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Lactate dehydrogenase is found in the cytoplasm of every cell. LD is present in the serum at a level of 100-190 U/L. The serum LD level will rise during increased cell damage.Persons with alpha thalassemia intermedia usually have an increased levels of lactate dehydrogenase (LD). This LD is of red blood cell origin, which leaks in to the plasma during hemolysis.

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

Purpose: To design a set of panel cells that may help you to rule out additional antibodies and lead to the identification of the antibody that is present in the patient's plasma.Benefit of running selected cell panel: Decreases the use of reagents and specimen. How to choose selected panel cells: If you suspect that a specific antibody is present, the cells you choose for the select panel should be negative for that antigen and positive for the antigen you are trying to rule out (homozygous state).

The composition of CSF has some similarities to plasma. However, the protein level of normal CSF is dramatically lower than that of plasma.

Specific gravity (SG) measured with the reagent strip method correlates well with gravimetric measurement, and, unlike the gravimetric or refractometer methods, does not need to be corrected for glucose or protein. Cloudy/turbid urines do not need to be clarified before measuring SG with the reagent strip method. It is the recommended method for determining SG if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the SG result on the reagent pad. If the result is being read visually, it is recommended that .005 be added to the SG result when the pH is alkaline. Most dipstick readers, however, will automatically adjust the SG reading for pH. A SG reading higher than the reagent strip range would need to be measured by another method, and may require dilution.

Normally, small amounts of conjugated bilirubin, regurgitate back from the bile duct and enter the blood stream, so small amounts of conjugated bilirubin can be found in the plasma. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma level is increased. Normally, no detectable amount of bilirubin (sometimes referred to as “bile”) is found in the urine.

Serum and plasma are the most common clinical specimens used for electrophoresis applications. Urine and cerebrospinal fluids (CSF) are also suitable. Other body fluids such as pleural fluid and pericardial fluid are analyzed less frequently. Some specimens require pretreatment before electrophoresis. Low concentrations of proteins normally in urine and CSF are concentrated in order to have enough proteins for detectable separations. Some body fluids require removal of pigments, salts, and other compounds that interfere with electrophoresis or the detection of separated solutes. In molecular diagnostic testing of DNA and RNA, the nucleic acids must first be isolated from the specimen and then purified before separation with electrophoresis.

Agarose gels are chemically purified forms of agar, a polysaccharide extracted from seaweed. The gel pores allow for separation of proteins based on their individual charge and mass. Agarose gel will naturally clear after drying the separated proteins.Common clinical uses of agarose gel electrophoresis (AGE) are separations of plasma proteins, hemoglobin variants, lipoproteins, and isoenzymes. The gels come prepackaged with a plastic template to lay over gel for sample application or slots etched in the gel for these samples.

Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.htmlAccessed June 23, 2009.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, Rochester MN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed June 23, 2009.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: Burtis CA, Ashwood ER. Bruns DE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.

Serum lipoprotein electrophoresis is usually performed using fasting serum or plasma. In a fasting sample, large chylomicrons are not normally present and therefore, will not obscure or confound the gel. Because electrophoresis relies on dye-binding and densitometry, samples should have cholesterol > 100 mg/mL. The results of this testing can be used in a variety of ways but typically a report of "type B" or "type A" is sufficient to inform physicians whether there is increased cardiovascular risk.

Fibrin strands woven into the clot structure are cleaved into soluble fibrin fragments, and then removed by macrophages. The action of fibrinolysis also serves to restore blood flow into the area that had been sealed off, helping to promote further healing. Fibrinolysis is mediated by a proteolytic enzyme called plasmin. Plasminogen is the inactive precursor form of plasmin that is found in plasma. Plasmin takes on fibrinolytic properties after activation, digesting both fibrin and fibrinogen. Inhibitors act to control the process, serving as a check and balance system for fibrinolytic activities.

Performed after an unexpected, prolonged PT or APTT is encountered to determine if the problem stems from a factor deficiency or the presence of an inhibitor. To perform the test, the patients’ plasma is mixed with an equal volume of pooled normal plasma, and then a PT and APTT are performed off the mixture. If the addition of the pooled plasma brings the resultant values into normal range, then the pooled plasma contained factors the patient's sample was deficient in, and the patient has a factor deficiency. If the results are not “corrected” or brought back into normal range after the addition of pooled normal plasma, then an inhibitor may be present. The next step in the diagnostic sequence of events, if correction has occurred, is to perform a factor assay, to determine which specific factor is lacking.

Disseminated Intravascular Coagulation (DIC) is best described as a disorder of consumption, because clotting factors are depleted from the blood. Basically, clotting occurs randomly throughout the body, as opposed to just in the localized areas where vascular damage has occurred, consuming clotting factors and other components such as platelets in the process. Symptoms may range from a mild bleed, to severe, profuse bleeding, primarily dependant upon the availability of clotting factors. As more and more coagulation factors and components are consumed, the disorder progresses and symptoms worsen. Most heavily impacted are the levels of factors I, V, and VIII as well as the number of available platelets. Clinically, DIC is detected via an elevated (positive) FDP, positive D-dimer test, a prolonged PT and APTT, plus the manifestation of hemorrhagic episodes. DIC is diagnosed as two primary types, acute and chronic. Acute DIC manifests in a few hours or a few days, has a high mortality rate, and is seen in infections, obstetric complications, liver disease, and tissue injury. Chronic DIC is a secondary condition to some other disease state. Once you treat the primary disease, this type of DIC will go away. Treatment is often factor replacement therapy through the use of fresh frozen plasma and/or cryoprecipitate.

Pre-analytical variables are those that affect the specimen before the actual testing begins. Some of the pre-analytical variables to consider with molecular testing include those that are applicable to all clinical specimens but should be emphasized when discussing molecular methodologies; some of these include but are not limited to: Receipt of valid order Proper patient identification Proper venipuncture procedure for blood collection Use of correct anticoagulant Collection of correct specimen type (i.e.- plasma, serum, whole blood) Order of draw Proper storage Proper transport Procedures if there is a delay in testing and/or transport

Methodologies can be classified as to the target of interest, detection techniques, or the necessity for amplification.Targets of interest can include: DNA View Page

Once absorbed through the mucosal cells of the duodenum, iron is bound to a carrier plasma protein, transferrin (Tf), for movement to sites of utilization. Almost all iron in plasma is bound to Tf, and most Tf-bound iron is carried to the bone marrow to be incorporated into developing erythrocytes. Transferrin is normally about 20% to 40% saturated with iron. (5)Transferrin releases iron to specific transferrin receptors (TfRs) for movement into cells. Transferrin receptors are found on all cells, but are found in relatively high concentration in erythroid precursors, hepatocytes, and placental cells. When the capacity of plasma Tf to bind iron is exceeded, i.e., transferrin saturation (TS) is higher than normal, excess iron is taken up by hepatocytes and other cells. A brief summary of iron metabolism is illustrated.

Hereditary hemochromatosis (HH) is a genetic disorder characterized by iron overload as a result of increased iron absorption. As iron absorption increases, the amount of iron bound to transferrin and transported in the plasma subsequently increases.With no available mechanism for excreting excess absorbed iron, normal iron storage sites become overloaded, resulting in ferritin levels that far exceed normal. As a result, iron is deposited in the parenchymal cells of the liver, pancreas, pituitary, heart, synovium, and other tissues with high concentrations of transferrin receptors. Iron in excess of normal cellular ferritin stores contributes to the generation of free radicals and reactive oxygen intermediates that cause cell damage to organs and tissues. This process results in the clinical condition known as iron overload, a hallmark feature of HH.

In some laboratories the anticoagulated sample is used to prepare concentrated smears. Placing the fluid in a Wintrobe tube and centrifuging it separates the sample into four layers:fat and perivascular cellsplasmabuffy layer - myeloid and nucleated erythroid cellserythrocytesThe volume of each layer is measured using the scale on the Wintrobe tube and then the percentage of each layer is calculated. Next the plasma is removed and a smear is made from the buffy coat and top of the red cell layer. Either the manual push method or cytospin technique may be used to make the smears. They may be stained with a variety of cytochemical stains. Concentrated smears are used to examine cell morphology and demonstrate the presence of abnormal cells when the marrow is hypocellular. The smears cannot be used for differential counts or evaluation of cellularity.

Erythrocytes are produced in the bone marrow and released into the peripheral blood where they may remain for approximately 120 days before senescence.Their main function is the transport of the respiratory gases (oxygen and carbon dioxide) between the lungs and body tissues.Each erythrocyte can be thought of as an "envelope" containing hemoglobin.Each hemoglobin molecule contains iron which has a high affinity for oxygen.As a result, when an erythrocyte passes through one of the capillaries of the lungs, it picks up oxygen.The oxygen is transported through the blood to the tissues where it is released.Carbon dioxide from the tissues then diffuses into the RBC where it undergoes chemical changes.About 70% of the altered carbon dioxide diffuses into the plasma, 25% binds to the hemoglobin molecule, and 5% goes into simple solution within the red cell.In each of these three ways carbon dioxide is transported from the body tissues back to the lungs, where it is released.

Blood is composed of an isotonic fluid (plasma) in which various cells (hemocytes) are suspended. There are three major groups of these cells.

Hemolysis can easily be caused by improper phlebotomy techniques. Hemolysis occurs when RBCs are broken up and hemoglobin is released into the plasma, causing it to become pink rather than its natural straw color. Hemolysis can occur by using too small a needle, pulling a syringe plunger too rapidly, expelling blood vigorously into a tube, or shaking a tube of blood too hard. Hemolysis can cause falsely increased potassium, magnesium, iron, and ammonia levels, and other aberrant lab results.In this case, Marcie did not properly wipe the site with gauze after cleaning it with alcohol, and alcohol contacting the blood could have caused RBCs to break up or hemolyze. Marcie also squeezed the baby’s foot too hard, causing hemolysis.Relevant topics:Site selection and preparation, Heelstick: Puncture, Hemolysis, Causes of hemolysis

Cholesterol High density lipoprotein Low density lipoproteinTriglycerides Lipid profile is run on serum or plasma. It requires a 14 hour fast prior to collection.

Contain either sodium or lithium heparin.Used for tests requiring whole blood or plasma such as ammonia or whole blood potassium.

Blood clots when the coagulation factor proteins within the plasma are activated.Blood starts to clot almost immediately after it is drawn unless it is exposed to an anticoagulant.Clots within the blood specimen, even if not visible to the naked eye, will yield inaccurate results.

Plasma and formed elements stay mixed in circulating blood. When centrifuged (or spun down), blood is separated into plasma, and formed elements including red blood cells. The plasma separator tube shown here has a barrier to maintain separation of plasma and cellular elements during centrifugation and storage. The red cell layer also includes a relatively small amount of platelets and white blood cells, not visible in the photo on the right.

Water (HĢ0) makes up the majority of the blood plasma.

Electrolytes are salts dissolved in water, including:Sodium (Na) Potassium (K) Chloride (Cl) Bicarbonate (CO2). Calcium (Ca)

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.

Serum is the fluid that is left over the coagulum after the specimen is centrifuged (spun down). Serum contains all the same substances as plasma, except for the coagulation proteins, which are left behind in the blood clot.

Another example of rouleaux is seen in this slide taken from a patient with multiple myeloma. Frequently, the darkly stained macroscopic appearance of the slide will be a clue to the presence of rouleaux on the smear. Increased globulins in the plasma often cause the background of the stained smear to be somewhat bluer than the other slides stained at the same time.

Preanalytical Error What is it? How does it happen? What is the result? Hemolysis Red blood cells (RBCs) break and release contents of cell into plasma. Needle incorrectly positioned in vein; cells forced to squeeze through opening. Needle gauge too small; slow blood return into tube. Vigorous mixing or shaking of tube. Alcohol on skin that has not had sufficient time to dry. Some test results may be falsely elevated. (Potassium is especially affected by hemolysis.) Patient may have to be re-drawn. Clotted specimen Clumped or clotted cells in specimen that requires anticoagulated or whole blood Insufficient mixing of blood with anticoagulant in tube. Delay in mixing tube. Slow filling tube. Inaccurate test results for cell counts and clotting studies. Patient may have to be re-drawn. Tube filled to incorrect volume Too little or too much blood in tube. Tube removed from needle too quickly. Vacuum in tube has been compromised due to use of tube past the expiration date (Results in a short fill). Manual fill of tube may lead to over-fill. Test results may be unreliable due to dilution errors. Patient may have to be re-drawn.

Most blood collection tubes contain an additive that either accelerates clotting of the blood (clot activator) or prevents the blood from clotting (anticoagulant). A tube that contains a clot activator will produce a serum sample when the blood is separated by centrifugation and a tube that contains an anticoagulant will produce a plasma sample after centrifugation. Some tests require the use of serum, some require plasma, and other tests require anticoagulated whole blood. The table below lists the most commonly used blood collection tubes. Tube cap color Additive Function of Additive Common laboratory tests Light-blue 3.2% Sodium citrate Prevents blood from clotting by binding calcium Coagulation Red or gold (mottled or "tiger" top used with some tubes is not shown) Serum tube with or without clot activator or gel Clot activator promotes blood clotting with glass or silica particles. Gel separates serum from cells. Chemistry, serology, immunology Green Sodium or lithium heparin with or without gel Prevents clotting by inhibiting thrombin and thromboplastin Stat and routine chemistry Lavender or pink Potassium EDTA Prevents clotting by binding calcium Hematology and blood bank Gray Sodium fluoride, and sodium or potassium oxalate Fluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium. Glucose (especially when testing will be delayed), blood alcohol, lactic acid

The p value is a statistical tool that increases the confidence that an antibody has been identified with a scientifically acceptable level of uncertainty (0.05). As applied to antibody identification, it is computed using Fisher's exact test. Tidbit: This is the same Fisher who helped developed the Fisher-Race theory of Rh inheritance.The p value is calculated using the number of cells that are positive and negative with the patient's plasma. Calculating p values is beyond the scope of this case study but basic understanding of p values at the conceptual level is covered.

In immunohematology textbooks, the "rule of three" is sometimes presented as follows:1. If a patient plasma or serum gives positive results with a minimum of three antigen-positive cells and negative results with a minimum of three antigen-negative cells, concluding that the serum contains an antibody directed against the antigen has a p value of 0.05.2. Therefore, a p value of 0.05 requires at least three positives and three negatives.The first statement is correct but second statement is a misinterpretation of the p value.Three positives and three negatives are required to identify an antibody with a p value of 0.05 ONLY if you have only a 6-cell panel. It does not mean that you always need three positive cells and three negative cells to get p=0.05.For example: A 10-cell panel with eight Jk(a+) cells and two Jk(a-) cells gives a probability of 0.02 if all the positive cells and none of the negative cells react. A 10-cell panel with eight K- cells and two K+ cells gives a probability of 0.02 if all the positive cells and none of the negative cells react. Learning point: You do not need three positive cells and three negative cells to get an acceptable p value of 0.05.

The patient's post-transfusion plasma was also retested with the 6 RBC that tested positive initially. Like the antibody panel done on the post-transfusion plasma, they are now all negative by gel IAT.Unfortunately, the panel results with the patient's post-transfusion eluate do not give clear results (only cells #1 and #9 react) and the antibody remains unidentifiable. Suppose that the physician had decided to continue transfusing the patient at this stage. Take a moment to think about what you would advise regarding the compatibility of such transfusions, all of which appear to be compatible in the crossmatch. When you have considered the options, continue to the next page.

In this case the patient's antibody has disappeared from the plasma by adsorbing to transfused donor red cells. It is detectable but unidentifiable in the post-transfusion red cell eluate. Several trial and error procedures exist to enhance weak antibodies. Which methods will enhance the reactivity of a given antibody depend on its characteristics. Methods to investigate weak antibodies include: Use a higher plasma to red cell ratio (add more antibody-containing plasma or eluate) Increase incubation time (if consistent with manufacturer instructions, if applicable) Use enzyme-treated panel red cells (enzymes enhance IgG antibodies in Rh and Kidd blood systems but denature some antigens, e.g., Fya, Fyb, S) Try alternative antibody detection methods, e.g., if using LISS routinely, try polyethylene glycol (PEG) or column agglutination methods such as gel, providing they have been validated for use in the TS laboratory.

Granular casts are composed of plasma protein aggregates and cellular remnants. Granular casts appear as cylinders of coarse, or fine, highly refractive particles. Coarsely granular casts (yellow arrow) contain large, coarse dark yellow or dark brown granules. They are very irregular in shape as shown in this high power magnification under the brightfield microscope. A hyaline cast can be seen just to the left of the coarse granular cast (blue arrow).