Antibody Information and Courses from MediaLab, Inc.

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

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) .

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

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.

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)

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.

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.

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.

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).

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.

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

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.

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

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.

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.

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

The 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.

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.)

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

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 cells 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 white blood cells. 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 clumps are 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.

Heparin-induced thrombocytopenia (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.

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

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.

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.

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.

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

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.

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.

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.

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

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?

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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

The 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.

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.

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.

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.

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.

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

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 C. difficile infection, 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 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 C. difficile infection (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.

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).

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).

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.

TORCH panelToxoplasma antibody Rubella antibodyCMV antibody Herpes antibody

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

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.Many 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 optional only 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.

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

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.

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.

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

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.

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.

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

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

If the physician had decided to continue transfusing the patient at this stage, the following information should be 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 RBC. Given that 6 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 TS medical director before any decision to transfuse is made. Transfusing RBC before tests are complete requires physicians to sign an emergency release form in which they assume full responsibility.

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

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 results 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 rr 0 0 0 + + 0 0 + 0 + 0 + 0 0 + + + +S + 0 0 1+ 1 2 rr 0 0 0 + + 0 0 + 0 + 0 + + 0 + + + +S + 0 0 w+ 2 3 rr 0 0 0 + + 0 0 + 0 + + 0 + 0 + + 0 + 0 + 0 0 3 4 r"r 0 0 + + + 0 0 + 0 + + 0 + 0 + 0 + + + 0 0 0 4 5 R2R2 0 + + + 0 0 + 0 0 + + + + + 0 + 0 + 0 + 0 w+ 5 6 R2R2 0 + + + 0 0 + + 0 + + + + + 0 + 0 + 0 + 0 w+ 6 7 R1R1 + + 0 0 + 0 0 + 0 0 + 0 + + 0 + 0 +S 0 + + 0 7 8 R1R1 + + 0 0 + 0 0 + 0 0 + + 0 + 0 0 + + + 0 0 1+ 8 9 RZR1 + + + - + 0 + + 0 + 0 0 + + 0 0 + + + 0 0 0 9 10 r'r + 0 0 + + 0 0 + 0 + 0 0 + + 0 + 0 +S 0 + 0 0 10 11 Auto 0 11

The antibody in the pretransfusion specimen (prior to the patient being transfused with two units of unmatched group O Rh-negative RBC) reacted 2+ and 3+ with antibody screen and donor cells.If Jk(a+), the transfused donor RBC 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 "mop up" (adsorb) 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-) RBC and w+ with Jk(a+b+) RBC.This effect is called dosage. Learning points When 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 rbc and been removed by EVH.~

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.

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 hemoglobulinemia and hemglobinuria 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.

Acute hemolytic transfusion reactions (AHTR) are caused when red cells are transfused to a patient with a pre-existing antibody that destroys the transfused incompatible red cells through intravascular or extravascular hemolysis. Life threatening acute hemolytic reactions most commonly occur 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. AHTRs feature rapid destruction 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. Fever is the most initial symptom followed by the chills. These reactions are mostly associated with the transfusion of ABO-incompatible red cells. Causes include clerical errors, such as mislabeled patient samples and mislabeled blood products. Although acute hemolytic reactions are rare with an incidence of 1 to 9 in 100,000 transfusions, they are the most dangerous and are severely life threatening.

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.

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.

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.

Post-transfusion purpura (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 soluable 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 (AABB Technical Manual, p. 744). 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 occurr even after the transfusion of deglycerolized rejuvenated or washed red cells, so these processes do not prevent a reaction.