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Antibody Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Antibody and links to relevant pages within the course.

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Antibody Detection and Identification (retired 12/6/2013)
Case Study: Immune Alloantibody

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.

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Initial Steps for Identifying an Antibody

The antibody screen can provide sufficient data to make initial hypotheses regarding the likely antibody specificities and may be useful to presumptively rule-out some antibody specificities. When analyzing the antibody screen data, the strength/characteristics of reactions (for example, mixed-field or weak versus strong), the phase of testing (for example, room temperature versus AHG), and the pattern of reactivity (which cells react and which do not) are all important factors that will provide clues about the possible identification of the antibody(ies). Many antibodies exhibit dosage, that is, they react more strongly with homozygous cells than with heterozygous cells. If a tube method is used, reactions are usually read at immediate spin and AHG phase. If a gel method is used, reaction readings are done only at AHG phase. Reactions occurring only at immediate spin phase could indicate a possible IgM antibody, cold agglutinin, or rouleaux. Reactions occurring in the AHG phase could indicate a possible IgG antibody. Reactions occurring in both reaction phases could indicate a combination of both IgM and IgG antibodies or a strong IgM antibody that carries through to AHG phase. The presence of multiple antibodies should be considered if reactions vary in strength or there are two separate reaction patterns in the IS and AHG phases.

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Initial Observations of Antibody Panel

Look at the phase in which reactions are occurring. Reactions at immediate spin (IS) usually are not clinically significant. Reactions at AHG are clinically significant. Check for a match in the reactivity pattern by comparing sample reactions and individual antibody reactions Varying strengths of reactions could indicate dosage. Dosage means that there are two "doses" of the same antigen present on the red cells . Antibodies that exhibit dosage react more strongly with homozygous cells (e.g., Jkb Jkb ) than with heterozygous cells (e.g., Jka Jkb) .

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Cold antibodies

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 .

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Effect of Enzymes and Dithiothreitol (DTT)

Antibody Effect of Enzymes Effect of DTT Anti- D Ficin and Papain Enhanced DTT Resistant Anti-C Ficin and Papain Enhanced DTT Resistant Anti-c Ficin and Papain Enhanced DTT Resistant Anti-E Ficin and Papain Enhanced DTT Resistant Acid Resistant Anti-e Ficin and Papain Enhanced DTT Resistant Anti-Cw Ficin and Papain Enhanced DTT Resistant Anti-K Ficin and Papain Resistant DTT Sensitive Anti-k Ficin and Papain Resistant DTT Sensitive Anti-Kpa Ficin and Papain Resistant DTT Sensitive Anti-Jsa Ficin and Papain Resistant DTT Sensitive Anti-Fya Ficin and Papain Sensitive DTT Resistant Anti-Fyb Ficin and Papain Sensitive DTT Resistant Anti-Jka Ficin and Papain Enhanced DTT Resistant Anti-Jkb Ficin and Papain Enhanced DTT Resistant Anti-Lea Ficin and Papain Enhanced DTT Resistant Anti-Leb Ficin and Papain Enhanced DTT Resistant Anti-P1 Ficin and Papain Enhanced DTT Resistant Anti-M Ficin and Papain Sensitive DTT Resistant Anti-N Ficin and Papain Sensitive DTT Resistant Anti-S Usually Ficin and Papain Sensitive; Some Variable DTT Resistant Anti-s Usually Ficin and Papain Sensitive; Some Variable DTT Resistant

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Is It a Cold or a Warm Autoantibody?

Cold antibody Immediate spin screen and panel cell reactions will be positive (W+ to 4+). The auto control may also be positive. AHG reactions may be weakly positive if the cold antibody is bound strongly to the red cells. Prewarming should prevent binding from occurring. So, prewarm panels and tests should have negative reactions.Warm antibody Immediate spin screen, panel cell and auto control usually not positive. AHG reactions will be positive including auto control (W+ to 4+). Prewarming of sample and reagents will not change positive reactions since they react best at 37°C and AHG phase. So, reactions will still be positive. Elution and autoadsorption techniques may be used to help further identify the antibody or to help identify other clinically significant antibodies that may be present.AutoadsorptionAutoadsorption is a technique that involves adsorbing unbound autoantibody from the patient's serum using the patient's own red cells. Once the autoantibody is removed, then testing can be performed to determine if any clinically significant antibodies are present.

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Case Study Two

1. Based on these reactions, which antibody or antibodies could be present?2. Based on these reactions, which antibodies can be ruled out?3. What further testing, if any, should be done to assist in the antibody identification? NOTE: A PDF of this page is available for printing. This can be used as a worksheet if preferred.

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Case Study Two- Explanation

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.

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Example of Dosage and/or Multiple Antibodies Influencing the Strength of Reactions

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)

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Multiple Antibodies: Example

In this example the patient's plasma tests positive with both screening cells at a strength of 4+. In the panel below, reaction patterns show varying strengths, 2+ to 4+ (highlighted in green).4+ could indicate one strong antibody or a combination of several antibodies that increases the strength of the reaction.3+ could indicate the presence of just one strong antibody.2+ could indicate a weaker reaction of an antibody that commonly exhibits dosage if the panel cell is in the heterozygous state.Since Cw, Kpa, Jsa, Lua are not present on the testing cells, they are probably not causing these reactions. Perform rule outs using panel cells 5 and 7 (sample had no reaction in any phase with these panel cells)Antibodies that can probably be ruled out at this point because the corresponding antigens are present on cell 5 and/or 7: C, c, e, k, Kpb, Jsb, Fya, Jkb, Lea, M, N, s, P1, LubAntibodies that could not be ruled out with this panel: D,E, K, Fyb, Jka, Leb, SPredominant pattern of 4+ in panel cells 1,2,4,10 matches anti-D Varying strengths in reactions indicates a possible second antibody so selected cells should be picked to aid in identificationFind panel cells that do not contain D (antibody you suspect) and are homozygous positive for the antibodies you are trying to rule out.

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Multiple Antibodies Example, continued: Selected Cell Rule-Out Panel

Rule-outs are highlighted in blue.Second antibody identified based on panel cells 2 and 5 : S Refer to the following page for an explanation on varying strengths of S.

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Cold Autoantibody Example

Reactions are occurring strongly in IS phase but are weaker in AHG phase which could be due to some disassociation of the cold antibody occurring at the warmer testing phase.Prewarming of all reagents and sample will prevent binding of cold antibody. If the W+ reactions at AHG are due to residual cold antibody, the reactions should be negative with a prewarmed panel. No IS phase reading is performed. Prewarmed Panel

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Example- Choosing Selected Cells

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.

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Case Study Three - Selected Cells

List panel cells to test for ruling-in or ruling-out remaining antibodies in Case Study Three. These would be your selected cells. For rule-out, selected cells should be negative for the antigens that correspond to the antibodies you have possibly identified. In this case, the selected cells for rule-out should be antigen-negative for K and Fya. If you are trying to rule in a possible antibody like K, then the panel cell should be positive for that corresponding antigen so that reactions will occur if the antibody is present.Panel cells 1 and 7 could be used for rule-in of K.Panel cells 2, 4, 5, 6, and 9 can be used for rule-outsPanel cell 2: to rule out C, e, Fyb, Jka, N, s Panel cell 4: to rule out Jka, Lea, N, SPanel cell 5: to rule out C, e, Jkb,MPanel cell 6: to rule out E, Jkb, Lea, N, and sPanel cell 9: to rule out M, S

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Case Study Three

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Case Study Three Rule-Outs Key

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+.

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Ruling Out Procedures, continued: Selecting Additional Rule-Out Cells

Once an antibody hypothesis is generated, most laboratories will select additional cells to rule-out any other commonly encountered antibodies that could not be ruled-out with the initial antibody screen and panel. Cells should be selected that are negative for the antigen(s) that correspond to the hypothesized antibody and positive for the antigen (s) to commonly encountered antibodies that have not been ruled out. If not ruled-out most laboratories will select cells for at least the following: anti-D, anti-C, anti-c, anti-E, anti-e, anti-K, anti-k, anti-Fya, anti-Fyb, Anti-Jka, anti-Jkb, anti-Lea, anti-Leb, anti-P1, anti-M, anti-N, anti-S, and anti-s. Antibodies to antigens of very low incidence (for example, anti-Jsa) are generally not eliminated in initial testing, but in most settings it is not feasible to try and find rule-out cells. In these cases, it is important for the technologist to understand that these antibodies HAVE NOT been ruled-out due to limitations in the test system.

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Use of Heterozygous Cells for Rule-Out

Sometimes with Rh or K antibodies present, it may be difficult to find enough homozygous cells to use for rule out. In these cases, heterozygous cells can be used for rule out as long as you have at least one homozygous rule out reaction for that antibody. Only do this if you have checked all other available panels and your screen cell anagram reactions for possible homozygous cell reactions to use for rule out.If potential clinically significant antibodies cannot be ruled out completely with the first panel tested, then cells from other panels will need to be selected for testing.

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Selected Cell Panels

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).

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Picking Selected Panel Cells Conservatively

Choose cells that can help rule out more than one antibody at a time in order to help decrease supply usage and tech prep time. Example: Ruling out C, Fyb, and M if you have a suspected JkacCFyaFybJkaJkbMNPanel cell 90+0+0++0Panel cell 100+++0+0+Panel cell 11++0+0++0Panel cell 12++++0++0Instead of running 3 separate cells to rule out the antibodies, you can choose one that is homozygous positive for M, C, Fyb and negative for Jka. Panel cell 9 works in this case.If the only antibody that is present is Jka, then your test results should be negative. If the results are positive then further rule outs will be needed to determine what is present.

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Rule-Out Procedures

Rule-out (also referred to as exclusion or cross-out) is a process by which antibodies are identified as being unlikely in a given sample because of the absence of an expected antigen-antibody reaction. In other words, the absence of a reaction is noted with a cell that is positive for the corresponding antigen. Rule-out, while very useful, can lead to error. Ruling out an antibody should be combined with other supporting data to increase confidence in the solution; the more data collected, the higher the probability that the final solution is correct.Non-reactive cells are selected for rule-out. To be classified as non-reactive, a cell must NOT have reacted in any phase of testing in a given panel or screen. In the case of cold antibodies: if reactions are only occurring at immediate spin and are negative in the AHG phase, then that panel cell can be used as a rule out cell for IgG reactive antibodies but not for antibodies that react at immediate spin (IgM).If there is no reaction with a panel cell then it is possible that antibodies to the antigens on that cell are not present in the sample being tested.

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When to Suspect Dosage

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.

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Case Study Four

A 72 year old male with a previous history of a cold antibody is in the ICU with an abdominal aortic aneurysm. The doctor has ordered 8 units of packed red cells to be crossmatched at all times for this patient.The patient's ABO/Rh is B positive.The current antibody screen is positive in both the immediate spin phase and the AHG phase.Anagram resultsResults of the anagram show the presence of an antibody that reacts at immediate spin and AHG. Because Screen cell 1 is negative at the IS phase and positive at AHG, there are possibly multiple antibodies present (IgM and IgG).Antibody PanelSelected Cell Panel 1Enzyme PanelSelected Cell Panel 2

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Case Study Four- Antibody Panel

Antibody panel resultsResults of the antibody panel show reactions at immediate spin and AHG with varying strengths. The pattern at IS matches P1. Remember that varied strengths can mean multiple antibodies, dosage or both.There are not enough rule-out cells to rule anything out with 3 negative reactions. You can use panel cells that reacted at IS and are negative at AHG for rule-out. Use cells 4 and 10 for rule-outs. Antibodies that have no rule-outs from this panel are: C, E, Cw, Kpa, Jsa, Fya, Lea, M,s, P1, and Lua. Cw, Kpa, Jsa, and Lua are usually not present on panels and fall under the "unable to rule out" catagory. C,E, Fya and s are clinically siginificant and should have further testing done to rule-out or rule-in these antibodies. Lea , P1 and M tend to react at IS, so if the pattern of reactivity is compared to the reaction pattern at IS, there is a match for P1.A selected cell panel was then performed.

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Naturally Occurring Antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after stimulation by a specific antigen. The antibody formed binds to the specific antigen in order to mark the antigen for destruction.The type of antigenic exposure occurring in the body determines if the antibody is a naturally occurring or immune antibody.Naturally occurring antibodies can be formed after exposure to environmental agents that are similar to red cell antigens, such as bacteria, dust or pollen. Sensitization through previous transfusions, pregnancy or injections is not necessary. These antibodies are usually IgM and react best at room temperature or lower. Most of these antibodies are not clinically significant with the exception of ABO antibodies. Examples of naturally occurring antibodies include anti-A, anti-B, anti-Cw, anti-M, and antibodies in the Lewis and P system.

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Example Of A Naturally Occurring Antibody

In this example, anti-M can be identified as the possible antibody by looking at the patterns of reactivity. Reactions are only occurring at immediate spin, so this would not be considered a clinically significant antibody. Clinically significant antibodies are usually IgG and react at 37°C and at the AHG phase.IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control

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Example of Clinically Significant Immune Antibody

The panel below shows reactions in the AHG phase only (clinically significant). Pattern reactivity of sample matches the pattern displayed by C on the panel. Anti-C is a clinically significant antibody that can cause both hemolytic disease of the newborn (HDN) and hemolytic transfusion reaction (HTR).ND= not done

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Products Used to Facilitate Antibody Identification

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.

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Test Methods

The three most commonly used methods in antibody detection and identification are tube, gel and solid phase.Increased sensitivity in detection of antibodies is seen when using the tube method with PEG or the gel method (especially for mixed field reactions). Solid phase testing has a better sensitivity than just using the test tube method with LISS.

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Course Introduction

Antibody screening and antibody identification are critical components in blood bank testing. Clinically significant antibodies must be identified so that appropriate blood products are selected for transfusion and the risk of adverse reaction is minimized. Clinically significant antibodies are capable of causing transfusion reactions, hemolytic disease of the newborn and in severe cases, death.This course will discuss the techniques that are used by blood bank technologists to detect and identify various types of antibodies.

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Significance of Reactions at Different Phases of Testing

Antibodies have optimum temperatures for reactivity. Reaction readings can be made at different phases: after immediate spin, after incubation at 37°C, and after the addition of antihuman globulin (AHG) and centrifugation. Reactivity in a certain phase will help to determine whether the antibody is cold reacting (IgM) or warm reacting (IgG). It will also help to distinguish between antibodies that are clinically significant and not significant. Clinically significant antibodies that are capable of causing acute and delayed hemolytic transfusion reactions (HTR) or hemolytic disease of the newborn (HDN) are usually IgG and react best in the AHG phase.Readings can be done at all three phases if a tube method is used. If a gel method is used, readings are done only at AHG. Immediate spin: Antibodies reacting in this phase tend to be cold reactive. They are usually IgM class and not clinically significant (with the exception of the A and B antibodies). 37°: Antibodies that react in this phase include strong IgM or IgG antibodies. After incubation, the tubes are examined for the presence of hemolysis. If complement was bound during incubation then hemolysis could be seen. NOTE: This reaction would only occur in serum samples. If EDTA plasma samples are used for testing, the complement cascade has been halted. Magnesium and calcium ions are not available for complement to be activated. AHG:Antibodies reacting in this phase are considered clinically significant. They are usually warm reactive and IgG.

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Antibodies to Low- and High-Incidence Antigens

Low-incidence antigens are antigens that occur in less than 1% of the population.Antibodies to low-incidence antigens Low-incidence antigens are not usually found on screen cell and antibody panels. Antibodies are hard to test for, but it is usually not difficult to find compatible blood. Suspect this antibody if an AHG crossmatch is incompatible and other causes have been ruled out, such as a positive donor DAT or ABO incompatibility. Examples of low-incidence antigens include: Cw, V, Kpa, Jsa. When going through the process of Ruling Out, antibodies like anti-V, anti-Cw, anti-Lua, anti-Kpa, and anti-Jsa usually fall into the "unable to rule out" category. High-incidence antigens are antigens that occur in greater than 99% of the population. Antibodies to high-incidence antigens Antibodies are rare and may be difficult to identify due to lack of negative panel cells for other high-incidence antigens (difficult to rule out). Reactions with screen and panel cells will all be positive (same strength and same phase). Auto control will be negative. Difficult to find antigen-negative compatible blood. Examples of antibodies to high-incidence antigens are: anti-k, anti-Kpb, anti-Jsb, and anti-Lub. If an antibody to either a high- or low-incidence antigen is present, it may be difficult to identify and may require further testing in a reference blood bank.

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Examples of Antibodies to High-Incidence Antigens

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)

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Examples of Antibodies to Low-Incidence Antigens

Antibodies to low-incidence antigens will be difficult to test for since most screen and panel cells do not have these antigens on the testing cells. Further testing may be needed at a reference laboratory where a larger selection of antibody panels are available to locate cells positive for these antigens.Suspect an antibody to a low-incidence antigen if: AHG crossmatch is incompatible and Other causes have been ruled out (positive donor DAT, ABO incompatibility) Examples of antibodies to low-incidence antigens are: anti-V, anti-Cw, anti-Kpa, anti-Jsa, and anti-Lua.

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Antinuclear Antibody Testing: Methods and Pattern Interpretation
Antinuclear Antibody Test

The antinuclear antibody test (ANA) is a test used to screen for the presence of autoantibodies that are directed toward components in the nucleus of the cell. Clinicians use the ANA test to assess the likelihood that a given patient has a SARD. The results of the ANA test alone are not diagnostic for the SARD. The patient must also have clinical evidence of the disease as well. Because the early clinical presentation for many of the SARDs are nonspecific, the results of the ANA test and subsequent follow-up testing are key pieces to making the correct diagnosis.Rheumatoid arthritis (RA) is the most prevalent disease in this group; however, the ANA assay is not the primary laboratory test for RA. Instead, the test for RA looks for the presence of rheumatoid factor (RF) or more recently, cyclic citrullinated peptide antibodies (anti-CCP).For the other diseases in the SARD group, especially SLE and SSc, the results of the ANA test can be useful in determining a correct diagnosis. The utility of the ANA test is to detect the antibodies early in the disease process.The ANA results in conjunction with clinical presentation give the clinician solid evidence to intervene with an appropriate treatment. Studies have shown that once treatment is started, the formation of new antibodies slows or even halts.(Ref3)Currently there are no cures for the SARDs. Treatments primarily focus on keeping the patient comfortable and the immune response in check. Treatments can vary from non-steroidal anti-inflammatory drugs, to immuno-suppressive drugs, to stem cell transplants. Individual treatment is often dependent on the severity of the disease and the response to the selected drug regimen.

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Fluorescent ANA Testing

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.)

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Colorzyme®

A similar procedure that is also widely used is called Colorzyme®.(Ref7) This system uses horseradish peroxidase rather than FITC as the marker on the secondary antibody. This technique offers the same advantages as the IFA procedure but also has the added benefits of being more photo-stable and not requiring a fluorescent microscope. The Colorzyme® ANA Test utilizes the indirect enzyme antibody technique. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANA's 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 reagent conjugated to horseradish peroxidase. When results are positive, there is the formation of a stable three-part complex consisting of enzyme antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized by incubating the slide in an enzyme specific substrate. The reaction between the enzyme labeled antibody and enzyme specific substrate results in a color reaction on the slide visible by standard light microscopy. In positive samples, the cell nuclei will show a bright bluish purple staining with a 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 staining. The cytoplasm may demonstrate weak staining while the non-chromosome region of the mitotic cells may demonstrate a darker 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.)

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ANA Testing Scheme

Most laboratories use a two-stage approach for ANA testing with the first stage being an initial screen at a 1:40 or 1:80 dilution of the patient sample. Negative samples are reported out as such and positive samples are titered. There are two reasons for titering ANA-positive samples. One is to determine the amount of antibody present and the other is to look for multiple patterns. In the case of ANA testing there is no correlation between titer endpoint and disease activity or severity. However, the higher the titer the higher the likelihood the patient has one of the SARDs. Often more than one ANA pattern present in the sample; titering facilitates the identification of these mixed patterns. (Identification of mixed patterns will be covered later).The titering scheme most frequently used in ANA testing is two-fold dilutions starting at the initial screening dilution. For example: 1:40, 1:80, 1:160... While there is no consensus on how far to titer samples, informal communications with laboratories suggest most stop titering at about the 1:2560 dilution (plus or minus one dilution). By this dilution the samples are often negative and/or no longer contain mixed patterns.For ANA positive results the sample is reported as: ANA positive, the pattern that is indicated along with the titer endpoint. For example: Sample 12345: ANA positive, speckled, titer: 1:640.If more than one pattern is present all patterns are reported along with their respective endpoints. Additionally, many labs will suggest appropriate follow-up testing to identify the antibody(ies) present in the sample. By identifying the specific antibodies present in the sample, the clinician may gain further insight into which of the SARDs the patient has and what other symptoms the patient may develop.

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Antigen Chart

Pattern observed by indirect immuno fluorescence Antigen Disease(s) in which antibodies are seen Routine tests used to confirm specific antibody Homogeneous Double stranded DNA (dsDNA) Characteristic of SLE, lower levels in other rheumatic diseases IFA or CZ using Crithidia luciliae, RIA, ELISA, Addressable Laser Bead Assay (ALBIA) Nucleosome or Chromatin SLE, Drug-induced LE ELISA Histone Drug-induce LE, SLE ELISA, ALBIA Unusual Homogeneous Nuclear Membrane Lupoid hepatitis ELISA for gp-210 Speckled Sm (Smith) Marker antibody for SLE Immunodiffusion (ID), ELISA, ALBIA U1-RNP High levels in MCTD and SLE, low levels in other rheumatic diseases ID, ELISA, ALBIA Speckled (and/or SSA pattern if using HEp-2000®) Can also be ANA negative SS-A/Ro High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases With HEp-2000 characteristic ANA pattern is confirmatory, others confirm with ID, ELISA, ALBIA Fine speckled or ANA negative Ro52 Sjögren syndrome, myositis, Neonatal Lupus ELISA, ALBIA Fine speckled (sometimes with nucleolar staining as well) SS-B/La High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases ID, ELISA, ALBIA Fine speckled, Homogeneous, Nucleolar Scl-70 Marker antibody for Scleroderma ID, ELISA, ALBIA Cell Cycle Dependent Speckled PCNA Marker antibody for SLE ID, ELISA, ALBIA Coarse Speckled Nuclear Matrix Seen in some patients with evolving connective tissue disease NONE 3-20 dots NSp I, sp-100, MND, PBC 95 Associated with Primary Biliary Cirrhosis ELISA, ALBIA Cell Cycle Dependent Speckled with speckling in metaphase mitotics NSp II, CENP F Some association with malignancies NONE Staining in cleavage furrow between dividing cells Midbody Unknown Confirm by staining pattern Centromere CENP A, CENP B, CENP C Seen in 57-82% of patients with limited form (CREST) of scleroderma and Raynaud phenomenon Confirmed by staining pattern ELISA, ALBIA Nucleolar Fibrillarin (Clumpy nucleolar) Scleroderma ELISA, ALBIA RNA polymerase I, NOR-90, others? (Speckled nucleolar) Scleroderma and other connective tissue diseases ELISA, ALBIA PM-1 (PM/Scl), others?(Smooth nucleolar) Polymyositis/Scleroderma overlap ELISA, ALBIA

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Why are ANA positive samples titered?View Page
The image on the right represents the result of a fluorescent antinuclear antibody (ANA) test. What pattern should be reported?Note: (a) points to the nuclei of several interphase cells, the primary consideration for discerning the ANA pattern and (b) indicates a metaphase mitotic cell. Observing the chromosomal area and cytoplasm of the metaphase cell may assist in identification of the ANA pattern.View Page
References

American College of Rheumatology, Committee on Rheumatologic Care, Position Statement, Methodology of Testing for Antinuclear Antibodies; Feb, 2009. Available at http://www.rheumatology.org/search/search.asp accessed on June 16, 2010Anuradah V, Chopra A, Sturgess A, Edmonds J. Cost-effective screening method for antinuclear antibody detection. Asian Pacific League of Associations for Rheumatology. 2004(7):13-18.Arbuckle MR, McClain MT, Rubertone MV, et al. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct 16 2003;349(16):1526-1533.Bossuyt X, Frans J, Hendrickx A, Godefridis G, Westhovens R, Marien G. Detection of Anti-SSA Antibodies by Indirect Immunofluorescence. Clin Chem. 10 7 2004;50(12):2361-2369.Clinical and Laboratory Standards Institute (formerly NCCLS); Quality Assurance of Laboratory Tests for Autoantibodies to Nuclear Antigens: (1) Indirect Fluorescence Assay for Microscopy and (2) Microtiter Enzyme Immunoassay Methods; Approved Guidelines - Second Edition. CLSI I/LA2-A2. 2006;26(13).Fritzler MJ, Hanson C, Miller J, Eystathioy T. Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate. J.Clin.Lab.Anal. 2002;16:103-108.Fritzler MJ, Miller BJ. Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells. J.Clin.Lab.Anal. 1995;9:218-224.Fritzler MJ, Wall W, Gohill J, Kinsella TD, Humbel RL. The Detection of Autoantibodies on HEp-2 Cells Using an Indirect Immunoperoxidase Kit (Colorzyme®). Diag Immunol. 1986;4:217-221. Keech CL, Howarth S, Coates T, Rischmueller M, McCluskey J, Gordon TP. Rapid and sensitive detection of anti-Ro (SS-A) antibodies by indirect immunofluorescence of 60kDa Ro HEp-2 transfectants. Pathology. 1996;28:54-57.Keech CL, McCluskey J, Gordon TP. Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells. Clin.Immunol.Immunopathol. 1994;73:146-151.Kroshinsky D, Stone JH, Bloch DB, Sepehr A. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash and numbness and pain in the legs. N Engl J Med. Feb 12 2009;360(7):711-720. McCarty, G.A., Valencia, D.W., and Fritzler, M.J., Antinuclear Antibodies-Contempory Techniques and Clinical Application to Connective Tissue Disease. New York: Oxford University Press, Inc. 1984. Murray DL, Homburger HA, Horvat RT, Snyder MR, College of American Pathologists; S-C 2009: Antinuclear Antibody Screening Methods; CAP Surveys S-C Diagnostic Immunology;2009 Pollock W, Toh BH. Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A. J.Clin.Pathol. 1999;52:684-687.Singer, M. and Berg, P., Genes & Genomes-A Changing Perspective. Mill Valley, CA: University Science Books. 1991.Sullivan KE. The complex Genetic Basis of Systemic Lupus Erythematosus, Reprint from 1999 and 2000; Lupus Foundation, Available at http://www.lupus.org/education/articles/geneticbasis.html Accessed June 16, 2010.Wallace DJ. New methods for antinuclear antibody testing: does it cut costs and corners without jeopardizing clinical reliability? Nat Clin Pract Rheumatol. Aug 2006;2(8):410-411.Willcocks LC, Carr EJ, Niederer HA, et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus. Proc Natl Acad Sci U S A. Apr 27 2010;107(17):7881-7885.

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Authentic and Spurious Causes of Thrombocytopenia
Pseudo-thrombocytopenia: Platelet Satellitism and Platelet Clumping

Platelet satellitism and platelet clumping can cause pseudo-thrombocytopenia. Platelet satellitism was first reported in the early 1960's. It is a rare condition that occurs when an IgG antibody forms in the presence of EDTA, the anticoagulant that is used for the collection of hematology blood specimens. The IgG antibody is directed against the glycoprotein IIb/IIIa complex on the platelet membrane. As the antibody coats the platelets, the platelets rosette around segmented neutrophils, bands, and sometimes around monocytes. Antibody-coated platelets that are huddled around white blood cells (WBCs) will not be counted as platelets by automated equipment and the platelet count will be falsely decreased. If a peripheral blood smear is reviewed, platelets will be observed attached to WBCs. The image on the right illustrates platelet satellitism with platelets adhering to a neutrophil. Platelet clumping can also occur in the presence of EDTA and the platelet count again will be falsely decreased. The count will probably be flagged by the analyzer for platelet clumps or giant platelets. If either platelet satellitism or platelet clumping is observed on the peripheral smear, the sample could be recollected using sodium citrate as the anticoagulant. Platelets can then be counted using the automated method. The platelet count from a tube that contains liquid sodium citrate will need to be corrected for the dilutional effect of the citrate. This can be accomplished by multiplying the platelet count that is obtained from the automated analyzer by 1.1.

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Which of these conditions is associated with a pseudo-thrombocytopenia?View Page
Pathophysiology of Heparin-Induced Thrombocytopenia (HIT)

HIT is a complication of heparin use that was first recognized in 1969. When heparin is administered to some patients, it forms an immune complex with platelet factor 4 (PF-4) that is released from the alpha granules in platelets. The body's immune system recognizes this complex as a foreign substance and forms an antibody against it. The antibody binds to this complex and the platelets are destroyed. Thrombocytopenia occurs in approximately 3% of patients who receive heparin therapy. It usually takes 5 - 14 days for the platelet count to decrease after heparin therapy begins. For this reason, patients need to have a baseline platelet count upon initial heparin use and should then be monitored with regular platelet counts for the duration of therapy. HIT has been associated with both unfractionated heparin and low molecular weight heparin treatment.

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Drug-induced thrombocytopenia

Some drugs may cause thrombocytopenia in a small number of patients that take them. Drugs that have been implicated in development of thrombocytopenia include heparin, quinidine, quinine, and sulfonamides, among others. Thrombocytopenia is most likely the result of an antibody forming against the drug. This antibody binds to a glycoprotein on the platelet and then the platelet is taken out of circulation by the reticuloendothelial (RE) system. The platelet count will rise once the drug is discontinued.

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Blood Banking Question Bank - Review Mode (no CE)
The diagram on the right shows two antibody response curves. Match the terms on the left with the corresponding numbers in the diagram.View Page
Anti-Rho immune serum is administered to:View Page
Rh immune globulin therapy in postpartum women provides:View Page
In HDN which of the following antigen-antibody reactions is occurring:View Page
Which of the following best describes the direct antiglobulin test principle:View Page
When AHG or Coombs serum is used to demonstrate that red cells are antibody coated in vivo, the procedure is termed:View Page
The prozone effect can be described by all of the following except:View Page
Match each blood type with the corresponding antibody you would find in its serum:View Page
Avidity is best described by which of the following statements:View Page
Which of the following options gives in order from most to least important, the factors you would use to select blood for a transfusion:View Page
The use of cells with known blood groups to confirm ABO typing is known as:View Page
Essential components of compatibility testing include all of the following except :View Page
Patients with antibody to the following antigen are immune to Hepatitis B:View Page
What is Coombs sera comprised of:View Page
The Western Blot Assay is used as a confirmatory test for which of the following:View Page
Autologous blood must be tested for which of the following before transfusion:View Page
Which of the following might cause a false positive indirect antiglobulin test:View Page
A solution of gamma globulins containing anti-Rh (D) is given to an Rh (D) negative mother to:View Page
To detect the presence of blocking antibodies fixed on the red cells of a newborn infant:View Page
In preparing red cells for any elution method , one must be particularly careful to:View Page
Which of the following is the proper temperature to use when crossmatching in the presence of a cold antibody:View Page
A primary immune response is generally associated with which of the following antibodies:View Page
Match antibody group with corresponding descriptor:View Page
Which of the following antibodies is predominantly associated with the secondary antibody response:View Page
Which of the following antigen groups is closely related to the ABO system:View Page
A false-negative reaction while performing the DAT technique may be the result of:View Page
The two or three reagent cells used for antibody screening will detect which of the following:View Page

Cardiac Biomarkers (retired 12/6/2013)
Troponin Measurement and Ranges

Rapid immunoassays provide concentration levels of cTnI and cTnT that are approximately 96% sensitive and 94% specific for cardiac injury.Each diagnostic company develops their unique antibody against epitopes on the proteins. There is only one assay available for cTnT. However, there are several different antibodies that are used by manufacturers to detect cTnI. Consequently, different assay methods may not correlate well. Standardization is needed for intra-laboratory comparisons. Reference Ranges for an adult: cTnT <0.01 ng/mL No cardiac injurycTnI references may vary with different assay methods, but approximate these values: Equal to or < 0.03 ng/mL -- No detectable cardiac injury 0.04-0.49 ng/mL -- Cardiac muscle injury Equal to or > 0.5 ng/mL-- Myocardial infarction

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Case Studies in Clinical Microbiology
The carbohydrate utilization reaction seen in the QuadFerm system shown in the image provides a definitive identification of N. gonorrhoeae.View Page
Review 1

Smith KR, Fisher HC III, Hook, EW III: Prevalence of fluorescent monoclonal antibody-nonreactive Neisseria gonorrhoeae in five North American sexually transmitted disease clinics. J Clin Microbiol 34:1551-1552, 1996 We compared a direct fluorescent monoclonal antibody (DFA) test with alternative enzymatic and fermention tests for identifying presumptive gonococcal isolates in a systematic sample from patients attending five sexually transmitted disease clinics in five cities. Fourteen (2.5%) of 556 isolates from three clinics were nonreactive with the DFA confirmatory reagent and reactive by both the Quad-Ferm and Rapid NH tests. The prevalence of DFA-nonreactive Neisseria gonorrhoeae isolates varies geographically and is independent of local methods for the identification of possible gonococci. On the basis of our findings, we recommend that for use in medicolegal and other instances in which a diagnosis of gonorrhea has the potential to have far-reaching effects, it is appropriate to test DFA reagent-nonreactive, oxidase-positive, gram-negative diplococci by alternative methods of gonococcal confirmation. Although the prevalence of such isolates could change, the fluorescent monoclonal antibody confirmation reagents remain useful for many clinical situations. Their ease of use and ready applicability for screening large numbers of isolates make them useful for many laboratories.

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Pneumococcal vaccine is particularly effective in children less than two years of age.View Page

Detecting and Evaluating Coagulation Inhibitors and Factor Deficiencies (retired 5/27/2014)
Mixing Study: Specimen Requirements

The specimen drawn for a mixing study must meet the following conditions for accurate testing: A properly filled 3.2% sodium citrate tube must be collected. Proper centrifugation to create platelet-poor plasma for analysis must occur; the presence of platelet phospholipids can interfere with the mixing study if an anti-phospholipid antibody is present. Testing must be performed within 4 hours of collection.

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Lupus Anticoagulant

No single screening test can detect all lupus anticoagulant-positive (LA-positive) patients. Several tests are available and at least two should be employed to verify the presence of LA. Before any LA screening test is done, a thrombin time (TT) should be performed to rule out therapeutic heparin or the presence of a thrombin (factor-II) inhibitor.These are some of the LA screening procedures that can then be used: Dilute Russell's Viper Venom time (DRVVT). This test utilizes a reagent containing venom from the viper Vipera russelli (which activate factor V and X), low levels of phospholipids, and calcium ions in a clotting time test. The DRVVT test principle is based on the idea that the reagents can help to identify the antibody's dependence on phospholipids . Platelet neutralization procedure. This assay will show the dependence on phospholipids for the lupus anticoagulant to take effect. This can be performed using the aPTT based technique, with the DRVVT test, or using Taipan snake venom time tests. Kaolin clotting time or silica clotting time Hexagonal Phospholipid test (HPP). This is a similar assay to the platelet neutralization procedure, but thought to be more sensitive.

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Further Analyses for Coagulation Inhibitors (cont.)

When considering the presence of a coagulation factor inhibitor other than the common lupus anticoagulant, specific inhibitors such as anti-Factor VIII should be considered. Titers can be performed for the antibody in question to quantify the inhibitor and determine the extent of the antibody proliferation. These tests are called Bethesda titer assays. These tests use a universal inhibition unit in measuring specific-factor antibody activity. The appropriate dosing of medications may be measured based on these titer results.

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Further Analyses for Coagulation Inhibitors- Lupus Anticoagulant

For the diagnosis of lupus anticoagulant, the International Society on Thrombosis and Hemostasis has set a protocol of diagnostic criteria that should be met. This includes the following requirements: The patient sample must show abnormal phospholipid-dependent reactions in the coagulation lab. The patient sample must show inhibition of clotting after the mixing study test has been performed. The patient sample must be proven to have an inhibitor and not a factor deficiency. The patient must have a definitive phospholipid-dependent antibody and not a specific factor inhibitor.

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Treatment for Coagulation Inhibitors

Currently, there are several treatment options for patients suffering from coagulation inhibitors. Treat the patient by administering recombinant factor replacements. For example, using a recombinant factor VIII or factor IX for the treatment of acquired hemophilia due to coagulation inhibitors. Treat the patient with immunosuppresants, such as prednisone, to prevent large amounts of coagulation antibodies from forming. Then factor replacement can be given to the patient. If lupus anticoagulant is suspected, anticoagulants may be ordered to prevent thrombotic episodes. Often for treatment purposes, a patient is given a very high level of the coagulation factor that the antibody is targeting. The goal is to overwhelm the antibody with excess factor so that the antibody is neutralized and the residual factor can participate in the normal coagulation process.

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Introduction: Coagulation Inhibitors

As the name implies, coagulation inhibitors (also called circulating anticoagulants) interfere with normal blood coagulation. Coagulation inhibitors may be congenital or acquired (developing in patients during the course of a disease) and are almost always immunoglobulins, either IgG or IgM. There are two types of inhibitors: those directed toward a coagulation factor (or multiple factors) and the lupus anticoagulant. Lupus anticoagulant is one of the more commonly encountered coagulation inhibitors. It is also known as antiphospholipid antibody because it is directed toward phospholipids. Lupus anticoagulant is usually an IgG antibody. It differs from factor-specific inhibitors in that lupus anticoagulant causes thrombosis and abnormal clotting while factor-specific inhibitors cause serious bleeding.

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Coagulation Inhibitors and Coagulation Screening Tests

Patients with factor-specific coagulation inhibitors will have prolonged prothrombin time (PT) and/or aPTT test results (depending on the coagulation factor that is targeted by the inhibitor). Clinically, this is associated with abnormal clotting and bleeding complications. A prolonged aPTT, and sometimes PT, is seen with lupus anticoagulant. The antibody combines with the phospholipids on the surfaces of test reagents that are used in the aPTT test, and sometimes in the PT test, prolonging the test result(s). Clinically, lupus anticoagulant is associated with thrombosis and not with bleeding symptoms.Click here to read an important note regarding lupus anticoagulant

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Diabetes and the Current American Diabetes Association Guidelines
Insulin Antibodies

Since type 1 diabetes is caused by an autoimmune destruction of pancreatic tissue, sometimes antibody measurements are used to gain more information about a type 1 diabetic. Like insulin and C-peptide, insulin antibodies are not measured to diagnose or monitor a diabetic patient.

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Electrophoresis
Electroimmunoassay Electrophoresis

In electroimmunoassay electrophoresis, the antiserum is mixed in the gel during preparation. In the electrophoresis of the serum sample, the voltage drives the sample antigen into the antiserum creating a precipitin line in the shape of a rocket. This line is proportional to the concentration of the antigen, which is the protein to be detected. Each gel contains several serum samples, one antibody suspended in the gel, and standards of known concentration of antigen. Quantitation of the unknown antigen is derived from the height of the sample rockets compared to the height of the standard rockets. Electroimmunoassay electrophoresis is often referred to as rocket electrophoresis.

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Blotting Techniques

Blotting techniques were developed to discriminate fragments of nucleic acids. These techniques involve several processes; electrophoresis is one of the processes and is used to separate fragments of DNA and RNA. In Southern blotting (named after Edward Southern) restriction enzymes cut fragments of DNA are separated by AGE or PAGE, transferred to a membrane or blot, and visualized by hybridization with labeled probes.Northern blotting (not named after an inventor but by analogy to Southern blotting) separates RNA. RNA molecules are shorter and have defined lengths; cutting by restriction enzymes is not required. Denaturing conditions are required because of RNA secondary structures. After membrane blotting, the separated types of RNA are visualized with staining or labeled probes.Western blotting (again not named after an inventor but by analogy to Southern blotting) does not separate nucleic acids; it separates proteins in a mixture. The proteins are usually separated with PAGE, transferred to the membrane and visualized with a labeled antibody against the proteins of interest.

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Emerging Cardiovascular Risk Markers (retired 12/6/2013)
Oxidized LDL Tests

There are currently two antibodies that have been developed that target oxidized lipids; the 4E6 antibody and EO6 antibody. The 4E6 antibody is directed against an oxidized epitope on the ApoB-100 protein on LDL. The EO6 antibody recognizes oxidized phospholipids and the assay measures the content of these oxidized phospholipids on lipid particles that contain ApoB-100.

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Fundamentals of Molecular Diagnostics (retired 2/12/2013)
Targets

Molecular based clinical diagnostic test methodologies differ according to the target of interest. For example, patients suspected of having different diseases will require the identification of different targets. These targets might be found in different cells of the body and may therefore require different specimens to provide the answers. Patient A suspected of having Disease 1: Requires the identification of a target of missequenced DNA - might require specimen of whole blood Patient B suspected of having Disease 2: Requires identification of a target of antibody production -methodology might require specimen of serum Using this specific approach of disease diagnosis based on unique target identification, tests can provide answers that are more:Rapid Sensitive Specific

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General Laboratory Question Bank - Review Mode (no CE)
The prozone effect ( when performing a screening titer) is most likely to result in:View Page
Which of the following antibody types is chiefly seen in the primary immune response:View Page
Which of the following antibody types is chiefly seen in the secondary immune response:View Page
A hapten is only antigenic when it is coupled with which of the following:View Page
The reaction that occurs when a soluble antigen is mixed with its specific antibody is termed:View Page
The term TITER ( as it applies to the measurement of antibodies) is best defined as:View Page
This question refers to results of the classical complement fixation test; match the result on the left with the presence or absence of hemolysis on the right.View Page
Which of the following would be considered most significant as it relates to serological testing:View Page

Hematology / Hemostasis Question Bank - Review Mode (no CE)
What principle(s) of flow cytometry are employed when performing immunophenotyping:View Page

Hemolytic Disease of the Fetus and Newborn
Advance Organizer

Before beginning the course take some time to review and think about what you already know about HDFN. For example, jot down brief notes to answer the following questions: Which antibody causes the most severe HDFN? Antibodies in which blood group system are the most common cause of positive direct antiglobulin tests (DATs) in newborns but rarely cause clinically significant hemolysis? Should DATs be performed on all newborns regardless of maternal ABO and Rh blood groups? What is Rh immune globulin (RhIg), its source, constituents, purpose, and mechanism of action? Which tests are used to determine postnatal RhIg dosage? Which type of D variant can produce anti-D? What follow-up tests are typically indicated if a pregnant female has a positive antibody screen when initially tested? Which laboratory findings would suggest that an infant may have ABO HDFN? How can the clinical status of fetuses at risk for HDFN be monitored? What are the characteristics of red cells suitable for intravenous transfusion to fetuses suffering from severe HDFN due to anti-D?

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Introduction

Although HDFN can be life threatening, in the case of anti-D it is a disease that can be prevented. Regardless of causative antibody, HDFN's serious consequences can be lessened by early laboratory diagnosis and treatment. This course begins with an in-depth review of HDFN and later discusses its prevention in detail. In reviewing HDFN, key questions to be answered include: What are the typical signs and symptoms of severe HDFN? Which serologic tests does the transfusion service laboratory use to diagnose HDFN? How is severe HDFN treated? Which development dramatically changed the incidence of HDFN due to anti-D? Other than the causative antibodies, what are some of the main differences between ABO HDFN and HDFN due to anti-D and other antibodies?

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Typical Case of Rh HDFN (Prior to RhIg)

RhIg became widely available in 1968. Prior to that, HDFN due to anti-D typically developed as described below. Cases were much more prevalent in Caucasians due to the relative incidence of the D antigen in various populations, For example, approximate incidence of D+ individuals: Caucasians (European ancestry): ~85% African-Americans: ~93% Asians: ~99%In the first pregnancy, Rh positive fetal red cells enter the maternal circulation during the pregnancy and/or at delivery. The mother has a 1o immune response in which mainly IgM antibody is produced, with lower levels of IgG anti-D produced. Thus the first infant is rarely affected because: Larger fetal bleeds occur at delivery and these are more likely to cause antibody production than smaller antenatal bleeds. Antibody is produced slowly and is mostly IgM. In the second pregnancy, if the fetus is again D-positive, when fetal cells enter the mother, they cause a 2o immune response in which higher levels of IgG anti-D are produced. Depending on the antibody titer, the second child may suffer mild to severe HDN. If a third or fourth pregnancy results in D-positive infants, these infants (by also bleeding into the mother) cause the production of even higher titers of IgG anti-D and offspring will be more severely affected, perhaps dying in utero or soon after birth, if untreated.

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Primary versus Secondary Response

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).

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Symptoms and Laboratory Findings in Severe HDFN Due to Anti-D

Anti-D causes the most severe HDFN. Symptoms and laboratory findings in HDFN due to anti-D typically include:1. Anemia: Cord Hb can be less than 10 g/dL (100 g/L) and as low as 3–5 g/dL (30–50 g/L).2. Jaundice (icterus gravis): Jaundice occurs after delivery, as fetal bilirubin is cleared by the mother during pregnancy. Extravascular fetal red cell destruction by maternal antibody produces high bilirubin levels. The newborn, who is unable to produce adequate amounts of the liver enzyme glucuronyl transferase, is unable to conjugate the bilirubin into its water-soluble, excretable form.3. Kernicterus: If indirect bilirubin levels reach approximately 20 mg/dL (340 mmol/L) the fat soluble unconjugated bilirubin deposits in the fat-rich brain cells causing brain cell damage. Cerebral palsy, deafness, mental retardation, and other serious disorders can result.4. Hydrops fetalis: Gross edema occurs in severely affected infants, and often results in stillbirth or death soon after birth. Liver failure and hypoproteinemia also play a role in this syndrome.5. Enlarged organs, e.g., liver, spleen and heart6. Laboratory findings include a positive direct antiglobulin test (DAT), low hemoglobin (as above), increased reticulocyte count, and increased nucleated red cells.

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Postnatal Treatment: Exchange Transfusion

Whenever possible, a hallmark of HDFN treatment is to induce labor as early as possible once lung maturity has been attained so that the newborn will be able to survive. Once the infant is born, the main treatment for severe HDFN due to anti-D (and other antibodies causing severe disease) is exchange transfusion. In exchange transfusions, up to 85–90% of the infant's blood can be exchanged with donor blood by a process of removing 5–20 mL of blood at a time, and injecting an equivalent amount until the exchange is complete. An exchange transfusion accomplishes the following: Removes bilirubin and thus helps prevent kernicterus; Removes sensitized red cells that have not been broken down yet; Removes circulating maternal antibody; Provides antigen-negative red cells that will not be destroyed by the maternal antibody, thus will survive and provide oxygen to the tissues.

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Other Postnatal Treatment

Besides exchange transfusion, postnatal treatment of HDFN may include the following:RBC TransfusionMany infants who have received IUTs also require simple RBC transfusions in the first few weeks of life to treat ongoing hemolysis caused by persistent maternal antibody in the newborn's circulation.Phototherapy Phototherapy is used to treat jaundice in preterm infants without HDFN and in infants with mild HDFN. Intensive phototherapy has also been used to treat moderate and severe HDFN and decrease the need for exchange transfusion. The newborn is placed under a "blue light" which chemically alters the bilirubin in the surface capillaries to a harmless substance. Human Serum AlbuminHuman serum albumin can also be transfused, either separately or as part of an exchange transfusion in place of FFP. Albumin binds unconjugated bilirubin, thus preventing its deposition in the fat-rich brain cells. Albumin must be used judiciously, because it can aggravate congestive heart failure.

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ABO HDFN - Etiology and Symptoms

ABO HDFN is the most common type of HDFN, in that anti-A is the antibody most often found bound to the red cells of a newborn. While the disease is usually so mild as to not require treatment, severe HDFN is possible. EtiologyABO HDFN is caused by maternal IgG anti-A or anti-B, which can be produced as a result of prior pregnancy or prior inoculation (some common inoculations contain A or B substances). In Caucasians, most often the mother is group O and the child is group A, although other combinations are possible. Group O people tend to produce IgG ABO antibodies more commonly than other blood groups.Just as in other types of HDFN, maternal IgG antibody crosses the placenta and destroys fetal red cells.SymptomsTypical symptoms of ABO HDFN include mild anemia and especially jaundice appearing in the first 24 hours. In rare severe cases the infant can have the more severe symptoms of Rh HDFN, except that prenatal death is unlikely. Rationales to explain the mild nature of ABO HDFN include Fewer A and B antigens on fetal cells Poorly developed fetal A and B antigens Presence of A and B antigens on cells and tissues other than red cells

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HDFN Due to Other Antibodies

After anti-D, the antibodies that are most often associated with HDFN include: anti-K anti-c anti-E anti-Fya (rarely) anti-Jka (rarely) anti-M,-N,-S,-s,-U (all rarely)Of these antibodies, anti-K, anti-c, and anti-E are more common causes. Anti-K typically causes more severe HDFN (hydrops and neonatal death) than the others. Anti-c has also been known to cause severe HDFN.Antibodies to low frequency antigens have also been known to cause HDFN, albeit rarely. Examples include anti-Mia, -Dia, -Wra and anti-Rd. In such cases the maternal antibody screen is usually negative and the only unexpected test is a positive DAT on the newborn. In theory any IgG antibodies directed against antigens that are well developed on fetal red cells can cause HDFN. The complete list of antibodies documented to cause HDFN is long and will not be covered in this survey course.

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ABO HDFN - Diagnostic Tests

Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, for example, erythrocyte membrane defects or red cell enzyme deficiencies. The diagnosis of ABO HDFN in the laboratory differs from diagnosing Rh and other types of HDFN in which clinically significant antibodies must be identified. Diagnosis may be difficult, because the DAT on the newborn's red cells is unreliable. Indeed, many labs do not routinely do a DAT on infants born to Rh positive females, since many will be positive in the absence of clinically significant hemolysis. Cord blood is often retained (e.g., for 7 days) should the infant develop signs of HDFN and required testing.If ABO HDFN is possible, based on incompatible ABO blood groups and a positive DAT, and the mother's antibody screen is negative, many laboratories do not investigate the positive DAT as would be done for unexpected antibodies like anti-D or anti-K (the laboratory does not perform an elution on the newborn's red cells). Instead, the infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN.

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For the test results shown above, which of the following antibodies is most likely to be causing the newborn's positive DAT?View Page
ABO HDFN - Expected Findings

Diagnosis of ABO HDFN is supported by these findings: ABO incompatibility between mother and child, with mother typically group O; Maternal antibody screen negative; Cord DAT weakly positive or negative; Newborn hyperbilirubinemia with jaundice occurring in first 24 hours; Increased spherocytes and reticulocytosis in the newborn; Presence of IgG anti-A or anti-B in cord plasma / serum.

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Introduction

Today, severe HDFN is rare due to perinatal testing programs, which are designed to prevent HDFN due to anti-D.Perinatal testing programs have two main purposes:1. To detect, at an early stage in pregnancy, the presence of any IgG antibody that could cause HDFN in order to identify, monitor, and treat the infant as soon as possible.2. To determine which women are candidates for RhIg in order to try to prevent the production of anti-D. Testing programs include both Rh negative and Rh positive women, but because antibodies other than anti-D only rarely cause HDN, Rh negative females are tested more extensively.

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Routine Serologic Tests - Mother

Tests done routinely as part of perinatal testing programs vary from country to country and within countries. Below is one example of routine serologic tests typically done when pregnant females lack clinically significant antibodies. Other test protocols exist.Tests on Mother ABO, Rh*, and antibody screen at first prenatal visit; Test for weak D, if initial Rh typing appears to be D-negative (Optional -not mandated by blood safety standards); D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks gestation prior to administration of RhIg (depending on the country) and again at delivery.* The mother, putative father, and fetus can be typed for D using DNA methods, if available.

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Follow-up Investigative Tests (Mother)

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.

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Follow-up Investigative Tests (Fetus)

If a mother has a clinically significant antibody, fetal blood for phenotyping can be obtained by several procedures, depending on gestational age and the antigen involved. These include Amniotic fluid sampling* Chorionic villus sampling* Cell-free fetal DNA in maternal plasma* Percutaneous umbilical cord blood sampling (PUBS) / cordocentesis** * molecular genotyping / ** serologic testsAs noted, typing the fetus is warranted when the father's blood type is unknown or the father tests as heterozygous positive.

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Follow-up Investigative Tests (Newborn)

If the mother has a clinically significant antibody, routine serologic tests done on the newborn include ABO and Rh; Direct antiglobulin test (DAT). If the newborn's DAT is positive: Elution of newborn's red cells to prepare an eluate containing the sensitizing antibody (unless assessed to be passive anti-D from RhIg*); Antibody identification using eluate. Antibody in the eluate should correspond to at least one antibody found in the maternal serum.* The exception is a positive DAT in a newborn whose mother received RhIg antenatally and who has anti-D at delivery. If other maternal antibodies have been excluded, the positive DAT is assumed to be from RhIg and no elution is performed.

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Maternal antibody titer is a good indicator of severity of HDFN.View Page
Molecular Genotyping - Introduction

The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. Molecular methods exist for typing Rh (RHD and RHCE), Kell (K & k), Duffy (Fya & Fyb), and Kidd (Jka &Jkb) loci.In perinatal testing programs, molecular typing can determine the Rh type of the mother, father, and fetus and may be done if the mother has anti-D or another antibody known to cause HDFN. More specifically, if available, DNA methods are typically used in these circumstances: For women who type as weak D in serologic tests, to determine the Rh genotype of the mother to identify if she is partial D or weak D; For women who have made anti-D, to determine the Rh genotype of the father to see if fetal monitoring is needed; For women who have made anti-D, to determine the Rh type of the fetus if the father is heterozygous for RhD or unavailable for testing. Fetal blood typing can be done using fetal DNA from cells obtained by amniocentesis or by testing cell-free, fetal-derived DNA present in maternal plasma at 5 weeks gestation and later. Like all diagnostic methods, DNA typing has limitations and is not 100% sensitive and specific. For example: The blood group's molecular basis may be unknown; Not all alleles in ethnic populations are known; Rare mutations in the RHD and other genes may not be detected; Silencing changes (switching off of a gene) may affect antigen expression; Fetal typing using amniotic fluid may give false-negative results because of maternal cell contamination.

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When monitoring maternal antibody strength using a doubling dilution, an increase in titer from 16 to 32 is considered a significant rise in titer.View Page
Routine Serologic Tests - Father

Policies for typing fathers vary widely and usually testing is not done unless the mother develops anti-D or another clinically significant antibody. However, for Rh negative women, some labs consider Rh typing the father if paternity is certain. For example: Tests on Father ABO and Rh type; Test for weak D if initial Rh typing appears to be D-negative. If father is Rh negative, the fetus will be Rh negative and antenatal RhIg is not needed. The purpose of DCEce typing Rh positive fathers is to determine if the father is homozygous or heterozygous for D in order to predict whether the fetus is Rh positive. The father's actual Rh genotype can be determined by molecular methods, if available.

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Molecular Genotyping - Father and Fetus

Rh Genotype (Father and Fetus)As noted, usually molecular typing of the father is done only if the mother has anti-D or an antibody to another antigen for which molecular methods exist. In the case of a mother with anti-D and a father who is D+ using serologic methods, molecular typing can determine the father's RHD heterozygosity or homozygosity*. If the father is homozygous for the RHD allele, all of his offspring will be Rh positive, negating the need for fetal D testing, but indicating that the fetus should be monitored for HDFN. If the father is heterozygous for RHD, the Rh type of the fetus should be determined to see if HDFN is possible. * 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 (see later).

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Immunogenicity

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.

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Newborn Serologic Testing Protocols

Protocols for testing newborns vary internationally and within countries. The table below summarizes some of the more common protocols. Scenario Typical Newborn Testing Protocol Comments Mother is D-negative with no unexpected antibodies Newborn is tested at delivery for: ABO and Rh Test for weak D (mandatory) if initial Rh typing appears to be D-negative Direct antiglobulin test (DAT)* A positive DAT does not always mean that the newborn has clinically significant hemolysis. A positive DAT commonly occurs due to ABO incompatibility, yet infants seldom require treatment. Infants born to mothers who received antenatal RhIg sometimes have a positive DAT that does not cause clinically relevant hemolysis. Mother is Rh positive and a blood group other than group O Routine testing not performed Cord blood retained for a specified period of time (e.g., seven days) in the event that the mother has an unexpected antibody at delivery or the newborn develops signs of red cell hemolysis. Routine testing would result in many positive DATs due to ABO incompatibility- not clinically significant. Mother is group O Rh positive Newborn is tested- especially important if women and their infants are discharged within 24 hours since hyperbilirubinemia due to ABO HDFN may develop later. Optional only if there is appropriate surveillance and risk assessment before discharge and provided there is follow-up (American Academy of Pediatrics). *Policies for DAT testing of newborns whose mothers have received antenatal RhIg vary internationally. For example, the British Committee for Standards in Haematology guidelines state that a DAT should not be performed on cord blood routinely since in some cases it may be positive due to antenatal RhIg prophylaxis. A DAT is recommended only if HDFN is suspected because of a low cord blood hemoglobin or the presence of unexpected maternal antibodies. However in North America, DATs are always performed on infants born to Rh negative mothers who are RhIg candidates.

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RhIg 'Failures'

Numerous studies have shown that, if administered correctly, RhIg is effective at preventing D immunization. To work, RhIg must be given in sufficient dose, and it must be given before Rh immunization has begun.Unfortunately, despite RhIg's proven efficacy, some women continue to make anti-D in the perinatal period. Such 'failures' are mainly (but not totally) due to human error. Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis: Immunization to D occurred before the administration of RhIg, e.g., before 28 weeks gestation*; Immunization to D occurred after the administration of RhIg at 28 weeks and before delivery because an antenatal fetomaternal hemorrhage (FMH) occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIg administration; RhIg dosage was insufficient to clear a larger fetal bleed at delivery (e.g., FMH screen was not done or a false negative occurred); Incorrect calculation of RhIg dosage; RhIg administered too late , e.g., well after 72 hours of delivery; Antenatal RhIg not given, e.g., mother had no, or limited, access to prenatal care, or did not seek it, and a FMH occurred during pregnancy; Failure of physician to carry out prenatal blood testing; RhIg not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIg not given in cases such as abortions, ectopic pregnancies, and trauma (e.g., car accidents).* Because anti-D production before 28 weeks is rare (the order of 0.24% to 0.31%), RhIg's use earlier in pregnancy is not recommended. It is not cost effective and would expose most women to an unneeded blood product.

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Passive Anti-D following RhIg Administration

A scenario where anti-D is detected at delivery in a female who received RhIg during pregnancy raises the question, is the anti-D active or passive?Distinguishing between passive and immune anti-D is important clinically: If passive anti-D is misinterpreted as active, RhIg prophylaxis may be omitted, leading to D sensitization. If active anti-D is misinterpreted as passive, appropriate antibody investigation may be curtailed putting the fetus at risk of developing HDFN.When this occurs, two main serologic questions need to be answered: Are the reactions due to passive anti-D from RhIg or due to active anti-D? Are there other antibodies that need to be excluded?

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Protocols to Deal with RhIg-Derived Anti-D

Laboratories use different protocols to confirm anti-D from RhIg administration and simultaneously exclude other antibodies in pregnant females at delivery.The following protocols are examples only and assume that the patient has: Received RhIg (this needs to be confirmed); That the antibody screen is positive (2+ or less); Antibody reacts only in the IAT phase and only with D+ screen cells.In other words, the following protocols assume that the antibody looks like a relatively weak IgG anti-D consistent with RhIg administration.Antibody Exclusion Protocols1. Mini-panelProbably the most common protocol is to perform a mini-panel to exclude other antibodies and report "probable passive anti-D due to RhIg administration;" "passive anti-D consistent with recent RhIg administration" or similar.Some commercial panels indicate which panel cells are useful to rule out other antibodies in the presence of anti-D. 2. Full panelSome labs do a full antibody identification panel to confirm anti-D and exclude other antibodies. This protocol is acceptable, but may be overkill, given that the same results can be achieved with fewer red cells.Passive versus ImmuneUnfortunately, there is no definitive test to determine if anti-D is passive or immune. Some labs perform a titration with the assumption that an anti-D titer greater than 4 likelyrepresents active immunization. While a high titer can exclude passive anti-D, a low titer cannot.This issue is discussed in detail in Rh Negative Female with Anti-D at Delivery: A Case Study on Dealing with the Issues, a case study that complements this course.

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HIV Safety for Florida
Before a HIV antigen or antibody test can be ordered, informed consent must be obtained.View Page
Informed Consent

Informed consent must be obtained first before a HIV antigen or antibody test can be ordered.Informed consent must be preceded by: Explanation of the test subject's right of confidentiality. Notification that a positive HIV test result will be reported to county health department with enough information to possibly identify the test subject. Availability and location of sites where anonymous testing is performedInformed consent can be given by a legal guardian or other person authorized by law when the test subject is: not competent incapacitated a minor

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Human Papillomavirus (HPV) and Molecular Diagnostic Testing
Step 4 High-Risk HPV HC2 DNA Assay

Multiple antibodies for RNA:DNA hybrids conjugated with alkaline phosphatase are added. These labeled antibodies attach to the hybrids and form sandwiches. The three components of each sandwich are: capture antibody: hybrid : labeled antibodies. The microplate is washed to remove any unbound alkaline phosphatase-labeled antibodies. Since multiple labeled antibodies attach to each hybrid, the signal is amplified 3000 fold.

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Twenty-five samples of isolated DNA from liquid cervical cytology specimens, one negative control, and one positive control were assayed with the High-Risk HC 2 DNA assay. All patient samples and both controls were positive for HR-HPV types.Which one of the following is likely the cause of the false positives?View Page

Introduction to Flow Cytometry: Blood Cell Identification
Extrinsic Cell Characteristics

As each cell makes contact with the laser beam, the instrument determines cellular intrinsic and extrinsic properties. These extrinsic characteristics are reflected by the presence of a fluorescent signal emitted by monoclonal antibodies that are bound to specific cellular antigens on the cell surface. The cell image below shows a cell stained with two different monoclonal antibodies, each with a different fluorochrome. Each fluorochrome will emit a different color when it intersects the laser.The second image below is an example of a histogram display. It represents the monoclonal antibody for CD5 labeled with FITC dye. When this dye intersects a 488 nm argon ion laser beam, it emits a fluorescent signal in the green portion of the spectrum (don't be confused by the red peaks; that is simply how this particular flow cytometer is programmed to display every color on a single color histogram).

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CD19 and CD20

These images represent fluorescent monoclonal antibody data for CD19 and CD20 respectively. The peak to the left of the vertical line is negative and the peak to the right is positive. This means that the percentage of cells in the gated population (lymphocytes) that have CD19 cell surface antigens is 6.8% and CD20 positive cells comprise 6.7% of that population.A CD5 analysis further confirmed that the B-cell population was CD5-negative.The B-cell expression at this point is:CD5=0%CD19= 7%CD20= 7%

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Definitions

Important Flow Cytometry DefinitionsCluster designation or cluster differentiation (CD): A group of antibodies that recognize the same antigen.Fluorochrome (fluorophore): Fluorescent marker that is excited by light of one wavelength (generated by the laser of the flow cytometer) and emits light of a different wavelength (fluorescent light). Gate: A boundary that is set up around a subset of data points to segregate those events for analysis, or to exclude other events from the analysis.Immunofluorescence: Fluorescent expression of immune reactions between antigens and antibodies.Monoclonal antibody (MoAb): An antibody that is produced from a single clone of cells and therefore has high purity and reproducibility. A monoclonal antibody that is used in flow cytometry is directed against a single antigen. For example, the CD2 MoAb will bind to the CD2 antigen on the cell surface of a T cell.

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Sample Preparation

Sample preparationSamples for flow cytometric analysis are prepared based on the following procedural steps. Prepare the sample according to cell type. Bone marrow (BM) and peripheral blood (PB): Prepare a blood smear, stain with Wright's stain, and scan under the microscope to identify basic cell distribution and morphology. BM can contain spicules; these samples need to be filtered. Tissue: Mince and filter tissues in a sterile cell culture media to release cellular components from the solid tissue form. Fluid: Filter fluid, prepare a cytospin, and scan under a microscope to identify cellular components. Obtain a white blood cell (WBC) count. Unless red blood cells are the population of interest, they should be lysed with a mild agent that will preserve the integrity of the cells targeted for analysis. If the red blood cells are not lysed, they lead to false analytic results. Adjust the WBC count by concentrating the WBCs to optimize for ‘staining' with monoclonal antibodies (MoAbs). Incubate the prepared cell concentration with assorted monoclonal antibody concentrations to allow antigen-antibody complexes to form. Lyse red blood cells (RBCs) with ammonium chloride or equivalent that will preserve cellular viability and integrity. The purpose is to eliminate RBCs while maintaining WBC integrity. If RBCs remain in the sample, they will interfere with the cell scatter plot and skew the results. Centrifuge to precipitate cells. Pour off supernatant. Wash in phosphate buffered saline (PBS) or equivalent to eliminate cellular debris and unbound MoAbs, centrifuge, and decant. Resuspend cells in PBS. Analyze cells using flow cytometer.

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Introduction to the ABO Blood Group System
The History of the ABO System, continued

Landsteiner, knowing that none of his subjects had been immunized, realized that “natural” antibodies must develop which are directed against antigens not present on the red cells. Individuals with “A” antigens on their red cells had sera containing “Anti-B” antibody. Individuals with “B” antigens had sera containing “Anti-A.” “AB” individuals had sera with no ABO antibodies present and “O” individuals’ sera contained “Anti-A” and “Anti-B.” Sera from group O individuals may contain a separate antibody, “Anti-A,B.” Anti-A,B possesses serologic activity not found in mixtures of Anti-A and Anti-B. Anti-A,B sera will agglutinate A, B, and AB cells. It is particularly useful in detecting weak A and B antigens. See the table on the next page.

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Importance of Understanding the ABO System

The predictability of ABO antibodies appearing in serum lacking the corresponding antigens makes ABO typing a simple process in most cases. However, the importance of getting it right cannot be stressed enough when a patient will be transfused with blood from a donor. If a patient receives donor cells containing A or B antigens and the transfused patient's serum contains the corresponding antibody, the donor cells will be destroyed almost immediately, causing a severe (hemolytic) and sometimes fatal reaction. Therefore, it is of utmost importance to thoroughly understand the ABO blood group system. 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.

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ABO Antibodies

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.

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Immune ABO Antibodies

A person exposed to a specific immunizing event may produce “immune” ABO antibodies of the same specificity as the naturally occurring antibody, but with different biological behavior. Such immunizing events include pregnancy with an ABO incompatible fetus, or transfusion of ABO incompatible red cells. After immunization, the subject’s antibody may increase in titer and/or avidity, develop powerful hemolyzing properties, or become more active at 37°C.

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A1 and A2 Subgroups

The most common subgroups of group A phenotype are A1 and A2. These account for over 99% of individuals who are classified as Group A. Of this 99%, A1 comprises approximately 80%. Commercial anti-A typing serum does not differentiate between A1 and A2 cells. A1 cells contain "A" antigen and "A1" antigen. A2 is not really a unique antigen. It is thought to be simply "A" antigen with no "A1" antigen. Several preparations are available that will react with A1 cells, but not other subgroups of A. The most commonly used reagent is Anti-A1 lectin, an extract of the seeds of the plant, Dolichos biflorus, which has specific anti-A1 activity.Approximately 4% of individuals who are subgroup A2 have naturally occurring anti-A1 in their serum.

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Rare Subgroups of A

Other rare subgroups of A exist. These comprise less than 1% of the total pool of A genes. Of these rare types, A3 is the most common, but Ax, Aend, and Ael have also been identified. In subgroup A3, the red blood cells characteristically give a “mixed field” agglutination pattern when tested with anti-A and anti-A,B. Small clumps of agglutinated cells are present among large numbers of cells, which absorb the antibody to their surface but are not agglutinated by it. Mixed field reactions or weak reactions should be further investigated before the ABO type is interpreted.

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Why Knowledge of A Subgroups Is Important For Laboratorians

For the most part, subgroups are merely of academic interest, but occasionally they present clinical problems. The antigen may be so weak that it is not detected and the red cells are mistyped as group O. This is especially dangerous if the cells are those of a donor. Problems may arise because the serum of an A2 or A2B, A3, or Ax individual might contain anti-A1. This antibody may be detected in serum typing and cause confusion. You would not expect to find a person with A antigen on his/her red cells and anti-A in the serum. Anti-A1 is produced by about 4% of group A2 individuals and about 25% of group A2B individuals. Subgroups may be determined by reactions with antisera as seen in the table on the next page.

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Given the results below, what is the most probable ABO type for this individual?Forward (Cell) Grouping Reverse (Serum) Grouping Anti-AAnti-BAnti-A,BA1 CellsB Cells4+04+2+4+View Page
Agglutination Reactions

Antibodies of the ABO system cause agglutination of saline-suspended red cells at 4°C to 20°C. Heating to 37°C weakens the reaction. "Naturally" occurring ABO antibodies may not be strong enough to agglutinate cells without centrifugation. Thus, testing serum for the presence of anti-A or anti-B has classically been performed using the tube system in which serum and cells added to a test tube are centrifuged and then evaluated for agglutination. A slide test has also been performed for forward reactions. Although tube tests are still in wide use, newer systems utilizing other technology such as gel agglutination are becoming more prevalent. The image on this page illustrates agglutination reactions observed with the tube system, from 4+ in the topmost image, to 0 in the lowest image. ABO reactions should be strong. Weak or missing reactions occur, but must be "resolved" before blood products can be released.4+ agglutination: Red blood cell button is a solid agglutinate; clear background.3+ agglutination: Red blood cell button breaks into several large agglutinates; clear background.2+ agglutination: Red blood cell button breaks into many medium-sized agglutinates; clear background; no free red blood cells.1+ agglutination: Red blood cell button breaks into many small clumps barely visible macroscopically; background is turbid; many free red blood cells.Negative: No agglutinated red blood cells present; red cells are observed flowing off the red blood cell button during the process of grading.Other reaction which may occur are the mixed-field reaction, in which mixtures of agglutinated and unagglutinated red blood are present; and hemolysis, in which red cells are hemolyzed by the antibody. Both of these patterns are considered positive reactions.

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Automated Systems

An increasing number of transfusion services are using automated blood banking systems. These systems may employ either solid phase or gel techniques. Use of automation may increase productivity, reduce costs, and, by decreasing the number of manual steps in the testing process, potentially reduce errors.

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Example of an ABO discrepancy

The composite image shown on the right illustrates the ABO typing reactions that were obtained for a patient. This particular case illustrates an ABO discrepancy. An ABO discrepancy occurs when the results of forward and reverse typing do not match. The reactions shown are described below in descending order:Patient red cells with reagent anti-A: negative reaction.Patient red cells with reagent anti-B: 4+ agglutination.Patient red cells with reagent anti-D: 4+ agglutination.Patient serum with reagent A1 red cells: negative reaction.Patient serum with reagent B red cells: negative reaction.This patient forward types as a group B, but reverse types as a group AB. (A group B patient should have anti-A. This patient demonstrates neither anti-A nor anti-B, similar to an AB patient). Further workup is necessary to determine the ABO type since the forward and back typing do not match. In this case, incubation at 40 C demonstrated the presence of weakened anti-A. The patient was therefore typed as group B. This case is an example of an ABO discrepancy which was due to a "missing" anti-A antibody. This could be due to old age, severe illness, or immunosuppression. Although evaluation of ABO discrepancies is beyond the scope of this course, it is important to note that all ABO discrepancies must be resolved before blood products can be released for transfusion.This patient is Rh (D) positive, as evidenced by the strong agglutination of his cells with reagent anti-D antibody.

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel (retired 9/1/2010)
Description of Specialties (4)

Specialists in cytogenetics detect chromosome abnormalities and genetic disorders. Cytogenetics counseling may only be performed by an individual licenses in the cytogenetics specialty at the director level. Specialists in molecular genetics analyze DNA and RNA to find disease-related genotypes, mutations, and phenotypes in order to detect or predict disease and identify carriers. Specialists in histocompatibility test to determine tissue compatibility, prevent infections, and investigate and post-transplant problems. Techniques include blood typing, HLA typing, HLA antibody screening, disease markers, flow cytometry, crossmatching, HLA antibody identification, lymphocyte immunophenotyping, immunosuppressive drug assays, allogenic, isogeneic and autologous bone marrow processing and storage, mixed lymphocyte culture, stem cell culture, cell mediated assays, and assays for the presence of cytokines. Specialists in andrology and embryology examine gametes and embryos, including production, morphology, number, and motility, to address issues of fertility and infertility.

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Microbiology / Serology Question Bank - Review Mode (no CE)
Which one of the following viruses requires a complex lymphoblastoid cell culture, and is rarely if ever diagnosed by culture:View Page
The capsular material used to identify capsular subtypes of Pneumococci consists of:View Page
VDRL is an example of which of the following types of tests:View Page
The Quelling test is useful for which of the following :View Page
Which of the following tests would be used to directly document the presence of a specific organism in a clinical specimen:View Page

Molecular Methods in Clinical Microbiology
The Key Benefits: Improved Sensitivity of Detection, continued

Some organisms are present in infections in very low numbers, which may be undetectable with direct staining methods. These organisms may also prove to be difficult to recover with currently available culture methods.Although non-culture antigen methods have been developed to address some of these difficulties (examples include direct fluorescent antibody (DFA) and enzyme immunoassay (EIA) methods), the sensitivity of these methods has not always been desirable.Molecular methods offer the prospect of:Detecting nonviable organisms that did not survive transport Detecting organisms difficult/impossible to cultivate Detecting organisms present in low numbers Providing better detection capability than other non-culture methods

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Prior Traditional Methods and the Need for Change

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.

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Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
Treatment of Clostridium difficile Infection (CDI) and C. difficile Associated Disease (CDAD)

The first step in treating patients with CDAD is to discontinue the causative agent wherever possible. The choice for initial antibiotic therapy depends on the severity of disease. Oral vancomycin or metronidazole remain the mainstays of therapy for CDI, with vancomycin reserved for patients with more severe disease and/or those who have not responded to metronidazole. Metronidazole is currently favored in guidelines from the Centers for Disease Control and Prevention (CDC) on the basis of cost and concern that oral vancomycin promotes colonization with vancomycin-resistant Enterococcus. Oral fluids (water and electrolytes) may be necessary to counteract fluid loss as a result of excessive diarrhea, which can quickly lead to dehydration. Patients with fulminant disease and toxic megacolon may require colectomy. Recurrence of CDI is becoming an increasing problem. Most recurrences happen 7-14 days after completion of therapy, suggesting relapse rather than re-infection. If a patient develops a second episode of CDI following initial successful treatment, it is recommended that if possible, the same drug be used to treat the second episode. Contributing factors to recurrent CDI include:Continuing exposure to organisms either through re-infection (via contaminated environment or poor hand hygiene) or an endogenous source, such as C. difficile spores in GI tract. An inability to mount an adequate anti-Toxin A IgM and/or IgG antibody response (i.e., poor host immune response); a likely reason why CDI affects an increasingly elderly population. Unfortunately a vicious cycle can arise whereby the initial treatment prescribed, vancomycin or metronidazole, significally disrupts normal colonic flora reducing colonization resistance and leaving the patient vulnerable to the next recurrent episode.Other treatments, including the use of probiotics or anion-exchange resins to absorb toxins, may work in some cases but none work in every case.The goal of all treatment is to reestablish normal colonic flora so as to control C. difficile (over)growth.

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Normal Peripheral Blood Cells
Match the following cells with their corresponding characteristics:View Page
Glossary of Terms A through M.

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.

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Normal Peripheral Blood Cells (retired 6/20/2012)
Match the cells with their characteristics.View Page
Glossary of Terms A through M.

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.

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OSHA Bloodborne Pathogens
What Happens After HIV Infection?

Days to weeks after exposure, the patient may begin to complain of fever, headache, and fatigue. This may also be accompanied by a rash.For the first several months after the infection, the exposed individual may be HIV-antibody negative and the disease may not be detected. However, the individual is still infective and can transmit the disease during this period.The disease may remain silent in the patient for months to years, even with no treatment.When the immune system is weakened enough, the patient will develop opportunistic infections and be classified as having acquired immunodeficiency syndrome (AIDS).

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Packaging and Shipping Infectious Materials (retired July 2013)
IATA and US Postal Service Exempt Specimens

Laboratory specimens that are unlikely to cause disease and do not meet the criteria for category A or B substances are not subject to Division 6.2 regulations. Specimens for which the hazardous materials regulation (HMR) does not apply include human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) being transported for routine testing not related to the diagnosis of an infectious disease. This includes specimens that are being sent for:drug or alcohol testing cholesterol testing blood glucose level testing prostate specific antibody (PSA) testing testing to monitor kidney or liver function pregnancy testing tests for diagnosis of non-infectious diseases such as cancer biopsies The US Department of Transportation (DOT) has no "Exempt Specimen" classification and there are no DOT guidelines for packaging non-regulated specimens.* According to the DOT, in the U.S., if a package is marked as "Exempt Human/Animal Specimen" the understanding is that it contains no infectious substance. However, both IATA and the US Postal Service (USPS) have these requirements for packaging exempt specimens: Packaging IssueIATAUSPSType of packaging requiredTriple packagingTriple packagingOuter containerOne dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet One dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet Quantity limits: outer containerNone NoneQuantity limits: Primary receptacleNone500 mLQuantity limits: secondary packagingNone500 mL* Non-regulated specimens may become regulated because of preservatives, such as 10% formaldehyde (class 9) or 25% formaldehyde (class 8); or 25% ethanol (class 3).

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Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics (retired 10/15/2012)
PETINIA

Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) is a homogeneous competitive immunoassay.Antibody fragments and drug-latex particles will bind to form aggregates that increase the turbidity of the solution. Free drug from the sample competes for the antibody fragment, thereby decreasing the rate of particle aggregation. The rate of aggregation is inversely proportional to the concentration of drug in the sample.

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FPIA

Fluoresence polarization immunoassay (FPIA) is also a homogenous competitive immunoassay. In this system, fluorescein-labeled drug competes with unlabeled drug from the patient's serum sample for binding sites on an antibody reagent. The patient's sample, presumably containing the therapeutic drug that is being monitored, and the fluorescein-labeled drug are added to a chamber containing antibody for that drug. The labeled and unlabeled drug will compete for binding sites on the antibody. The greater the amount of drug in the sample, the fewer the number of binding sites that are available for the labeled analyte, leaving a greater number of small, free fluorescein-labeled molecules in the solution.When the chamber is excited with plane polarized light, fluorescein will absorb the light and emit it at a higher wavelength as fluorescent light. A small, free fluorescein-labeled drug rotates randomly and faster than it would if it were bound to antibody, interrupting the light and leading to less emission of light. The larger antibody-drug-fluorescein complexes rotate slower and emit more light in the measured plane. A lower level of drug in the patient's sample results in greater emission of polarized light because there are more antibody-drug-fluorescein complexes present to produce light in the measured plane. A higher level of drug in the patient's sample results in a lower emission of polarized light. This inverse relationship between the concentration of the drug and the polarization units (signal) is illustrated in the image below.

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Phlebotomy
TORCH panel

TORCH panelToxoplasma antibody Rubella antibodyCMV antibody Herpes antibody

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Acute hepatitis panel

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

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Obstetric panel

CBC Hepatitis B surface antigen Antibody, rubellaSyphilis test (RPR) Antibody screen Blood type, Rh and ABO

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Preliminary Identification of the Primary Select Agents of Bioterrorism
Toxins

Toxin Comment Most Likely Means of Dissemination Primary Route of Entry General Signs and Symptoms Laboratory Testing Botulism toxin: Gram stained image of C. botulinum courtesy of CDC Produced by Clostridium botulinum Could be purified and used in a bioterrorist event to contaminate food or aerosolized to cause disease Aerosol Food contamination Inhalation Ingestion Difficulty speaking or swallowing Blurred or double vision Drooping eyelids (ptosis) Dilated pupils Dry mouth, decreased gag reflex Weakening of the reflexes (hyporeflexia) Abnormal sensations such as numbness, tingling, and progressive arm or leg weakness Flaccid paralysis Culture, anaerobic Digoxigen-labeled IgG ELISA to detect A, B, E, and F toxins Mouse Bioassay for all toxin types and to confirm DIG ELISA Ricin toxin: Extracted from Castor beans Inhibits protein synthesis Causes death approximately 72 hours after initial exposure As an aerosol Inhalation Fever Cough Chest tightness Dyspnea Cyanosis Gastroenteritis Necrosis Antibody detection in clinical specimens Clinical testing not performed unless known exposure has occurred

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Rouleaux and Agglutination

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

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A 49-year-old male with pneumonia was treated with high-dose intravenous penicillin. He became jaundiced with yellow sclera. The image on the right is typical of other fields that were observed on his peripheral blood smear.Since penicillin may, in some individuals, cause autoimmune hemolytic anemia, the clinician requested a direct antiglobulin test (DAT) be performed. The DAT was positive, indicating that antibodies to the drug were produced, which then attached to the drug on the surface of the red cells. Hemolysis occured due to the drug-induced antibody attachment, leaving the patient with various abnormal red blood cell morphologies. Which of the following cell types would you report for this patient?View Page

Red Cell Morphology
Autoagglutination

A second type of irregular arrangement is erythrocyte agglutination. Agglutination is recognized by irregularly sized and spaced clumps of several to many erythrocytes. It may be impossible to visualize individual cells if a high degree of agglutination is present. Autoagglutination may indicate the presence of a cold reacting antibody in the patient sample, which reacts with erythrocyte antigens during slide preparation. In this case, warming the blood sample to 37oC before making a repeat slide may eliminate the problem. Agglutination is observable at a magnification of 400X. True rouleaux and agglutination may each be observed throughout all areas of the blood smear, from feathered edge to the start of the smear.

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Rh negative female with anti-D at delivery: A case study
Case Presentation

Patient A.D., a 30 year old female, was admitted to the hospital in active labor to deliver at 37 weeks gestation. Transfusion service (TS) records showed A.D. to be group O Rh negative with no record of unexpected red cell antibodies.Maternal history showed two prior pregnancies. Her first pregnancy four years ago ended in spontaneous abortion at 9 weeks gestation and she received a mini-dose (50 µg) of RhIg.In the second pregnancy, two years ago, the infant typed as Group A Rh positive, DAT negative. Patient A.D. was injected with RhIg within 72 hours of delivery. The laboratory also confirmed that in the current pregnancy RhIg was administered at approximately 28 weeks gestation subsequent to a negative antibody screen.After many hours of non-productive labor, the physician considered that labor had stalled and decided to do a cesarian section (C-section). According to hospital policy for C-sections, a type and screen was ordered.

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Introduction

This case concerns a common scenario in the transfusion service (TS) laboratory, the detection of anti-D at delivery in a female who has received Rh immune globulin (RhIg) during pregnancy.Distinguishing between passive and immune anti-D is important clinically:If passive anti-D is misinterpreted as immune, RhIg prophylaxis may be omitted leading to D sensitization. If immune anti-D is misinterpreted as passive, appropriate follow-up of the antibody may be curtailed putting the fetus at risk.Unfortunately, differentiating between immune and passive anti-D is often impossible. This case study presents an opportunity to review perinatal testing programs and the crucial role of RhIg in preventing hemolytic disease of the fetus and newborn (HDFN) due to anti-D. The case also examines practical aspects of routine serologic testing involving neonates and women who have received RhIg during pregnancy. In brief, the case will: Guide participants through laboratory findings that need to be interpreted and resolved; Examine current best practices in perinatal testing programs; Review the characteristics of RhIg and its use in pregnancy; Review and investigate key issues associated with detection of anti-D in women who have received antenatal RhIg; Discuss crossmatch and LIS policies related to RhIg-derived passive anti-D.

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RhIg 'Failures'

Numerous studies have shown that, if administered correctly, RhIg is effective at preventing D immunization. To work, RhIg must be given in sufficient dose, and it must be given before Rh immunization has begun.Unfortunately, despite RhIg's proven efficacy, some women still make anti-D in the perinatal period. Such 'failures' are mainly (but not totally) due to human error. Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis: Immunization to D occurred before RhIg was administered, e.g., before 28 weeks gestation*; Immunization to D occurred after the administration of RhIg at 28 weeks and before delivery because an antenatal FMH occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIg administration; RhIg dosage was insufficient to clear a larger fetal bleed at delivery (e.g., FMH screen or quantification was not done or a false negative occurred); Incorrect calculation of RhIg dosage; RhIg administered too late , e.g., well after 72 hours of delivery; Antenatal RhIg not given, e.g., mother had no or limited access to prenatal care, or did not seek it, and a FMH occurred during pregnancy; Failure of physician to carry out prenatal blood testing; RhIg not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIg not given in cases such as abortions, ectopic pregnancies, and trauma (e.g., car accidents). * Because anti-D production before 28 weeks is rare (the order of 0.24% to 0.31%), RhIg's use earlier in pregnancy is not recommended. It is not cost effective and would expose most women to an unneeded blood product.

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Antibody Exclusion Protocol (General)

Transfusion service (TS) laboratories use different protocols to exclude antibodies. For example:For antibodies whose corresponding antigens exhibit dosage, some laboratories exclude them based on a negative reaction with one homozygous cell.* Other laboratories require negatives with two homozygous cells to increase the confidence that the antibody is not present.If a homozygous cell is not available, some laboratories exclude such antibodies based on a negative reaction with two heterozygous cells.* Other laboratories require negatives with three heterozygous cells to increase confidence that the antibody is not present. * Homozygous and heterozygous do not refer to the red cells per se but to the red cell donors who are homozygous or heterozygous for the genes that determine the red cell phenotypes. See the general antibody exclusion protocol to be used in this case.

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Using the initial screen cell antigram below, which antibodies have not been eliminated? Include all antibodies even if they are unlikely to cause HDFN.Screen CellRhRhesusKellDuffyKiddMNSsPLewisResultsCellCDEceKkFyaFybJkaJkbMNSsP1LeaLebGelIAT1R1R1++00+0+++0++00++002+12R2R20+++0++0++++++++0+3+23rr000++0++00+0++0+S+003Auto0AutoView Page
Antibody Exclusion Protocols (RhIg)

Laboratories use different protocols to confirm anti-D from RhIg administration and simultaneously exclude other antibodies in pregnant females at delivery. The following are sample protocols (not all inclusive) with comments. All protocols assume that the patient has received RhIg (this needs to be confirmed); that the antibody screen is positive (2+ or less); reacts only in the IAT phase; reacts only with D+ screen cells.In other words, the following protocols assume that the antibody looks like a relatively weak IgG anti-D consistent with RhIg administration.1. Mini-panelProbably the most common protocol is to perform a mini-panel to exclude other antibodies and report "probable passive anti-D due to RhIg administration"; "passive anti-D consistent with recent RhIg administration" or similar. Some commercial panels indicate which panel cells are useful to rule out other antibodies in the presence of anti-D. A mini-panel of 5-7 carefully selected red cells is typically all that is needed.2. Full panel: Some labs do a full antibody identification panel to confirm ant-D and exclude other antibodies. This protocol is acceptable, but may be 'overkill' given that the same results can be achieved with fewer red cells.

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Factors Affecting RhIg Reaction Strength

Red cell reaction strengths at delivery from an antenatal RhIg injection at 26–30 weeks (usually 28 weeks) are typically 2+ or less, although stronger reactions are possible depending on the detection method, time since injection, and other factors. Multiple variables can affect the reaction strength of passive anti-D seen post-RhIg injection: Amount of RhIg injected (the greater the number of IU of anti-D administered, the stronger reactions will be); Titers of anti-D in the plasma pool used to manufacture RhIg (occasionally a donor with an exceptionally strong anti-D may be in the pool); Maternal physical size and related blood volume (a larger volume of maternal plasma will dilute RhIg more); Time between RhIg administration and testing (passive antibody will decrease in strength over time); Sensitivity of antibody detection method (e.g., gel-IAT and PEG-IAT may give stronger reactions than LISS-IAT); Volume of FMH (amount of D-positive fetal RBC available in the mother to adsorb anti-D); Route of RhIg administration: Some RhIg products can be administered IM only, whereas others can be given both IM and IV (see later). Peak levels of RhIg are reached faster with IV compared to IM administration (within hours with IV administration compared to days with IM administration). Also, with IV administration, higher levels of IgG anti-D are achieved. Operator variability (technologist techniques vary in removing cell buttons when reading IATs). Because of these variables, many laboratories consider 2+ or less reaction strengths to be consistent with passive anti-D.

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How Long Can RhIg Be Detected?

An issue related to reaction strength of RhIg in serologic tests is how long passive anti-D from RhIg can be detected post-injection. The half-life of IgG is 23 to 26 days. Following injection of RhIg, serologically detectable levels of anti-D peak within hours (IV injection) or days (IM injection).Although the half-life of passive anti-D from RhIg is approximately 3 weeks, it may be detectable by serologic tests for approximately 8 weeks by the indirect antiglobulin test (IAT) and up to 12 weeks or more by continuous flow analyzers used to quantify anti-D. Levels of passive anti-D will decrease over time.Immune anti-D becomes detectable later (e.g., ~4weeks after exposure to D+ red cells), and generally reaches a peak after 6–8 weeks. Levels of immune anti-D will remain constant for longer and will increase following exposure to another immunizing dose of fetal D+ cells. Depending on the many variables that can affect reaction strength (mentioned earlier), as detected serologically, passive anti-D from RhIg can be detected for about 8 weeks or longer by routine, sensitive antibody detection methods.Since RhIg is injected at about 28 weeks, it is routinely detected at delivery, which could occur well before the ~40 weeks considered to be normal gestation (37–42 weeks by the World Health Organization).

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How Much Testing?

As noted, policies for further testing to confirm anti-D, exclude other antibodies, and assess whether the anti-D is passive or immune vary among TS laboratories. Even though patient A.D. had a negative antibody screen at 28 weeks and her positive antibody screen appears to be anti-D from RhIg administration at 28 weeks, some TS laboratories may set up a full antibody identification panel to confirm the presence of anti-D. Others would proceed straight to a mini-panel of red cells specifically selected to exclude other clinically significant antibodies in the presence of anti-D.In this case the laboratory's protocol was to set up a mini-panel of six selected red cells (rr, r'r, and r'r cells), along with a positive Ror control and an autocontrol.

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Passive or Active Immunization?

Unfortunately, there is no definitive test to determine if anti-D is passive or active. Tests can be done that will suggest whether the anti-D is probably passive or active. However, many laboratories do not perform such testing routinely since it's both safe and efficient to consider a post-RhIg anti-D to be passive without further testing, thereby triggering a post-natal RhIg injection. Further testing would be done if serologic test results suggest an immune anti-D, e.g., 4+ reactions with D+ red cells.One test that may be used to try to interpret if anti-D is passive or active is antibody titration.

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Antibody Titration

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.

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Which of the following factors is most likely to cause a false negative antibody screen following RhIg injection?View Page
A pregnant female has been injected with RhIg antenatally and has a positive antibody screen at delivery. If the antibody has been confirmed as anti-D alone and reacts only weakly (1+ in the indirect antiglobulin test), the anti-D is definitely passive.View Page
A pregnant female who received RhIg at 28 weeks gestation has a positive antibody screen at delivery. If the antibody has been confirmed as anti-D alone and reacts 1+ in the indirect antiglobulin test with D+ red cells, performing a titration to investigate if the anti-D is immune is good practice.View Page
Introduction

As noted earlier, in this case study the laboratory's protocol is to set up a mini-panel, providing these criteria are met: Mother is Rh-negative and has been tested on two separate occasions; Laboratory has confirmed administration of RhIg prophylaxis; Result of current antibody screen is positive and typical of anti-D due to RhIg; There is no record or history of an unexpected antibody. All criteria were met and a selected mini-panel was set up to confirm the presence of anti-D and exclude possible co-existing maternal antibodies. Other clinically significant antibodies have implications for possible HDFN and for transfusion to both the mother and newborn, thus must be excluded.

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Mini-Panel Antibody Exclusion

Below are the results of a mini-panel of red cells specifically chosen to exclude other clinically significant antibodies in the presence of anti-D. Besides an autocontrol, a positive control (Ror) was included to confirm that the mother's plasma containing the probable anti-D was reactive at the time of testing. Recall that the results of the initial antibody screen showed that the possible (unexcluded) antibodies were anti-C, D, E, K, Fyb, Jka, M, s, Leb(with anti-M less likely as a cause of HDFN and anti-Leb not a cause).Antibodies excluded by Screen Cell #3 included anti-c, e, Fya , Jkb, N, S, P1 and anti-Lea.Before proceeding to the next page, assess whether the unexcluded antibodies from the initial antibody screen have been excluded by the mini-panel below using the guidelines in the antibody exclusion protocol.Mini-Panel ResultsCellRhRhesusKellDuffyKiddMNSsPLewisResultsCDEceKkFyaFybJkaJkbMNSsP1LeaLebGel IAT*1rr000+++++0+00++0+S+002rr000++0+0++0++0++S+003r'r+00++0++00++0+00+004r'r+00+++++++++0+++0+05r"r00+++0+0+0+++0+++006r"r00+++0++++++++++0+07Ror0++++0++++++++++0+2+8Auto0* IAT = indirect antiglobulin test All panel cells are negative for low frequency antigens and positive for high frequency antigens unless noted otherwise. All cells are also negative for Cw, Kpa, and Lua.

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Using the guidelines in the antibody exclusion protocol, all unexcluded antibodies (anti-C, E, K, Fyb, Jka, M, s, Leb) have been excluded by the mini-panel and the Ror control cell confirms reactivity of anti-D.View Page
Is the mother a candidate for RhIg? (type Y for yes or N for no)View Page
Crossmatch Issues

In this case the mother did not require transfusion. For reference, the TS laboratory routinely uses an electronic crossmatch to detect ABO incompatibility for cases where patients do not have unexpected clinically significant antibodies in current antibody screen tests nor a history of clinically significant antibodies. When the laboratory information system (LIS) is down, the lab performs an immediate spin crossmatch.Should transfusion have been needed, these questions arise:1. Does a mother with a detectable passive anti-D due to RhIg qualify for an immediate spin (IS) or electronic crossmatch should transfusion be necessary?The issue also extends to the future:2. Should having a record of passive anti-D that is no longer detectable disqualify a woman from being a candidate for an immediate spin or electronic crossmatch?Before proceeding, consider the policies used in your TS laboratory and which rationales are used to support them.

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Crossmatch Implications of RhIg-associated Passive Anti-D

Once again, policies vary from laboratory to laboratory since the issue is not directly addressed by blood safety standards. For example, AABB and other standards require a version of the following: When clinically significant red cell antibodies are detected or the recipient has a history of such antibodies, RBC components shall be prepared for transfusion that lack the corresponding antigen and are serologically crossmatch-compatible, where serologically is taken to be an IAT at 37oC. If no clinically significant antibodies were detected in antibody screen tests and the patient has no record of such antibodies, detection of ABO incompatibility is required, which can be accomplished by immediate spin crossmatch or an electronic crossmatch. The key issues are whether detectable passive anti-D from RhIg or a record of passive anti-D from RhIg should be considered clinically significant for crossmatch purposes. Because standards do not directly address these issues, TS laboratories are left to interpret what is required to meet the standards. Practices may be further complicated because of the transfusion service's laboratory information system (LIS).

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Crossmatch Practices Related to RhIg - Introduction

Crossmatch practices for RhIg-derived passive anti-D vary significantly. Inclusion here does not indicate endorsement or lack thereof for a particular policy, but merely documents that such policies exist. All policies assume the following scenario: An Rh negative female has received antenatal RhIg; She has a positive antibody screen consistent with passive anti-D; A mini-panel has been done to exclude other clinically significant antibodies and shows only anti-D; If performed to detect possible ABO incompatibility, the electronic crossmatch or immediate spin (IS) crossmatch is done with Rh negative RBC whose D typing is confirmed in-house.

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LIS Issues Related to RhIg

Before discussing crossmatch policies for women with passive anti-D likely due to RhIg, LIS-related issues will be outlined. A transfusion service's LIS and how it is configured determines under which circumstances an electronic crossmatch is possible.Regardless of crossmatch policy, almost all laboratories use a special designation or code in their LIS for anti-D likely due to RhIg. Often this designation is entered in the patient history comment field and not the antibody field, thus eliminating the need to remove the passive anti-D from the antibody field when the antibody disappears. Using this policy, once the passive antibody no longer reacts, the patient becomes eligible for an electronic crossmatch without the need to remove the antibody history.In essence, using a special designation for passive anti-D allows the lab to bypass the LIS's normal requirements for patients with clinically significant antibodies, i.e., allows them to omit doing an IAT crossmatch. Examples of how RhIg-derived anti-D is designated in lab information systems: Passive anti-D (eg., code 'PD', 'DPAS', etc.); Probably passive anti-D; Anti-D consistent with RhIg; Anti-D due to RhIg.Depending on the LIS, other variations are possible.

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Perinatal Testing Programs

Before proceeding with the case, let's review perinatal testing programs, also called 'Rh prevention programs' since they are designed to prevent HDFN due to anti-D.Perinatal testing programs have two main purposes:1. To detect, at an early stage in pregnancy, the presence of any IgG antibody that could cause HDFN in order to treat the infant as soon as possible.2. To determine which women are candidates for RhIg in order to try to prevent the production of anti-D. [RhIg will be discussed in detail later.]Testing programs include both Rh negative and Rh positive women, but because antibodies other than anti-D only rarely cause HDN, Rh negative females are tested more extensively.

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Routine Serologic Tests - Mother

Tests done routinely as part of perinatal testing programs vary from country to country and within countries. Below is one example of serologic tests typically done when pregnant females lack clinically significant antibodies. Other test protocols exist.Mother ABO, Rh, and antibody screen at first prenatal visit; Optional (not mandated by blood safety standards): Test for weak D, if initial Rh typing appears to be D-negative; D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks gestation prior to administration of RhIg (depending on the country) and again at delivery. Note: The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. When available, the mother can be typed for D using molecular methods, but this is usually not done unless she is weak D. The purpose is to determine using molecular methods which D variant the mother has, weak D or partial D, since the latter can produce anti-D. (see Further Reading)

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Routine Serologic Tests - Newborn Protocols

Protocols for testing newborns vary internationally and within countries.If the mother is D-negative and has no unexpected antibodies, newborns are always tested at delivery.Some labs do not test all newborns if the mother is Rh positive and especially do not test if the mother is a blood group other than group O. If all infants born to Rh positive women were tested, many positive DATs due to ABO incompatibility would be detected that are of no clinical significance. Instead cord blood is retained for a period (e.g., 7 days) should it be needed, for example, if the mother has an unexpected antibody at delivery or if the newborn develops signs of red cell hemolysis.However, some clinical practice guidelines, such as those of the American Academy of Pediatrics specify that testing infants born to group O Rh positive mothers is optionalonly if there is appropriate surveillance and risk assessment before discharge and provided there is follow-up. (See Further Reading) Not testing becomes an issue if group O women and their infants are discharged within 24 hours as occurs in some locations, since hyperbilirubinemia due to ABO HDFN may develop later. Therefore, some facilities where early discharge occurs require that all infants born to group O Rh positive mothers be tested.Typical protocols: Infants born to Rh negative mothers are tested; Infants born to Rh positive mothers who are group O are often tested, especially if early discharge is common (limiting surveillance); Infants born to Rh positive mothers who are not group O are often not tested and this is acceptable good practice. Cord blood is typically retained for a period should it be needed for testing later.

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Immunogenicity

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.

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ABO, Rh, and Antibody Screen

ABO and Rh typing ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D* 0 0 4+ 4+ 0 * Transfusion medicine standards used in the hospital's region do not require weak D testing on D-negative pregnant patients and none was done.Antibody screen Cells Gel IAT* Screen Cell I (R1R1) 1+ Screen Cell II (R2R2) 2+ Screen Cell III (rr) 0 * IAT = indirect antiglobulin test

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Which of the possible causes is the most likely cause of the positive antibody screen?View Page
Why might screen cell #2 be reacting stronger than screen cell #1?View Page
Antigram to explain prior question

The antigram below explains possible reasons for cell #2 reacting stronger: The patient may have anti-D and another antibody whose corresponding antigen is on cell # 2 (e.g., anti-E or anti-K). The patient has an antibody other than anti-D (e.g., anti-Jka) and cell #2 has a double dose of the antigen but cell #1 has only single dose. Screen Cell Rh Rhesus Kell Duffy Kidd MNSs P Lewis Lu Results Cell C D E c e Cw K k Kpa Fya Fyb Jka Jkb M N S s P1 Lea Leb Lua Gel IAT 1 R1R1 + + 0 0 + 0 0 + 0 + + + + 0 + 0 + + + 0 0 2+ 1 2 R2R2 0 + + + 0 0 + + 0 0 + + 0 + + + + + 0 + 0 3+ 2 3 rr 0 0 0 + + 0 0 + 0 + 0 0 + + 0 + 0 +S 0 + 0 0 3 Auto 0 Auto

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Which of the following are possible causes of the positive antibody screen?View Page

The Disappearing Antibody: A Case Study
Evaluating Inconsistencies

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.

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Unexpected Anomaly

3. Do the results of the initial antibody screen (shown below) support the presence of the identified antibody?No: All 3 screen cells reacted in the initial screen. Upon review, however, only Screen Cells 1 and 3 were Jk(a+); Screen Cell 2 reacted but was Jk(a-).This anomalous result was investigated by a reference laboratory. It was discovered that the patient had anti-Rd, an antibody to the low frequency antigen Radin (Rd). By chance, Screen Cell 2 was Rd-positive. Radin has a frequency of less than 0.5% in several populations tested. If the screen cell manufacturer was notified, they would likely confirm that the cell was Rd-positive, make their clients aware of it, and document it in future antigrams.

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Reflecting on Probability of the Solution

Similar to evaluating inconsistencies, one of the post-analytic tools for confirming that the serological data fit the solution is to consider the "big picture." For example:Is there a likely red cell stimulus (prior transfusion or pregnancy) for IgG antibodies such as anti-Jka? Can different reaction strengths with panel cells be explained by the identified antibody (e.g., dosage) or by the presence of more than one antibody? Is the antibody unusual for a patient of a particular race? For example, anti-Dib is more likely to occur in Native Americans than in Caucasians.

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Using Probability (p) Values

The p value is a statistical tool that increases the confidence that an antibody has been identified with a scientifically acceptable level of uncertainty (0.05). As applied to antibody identification, it is computed using Fisher's exact test. Tidbit: This is the same Fisher who helped developed the Fisher-Race theory of Rh inheritance.The p value is calculated using the number of cells that are positive and negative with the patient's plasma. Calculating p values is beyond the scope of this case study. However, a basic overview of p value, its relationship to antibody identification in general, and its application in this Case Study is provided in the attached PDF, if you would like more information.

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Antibody Identification Checklist

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 ChecklistYes/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?

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As discussed earlier, one of the post-analytic tools for confirming that the serologic data fit the solution is to consider the big picture, as presented below. Think of how you would reply to each question in this case and then click each question to see sample responses.View Page
Using p values in antibody identification

When p values are calculated for antibody identifications, we think of the null hypothesis as meaning, "the relative proportions of one variable (panel cell being antigen-positive) are independent of the second variable (patient's plasma reacting with the cell). In other words, the results could be due to another cause (different antibody, combination of antibodies, or spurious reactions), not the antibody that we have identified as being probable.Therefore, a p value of 0.05 can be interpreted as meaning that the same results produced by another antibody or cause would be expected to occur by chance alone only one in 20 times (5% of the time), given the number of cells tested. By scientific tradition, this is an acceptable level of uncertainty.A p value of 0.05 does not mean that we have identified the correct antibody.

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Which statement best describes a p value of 0.05 generated by a panel that identifies anti-K as the probable antibody?View Page
The p value in this case

This CaseWith the panel done 2 weeks post-transfusion, 5 panel cells that were Jk(a+) reacted and 5 that were Jk(a-) did not. This yields a p value of 0.004, which is less than the standard of 0.05, and therefore is more than acceptable statistically. In other words, an antibody other than anti-Jka would be expected to produce these panel results only 4 times in 1000 (which is pretty unlikely).Th true p value is much lower because many more cells were tested than in the panel alone.Concluding that the antibody is anti-Jka is further strengthened because the patient's red cells type as Jk(a-).Learning points: The most important things to know about statistical tools such as p values are that they: Relate to the probability of getting the observed results if the null hypothesis were true (the panel results were due to another antibody)Do not substitute for technical and clinical judgment.

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Understanding the "rule of three"

In immunohematology textbooks, the "rule of three" is sometimes presented as follows:1. If a patient plasma or serum gives positive results with a minimum of three antigen-positive cells and negative results with a minimum of three antigen-negative cells, concluding that the serum contains an antibody directed against the antigen has a p value of 0.05.2. Therefore, a p value of 0.05 requires at least three positives and three negatives.The first statement is correct but second statement is a misinterpretation of the p value.Three positives and three negatives are required to identify an antibody with a p value of 0.05 ONLY if you have only a 6-cell panel. It does not mean that you always need three positive cells and three negative cells to get p=0.05.For example: A 10-cell panel with eight Jk(a+) cells and two Jk(a-) cells gives a probability of 0.02 if all the positive cells and none of the negative cells react.A 10-cell panel with eight K- cells and two K+ cells gives a probability of 0.02 if all the positive cells and none of the negative cells react.Learning point: You do not need three positive cells and three negative cells to get an acceptable p value of 0.05.

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When performing an antibody investigation, which of the following would indicate an inconsistency that needs to be further investigated? (Select all that apply)View Page
Case Study Summary

This case study presents a scenario in which a patient had an unexpected antibody that disappeared after he was transfused with unmatched group O Rh negative Red Blood Cells. The patient developed a positive direct antiglobulin test (DAT) with mixed field agglutination (MFA). An antibody identification using the post-transfusion red cell eluate was inconclusive, making the antibody unidentifiable. Fortunately, the patient improved and further transfusion was not required. Ultimately, the patient's antibody was identified as anti-Jka, with a second antibody to a low frequency antigen (Radin) also unexpectedly present.The case illustrates the risks involved in using unmatched blood.

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Risks of Transfusing Unmatched Red Blood Cells

We often "get away" with transfusing unmatched Red Blood Cells 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.

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Balancing the Risks

Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative Red Blood Cells for the K antigen, since anti-K is a relatively common clinically significant antibody.

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References

Dutton 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 May 8, 2014.Reid ME, Lomas-Francis C. The Blood Group Antigen Facts Book. 2nd ed. London: Elsevier – Academic Press: 2004.Roback JD, Combs MR, Grossman BJ, Hillyer CD, eds. Technical Manual. 16th ed. Bethesda, MD: AABB Press; 2008.Rudmann SV, ed. Textbook of Blood Banking and Transfusion Medicine. 2nd ed. Philadelphia, PA: Elsevier – Saunders; 2005.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 customersQuick calsOnline calculator of p values for Fisher's exact test)

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ABO, Rh, and Antibody Screen

These ABO, Rh, and antibody screen results were obtained by the TS using the blood specimen that was collected prior to starting the emergency transfusion with O Rh-negative RBCs. ABO and Rh typingABO Forward GroupABO Reverse GroupRh anti-Aanti-BA1 cellsB cellsanti-D004+4+3+Antibody screen Cells Gel IAT*Screen Cell I 3+Screen Cell II 2+Screen Cell III 2+* IAT = indirect antiglobulin test

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The antibody screen is positive but the transfusion of the O Rh-negative RBCs is already in progress. What are the transfusion service (TS) laboratory's priorities in this case?Place the following procedures that will be followed by the TS in the appropriate order of priority.View Page
Which of the following statements about mixed-field agglutination (MFA) are true? Select all that are correct.View Page
In this case, which red blood cells (RBCs) are agglutinating in the DAT and why?Antibody screen Cells Gel IAT*Screen Cell I 3+Screen Cell II 2+Screen Cell III 2+Patient CellsDATCCPost-transfusion2+ mixed field agglutination (MFA)N/AView Page
Antibody Investigation

Immediate post-transfusion antibody identification resultsCellRhRhesusKellDuffyKiddMNSsPLewisLuResults1Results2CDEceCwKkKpaFyaFybJkaJkbMNSsP1LeaLebLuaGel IATGel IAT1rr000++00+0+0+00++++S+000w+2rr000++00+00+++0++++S+00003rr000++00+0+00+0++0+0+0004r"r00+++00+0++0+0+0+++00005R2R20+++00+00+0+++0+0+0+0006R2R20+++00++0+++0+0+0+0+0007R1R1++00+00+00++0+0+0+S0++008R1R1++00+00+00++++00+++00009RZR1+++-+0++0+0+0+00+++000w+10r'r+00++00+0+0+++0+0+S0+00011Auto2+MFA2+MFAResults1:Patient's post-transfusion plasma Results2:Patient's post-transfusion red cell eluateNOTE: Tests were performed using blood specimens that were collected AFTER the patient had been transfused with O Rh-negative RBCs.

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Which of the following most likely accounts for the patient's post-transfusion plasma giving negative panel results?View Page
Other Post-Transfusion Tests

The patient's post-transfusion plasma was also retested with the six Red Blood Cell units that tested positive initially. Like the antibody panel performed on the post-transfusion plasma, all six units are now negative by gel IAT.Unfortunately, the panel results with the patient's post-transfusion eluate do not give clear results (only cells #1 and #9 react) and the antibody remains unidentifiable. Once the clinical staff was alerted to the presence of an unexpected antibody, the transfusion was stopped. However, suppose that the physician had decided to continue transfusing the patient at this stage. Take a moment to think about what you would advise regarding the compatibility of such transfusions, all of which appear to be compatible in the crossmatch. When you have considered the options, continue to the next page.

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Consulting the Patient's Physician

The transfusion was immediately stopped after the clinical staff was alerted to the presence of an unexpected antibody, but not before most of the O negative Red Blood Cells had been transfused. If the physician had decided to continue transfusing the patient at this stage, the following information should have been communicated:Although all donors appear to be compatible in the post-transfusion crossmatch, they are not.The results are false-negatives. The patient's antibody has been "mopped up" by adsorbing to the incompatible transfused O Rh-negative Red Blood Cells.Given that six donors were positive using the pretransfusion plasma, the antigen is a higher frequency antigen and most donors would likely be antigen-positive and incompatible.The patient's physician should consult the transfusion services medical director before any decision to transfuse is made.Transfusing Red Blood Cells before tests are complete requires a physician to sign an emergency release form in which the physician assumes full responsibility.

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Follow-up With Clinical Staff

The patient's physician was notified that compatible blood was unavailable and that the patient's antibody was still being investigated.When asked whether or not the patient was experiencing a transfusion reaction due to the transfusion of the unmatched and incompatible O Rh negative Red Blood Cells, the nurse in the OR stated that the patient was undergoing surgery and completely sedated. A transfusion reaction was not apparent but they would investigate and closely monitor.Hemolytic Transfusion Reactions (HTR)Before proceeding to the next page, make a short list of signs and symptoms associated with immediate hemolytic transfusions reaction and another list associated with delayed hemolytic transfusion reactions.

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Cause of Delayed HTR

Delayed HTR result from a secondary (anamnestic) immune response causing a weak, undetectable antibody to become stronger.Upon re-stimulation by donor red blood cells 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.

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Investigating Weak Antibodies

In this case study the patient's antibody has disappeared from the plasma by adsorbing to transfused donor red cells. It is detectable but unidentifiable in the post-transfusion red cell eluate. Several trial and error procedures exist to enhance weak antibodies. Which methods will enhance the reactivity of a given antibody depend on its characteristics. Methods to investigate weak antibodies include:Use a higher plasma-to-red cell ratio (add more antibody-containing plasma or eluate) Increase incubation time (if consistent with manufacturer instructions and if applicable) Use enzyme-treated panel red cells (enzymes enhance IgG antibodies in Rh and Kidd blood systems but denature some antigens, e.g., Fya, Fyb, S) Try alternative antibody detection methods. For example, if LISS is used routinely, try polyethylene glycol (PEG) or column agglutination methods such as gel, providing they have been validated for use in the transfusion services laboratory.

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Antibody Identification (2 weeks post-transfusion)

Fortunately, the patient's condition stabilized and additional transfusions were not required. Two weeks later, new patient specimens were drawn for antibody studies. Antibody identification resultsCellRhRhesusKellDuffyKiddMNSsPLewisLuResultsCellCDEceCwKkKpaFyaFybJkaJkbMNSsP1LeaLebLuaGel IAT*1rr000++00+0+0+00++++S+001+12rr000++00+0+0++0++++S+00w+23rr000++00+0++0+0++0+0+0034r"r00+++00+0++0+0+0+++00045R2R20+++00+00+++++0+0+0+0w+56R2R20+++00++0+++++0+0+0+0w+67R1R1++00+00+00+0++0+0+S0++078R1R1++00+00+00++0+00+++001+89RZR1+++-+0++0+00++00+++000910r'r+00++00+0+00++0+0+S0+001011Auto011

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Antibody Exclusion Protocol

Use this antibody exclusion protocol to identify the antibody or antibodies present.

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Using the guidelines in the Antibody Exclusion Protocol, which antibodies are possible (have not been excluded) using this panel? Select all that apply.NOTE: A PDF of the panel is provided on this page and can be printed to assist in working through the antibody exclusion process.Antibody identification resultsCellRhRhesusKellDuffyKiddMNSsPLewisLuResultsCellCDEceCwKkKpaFyaFybJkaJkbMNSsP1LeaLebLuaGel IAT*1rr000++00+0+0+00++++S+001+12rr000++00+0+0++0++++S+00w+23rr000++00+0++0+0++0+0+0034r"r00+++00+0++0+0+0+++00045R2R20+++00+00+++++0+0+0+0w+56R2R20+++00++0+++++0+0+0+0w+67R1R1++00+00+00+0++0+0+S0++078R1R1++00+00+00++0+00+++001+89RZR1+++-+0++0+00++00+++000910r'r+00++00+0+00++0+0+S0+001011Auto011View Page
Which of the following cells would be the one most useful cell to exclude both anti-E and anti-K in this patient?View Page
Variations in Antibody Strength

The antibody in the pretransfusion specimen (prior to the patient being transfused with unmatched group O Rh-negative RBC) reacted 2+ and 3+ with antibody screen and donor cells.If Jk(a+), the transfused donor red blood cells would have stimulated increased antibody production and the patient's plasma would be expected to react even more strongly with Jk(a+) red cells than in the pretransfusion specimen.However, the expected increase in antibody strength did not happen. Because Jk(a+) donor cells "mopped up" (adsorbed) the patient's anti-Jka initially, the anti-Jka decreased in strength. Later, once donor red blood cells are no longer present to adsorb the antibody, the anti-Jka would be expected to become stronger.Currently, (2-weeks post-transfusion) the patient's plasma is only reacting 1+ with Jk(a+b-) red blood cells and w+ with Jk(a+b+) red blood cells.This effect is called dosage. Learning pointsWhen a secondary immune response occurs, antibody first decreases before it increases. The expected increase in antibody strength will vary depending on the amount of excess antibody available in the patient's plasma at the time of testing versus the amount that had adsorbed to donor red cells and been removed by extravascular hemolysis.~

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Antigen Phenotyping

A standard follow-up to antibody identification is antigen phenotyping. Phenotype the following: 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?Consider possible antigen typing problems and how to overcome them before proceeding to the next page.

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Which of the following antibodies in this scenario could explain all reactions by itself?Antibody identification results CellRhRhesusKellDuffyKiddMNSsPLewisLuResultsCell CDEceCwKkKpaFyaFybJkaJkbMNSsP1LeaLebLuaGel IAT* 1rr000++00+0+0+00++++S+001+1 2rr000++00+0+0++0++++S+00w+2 3rr000++00+0++0+0++0+0+003 4r"r00+++00+0++0+0+0+++0004 5R2R20+++00+00+++++0+0+0+0w+5 6R2R20+++00++0+++++0+0+0+0w+6 7R1R1++00+00+00+0++0+0+S0++07 8R1R1++00+00+00++0+00+++001+8 9RZR1+++-+0++0+00++00+++0009 10r'r+00++00+0+00++0+0+S0+0010 11Auto011View Page

The Influenza A Virus: 2009 H1N1 Subtype
Laboratory Tests

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.

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Transfusion Reactions
In Vivo Red Cell Destruction

Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include:Antibody binding to red blood cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of complement The end result of complement activation is red cell lysis. Activation of mononuclear phagocytes and cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobinemia and hemoglobinuria, which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system.

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Additional Testing

If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pretransfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pretransfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process that affected the blood should be suspected, such as inappropriate warming or a faulty infusion pump. If bacterial contamination is suspected, the unit should be cultured. A positive culture indicates a reaction due to bacterial contamination.

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Causes of Acute Hemolytic Transfusion Reaction (AHTR)

AHTR may occur when red cells are transfused into a patient with a pre-existing antibody that destroys the transfused incompatible red cells. Life threatening AHTR most commonly occurs from the transfusion of ABO incompatible blood. Naturally occurring ABO antibodies bind complement on the red cell surface and have efficient lytic properties, which cause intravascular hemolysis. Extravascular hemolysis is characterized by antigen-antibody complexes, which do not activate complement. AHTR is characterized by rapid destruction of red cells immediately after transfusion. Rapid hemolysis of as little as 10 mL of incompatible red cells can produce symptoms of an AHTR. Signs and symptoms can occur within minutes after starting the transfusion. Clerical errors, such as mislabeled patient samples and mislabeled blood products, could result in AHTR. Therefore, a system must be in place and rigidly followed to ensure samples and blood products are correctly identified.Although acute hemolytic reactions are rare with an incidence of 1-9 in 100,000 transfusions, they are the most dangerous, often life threatening.

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Clinical Laboratory Tests

A post transfusion specimen should be sent to the laboratory for work-up. A clerical check should be performed to investigate possible errors in specimen labeling, blood product issuance, or patient identification. The plasma must be examined for hemolysis. A direct antiglobulin test must be performed. The patient's ABO, Rh and antibody screen should be repeated and confirmed. The blood product ABO/Rh can be confirmed. Other laboratory tests include: complete blood count (CBC), urinalysis, serum bilirubin, creatinine, coagulation profile, and disseminated intravascular coagulation (DIC) evaluation. The full laboratory work-up and details of other laboratory tests will be discussed later in the course.

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An acute hemolytic reaction may be caused by which of the following? (Choose all that apply)View Page
Febrile Nonhemolytic Transfusion Reactions: Definition, Manifestation, and Prevalence

A febrile non-hemolytic transfusion reactions (FNHTR) is defined as a temperature increase of 1°C over 37°C occurring during or after the transfusion of blood components. FNHTRs are more common in the transfusion of platelets. Multiply-transfused patients and multiparous women make up the largest populations experiencing this type of reaction. There are two mechanisms involved in the manifestation of an FNHTR. The first involves the presence of a white cell antibody in the patient's plasma that interacts with the white cells in the blood product. These antibodies may be directed against granulocyte antigens or human leukocyte antigens (HLA).This interaction causes endotoxins to be released, which act on the hypothalamus and stimulate a fever. The second mechanism involves the generation of leukocyte cytokines during product storage. The production of cytokines usually occurs during storage in warmer temperatures, which is why non-leukoreduced platelets are commonly implicated.

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Pathophysiology

The exact mechanism of lung injury in transfusion-related acute lung injury (TRALI) has not be identified. It is believed that the mechanism may vary from patient to patient. The most common finding is leukocyte antibodies in donor or patient plasma. Anitbodies to human leukocyte antigen (HLA) have been associated with TRALI. These anti-HLA antibodies can be formed in response to exposure to foreign antigens from transfusion or pregnancy. The source of the antibody is usually the donor not the patient. Transfused antibodies react with the recipient which results in leukocyte emboli aggregating in the lung capillary bed. Capillary damage triggers interstitial edema and fluid in the alveolar spaces, causing decreased air exchange and hypoxia.

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Evaluation of Donors Associated with Transfusion-Related Acute Lung Injury (TRALI)

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.

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Definition and Incidence

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.

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Diagnosis

The symptom most commonly associated with a delayed hemolytic transfusion reaction (DHTR) is unexplained decrease in hemoglobin and hematocrit. Patients may also present with fever and jaundice. Hemolysis occurs slowly and is primarily extravascular. Unlike an acute hemolytic transfusion reaction (AHTR), hemoglobinuria, acute renal failure, and disseminated intravascular coagulation (DIC) are not generally seen. On some occasions, patient's may not present with any symptoms. Serologic findings include a positive direct antiglobulin test (DAT) and/or a positive antibody screen in post-transfusion testing. In many cases, the physician will send a request for an additional transfusion because of the decreased hemoglobin levels, and not suspect a DHTR. The positive antibody screen will trigger an investigation including antibody identification. The DAT may have a mixed field appearance because of the antibody-sensitized transfused red cells and the non-sensitized patient red cells. Segments from the donor unit can be tested for the offending antigen once the antibody has been identified.Antibodies that are most often reported as the cause of DHTR are anti-Jka and anti- Jkb. Other antibodies that are also commonly implicated in a DHTR include Kell, Rh, and Duffy system antibodies.The patient's physician should be notified so that additional clinical and laboratory evidence can be evaluated.

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Prevention

The most critical aspect of prevention is for the transfusion service to document all clinically significant antibodies. One challenge in antibody detection is finding a rapid method that is sensitive enough to detect low titers of clinically significant antibodies without being too sensitive for insignificant antibodies. Preventing severe reactions in sickle patients can be done by phenotyping the patients. This is useful in providing phenotypically matched blood and solving complex antibody identification problems.

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Delayed hemolytic transfusion reactions (DHTR) typically occur 3 hours after transfusion.View Page
Pathophysiology, Treatment, and Prevention of Post-Transfusion Purpura (PTP)

PTP is caused by platelet-specific antibodies in a patient who has been previously exposed to platelet antigens through pregnancy or transfusion. The most frequently identified antibody is Anti-PLA1, which reacts with platelet antigen HPA-1a. The platelet antibody binds to the platelet surface, which allows for extravascular removal through the liver or the spleen. The patient's own platelets are destroyed as well, thus aggravating the thrombocytopenia. Three theories are suggested regarding the destruction of autologous platelets. One suggests that immune complexes bind to the platelets through the Fc receptor and cause destruction. The second theory proposes that the patient's platelets absorb a soluble platelet antigen from the donor plasma. The third hypothesis, which has the most support, states that the platelet alloantibody has autoreactivity that develops when the patient is exposed to the foreign platelet antigen. Platelet transfusion is NOT a treatment option. Steroids, whole blood exchange, and plasma exchange are accepted options for treatment. According to the AABB, intravenous IgG (IVIG) is the treatment of choice. Most patients will respond to treatment within several hours to four days. PTP does not usually re-occur but it is recommended that patient's with a previous reaction be transfused with antigen-matched components. Autologous donations or directed donations from antigen-matched family members may be the best sources of blood. PTP has been known to occur even after the transfusion of deglycerolized rejuvenated or washed red cells, so these processes do not prevent a reaction.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Platelet satellites (see image to the right) may account for low platelet counts as determined by electronic counters. Satellitosis is initiated by:View Page


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