrates. Also, there are rare reports of fresh frozen plasma, not normally matched for Rh(D), causing anti-D production.Volume of fetal bleed: In general, the larger the fetal bleed, the more likely the mother is to produce anti-D. Approximately 1 pregnancy in 400 result in a fetomaternal hemorrhage (FMH) of 30 mL or greater. ABO incompatibility between mother and fetus: If fetal red cells are ABO incompatible with the mother, maternal anti-A or anti-B will rapidly remove fetal cells from the circulation before anti-D can be produced. This protection decreases the chance of anti-D being produced but does not eliminate it entirely.

If a pregnant woman is found to have an unexpected clinically significant antibody, routine antenatal serologic tests on the mother include Antibody identification to detect clinically significant antibodies. Antigen typing: Once the antibody is identified, the mother is tested for the corresponding antigen, which she should lack. Antibody titration: Laboratories have different protocols. Depending on the antibody titer, titration may be performed at 2 or 4 week intervals after 18 weeks gestation.Notes (titration): Maternal antibody titer is an unreliable indicator of fetal disease and is mainly done to determine if clinical fetal monitoring is warranted, e.g., Doppler ultrasonography of fetal cerebral blood flow or, more rarely, invasive monitoring such as amniocentesis. Careful quality control is needed for titrations. QC includes using red cells from donors with the same phenotype or likely genotype (e.g., R2r or R2R2) and titrating the new sample in parallel with the prior sample. A two-tube rise or more in a doubling dilution is considered a significant rise in titer. In the case of anti-D, a predetermined critical titer (often 16 or 32 for anti-D depending on the method) indicates the need for clinical fetal monitoring.

Investigative tests on the father depend on which maternal antibodies are present.1. Mother has anti-D ABO and Rh typing with anti-D, -C, -E, -c,-e to determine probable Rh genotype* to predict the chance the fetus has of being Rh positive and affected by HDFN; Test for weak D if initial Rh typing appears to be D-negative. * For D+ fathers, the probable Rh genotype can be determined using serologic tests, i.e., DCEce typing to determine if the father is probably homozygous or heterozygous for D.2. Other maternal clinically significant antibodies Phenotype father for the corresponding antigen and its antithetical antigen (e.g., K and k)

Mother's Rh Type - Weak D or Partial DRecall that some individuals have a variant RHD gene that encodes a reduced concentration of D antigen (weak D) or a D antigen with missing D epitopes (partial D). Various anti-D reagents react differently with these red cells and interpreting Rh(D) type can vary with the method used, e.g., tubes, solid phase, gel. Differentiating between weak D and partial D is important in pregnant women. Those with partial D, but not usually weak D, may make anti-D and should be considered D negative for transfusion and as RhIg candidates. Currently, serologic reagents cannot distinguish the two D variants, but RHD genotyping can.

Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking, assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k.

Policies for administering RhIg when the mother's Rh type is weak D vary among countries and within some countries. To understand the issues involved, we need to review the genetics of the Rh D antigen and the types of weak D (formerly Du). The Rh system is complex and only a basic overview will be given.In brief, Rh system inheritance is determined by two sets of genes: RHD codes for the proteins carrying D expression. In most people, the presence or absence of the RHD gene results in being Rh positive or Rh negative, respectively. RHCE codes for different combinations of the proteins carrying CcEe expression and Rh-associated glycoprotein (RhAG). RhAG is needed for the expression of Rh antigens. The RHCE locus is adjacent to RHD on chromosome #1. See a model for the Rh locus (NCBI)D variantsFor a small percentage of people, the inheritance of the RHD gene and the expression of the D antigen may be altered leading to several variants of D encoded by more than 100 RHD alleles

As noted, policies for administering RhIg to mothers with a variant of D vary among countries and within some countries. An Rh(D) red blood cell phenotype with a weak or variant expression of the D antigen occurs in 0.2% to 1% of whites and is slightly more common in African Americans. The phenotype is routinely called weak D, although several variants exist. A simple model includes these D variants: 1. Weak DMultiple weak D variants exist. Red cells have fewer D antigens/red cell (quantitative difference) and only minor variations in D antigen proteins. Some, but not all, weak D phenotypes are detected by today's Rh typing sera and may be classified as Rh positive or Rh negative by routine testing but will be positive when a weak D test (IAT with anti-D) is done. An extreme form of weak D is the Del phenotype, in which the D antigen is so weakly expressed that it may be demonstrated only by adsorption and elution of anti-D. Weak D individuals do NOT produce anti-D and can be considered to be Rh positive for transfusion and RhIg purposes.2. Partial DPartial D variants have altered Rh(D) proteins that differ sufficiently from normal D antigens (qualitative difference) to allow anti-D production. Partial D red cells may react with some but not all anti-D typing reagents. There are many categories of partial D antigens (e.g., DIIIa, DVI, DAR), each with a unique genetic basis.Some persons with partial D have weakly expressed D epitopes and are designated "partial weak D."In practice, partial D and weak partial D can be considered similarly, i.e., ideally they should be transfused with Rh negative RBC and are candidates to receive perinatal Rh immune globulin depending on the policy in their location.

The following basic associations can be made between certain surface markers and cell types. Please note that only the most basic associated cell types are addressed as this is an introductory flow cytometry course. A PDF file of this table is also available on this page and can be printed to use as a reference for case studies presented later in this course. Surface Marker Associated Cell Type CD2 Pan (across all) T cells; natural killer cells CD3 Pan T cells CD4 T-helper cells CD5 Pan T cells and B cell abnormalities (e.g., B-chronic lymphocytic leukemia (B CLL) and mantle cells) CD7 Pan T cells (earliest marker) CD8 T-suppressor cells (cytotoxic T cells) CD10 also known as common acute lymphocytic leukemia antigen (CALLA) Early T and B cells, mature follicular cells CD19 Pan B cells (earliest marker) HLA-DR B cells (also present on activated T cells) CD20 B cells CD23 B cells (present in B CLL and not present in mantle cells) Kappa or Lambda light chain immunoglobulin (not antigen) B cells --A mature B cell should express either one or the other and, across a B-cell population, there should be a good mix of both Kappa and Lambda. In a clonal (cancerous) population, one cell line will proliferate and that line will exhibit one of these light chains. This will indicate clonality. CD45 also known as the leukocyte common antigen Pan white blood cells (May vary in staining intensity between mature and immature white blood cells).

Commercially available antibodies that are used in flow cytometry are grouped according to the antigens that they recognize into clusters of differentiation (CD).Cluster of differentiation 3, (CD3), binds to all mature T cells (helper and suppressor) while CD4 binds to helper T cells only.When stained with the fluorochromes, fluorescein isothiocyanate (FITC) and phycoerythrin (PE): Suppressor T cells (CD3-bound) will fluoresce orange, but not green Helper T cells (CD3- and CD4-bound) will fluoresce both orange and green B cells, monocytes, and neutrophils should not express either antigen; thus they should not emit a fluorescent signal when they intersect the laser.

In this example, the T-cell that is shown is a T-suppressor cell. A T-suppressor cell has CD8 antigen, but no CD4 antigen. Consider the possibility that there were three MoAbs used in this tube. The orange labeled MoAbs are specific for the CD3 antigen on T cells, the green labeled MoAbs are specific for the CD4 antigen on T cells and the blue labeled MoAbs are specific for the CD8 antigen on T cells.A true T-suppressor cell would emit both orange and blue colors after it passes through the laser as shown in the image to the right. The T-helper cells, as shown on the previous page, would fluoresce orange and green. MoAbs in any combination can be used in the same staining tube, as long as the fluorochromes can be separated into their respective fluorescent channels.

Commercially prepared controls come in either assayed or unassayed forms. Assayed controls are tested by multiple methods before sale, and come with measuring system-specific values that are meant to be used as target values for the laboratory using the controls. Assayed controls:Are more expensive than unassayed controlsAre used to evaluate accuracy and precisionMay only be suitable for specific method systems Unassayed controls have no assigned analyte values provided by the manufacturer. The control values for these materials must be determined by the individual laboratory. Unassayed controls:Are less expensive than assayed controlsAre used to evaluate accuracy and precisionAre not linked to specific method systems Note: although commercially available control materials are screened for hepatitis antigens and HIV antibodies, control materials should still be handled with precautions, since they contain biological materials and could contain infectious agents.

In 1900, a German scientist, Karl Landsteiner, discovered that blood groups differ from one individual to another. He took blood samples from five associates and himself, allowed them to clot, and then separated the serum from the cells. Landsteiner found that when he mixed the serum and red cells from different individuals, some samples clumped and some didn’t. Our present day classification of the ABO system is based on Landsteiner’s realization that agglutination occurred because of highly reactive antigens present on the red blood cell which corresponded to antibodies present in the serum. Landsteiner isolated and named the red cell antigens “A” and “B” and the corresponding antibodies “Anti-A” and “Anti-B.” If the red cells contained neither antigen, he called these cells “O,” representing zero antigens present. The fourth type of red cells, “AB” was discovered in 1902 by Von Decastello and Sturli, associates of Landsteiner. “AB” cells contained both A and B antigens on their surface.

Antigen on Red Cells Antibodies in Serum ABO Blood Group A Anti-B A B Anti-A B Neither A nor B Anti-A, Anti-B, Anti-A,B O A and B Neither Anti-A nor Anti-B AB

Antibodies occur predictably in the sera of all normal adults in association with the ABO antigens. Demonstration of these antibodies is necessary for definitive classification of an individual's ABO cell type. The individual's serum is therefore tested against reagent red cells containing known antigens. Patient ABO Blood GroupPatient Serum Tested with Known Reagent CellsA CellsB CellsA0 3-4+ B3-4+ 0 O3-4+ 3-4+ AB0 0 + = agglutination (graded 1+ to 4+)0 = no agglutination or hemolysis

In addition to red cells, ABO antigenic determinants (epitopes) are found in many tissues, body fluids, and other cells including endothelial cells and platelets. Because ABO antigens are so widely expressed, ABO antigens are also a major consideration in solid organ and bone marrow transplants.

Another sugar, fucose, must be attached to the galactose in a specific configuration for further antigen development to take place. This "galactose-plus-fucose" configuration has antigenic activity called "H".

Three separate loci (ABO, Hh, and Se) contain the genes that control the location and occurrence of the A and B antigens. Hh and Se genes are closely linked on chromosome 19. The precursor substance is acted upon by the H gene and is converted to H substance. The product of the H gene is an enzyme fucosyltransferase, responsible for attaching fucose to the terminal galactose of the precursor substance on the RBC membrane and thus forming H substance. There are only two recognized alleles at this locus: the active form, H, and an amorph, h. The H gene is a high-incidence gene. People who inherit hh are extremely rare. Since the h gene is amorphic, it does not act on the precursor substance.

As mentioned previously, the A and B genes cannot act directly on the precursor substance. Thus, since individuals with the Bombay phenotype have only the precursor substance and no H antigen, they cannot have A or B antigens, even if they have the A and/or B gene.

The presence of A and/or B antigen on the red cells can be recognized by serological tests with the appropriate antisera so that the presence of the gene that controls its production can be deduced in the absence of both A and B genes (when no A or B antigen is present on the red cells).

In most other blood group systems, antibody may be formed after an individual has been immunized by an antigen that is missing from his or her red cells; perhaps as the result of pregnancy or transfusion. In the ABO system, when the antigen is missing from the cells, the corresponding antibody will predictably be found in the serum and must be found before determining the ABO type. There are few exceptions to this rule and any exception must be explained before the true ABO blood type can be determined.

The predominant immunoglobulin class for the B antibodies produced by individuals with group A phenotype and the A antibodies produced by individuals with group B phenotype is IgM. Small quantities of IgG and IgA may also be present.The ABO antibodies found in the serum of group O individuals include anti-A and anti-B. An antibody designated anti-A,B is also present. Anti-A,B in group O individuals tends to be predominantly IgG, although IgM and IgA components are also present.Infants of group O mothers are at higher risk for hemolytic disease of the fetus and newborn (HDFN) than those born to mothers with group A or B because IgG immunoglobulins readily cross the placenta. IgM molecules do not cross the placenta because of their larger size. However, the HDFN that results is usually mild and often subclinical. Infants generally survive with little or no intervention.It is important to note that immune antibodies are usually IgG. Both naturally occurring and immune ABO antibodies are critically important in transfusion since both sensitize, and usually hemolyze, red cells with the corresponding antigen.

The strength of the A antigen can vary considerably, and although most A cells react strongly with anti-A and anti-A1B, some cells have been found that are very weakly reactive. The blood group has been divided into subgroups and is classified not only by the strength of the A antigen but also by certain other serologic characteristics.

A subgroup antigens are inherited, as are other ABO antigens with A1 being dominant over A2. Individuals who are phenotypically A1 may be genotypically A1O, A1A1, or A1A2. A phenotypically A2 individual may be genotypically A2A3. These alleles are passed to offspring in the same manner as other ABO antigens. Weak variant forms of the B antigen (B3, Bx, and Bel) also exist, but are very rare.

Patient Red Cells Tested With Known AntiseraABO Antigens Present on Red CellAnti-AAnti-BAnti-A,B3-4+03-4+A03-4+3-4+B000Neither A nor B3-4+3-4+3-4+A and B+ = agglutination (graded 1+ to 4+) 0 = no agglutination or hemolysis

Specialists in microbiology perform testing to diagnose and stop the spread of infectious organisms, including bacteria, viruses, and parasites. Specialists should be able to isolate and identify a wide variety of these organisms. Testing procedures include direction examination and antigen detection methods. Specialists in serology and immunology measure antibodies to infectious organisms. Specialists should be familiar with all serology techniques (except those specific to immunohematology). This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in hematology must be able to identify and evaluate cells in blood and bone marrow and identify disorders of these cell. Specialists should be familiar with routine and special tests to determine the number, morphology, and function of cells in body fluid.

Once relegated to the domain of research laboratories, molecular methods for the diagnosis of infectious disease had little, if any place, in a clinical diagnostic laboratory prior to 1985. Procedurally, molecular methods were very complex and required specialized instrumentation and dedicated laboratory space. They were also susceptible, initially, to the influence of variation of technique. Although they represented valuable research tools, and were helpful as esoteric testing for unique clinical situations, their performance characteristics simply did not fit well into most clinical laboratories.Certain pathogens were logical targets for development. Organisms that were of concern for significant patient populations, were difficult to sustain in transport, and/or were difficult to cultivate and detect by traditional methods represented some of the first targets of commercially offered molecular based assays.Sexually transmitted diseases, affecting significant numbers of people, with key pathogens affected by lability in transport or poor sensitivity with traditional cultivation or antigen detection methods, were among the first targets for development.

There are three key benefits that molecular methods can offer, in contrast to traditional culture methods:Improved sensitivity of detectionImproved specificity of identificationReduced turnaround timeImproved sensitivity of detectionSuccessful cultivation or detection of an organism depends on many factors, including the:Ability of the organism to survive transport Fastidious nature of the organism/its ability to grow in available culture media/systems Number of organisms present in a specimen Ability of a staining/culture system to visualize/recover low numbers of organisms Sensitivity of non-culture (antigen detection) methodsOrganisms that have been shown to be very labile are difficult to cultivate even when they are present in significant numbers. The immediacy of transport, plating, and incubation are critical factors that frequently cannot be controlled in a positive direction.Even under the ideal transport conditions, some organisms may require culture media and conditions that are not routinely available in every laboratory, which reduces the likelihood of successful cultivation.

Rapid detection of influenza was a key focus for method development for many years. Traditional viral culture methods require special transport mediums, appropriate cell culture lines, and staff well versed in the manipulation of these cultures. Although the introduction of shell vial cultures and monoclonal fluorescent staining provided some improvement, the availability of results did not always meet the clinical need.Direct fluorescent antibody (DFA) staining of specimen smears can provide more immediately available results; however the availability of trained staff to interpret these smears is an obstacle for many laboratories. Antigen detection kits employing enzyme immunoassay (EIA) or immunochromatographic membrane principles did provide easily performed alternatives that fit well in most laboratory settings and provided more immediate results. Despite the fact that published studies demonstrated less than desirable sensitivity, these assays had found a niche and remained in place, even as molecular methods began to target these viruses.

Judicious use of antimicrobials, especially in outpatient settings, can help control the emergence of CA-MRSA and limit the acquisition of additional resistance by existing strains. Regardless of origin, minimizing antibiotic selective pressure that favors the development of resistant strains is essential to controlling the emergence of these strains in both hospital and community settings.The development of vaccines to prevent S. aureus infection in both healthcare and community settings holds great promise. Recently (2007) a vaccine based on an immunotherapeutic licensed to Merck has shown promising results in a clinical trial against hospital acquired S. aureus infections, while Nabi Biopharmaceuticals, Ft. Lauderdale, FL, are developing the "next generation" of StaphVax, which will contain antigen against S. aureus detoxified Panton Valentine Leucocidin and the cytolytic alpha-toxin.

Some literature in recent years has suggested that assays for GDH (glutamate dehydrogenase - an enzyme produced by C. difficile) could provide a more sensitive means of screening for C. difficile.Published studies have indicated that toxin immunoassays, by themselves, may not provide adequate sensitivity of detection. Several investigators have examined the utilization of a two step algorithm. This first step is an enzmye immunoassay for the GDH antigen.A negative result for GDH has been associated with a high value for prediction of a true negative result; however, a positive result is not necessarily associated with a toxin producing strain. A second assay on positive samples for detection of toxin production is required in these algorithms.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

Humoral immunity involves the production of antibodies (immunoglobulins), and is brought about by lymphocytes which we call B-cells. B-cells are bone-marrow derived lymphocytes. After B-cells are stimulated by an antigen, they proliferate and transform into plasma cells which produce specific antibodies.

Acute hepatitis panel: Hepatitis A antibody (IgM) Hepatitis B core antibody, IgM (HBcAb) Hepatitis B surface antigen (HBsAg) Hepatitis C antibody

Cell TypeImageCellular DescriptionAssociated Diseases and ConditionsRouleauxRed blood cells (RBCs) appear as "stacked coins."Cells overlap each otherStacked-coin morphology is noted throughout the peripheral blood smear Conditions associated with increased concentrations of globulins and/or fibrinogenHyperparaproteinemiasWaldenstrom's ,macroglobulinemiaMultiple myelomaChronic inflammatory disordersAgglutinationClusters of RBCs due to antigen/antibody reactions in vivioCannot distinguish the outlines of individual RBCsCold agglutinins (most often IgM antibodies)Paroxysmal cold hemoglobinuria

As applied to pregnancy, RhIg's purpose is to prevent immunization to the D antigen in the perinatal period and thus prevent HDFN due to anti-D. If the mother has already produced anti-D, RhIg is of no use.Accordingly, RhIg is routinely administered to Rh negative women* not previously sensitized to the D antigen under the following circumstances:1, Antenatal. Antepartum prophylaxis of 300 µg (1500 IU) at about 28 weeks gestation in the USA and Canada, which could be weeks later, depending on how physician appointments are scheduled. To illustrate variation in antenatal international practice, in the UK smaller doses of RhIg (e.g., 500 IU) may be given at 28 weeks and 34 weeks, although many UK facilities issue a 1500 IU dose at 28–30 weeks. With antenatal administration, the Rh of the fetus is usually unknown. Some transfusion services recommend a further antenatal dose if the infant is undelivered after 40 weeks.2. Postnatal. Prophylaxis of 300 µg (1500 IU) at delivery of an Rh positive or weak D infant, preferably within 72 hours of delivery but can be given up to 28 days later if administration is delayed. If RhIg administration is delayed beyond 72 hours, laboratory policies differ as to when it would no longer be administered.* Policies related to women who are weak D (formerly Du) are discussed later.Note: Because RhIg contains IgG anti-D, when given antenatally, it can cross the placenta and sensitize fetal D-positive red cells. Occasionally the fetus may be born with a weakly positive DAT, but significant hemolysis does not occur.

When first developed in the 1960s, RhIg was believed to work by a simple clearance mechanism, i.e., by coating D-positive fetal red cells with IgG anti-D, which resulted in clearance of the sensitized cells in the spleen by macrophages with receptors for IgG.Current research shows that a simple model of antigen clearance by antibody-sensitized D-positive RBC is not the mechanism of anti-D suppression by RhIg. More is involved at the molecular level, possibly involving a down-regulation of antigen-specific B cells and related mechanisms (see Further Reading).

Clinically relevant information on D phenotypes can be summarized as follows: D phenotype D antigen expression Rh(D) typing Produce anti-D RBC to transfuse RhIg recommended** D+ normal direct agglutination no D+ or D– no Weak D normal but weak IAT no D+ or D– no Partial D altered direct agglutination* & IAT yes D– yes Partial weak D altered & variable direct agglutination* & IAT yes D– yes D– none IAT yes D– yes * Depending on anti-D reagent used ** USA, UK and parts of Canada

Some TS laboratories try to determine if anti-D is passive or immune by performing titrations to determine the titer of the anti-D. Such a protocol usually assumes that an anti-D titer greater than 4 likely represents active immunization. Unfortunately, a titer of 4 or 8 could be active or passive, although a high titer (e.g., 64 or more) almost certainly means the anti-D is immune.Titration results can be affected by several variables: Red cell phenotype; Donor antigen variability (even if the same phenotype); Method used; Operator variability.Because lower titers could be due to both passive and immune anti-D, in the absence of test results that suggest immune anti-D, routine antibody titration is not a good use of time compared to assuming that anti-D is passive. Most transfusion medicine best practice guidelines do NOT recommend routine titration for women known to be injected with RhIg and exhibiting a 2+ or less reaction with D+ red cells, i.e., test results consistent with RhIg-derived passive anti-D.

Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k.

Once an antibody has been identified and other clinically significant antibodies have been excluded, the case must be looked at as a whole to confirm the logical consistency of all results and data.This process includes assessing any inconsistencies.For example:1. Is the patient negative for the corresponding antigen? Yes: The patient is Jk(a-).2. Is the antibody specificity consistent with the typical phase(s) of reactivity for the antibody? Yes: Kidd antibodies are IgG and react in the antiglobulin phase.

To improve the quality of conclusions when identifying antibodies, a checklist is a simple quality control tool to increase transfusion safety. If a specific antibody pattern cannot be identified with acceptable confidence, or if significant serologic or non-serologic data are inconsistent and cannot be rationalized, further testing will be required.Before concluding that the investigation is complete, unless not applicable, mentally reply to each question in the checklist. If any answer is no, has it been resolved? Antibody Identification Checklist Yes/No/NA 1. For a single antibody, does the reaction pattern fit only one antibody specificity? 2. Is antibody specificity consistent with the results of the initial antibody screen? 3. Are reaction phases consistent with antibody specificity? 4. If multiple antibodies are present, can all reactions be explained by the antibody combination? 5. If the autocontrol is negative, are patient red cells negative for the corresponding antigen(s)? 6. Have additional possible antibodies been excluded by selected red cells? 7. Can all variable reaction strengths be explained? 8. If tested, are antigen-negative donor cells compatible by antiglobulin crossmatch? 9. If there are data that do not fit antibody specificity or if there are results that are improbable, are they explainable? 10. Have all results and conclusions been systematically evaluated for consistency?

We often "get away" with transfusing unmatched RBC because the incidence of unexpected antibodies in patients experiencing medical emergencies is thought to be relatively low ( ~3-5% is sometimes cited, but with little solid evidence).Antibody incidence may vary according to several factors: Genetic disposition Patient's underlying disease Number of prior transfusions Gender (females may get exposed to foreign antigens via fetomaternal bleeds as well as transfusion) Concordance of antigen phenotypes of patients vs blood donors in a given locale.In general, antibody incidence increases with the number of transfusions that are given, although most antibody producers will respond within the first 3 - 4 transfusions. Antibody incidence in transfusion-dependent patients, such as those with sickle cell anemia or thalassemia, is very high. Regardless of likelihood, transfusing uncrossmatched blood to a patient with unexpected antibodies can result in a serious hemolytic transfusion reaction.

LiteratureDutton RP, Shih D, Edelman BB, Hess J, Scalea TM. [abstract]. Available at: Safety of uncrossmatched type-O red cells for resuscitation from hemorrhagic shock.J Trauma. 2005 Dec;59(6):1445-9. Accessed November 5, 2012.Johnson ST, Rudmann SV,Wilson, SM. Serologic problem solving strategies:a systematic approach. Bethesda, MD: AABB, 1996.Online resourcesThe following are online examples of good practice. The information should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.Transfusion reactions: Transfusion complications (Canadian Blood Services)Education website for CBS's hospital customersREACT (Sunnybrook HSC, Toronto, ON, Canada) Pocket reference card for nurseson signs and symptoms of transfusion reactionsQuick cals (online calculator of p values for Fisher's exact test) Use a one-tailed test (since we would expect an antibody to react with red cells that are positive for the corresponding antigen)

Delayed HTR result from a secondary (anamnestic) immune response causing a weak, undetectable antibody to become stronger.Upon re-stimulation by donor RBC positive for the antigen corresponding to the patient's antibody:* Patient's memory B cells differentiate into antibody-producing plasma cells.* As new IgG antibody is produced, it sensitizes antigen-positive transfused donor red blood cells.* The IgG-sensitized donor red blood cells are then removed by extravascular hemolysis (EVH) mainly in the spleen.

A standard follow-up to antibody identification is to antigen phenotype: Patient's red cells (expecting them to lack the corresponding antigen) Donor red cells (in this case, those transfused before an antibody was identified, or, more typically, to find suitable antigen-negative donors to crossmatch prior to transfusion).If you had wanted to type the patient for any antigens at this point in the investigation (2-weeks post-transfusion), which specimen would you have used? Think about any antigen typing problems and how to overcome them before proceeding to the next page.

The patient's pretransfusion red cells and all donor red cells involved in the case (two group O Rh-negative RBC and four group O Rh-positive red cells initially crossmatched) were phenotyped for Jka.As expected, the patient typed as Jk(a-). The six donor RBC that were incompatible in the initial crossmatch were Jk(a+).The frequency of the Jka gene in Caucasians is ~77%, with most Caucasian red cells (50%) typing as Jk(a+b+).

A variety of tests are available for the detection of influenza A viruses, including the 2009 H1N1 strain. These tests include: rapid antigen tests, direct fluorescent antibody tests to detect the presence of virus in patient specimens, shell vial cell cultures, classical tube cell cultures, and reverse transcriptase PCR (RT-PCR), which detects influenza-specific viral genes. These tests differ in sensitivity, specificity, availability, and the ability to distinguish between different influenza strains and subtypes, such as influenza A 2009 H1N1.The rapid tests, such as the direct rapid antigen tests or immunofluorescence assays, have lower sensitivity and specificity compared to cell culture and the RT-PCR based tests. Rapid tests vary in their ability to detect the 2009 H1N1 virus. The range of sensitivity is 10% to 70% and none of the rapid tests that are currently available are specific for H1N1. However, results of rapid tests are available within 30 minutes to one hour so that a positive test will provide further information toward a diagnosis when it is coupled with a patient's symptoms. A few FDA-cleared RT-PCR kits are available for the detection of influenza A viruses. For the subtyping of influenza A viruses, such as Influenza A seasonal H3N2, and 2009 H1N1, the FDA has given the status of "Emergency Use Authorization" (EUA) to a few of the RT-PCR kits; currently available kits under this emergency status category include those made by the CDC, ELITech, Prodesse, Focus Diagnostics, and Roche. (http://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm)State Departments of Health have been provided with RT-PCR kits from the CDC for the subtyping of influenza A viruses. This testing has also been FDA-reviewed and given the status of EUA. State and local health department guidelines determine which specimens should be submitted to public health laboratories for RT-PCR testing. In addition, several commercial reference laboratories, academic labs, and hospital labs have been able to perform influenza A subtyping for 2009 H1N1 under the same EUA status. Any laboratory that performs an EUA method would be required to perform an internal validation process.

Adverse complications of transfusions can be classified into several categories: Immune-mediated transfusion reactions are those that trigger a response from the patient's immune system. Many transfusion reactions are mediated by the recipient's immune system. These reactions occur as a result of antigen-antibody interactions. Antibodies involved include those with specificity towards antigens on red cells, white cells, or platelets. In general, the immune responses occur in three stages: the immune system detects foreign material (antigen) the immune system processes the antigen the immune system mounts a response to remove the antigen from the body Non-immune mediated hemolytic transfusion reactions are caused by the physical or chemical destruction of transfused RBCs, bacterial contamination, circulatory overload, or citrate toxicity. Acute reactions are those that occur during or within 24 hours after the transfusion. There is usually a rapid onset of symptoms and these reactions may be fatal. Delayed reactions occur weeks or months after the transfusion of blood or blood components.

The AABB published an interim standard in 2005 that states, "Donors implicated in TRALI or associated with multiple events of TRALI shall be evaluated regarding their continued eligibility to donate." A donor is associated with TRALI when one of his/her donor units is transfused 6 hours before the clinical presentation of TRALI in a patient. A donor is implicated in TRALI if he/she is found to have an antibody to an HLA class I or II antigen and the antibody is specific for an antigen on the recipient's leukocytes or a positive crossmatch is obtained.*It is suggested that donors at greatest risk of developing HLA antibodies be tested, such as multiparous women. It has also been suggested that donors that present with demonstrable antibodies and have been implicated in TRALI be permanently deferred from donating. Studies have shown that donors implicated in TRALI reactions may present a future danger to transfusion recipients. Although, there are some instances where donors with HLA antibodies have not caused TRALI reactions. Another option would be to wash all red cell products from these donors in special circumstances such as rare donors. Reference: Association bulletin #05-09. AABB; August 2005. Available at: http://www.aabb.org/resources/publications/bulletins/Pages/ab05-09.aspx. Accessed November 12, 2010.

Delayed hemolytic transfusion reactions (DHTR) are reactions that occurs 3 to 10 days after the transfusion. Usually, the blood appears serologically compatible at initial testing. Delayed reactions are common in patients who have been immunized to a foreign antigen from a previous transfusion or pregnancy, but the antibody titers decrease over time and the antibody is not detectable during pre-transfusion testing. The transfusion leads to a secondary (anamnestic) response, causing increased antibody production that sensitizes antigen-positive donor red cells. Hemolysis is extravascular. Sensitized cells are removed from circulation by the reticuloendothelial system, also called the monocyte-macrophage system. Because there is a delay in the presentation of symptoms, DHTR is not usually considered as a cause of the clinical presentation. The transfusion service usually initiates investigation of a DHTR because of serologic findings in a post-transfusion specimen. DHTRs occur more frequently than acute hemolytic reactions. Approximately 1:2500 transfusions result in a DHTR.

Post-transfusion purpura (PTP) is a very rare complication of blood transfusion. It has been most commonly associated with the transfusion of red blood cells (RBCs) and whole blood, but has also been seen in platelet and plasma transfusions. It is characterized by a rapid onset of thrombocytopenia, or decreased platelet count, which results from the product of a platelet alloantibody. Platelet counts are usually less than 10,000/uL. Reactions occur around 7 to 14 days post-transfusion. Patients present with purpura, bleeding from the mucous membranes, gastrointesinal ,and/or urinary tract bleeding. Melena and vaginal bleeding have also been reported. The thrombocytopenia is usually self-limiting. Platelet counts and coagulation studies aid in the diagnosis. Patients can also be tested for platelet specific antibodies, human leukocyte antigen (HLA) antibodies and lymphocytotoxic antibodies. The differential diagnosis includes other causes of thrombocytopenia.

A 14-year-old boy came to the physician's office with a sore throat that progressively worsened over a three-day period. His posterior pharynx was swollen, shiny and erythematous. The boy complained of pain on swallowing. His temperature was 98.5°F. A rapid direct streptococcal antigen test was positive. However, his symptoms did not subside over the next two days while on antibiotic therapy. Anorexia and nausea were persistent and compounded by a frontal headache. Cervical lymph nodes became noticeably enlarged. A complete blood count (CBC) was ordered. The results of the CBC were: WBC 11.9 x 109/L ( Reference interval= 3.8 - 9.8 x 109/L) with: 17% segmented neutrophils 5% band neutrophils 72% lymphocytes 6% monocytes All red cell findings were normal. The automated differential flagged for atypical cells, presumptively atypical lymphocytes. A peripheral blood smear was prepared. The image on the right is a representative field from the Wright-Giemsa stained smear (1000X magnification).A rapid qualitative test for infectious mononucleosis was positive. This is a case of group-A streptococcal infection superimposed on infectious mononucleosis. Symptoms subsided in three weeks following completion of the antibiotic therapy.