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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.

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

A Pareto chart is a line and bar graph that provides a visual illustration of a problem. Pareto charts can be created using Microsoft Excel or Open Office. The Pareto chart ranks the problem based on occurrences from left to right. The Pareto chart is used extensively in quality control; it can help a team focus improvement efforts on the most critical problem. Here is an example of a Pareto chart that ranks different types of errors that occurred in a blood donor center. From the chart, the most frequent error is "no collector initials." While this information is helpful, it is probably not specific enough. To achieve more meaningful data one can create another Pareto chart, breaking the "no collector initials" further down by site (mobile blood drive, donor center location) or by procedure type (apheresis, whole blood collection, autologous donation, etc).

This is a higher power view of this same smear demonstrating a neutrophil that is filled with Toxopasma gondii tachyzoites (blue arrow).There are a few free organisms in this image well, indicated by the red arrows. Again, the typical morphology for toxoplasmsa organisms is lavender cytoplasm with a red granular cluster in the center of each parasite.This patient was negative for Toxoplasma gondii prior to a transplant but had received 15 units of blood products due to cytopenias.It is believed that a donor for one of the transfused units had been exposed to Toxoplasma gondii either through cats or contaminated food and had transient circulating Toxoplasma gondii in his or her blood when the donation was made. In this case, the recipient was profoundly immunocompromised, which lead to rapidly developing systemic disease.

The last type of collection is the observed collection. In this type of collection both the donor and "observer" enter the collection stall. Observed collections afford less privacy in order to guard against the donor using items which are designed specifically to beat the testing process. Under the Department of Health and Human Services mandatory guidelines for Federal workplace drug testing the observer must directly watch the urine go from the donor's body into the collection container. The use of mirrors or video cameras is not permitted.The observed collection is expanded under the Department of Transportation's 49 CFR § 40. After entering the stall the observer requests the donor to: Raise shirt, blouse, dress / skirt as appropriate, above the waist, just above the navel, Lower clothing and underwear to mid-thigh, and, Then turn around to show the observer that the donor does not have a prosthetic device. After the observer has determined that the donor does not have a prosthetic device, the donor is permitted to return clothing to proper position. The observer must personally watch the urine go from the donor's body into the collection container. The use of mirrors or video cameras is not permitted.

After the donor has given the specimen to the collector, the collector must check the box for an observed collection on the Custody and Control form and write in the REMARKS section the name of the "observer" and the reason why an observed collection had to be done. It is important to remember that if the observer is not the collector, it is the responsibility of the observer to maintain the integrity of the collection process and security of the specimen until it is delivered to the collector. Regardless whether the observer is the collector or not, the observed collection must be performed in a professional manner that minimizes, as much as possible, any discomfort to the donor. If the collector learns after the donor has been dismissed that a directly observed collection should have been done but was not, it is the responsibility of the collector to notify the employer that the employee must be directed to immediately return to the collection site so that a directly observed collection may be done. More about observed collections later.

Regardless whether you are collecting a Federally Regulated or a Non-Federally Regulated urine drug screen, there are five areas which demand specific prerequisites or conditions prior to performing a proper collection. These are: Requirements for the collection site. Supplies needed to conduct a collection. Criteria that must be met by collectors. Complete and accurate documentation. Proper identification of the donor.We will explore each of these in more detail over the next several pages.

All collection sites must meet the following security requirements: Must be able to prevent unauthorized access to the site during collection. Ensure that the donor does not have access to items that could be used to adulterate or dilute the specimen (e.g. soap, water, cleaning agents, etc.) Secure faucets, toilet tank tops, and other appropriate areas with tamper-evident tape if necessary. Ensure that the donor is at all times under the supervision of the collector or other collection site personnel. Provide for the secure handling and storage of specimens. (Specimens should be stored at 4-6° C. The refrigerator used should not be readily accessible to the general public and should be used only for the storage of urine drug screens and other clinical specimens. The refrigerator should be marked with a biohazard sign. No food or drink should ever be placed in the refrigerator.)

As collector: You can process only ONE donor at a time. You may not act as the collector for anyone whom you immediately supervise unless no other qualified collector is immediately available. You can not collect your own urine specimen. You should have appropriate identification available should the donor request it. This identification is limited to your name and the collection company where you work. You are not required to show documentation of training unless requested by a DOT representative, state government representative, or an employer. You should keep a file of the names and telephone numbers of Designated Employee Representatives (DER) to contact about any problems or issues that may arise during the collection process.

There are rare situations in which the donor cannot produce proper identification, such as a self-employed individual. In these cases, the collector should make a note in the Remarks section of the CCF that the positive identification was not available. The individual is then asked to provide two items of identification bearing his or her signature. The collector then proceeds with the collection. When the donor signs the certification statement (pink copy of the CCF), the collector compares the signature on the CCF with the signature on the identification presented. If the signatures appear consistent, the collection process continues. If the signature on the CCF does not match the signatures on the identification presented, the collector must make an additional statement in the Remarks section noting that the signature identification is unconfirmed.

Let the donor select a collection kit. There should be 10 or more kits available from which the donor may chose. The kit is comprised of a collection cup, leak resistant plastic bag, and two specimen vials. You or the donor may open the collection kit in the other's presence. The donor is given the collection cup. It is the only item that can be taken into the restroom. The specimen vials remain with the collector. Do not open these vials until you are ready to disperse the urine specimen.

After the donor has handed the specimen to the collector, the donor is given permission to wash his or her hands and to flush the commode. While the donor is accomplishing these tasks, the collector may be making the necessary observations discussed previous.It is important to remember that once the donor has handed his or her specimen to the collector, the donor must always be able to have visual contact to the greatest extent possible with the collector and the specimen.

The collector now directs the donor to read, sign, and date the certification statement located on the MEDICAL REVIEW OFFICER COPY (page 2 or "pink" copy) of the custody and control form. The donor must provide printed name, signature, date of birth, and day and evening contact telephone numbers. If the donor refuses to complete this section, the collector is to note the fact under the remarks section on page 1 (TEST FACILITY COPY).

After dispensing the urine specimen into the specimen vials, the collector, not the donor, removes the tamper-evidence seals from the control form and places them on the specimen vials. Seal "A" goes over the primary vial containing 30 mL; seal "B" goes over the secondary vial containing 15 mL. (When doing a Non-Regulated drug screen, since only one vial would be used, "A" would be the appropriate tamper-evidence seal to use.)The seal must be centered over the lid and down the sides of the vial to ensure that the lid cannot be removed without destroying the seal.

After a positive identification has been made, invite the donor into the area where the collection will be conducted. Be pleasant, but professional. Introduce yourself and generally explain the collection procedure. Be prepared to accommodate donors who do not speak English. Never argue with the donor or be judgmental. Always remember that you are a professional. Conduct yourself in that manner. Ask the donor to remove any unnecessary out clothing such as a coat, jacket, hat, etc., and to leave any briefcase, purse, or other personal belongings with the outer clothing. The donor may retain his or her wallet. If the donor asks for a receipt for any belongings left with the collector, the collector must provide one. Direct the donor to empty his or her pockets and display the items to ensure that no items are present that could be used to adulterate or dilute the specimen or be used as a substitute. If nothing is there, the donor may return the items to his or her pockets.

Instruct the donor to wash and dry his or her hands. Observe this step. Tell the donor not to wash or dry hands again until after the donor has provided and handed you a specimen. The donor may use soap to wash hands but it must be a liquid. Solid or bar soap gives the donor a chance to conceal soap shavings under the fingernails and subsequently use them to attempt to adulterate the specimen.

Make sure that all copies of the Custody and Control Form are legible and complete.Place the specimen vials and white copy of the CCF inside the appropriate pouches of the leak resistant plastic bag. Make sure to seal both pouches. The donor may now leave the collection site.

When a donor behaves in a confrontational way that disrupts the collection process, such as refusing to empty pockets or refusing to wash hands after being directed to do so by the collector, this is considered interfering with the testing process and is considered a refusal to test. The collector must stop the collection then contact the Designated Employer Representative (DER) regarding the refusal either by telephone, email, or secure fax to ensure that notification is immediately received. The collector must document the refusal to test on the Federal Custody and Control Form (CCF) and send all copies of the CCF to the DER.If a donor makes an attempt to provide a specimen and the quantity is not sufficient (QNS) and the donor refuses to make a second attempt to provide another urine specimen or leaves the collection site before the collection process is completed, this is considered a refusal to test. The collector must stop the collection and discard any urine collected. The collector must notify the DER as outlined above, document the refusal to test on the Federal CCF and forward all copies of the CCF to the DER.

Refusal to Complete Donor Certification on Pink Copy of CCF or Initial Security Strips.If the donor refuses to complete the donor certification on the pink copy of the CCF, or refuses to initial the security strips after they have been affixed to the specimen vials, this is not considered a refusal to test. Do not debate with the donor. It is the responsibility of the collector to note the fact in the "Remarks" section of the CCF. Failure to do so may result in a Fatal Flaw. The MRO may not release the results of the drug screen unless the collector has noted in the "Remarks" section why the donor certification was not completed.Refusal to Provide ID or Social Security NumberIf the donor refuses to provide the collector with an ID or Social Security Number, this is not considered a refusal to test. The collector must make a notation of the fact in the "Remarks" section of the CCF. Failure to do so may result in a Fatal Flaw. After making the notation, the collector continues with the collection.

Scenario 1:A donor was asked to wash her hands prior to picking out the drug screen collection kit. The collector noticed that the donor was washing only one hand.Collector's response:The collector tells the donor that not washing both hands is an indication of possible interference with the testing process and that it could be interpreted as a refusal to test. If the donor still refuses to wash both hands, the collector must stop the collection process, note the refusal on the CCF and notify the DER.Scenario 2:The donor was asked to remove his hat before going into the restroom. As he reluctantly did so, it was noticed that he was trying to conceal a container that was hidden inside the hat.Collector's response:The collector first explains the circumstances to a supervisor. If the supervisor concurs that an observed collection should be done, the collector then tells the donor that a directly observed collection will be conducted because his conduct indicated a possible attempt to adulterate, substitute, or dilute the specimen. The collector marks on the CCF that the collection was observed and notes under Remarks why it was observed.

If a security seal is broken while being removed from the CCF or during the application of the first seal on the primary specimen vial, the collector should transfer the information to a new CCF and use the seals from the second CCF.If one seal is already in place on a specimen vial and second seal is broken while being removed from the CCF or is broken during application on the second specimen vial or while the employee is initialing either seal, the collector should initiate a new CCF and note in the "Remarks" section how the seals were broken. The seals from the second CCF should be placed perpendicular to the original seals to avoid obscuring information on the original seals. The donor must initial the second set of seals also. The initials on all the seals must match. The collector should then draw a line through the specimen ID number (and bar code if present) on the original seals to ensure that the laboratory does not use that number for reporting the results. The collector must not pour the specimen into new vials.

In an observed collection, the observer must be the same gender as the donor. If an observed collection must be made, and the collector is not the same gender as the donor, it is the responsibility of the collector to locate someone who is the same gender as the donor to act as the observer. If the collector is not the observer, the donor can not hand the specimen to the observer but must only hand it to the collector. It is, however, the responsibility of the observer to make sure the donor directly hands the specimen to the collector.

Scenario 5: The collector notices that the urine the donor just handed to her has a very strong smell like that of a cleaning product such as bleach. Collector's response: The collector completes the collection in the usual manner and prepares the specimen for shipment. The collector explains the situation to a supervisor. If the supervisor concurs that an observed specimen should be collected, the collector explains to the donor that because of the strong, unusual smell, the first specimen is suspect for adulteration and that a directly observed collection will be done. A new CCF is initiated. The collector marks on the CCF that the collection is observed and notes under Remarks why it is observed. The collector also notes the control number of the suspect collection. The observed specimen along with the suspect specimen are both shipped to the laboratory in separate plastic tamper-resistant bags. In addition to an unusual smell, other indications of adulteration might be an unusual color that cannot be explained by medication, particles or debris in the urine, and a heavy or thick foam that is inconsistent with urine.

You, as a collector, have a great deal of responsibility in the collection of urine for drug testing. It is imperative that you know, understand, and stay current with the rules and regulations. Do the very best you can to make every collection "error free." A collection site must be ready to demonstrate that it satisfies all requirements. Guidelines now mandate that "Federal agencies" inspect each year up to five percent of randomly selected sites used by the agency.

Bone marrow transplants may be a cure but currently the risks are too high. Impediments to transplantation include the lack of matched sibling donors and prior transfusions, which have exposed the patient to donor antigens.The best candidates for bone marrow transplants are children less than 16 years old. Two umbilical cord blood transplantations have been performed that reportedly have not remanifested with sickle cell disease.Gene therapy may become an option in the future that may alter the expression of the sickle gene.

Prenatal treatment of severe HDFN due to anti-D consists of in utero transfusions. Because of significant risks, transfusion is indicated only if fetal monitoring suggests significant hemolytic disease. 1. Intrauterine Transfusion (IUT)IUTs are done when fetal monitoring indicates severe HDFN and the fetus is too premature for early delivery. IUTs involve the intraperitoneal infusion of packed red cells. The success of the procedure depends on absorption of the red cells through the subdiaphragmatic lymphatic vessels of the fetus. 2. Intravenous transfusion (IVT)Because there may be erratic and inconsistent absorption of intrauterine transfusions in severely hydropic fetuses, IVTs were developed. IVTs involve transfusing donor RBC directly into the umbilical vein.

Prenatal treatment Prenatal management and treatment of ABO HDFN is not routinely done because: Titers of anti-A and anti-B do not correlate well with severity of disease; The risks of fetal monitoring (e.g., amniocentesis, cordocentesis) and fetal transfusion are greater than the risk of ABO HDFN since it is usually mild and subclinical. However, if a woman has a history of infants with moderate to severe ABO HDFN requiring treatment, she may be monitored so that the infant can be treated for possible HDFN as soon as possible. Postnatal TreatmentTreatment of ABO HDFN usually consists of phototherapy in which the newborn is placed under a "blue light" that chemically alters bilirubin in the surface capillaries to a harmless substance.For more severe cases, exchange transfusion may be performed. Donor RBC for exchange transfusion in cases of ABO HDFN must meet these criteria: Group O; Rh compatible with infant; Less than or equal to 7 days old (or fresher); Reconstituted with AB FFP to obtain a prescribed hematocrit; CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from sickling under conditions of reduced oxygen concentration in the newborn; Irradiated to prevent graft-versus-host disease. Exchange transfusion is also discussed later in the course in the section related to HDFN due to anti-D and other antibodies. Red Blood Cells are crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

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

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.

Specialists in immunohematology perform all testing prior to blood transfusions and work to prevent transfusion infections. They also investigate any post-transfusion reactions. This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in clinical chemistry analyze body fluids such as blood, urine, and spinal fluid to determine the chemical makeup, including the amount of carbohydrates, proteins, enzymes, and trace elements. The special covers urine microscopics and chemical evaluation of the liver, kidneys, lungs, heart, and other vital organ systems. This specialty also covers all testing performed in the specialties of radioassay and blood gas analysis. Specialists in blood banking can perform all immunohematology testing as well as testing from the specialties of clinical chemistry, hematology and serology/immunology that relates to donor blood. Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, and serology / immunology may perform all tests in the blood banking specialty.

If a urine specimen is require, instruct the donor on its collection.Escort him or her to the restroom.The donor breaks the seal on the urine container and produces a specimen.

You may be required to measure the temperature of the urine, and to check it visually for tampering.Apply a tamper-evident seal to the specimen, and label it appropriately in the presence of the donor individual.

Fluorescence resonance energy transfer (FRET) is the process that permits probes to emit fluorescence only after hybridization and describes the energy transfer between two fluorophores. Each probe consists of both a donor and a receptor fluorophore with the donor being on the 3' end and the reporter on the 5' end. The proximity of the two fluorophores to each other allows for the donor to reduce the fluorescent state of the reporter. When the probe attaches to the single-stranded piece of DNA, the fluorescence is still being suppressed. As the polymerase takes effect and begins to synthesize the new complementary strand of DNA, the reporter and donor fluorophores are separated. This separation allows for the reporter to emit its energy, which is picked up by the computer. The more denaturing and annealing that takes place, the higher the fluorescence in the tube. The process of FRET can be compared to the classic science experiment using tuning forks. If you strike one tuning fork so that it rings and hold it near a tuning fork that has not been struck, it will begin to ring by the force of the resonating waves from the initial tuning fork.

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.

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?

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

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 5 2+ Donor 6 3+ * IAT = indirect antiglobulin test ** O Rh-negative RBC (Donors 3 - 6 are O Rh-positive)

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 often go undetected as the symptoms are usually mild and subclinical (death has occurred, but rarely). Symptoms may not occur until days after transfusion when the patient has left the hospital. Donor red cell destruction is usually by extravascular hemolysis (EVH). Signs and symptoms can include: Fever with or without chills Unexplained drop in hemoglobin and hematocrit Transient jaundice due to elevated serum bilirubin

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.~

There are two potential problems in typing a recently transfused patient who develops a positive DAT: There will be two cell populations, patient and donor red blood cells. If the typing sera reacts by IAT, the positive DAT will cause false positives. In the case presented, the DAT has become negative. This also suggests that most (if not all) transfused donor red cells have been removed from the patient's circulation.Regardless, to be on the safe side, the patient's initial pretransfusion specimen, which was DAT negative and consisted of only the patient's red blood cells, should be used for antigen phenotyping.

The US Food and Drug Administration's (FDA) worked with the CDC and other health agencies, both in the United States and globally, to protect public health during the H1N1 virus outbreak. The FDA ensures the safety, effectiveness, and supply of antiviral medications and the H1N1 vaccine that is produced and/or distributed in the United States; it has the responsibility of approving medical devices for the serologic testing of the 2009 H1N1 virus. The FDA also performs other roles such as ensuring the production of an adequate supply of respiratory protection and other personal protective equipment. The FDA also monitors the safety of the blood supply, and, although no cases of transmission of H1N1 virus through blood have been reported, the guidelines for donor deferral that have been established by the FDA further ensure that this would not occur.

Even though blood components are tested rigorously for certain infectious diseases, bacterial, viral, parasitic, and prion pathogens continue to evolve. If they are not detected, they can cause harm to the patient and even death. Donors must pass a medical screening and questionnaire. They are also tested for hepatitis B and C, human immunodeficiency virus (HIV) 1 and 2, human T-cell lymphotrophic virus (HTLV) I and II, West Nile virus and syphilis. The table to the right describes the screening tests performed on all blood donors in the United States. It is not yet possible to eliminate the risk of infectious disease transmission through transfusions. There are many other organisms that may be transfusion-transmitted which are not routinely tested for in the blood supply. These include the Epstein-Barr virus, cytomegalovirus (CMV), bacteria, and parasites such as malaria, Babesia microti, and Trypanosoma cruzi which is responsible for Chagas disease, and prions such as variant Creutzfeldt-Jakob disease (vCJD). Selection of eligible donors is a critical part of ensuring the safety of the blood supply. Donors with certain lifestyles, medical conditions, travel histories, immigration backgrounds, or specific physical findings are deferred, either for a specific period of time or indefinitely. This minimizes the risk that a transmittable agent will be present in the donors blood. Click here to learn more about donor eligibility criteria from the American Red Cross.Click here to learn more about Babesia microti. Click here to learn more about Chagas Disease. Click here to learn more about vCJD.Click here to learn more about malaria.

The first component of therapy is to stop the transfusion immediately. Vital signs must be closely monitored. Management involves treatment of hypotension and disseminated intravascular coagulation (DIC). It is essential to maintain blood volume and adequate renal blood flow. Diuretics, substances that increase urine output, may be administered. If the patient enters renal failure, dialysis must be initiated rapidly. It is impossible to prevent all hemolytic transfusion reactions. The purpose of pre-transfusion compatibility testing is to decrease the probability of a hemolytic transfusion reaction by performing ABO/Rh testing, detecting and identifying alloantibodies, and crossmatching compatible blood. Human error, the most common cause of hemolytic transfusion reactions, cannot be completely eliminated. Steps must be taken to reduce the possibility of human error in identification of patient samples, donor units, and recipients. Each person involved in the transfusion process, from collection of the blood sample to administration of the donor unit, must carefully adhere to each step outlined in the standard operating procedures. All appropriate protocols must be followed. Some examples are: Technologist checks blood sample to ensure proper labeling. Patient's previous transfusion records are examined and all transfusion testing is performed correctly and accurately. Technologist ensures correct unit is released from the blood bank. Transfusionist ensures the recipient is correctly identified.There must be a mechanism in place to train and assess all personnel involved in the transfusion process.

Mild allergic reactions result from a patient's hypersensitivity to soluble allergens in the plasma of the donor unit. The blood recipient forms antibodies to these allergens which are bound to IgE on mast cells and causes the release of histamines. Histamines increase vascular dilation and permeability which allows vascular fluids to escape into the tissues. Swelling occurs and itchy, raised, red welts appear. Allergen substances may be drugs or food consumed by the blood donor. Anaphylactoid and anaphylactic reactions (collectively referred to as anaphylaxis) result from the recipient's forming anti-IgA, which targets IgA proteins in the donor plasma. Recipients have a genetic IgA deficiency. It is also believed that these types of reactions may be caused by other substances in the donor blood such as a peanut allergen transfused to a patient with a peanut allergy.

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.

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

Although the risk of acquiring transfusion transmitted viral infections is low due to donor testing, bacterial infections are still reported. Platelets are the most implicated product in bacterial contamination reports because they are stored at room temperature (20-24oC) and provide a favorable environment for bacterial growth. Sespis occurs in about 1 in 25,000 platelet transfusions. It may be fatal in about 1 in 60,000 transfusions. Bacteria can be present in other components as well, such as red blood cells (RBCs), cryoprecipitate, and plasma. Contamination in red cell components is rare with events occurring 1 in 250,000 transfusions. This low incidence is due to the refrigerated storage requirements for red cells at 1-6oC. Because plasma and cryoprecipitate are stored frozen, they are least likey to contain bacteria. Contamination usually occurs when these products are thawed in a water bath that contains bacteria. Reactions range from minimal or no symptoms to fatal septic shock and death. Severity of the reaction depends on the bacterial species involved, the concentration and growth rate of the organisms, and the recipient's immune status. Septic reactions can present with a fever of higher than 38.5oC, rigors, and hypotension that begin during the transfusion. Patients may also have nausea, vomiting, dyspnea, and diarrhea. Septic shock, oliguria, and disseminated intravascular coagulation (DIC) are also complications.

Measures taken to reduce bacterial contamination of blood components include donor screening, improved skin disinfection, diversion of the first aliquot of blood, and pretransfusion bacterial detection. Screening of donors is done by questioning them about fever occurrence and dental or medical procedures that occurred days before donation. Donors who develop symptoms of an infection may be asked to notify the blood bank. Complete skin disinfection is not possible because of organisms living in places that are inaccessible, such as sebaceous glands and hair follicles. Factors affecting skin disinfection are the type and concentration of antiseptic, use or single or multiple antiseptics, method and steps of application, and contact time. Studies have shown that a two-stage method using a sponge scrub and ampule with tincture of iodine is the most effective method. The AABB recommends an initial 30 second scrub with a 0.7% iodophor solution followed by the application of a 10% iodophor compound, which must be allowed to dry for 30 seconds. To avoid normal flora contamination, blood may be diverted into a satellite bag at the beginning of donation. These bags are developed so that backflow is prevented. Blood contained in the satellite bag is used for blood grouping and infectious disease testing. Blood diversion is not a mandatory practice in the United States. The AABB requires that the transfusion service have a method to detect bacteria in all platelet components. Culture-based methods are used at blood collecting facilities near the time of collection. Hospital-based transfusion services use other less costly non-culture based methods such as gram staining or pH and glucose analysis prior to releasing the product for transfusion. Recently, a qualitative immunoassay for the detection of bacteria in platelets has been developed. This test detects antigens on the cell walls of the bacteria. It has been documented to be more sensitive than other non-culture based methods.

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.

Patients present with fever, a characteristic red rash from trunk or face to the extremities, watery diarrhea, nausea, vomiting, and hepatitis within seven to ten days following the transfusion. The rash may progress to blister-like lesions and erythroderma. Pancytopenia will develop due to the immune destruction of the recipient's bone marrow. The low platelet count causes hemorrhaging while a low white blood cell count can lead to infection. Most patients die within one to three weeks after the onset of symptoms. The diagnosis is often missed and is usually made too late or after death. Routine laboratory studies are not helpful. The only definitive method is the identification of donor lymphocytes in the circulation or tissues of the recipient which is accomplished through human leukocyte angtien (HLA) typing or cytogenic analysis.

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.