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Antiglobulin Information and Courses from MediaLab, Inc.

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

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Antibody Detection and Identification (retired 12/6/2013)
Immune Antibodies

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

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Blood Banking Question Bank - Review Mode (no CE)
Which of the following antibodies will most likely not be detected on immediate spin?View Page
Which of the following antibodies is detected primarily in the antiglobulin phase of the crossmatch: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
Essential components of compatibility testing include all of the following except :View Page
The use of the direct antiglobulin test is indicated in all the following except:View Page
False negative results may occur with both the direct and indirect antiglobulin tests as a result of all of the following except:View Page
What is Coombs sera comprised of:View Page
Which of the following might cause a false positive indirect antiglobulin test:View Page
To detect the presence of blocking antibodies fixed on the red cells of a newborn infant:View Page
IgG coated red cells are added to negative antiglobulin tests to detect which of the following sources of error:View Page
A false-negative reaction while performing the DAT technique may be the result of: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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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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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
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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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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Quantifying FMH

When a screening test such as the rosette test indicates a significant FMH, a quantitative test is required to determine the dose of RhIg required. Tests include Kleihauer-Betke acid elution test; Flow cytometry; Other techniques that are less commonly used, e.g.,fluorescence microscopy and enzyme-linked antiglobulin test.The Kleihauer-Betke and flow cytometry methods are briefly reviewed on the following pages.

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
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

Rh negative female with anti-D at delivery: A case study
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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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
Serologic Tests on Newborn

Based on the results of the mini-panel, the laboratory concluded that only anti-D was present and that it was consistent with administration of RhIg at 28 weeks.Patient A.D. delivered a 5 lb 13 oz female by C. section with serologic test results on cord blood as follows. Well washed cord red cells were used for ABO and Rh(D) typing to remove possible Wharton's jelly.Before proceeding to the next page, evaluate if the infant's ABO and Rh(D) types are valid. You will be asked questions that assess basic knowledge of blood grouping practices and test results for newborns.ABO Forward GroupABO Reverse GroupRh anti-Aanti-BA1 cellsB cellsanti-D*00NTNT3+NT = not tested / * monoclonal IgM anti-D DATReagentDATCCPolyspecific AHGw+2+W+ = microscopic positiveAHG = antihuman globulin serum CC = IgG sensitized cells Note: It is the lab's policy to add IgG sensitized cells to weak antiglobulin test results.

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Quantifying FMH

When a screening test such as the rosette test indicates a significant FMH, a quantitative test is required to determine the dose of RhIg required. Tests include Kleihauer-Betke acid elution test; Flow cytometry; Other techniques that are less commonly used, e.g.,fluorescence microscopy and enzyme-linked antiglobulin test.The Kleihauer-Betke and flow cytometry methods are briefly reviewed.

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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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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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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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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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Crossmatch Results

These are the results of the crossmatch that was being performed in the transfusion service laboratory while the patient was receiving the two units of O Rh-negative RBCs. Cells Gel IAT*Donor I**2+Donor 2**2+Donor 3 3+Donor 4 3+Donor 52+Donor 63+* IAT = indirect antiglobulin test ** O Rh-negative RBC (Donors 3 - 6 are O Rh-positive)

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Pretransfusion Direct Antiglobulin Test (DAT) Result

The laboratory obtained post-transfusion blood specimens in order to perform a serological investigation. Pretransfusion and post-transfusion DATs were performed. Patient cellsDATCCPretransfusion02+DAT = direct antiglobulin test with polyspecific antiglobulin serumCC = IgG sensitized RBC

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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
Which of the following statements about antigen phenotyping are TRUE? (Select all that apply)View Page

Transfusion Reactions
Preliminary Laboratory Investigation

When the laboratory receives notification of a transfusion reaction, the first step is a clerical check. The clerical check should be performed as soon as possible to identify any possible ABO incompatibility. The technologist will compare the component bag, label, paperwork, and patient sample and look for errors. If an error is found, the physician must be notified. Once the post-transfusion sample is received, the sample should be examined for the presence of hemolysis. Both the pretransfusion sample and post-transfusion sample can be compared. Destruction of red cells and release of free hemoglobin will result in a pink to red supernatant. Pink or red colored serum may indicate intravascular hemolysis. The patient's serum may appear icteric if the hemolytic process is extravascular. The ABO testing must be repeated on the post-transfusion specimen as well. Examination of a post-reaction urine sample made aid in the diagnosis of acute hemolysis. Free hemoglobin in the urine indicates intravascular hemolysis. A direct antiglobulin test (DAT) must be performed on the post-transfusion sample. An EDTA lavender top tube is the required specimen type. If the DAT is positive on the post-transfusion sample, then one should be performed on the pretransfusion sample. If the pretransfusion DAT is negative and the post-transfusion is positive, the presence of incompatible red cells should be suspected. All findings must be reported to the supervisor or medical director, who may request additional tests.

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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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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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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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