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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.

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

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

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

Ketone bodies are usually absent in urine. The presence of ketones in the urine probably indicates that the body is using fats rather than carbohydrates for energy. High levels of ketones may be present in the urine of individuals with uncontrolled diabetes because the body's ability to metabolize carbohydrates is defective. Detecting the presence of ketones in the urine is a valuable aid to managing and monitoring individuals with diabetes mellitus. Ketonuria is an indication that the insulin dose needs to be increased. Electrolyte imbalance and dehydration occur when ketones accumulate in the blood. If these conditions are not corrected by adjusting the dose of insulin, the patient may develop ketoacidosis and ultimately diabetic coma. Low levels of ketones may be detected during conditions of physiological stress such as fasting, rapid weight loss, frequent strenuous exercise or prolonged vomiting. The presence of ketones in these situations is due to either inadequate intake of carbohydrates or increased loss of carbohydrates.

The use of heparin is prophylactic. It is used either to prevent thromboembolism (a condition in which a blood clot forms inside a vessel) or used to limit a previous thromboembolism. Heparin inhibits thrombin. The degree of inhibition is dose-dependent. Low doses of heparin inhibit initial thrombin formation in the coagulation cascade and act to slow down overall thrombin generation. At higher doses, heparin can inhibit thrombin entirely, making blood coagulation impossible. Heparin is a potent anticoagulant. Accurate monitoring is essential. The activated partial thromboplastin time (aPTT), activated clotting time (ACT), and/or anti-Xa assays are used to monitor unfractionated heparin therapy.

Adverse drug reactions are a leading cause of morbidity and mortality in the United States. Drug metabolism is a process whereby drugs are delivered to the body, distributed, metabolized and then ultimately excreted. As there can be potentially significant differences between patient drug absorption, metabolism and excretion, molecular testing allows a physician to work with a patient's individual phenotype and/or genotype to deliver an optimum pharmaceutical selection and/or dosage.

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?

When first developed in the 1960s, RhIg was believed to work by a simple clearance mechanism, i.e., by coating D-positive fetal red cells with IgG anti-D, which resulted in clearance of the sensitized cells in the spleen by macrophages with receptors for IgG.Current research shows that a simple model of antigen clearance by antibody-sensitized D-positive RBCs is not the mechanism of anti-D suppression by RhIg. More is involved at the molecular level, possibly involving a down-regulation of antigen-specific B cells and related mechanisms.Regardless, if given soon enough following exposure to D+ red cells, and in a suitable dosage, RhIg has the ability to prevent immunization to D.

Using the estimated volume of fetal bleed determined by the Kleihauer-Betke test or flow cytometry, the number of vials of RhIg (300 µg) to inject is calculated as follows: Number of vials of 300 µg RhIg = volume of fetal bleed/30 mLIn the interests of safety some American organizations recommend the following to deal with decimal points: If the number to the right of the decimal point is <5, round down and add 1 vial (e.g., 1.4 = 1 +1 = 2 vials) If the number to the right of the decimal point is greater than or equal to 5, round up and add 1 vial (e.g., 1.7 = 2 +1 = 3 vials) Sub-calculations: Volume of fetal bleed: % fetal cells x maternal blood volume Maternal blood volume: 70 mL/kg x weight (kg) (assume 5,000 mL if maternal information is unknown) Note: RhIg dose calculators are available (see Further Reading: Paxton A. Bringing new rigor to RhIG calculations).

TDM provides a quantitative measure of the circulating concentration of a drug. The physician determines if the dosage of the drug needs to be adjusted based on this information.If a drug concentration is determined to be outside the therapeutic range, it may be for one of the reasons listed in the table below. Reason Discussion Noncompliance Patients may (intentionally or unintentionally) not take the drug. TDM can thus help monitor compliance. Dosing errors The dose may have been erroneous or inappropriate given the patient's condition. Malabsorption The TDM result will reveal if the drug cannot be absorbed well through the gut and an alternative route of administration will be needed. Drug interactions Many drugs interfere with the absorption or metabolism of other drugs. These interactions will be revealed by TDM. Kidney or liver disease Any pathology that affects elimination will cause an elevation in a drug level that will be unmasked by TDM. Altered protein binding Changes in serum proteins can lead to big changes in the amount of free drug in serum. Variations in the genetics of drug-metabolizing enzymes can also affect drug concentrations in the body. This is the field of pharmacogenomics that will be discussed later in the course.

In North America, a standard dose of RhIg is considered to be 1500 IU (300 µg). Note: 1 µg of anti-D = 5 IU.300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood (15 mL of packed rbc). If gestational age is known to be less than 12 weeks, a 600 IU (120 µg) dose may be sufficient.Depending on the gestation of the fetus, recommended dosages vary from country to country and within countries. Samples of recommendations that may change over time: USA: American Congress of Obstetricians and Gynecologists (1999, reaffirmed 2007): Antenatal RhIg dose of 300 µg (1500 IU) at 28 weeks and another 300 µg after delivery of a D-positive infant. Canada: Society of Obstetricians and Gynaecologists of Canada (2003): Antenatal RhIg dose of 300 µg (1500 IU)at 28 weeks (alternatively, 2 doses of 100–120 µg, one at 28 weeks and one at 34 weeks). After delivery of a D-positive infant, another 300 µg (alternatively, 120 µg IM or IV). UK: Royal College of Obstetricians and Gynaecologists (2002): Antenatal RhIg does of 100 µg (500 IU) at both 28 weeks and 34 weeks of gestation, and another 100 µg after delivery of a D-positive infant. All recommendations require testing to detect larger fetal bleeds, e.g., FMH larger than 30 mL of whole blood (for 300 µg doses) and FMH over 12 mL of RBC for 100 µg doses.

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

Using the estimated volume of fetal bleed determined by the Kleihauer-Betke test or flow cytometry, the number of vials of RhIg (300 µg) to inject is calculated as follows: Number of vials of 300 µg (1500 IU) RhIg = volume of fetal bleed/30 mLIn the interests of safety some organizations recommend the following to deal with decimal points: If the number to the right of the decimal point is <5, round down and add 1 vial (e.g., 1.4 = 1 +1 = 2 vials) If the number to the right of the decimal point is greater than or equal to 5, round up and add 1 vial (e.g., 1.7 = 2 +1 = 3 vials) Sub-calculations: Volume of fetal bleed: % fetal cells x maternal blood volume Maternal blood volume: 70 mL/kg x weight (kg) (assume 5,000 mL if maternal information is unknown) Note: RhIg dose calculators are available (see Further Reading: "Bringing new rigor to RhIG calculations").

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.

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.~

Transfusion-associated graft versus host disease (TA-GVHD) is generally unresponsive to medical treatment. Hematopoetic stem cell transplantation has been successful in rare instances. Gamma-irradiation of blood components containing viable lymphocytes is effective in preventing TA-GVHD. Irradiation is recommended for all Whole Blood, Red Blood Cell (RBC), Platelet, and Granulocyte transfusions to patients at risk. Patients at risk include neonates less than four months, patients with an acquired or congenital immunodeficiency, or patients receiving a directed donation from a family member. Irradiation prevents proliferation of donor lymphocytes with a required dose of 25 Gy to the mid plane of the blood container and a minimum of 15 Gy elsewhere. The dosage must not exceed 50 Gy to prevent harm to the patient from irradiation. Irradiation of blood can result in a decreased survival of red cells and a leakage of potassium from intracellular stores. Because of this, red cell units may only be stored for up to 28 days following irradiation. No reduction in storage time is required for platelets. Because Fresh Frozen Plasma (FFP) and Cryoprecipitate do not contain cells, irradiation is not required to prevent TA-GVHD in patients at risk.