Donor Information and Courses from MediaLab, Inc.

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 one strong antibody.2+ could indicate one the reaction between one weak antibody and the corresponding antigen that is present on with the other target antigen not present on that testing cell. If the panel cell is in the heterozygous state, the reaction of the antibodies present may be weaker if they commonly exhibit dosage. 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) Cells that have at least 1 out of the 3 rule outs needed: C, c, e, K,k, Kpb, Jsb, Fya, Jkb, Lea, M, N, s, P1, Lub Antibodies that could not be ruled out with this panel: D,E, K, Fyb, Jka, Leb, S Predominant pattern of 4+ in panel cells 1,2,3,4,10 matches D Varying strengths in reactions indicates a possible second antibody so selected cells should be picked to aid in identification Find a panel cell negative for D (antibody you suspect) and homozygous positive for the antibody you are trying to rule out. For example: D E e K k Fya Fyb Jka Jkb Lea Leb S s Donor cell 1 0 0 + 0 + 0 + + + 0 + 0 + Donor cell 1 could be used as a rule out test for e, k, Fyb and Leb. Reactions should be negative if these antibodies are not present.You should have a total of 3 negative reactions with panel or screen cells to rule out potential antibodies. If reactions with this panel cell are negative, then e and k can be ruled out with a total of 3 to rule out reactions. Selected cells should be picked for each antibody that needs to be ruled out in order to determine the identity of the other antibody

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.

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 dies 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. 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 4-6º 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 "pink" page of the control form. The donor must provide date of birth, printed name, and day and evening contact telephone numbers.The collector completes the collector's portion of the chain of custody on the control form by printing his or her name, signing where indicated, and recording the date and time of the collection. The collector must also enter the specific name of the delivery or courier service transferring the specimens to the laboratory if applicable.

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.

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 red cells and anti-A in his serum. Anti-A1 is produced by about 1-2% of group A2 persons and about 25% of group A2B persons. 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.

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.