Serum Information and Courses from MediaLab, Inc.

Normal urine contains very little protein, usually less than 10mg/dL, and the major serum protein that is found in normal urine is albumin. The presence of an increased amount of protein in the urine (proteinuria) can be an indicator of renal disease. The two mechanisms which can lead to proteinuria are glomerular damage or a defect in the reabsorption process of the tubules in the nephron. The concentration of protein in the urine is not necessarily indicative of the severity of renal disease.

It is important to test for urinary (and plasma or serum) ketones when any patient shows a greater than normal excretion of sugar or reducing substances. Screening for ketonuria is useful in following the effects of treatment for diabetes and in judging the severity of acidosis. Large amounts of ketones will appear in the urine before serum ketone levels are elevated.

Table III shows the unsorted raw data that will be used to make a frequency table. Note that the low and high results are highlighted. These data are continuous; however, the testing equipment rounds the data off to the nearest whole number of milligrams.Table IIIConcentration of Serum Glucose (mg/dL) in 130 Hospital Employees 100 83 80 114 100 80 85 81 101 80 95 108 79 81 97 77 84 88 78 86 81 77 98 85 92 105 85 108 90 89 84 94 84 81 82 78 84 82 98 86 87 74 79 104 89 91 85 72 92 90 93 87 90 99 96 110 107 97 84 76 83 80 101 75 84 76 73 86 71 84 70 79 91 86 86 91 87 96 96 97 106 104 65 81 103 83 90 70 80 80 75 82 83 76 81 87 84 86 93 86 103 76 112 102 93 89 67 78 84 82 91 86 82 82 87 89 95 90 73 103 75 113 93 86 77 95 94 99 87 92

Bar charts are preferred for discrete data.  The height of the bar between the "65" and "70" tick marks corresponds to the number of elements in the 65 - 70 class,  etc.Figure 3Frequency of Serum Glucose Levels in 130 Hospital Employees

The frequency polygon resembles a continuous curve, and is therefore appropriate for illustrating continuous data. Instead of bars, the class midpoints are plotted at heights corresponding to the class frequency. The midpoints are then joined by a line.Figure 5Frequency of Serum Glucose Levels in 130 Hospital Employees

The specimen of choice for coagulation testing is plasma. Venous blood is drawn into a 3.2% buffered sodium citrate tube (blue top tube), yielding a whole blood sample with a 9:1 blood to anticoagulant ratio. Inadequate filling of the collection tube will decrease this ratio, and may affect test results. A blue top tube used for coagulation testing should be drawn before any other tubes containing additives. This includes tubes containing other anticoagulants and/or plastic serum tubes containing clot activators. A serum tube that does not contain an additive can be collected before the blue top tube. If a winged blood collection set is used in drawing a specimen for coagulation testing, a discard tube should be drawn first. The discard tube must be used to fill the blood collection tubing dead space to assure that the proper anticoagulant/blood ratio is maintained, but the discard tube does not need to be completely filled. The discard tube should be a nonadditive or a coagulation tube. If a blood specimen used for coagulation testing must be collected from an indwelling line that may contain heparin, the line should be flushed with 5 mL of saline, and the first 5 mL of blood or 6-times the line volume (dead space volume of the catheter) be drawn off and discarded before the coagulation tube is filled.

Antigen on Red Cells Antibodies in Serum ABO Blood Group A Anti-B A B Anti-A B Neither A nor B Anti-A, Anti-B, Anti-A,B O A and B Neither Anti-A nor Anti-B AB

While the predictability of ABO antibodies in persons lacking the corresponding antigen makes the ABO blood group system an easy one for testing purposes, it can be treacherous as far as transfusion is concerned. If a patient receives cells containing A or B antigens and his/her serum contains the corresponding antibody, the donor cells will be destroyed almost immediately with severe and sometimes fatal transfusion reaction. It is, therefore, of utmost importance to thoroughly understand the ABO blood group system. Compatibility of the ABO system is essential for all other pre-transfusion testing.

Homozygous “hh” individuals do not form H substance and thus have no way for late sugars to attach. The blood group resulting from the homozygous “hh” condition is called the Bombay blood group (Bombay phenotype). Due to the presence of anti-H in the serum of a person with the Bombay phenotype, only blood from another person with the Bombay phenotype may be transfused.

ABO antibodies are not usually produced by an infant until 3 to 6 months of age. Antibodies found in the sera of newborns are almost always IgG, passively acquired from the mother. Thus, serum testing of newborns is not performed. Anti-A and anti-B titers are highest at ages 5-10 years and then they gradually decrease. Thus, in elderly patients, ABO antibodies may be difficult to detect. In patients with hypogammaglobulinemia, some leukemias, lymphomas or patients who are taking immunosuppressive drugs, the expected antibodies may be weak or even absent, reflecting the low levels of gamma globulin in the patient’s serum. As previously mentioned, these and other ABO typing discrepancies must be resolved before true ABO type can be determined.

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.

Antibodies of the ABO system cause agglutination of saline-suspended red cells at 4°C to 20°C. Heating to 37° weakens the reaction. “Naturally” occurring ABO antibodies may not be strong enough to agglutinate cells without centrifugation. Thus, testing serum for the presence of anti-A or anti-B has classically been performed using the tube system in which serum and cells added to a test tube are centrifuged and then evaluated for agglutination. A slide test has also been performed for forward reactions. Although tube tests are still in wide use, newer systems utilizing other technology such as gel agglutination are becoming more prevalent. The image on this page illustrates agglutination reactions observed with the tube system, from 4+ in the topmost image, to 0 in the lowest image. ABO reactions should be strong. Weak or missing reactions occur, but must be "resolved" before blood products can be released.4+ agglutination: Red blood cell button is a solid agglutinate; clear background.3+ agglutination: Red blood cell button breaks into several large agglutinates; clear background.2+ agglutination: Red blood cell button breaks into many medium-sized agglutinates; clear background; no free red blood cells.1+ agglutination: Red blood cell button breaks into many small clumps barely visible macroscopically; background is turbid; many free red blood cells.Negative: No agglutinated red blood cells present; red cells are observed flowing off the red blood cell button during the process of grading.Other reaction which may occur are the mixed-field reaction, in which mixtures of agglutinated and unagglutinated red blood are present; and hemolysis, in which red cells are hemolyzed by the antibody. Both of these patterns are considered positive reactions.

Patient Serum Tested With Known Reagent Red Cells Antibodies Present in Serum A1 Cells B Cells 0 4+ Anti-B 4+ 0 Anti-A 4+ 4+ Anti-A and Anti-B 0 0 No ABO antibodies present + = agglutination (graded 1+ to 4+) 0 = no agglutination or hemolysis

The composite image shown on the right illustrates the ABO typing reactions that were obtained for a patient. This particular case illustrates an ABO discrepancy. An ABO discrepancy occurs when the results of forward and reverse typing do not match. The reactions shown are described below in descending order:Patient red cells with reagent anti-A: negative reaction.Patient red cells with reagent anti-B: 4+ agglutination.Patient red cells with reagent anti-D: 4+ agglutination.Patient serum with reagent A1 red cells: negative reaction.Patient serum with reagent B red cells: negative reaction.This patient forward types as a group B, but reverse types as a group AB. (A group B patient should have anti-A. This patient demonstrates neither anti-A nor anti-B, similar to an AB patient). Further workup is necessary to determine the ABO type since the forward and back typing do not match. In this case, incubation at 40 C demonstrated the presence of weakened anti-A. The patient was therefore typed as group B. This case is an example of an ABO discrepancy which was due to a "missing" anti-A antibody. This could be due to old age, severe illness or immunosuppression. Although evaluation of ABO discrepancies is beyond the scope of this course, it is important to note that all ABO discrepancies must be resolved before blood products can be released for transfusion.This patient is Rh (D) positive, as evidenced by the strong agglutination of his cells with reagent anti-D antibody.

It is 11:00 PM and the specimen processing department is finishing up the night's accessioning and test requesting. A specimen processor is working on a requisition that has an order for a Hepatic Profile but there are two tubes of blood with the order, one of which is a lavender top tube. This is the fourth requisition from this same doctor's office and all of them have had a lavender top tube and serum tube with an order for a chemistry test and a CBC. No CBC is marked on the requisition or written on the tube. The specimen processor figures the office just forgot to mark the test and knows that the results will be delayed and the sample might not be any good if he doesn't order the CBC now. He is also under pressure from the technical departments to finish processing on time so they can get their work done on time for result printing in the morning. What should the processor do?Correct Answer: Look up the laboratory's policy for handling such a situation and follow the policy.Discussion: The laboratory is not permitted to change a doctor's order in any way. By ordering the CBC the processor is ordering a test that the doctor did not specifically order and therefore makes the laboratory subject to a violation of the False Claims Act. By reviewing and following the laboratory policy the processor assures that the laboratory, the physician and the patient's best interests are met.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

Most drugs are bound to proteins when they circulate in the body. Albumin is a major drug-binding protein in serum. Albumin is an alkaline protein, so acidic and neutral drugs primarily bind to it. If albumin binding sites become saturated, acidic and neutral drugs can bind to lipoproteins. Alkaline drugs tend to bind to globulins, particularly to the globulin, alpha-1 acid glycoprotein. Only free, unbound drugs are able to bind drug receptors and have therapeutic effects. An equilibrium exists in the systemic circulation between a free and protein-bound drug and between a free and receptor-bound drug. This is illustrated in the image to the right.

Ideally, a drug level would be monitored frequently and consistently, providing the clinician with a detailed pharmacokinetic profile over time. In reality, serum samples are often measured only during relatively infrequent clinic visits, meaning that many days or weeks may pass before a drug concentration 'snap-shot' is taken.

Immunoassay is the most common technique used by clinical laboratories for therapeutic drug monitoring. Antibodies that recognize drugs can be developed. Although most drugs are much too small to evoke an immune response, scientists can conjugate drugs to immunogenic proteins to produce antibodies that recognize drug-specific epitopes. There are several methods that utilize the principals of immunoassay for detection and quantification of therapeutic drugs in serum. Some of these methods are: Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) Fluorescence Polarization Immunoassay (FPIA) Chemiluminescent assays

Therapeutic Drug Monitoring (TDM) is a branch of clinical chemistry that specializes in the measurement of medication levels in serum. TDM requires quantitative measurements of drugs and/or their metabolites.

Most water-soluble drugs are eliminated from the body through hepatic metabolism. renal filtration, or a combination of the two.An alteration in renal function will have a major effect on the clearance of the drug or its active metabolite(s). Decreased renal function results in elevated serum drug concentrations.

Some drugs are more efficiently monitored by determining their effects rather than by measuring the serum drug level. Warfarin dosing, for example, is better monitored by measuring the Prothrombin time (PT) and International Normalized Ratio (INR).

Can we use therapeutic drug monitoring (TDM) to assess PGx?TDM of the drug in question can also tell us a good deal about a drug's metabolism and will also take into account all the other variables at play (co-medications, diet, impaired organ function, etc.) However, unlike genotyping and probe-drug testing, therapeutic drug monitoring must be performed during therapy, not before. So, in fact, TDM is not really used to predict therapy in PGx but serves as a confirmation of PGx findings. TDM and genotyping should be considered complementary and can be used in tandem to, first, predict and then verify appropriate serum drug levels.

Consists of an electrolyte panel, plus: Blood urea nitrogen (BUN), which a measure of renal function. Creatinine (Creat), which also measures renal function Glucose, the most important blood sugar, and Calcium. Run on serum or plasma

Blood is tested for the most important electrolytes (salts): Sodium (Na) Potassium (K) Chloride (Cl) Carbon dioxide (CO2)Can be run on serum or plasma.

Numerous types of proteins are dispersed in the plasma. These include: Coagulation proteins (blood clotting factors), which, if activated, will form a blood clot , and Serum proteins, which are left dispersed in liquid after the clot is formed. Serum proteins include: Albumin, a marker of nutrition, and Globulins, or antibodies.

Blood may be collected into either:Red top (clot) tubes.Speckle top tubes (serum separator tube).Gray top tubes specifically designed to preserve glucose levels. Gray top tubes contain additives such as sodium fluoride or potassium oxalate, which prevent metabolism of glucose by blood cells.

Controls must resemble as closely as possible the human samples they emulate.For hematology analyzers, controls need to have the same consistency and color as human blood. Likewise, serum controls need to have similar amounts of chemicals to those found in human serum.

Rouleaux formation correlates with an increased concentration of serum monoclonal proteins. Rouleaux may be seen as an artifact in the thicker portions of blood smears. The addition of a drop of saline to the blood smear will serve to disperse any artifactual rouleaux formation. The presence of rouleaux formation or RBC agglutination may result in a falsely decreased electronic red blood count and falsely increased MCV, as these clusters may be read as one cell.

In order to test collection containers for sperm collection, the sperm must be held in the container for several hours to ensure that neither the numbers nor motility are adversely affected. Numbers will decline if the sperm adhere to the container. Motility will decline if the container is toxic. One method of testing involves removing sperm from semen. The specimen would be centrifuged and the sperm pellet diluted in a small volume of culture medium containing an energy source and at least 0.5% of a protein, such as serum albumin. The processed sperm specimen would be placed in the container to be tested. Total count and motility of the sperm would be tested at the start of incubation and 24 hours later. The container is non-toxic if the motility at the end of 24 hours is no less than 50% of the original value.

Bilirubin crystals are seen in the urine when the serum bilirubin level is increased. The macroscopic appearance of urine with bilirubin crystals is orange to almost black in color. The crystals themselves appear as gold orange needle-like forms, or as amorphous material.