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Lactate dehydrogenase (LD) 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 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.

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).

Currently, the most effective treatment for TTP is therapeutic plasma exchange (TPE). Fresh frozen plasma (FFP), preferably cryoprecipitate-poor plasma (that lacks von Willebrand factor), is used as the replacement fluid in the treatment. The exchange takes place over several days until the patient's platelet count stabilizes above 100 x 109/L.The logic of TPE is to rid the circulation of plasma containing ultra-large vonWillebrand factor (vWF) multimers. vWF is a large multimeric protein that is made by megakaryocytes and endothelial cells. It is is a key factor in platelet adhesion and also is responsible for carrying Factor VIII into the circulation. vWF binds glycoproteins Ib, IIb, and IIIa. The largest multimer is called ultra-large vWF and in normal plasma, it is cleaved into smaller fractions (necessary for balanced coagulation activity) by an enzyme processed by the gene, ADAMTS13. In patients with TTP, the enzyme activity is < 5% of normal and therefore, these ultra-large vWF molecules get into circulation, resulting in excessive platelet aggregation and microvascular thrombus formation.Therapeutic plasma exchange has decreased TTP mortality rate from 90% to 15% since the treatment first came into use as the standard primary treatment of TTP in the 1970's. TPE does not cure TTP, but it arrests the manifestations of the disease until spontaneous remission occurs.

An ideal marker for cardiac disease should have these qualitites: Should be specific to myocardial tissue Be in low concentrations in normal peripheral blood Be rapidly released after myocardial injury Should be detected in low quantities with little interference from like compounds Should remain in circulation for a sufficient length of time for detection The plasma concentration of the marker should be directly related to the extent of injury. The test for the biomarker should be easily automated and relatively inexpensive to run, and results should be obtained rapidly.

Previously, screening for cardiovascular disease (CVD) focused on hyperlipidemia, obesity, and hypertension. However, approximately one half of AMIs occur in healthy men and women with normal or only slightly elevated plasma lipids. With new insights into cardiac disease and the ACS, novel biomarkers such as inflammatory markers, hormones, and other biomolecules indicating myocardial stress are required. Some new screening markers are in use today and many more are in study and evaluation for future use. New screening markers for CVD and ACS are: Highly Sensitive C-Reactive Protein (hs-CRP) Homocysteine Ischemial Modified Albumin (IMA) Myeloperoxidase (MPO)

There are a large number of other molecules being evaluated and studied to determine their use as reliable cardiac biomarkers. Future research will demonstrate if they provide additional helpful information in ACS and if they are helpful in screening for CVD in asymptomatic individuals. Some future markers under study are lipoprotein(a), oxidized LDL, metalloproteinases, lipoprotein-associated phospholipase A2, pregnancy-associated plasma protein A, and placental growth factor. With advances in molecular diagnostics and proteomics, an individual's plasma proteome, their fingerprint of proteins and peptides, may be a biomarker profile of current and future disease.

Specific gravity (SG) measured with the chemical 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 specific gravity with the reagent strip method. It is the recommended method for determining specific gravity if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the specific gravity result on the reagent pad. If the result is being read visually, it is recommended that .005 be added to the specific gravity result when the pH is alkaline. Most chemical reagent strip readers, however, will automatically adjust the specific gravity reading for pH. A specific gravity reading higher than the chemical reagent strip range would need to be measured by another method, and may require dilution.

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.

The order of draw for a capillary blood collection is slightly different than the order of draw for a venous blood collection.If capillary blood gases are ordered, they are drawn first to avoid introduction of room air as much as possible. A specimen for blood count is collected before tubes containing other anticoagulants and additives. This is to ensure that the blood will not begin to clot before this specimen is collected; clots will affect the accuracy of the blood count. The following order of draw is commonly used: Container Additive Use Lavender top EDTA For hematology blood counts Green top Lithium heparin Tests that require a heparinized plasma sample __ Other tubes containing anticoagulants Varied Red or gold top Clot activator Tests that require a serum sample Red top No additive Tests that require a serum sample but clot activator and/or gel may affect test

Improper collection of the blood specimen that is used for testing can cause false prolongation of PT or aPTT results. The following table covers several preanalytical variables that may affect PT or aPTT test results Preanalytical Variable Cause of False Elevation of PT and or aPTT Test Result Corrective Action Blood collection tube is inadequately filled. Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Recollect specimen ensuring proper fill to achieve a blood to anticoagulant ratio of 9:1. Patient has a hematocrit level above 55% Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Prepare a specimen collection tube that contains less anticoagulant. Refer to your laboratory's procedure for the proper amount of anticoagulant. Specimen is clotted. Coagulation factors have been activated; insufficient levels left in the plasma. PT and aPTT results will be affected. Recollect the specimen. Specimen collected from an arm with a heparin lock or from a heparinized vascular access device (VAD). Heparin contamination will prolong the aPTT. Collect the blood from a vein rather than a VAD. If blood must be drawn from the VAD, flush it first with 5 mL of saline, and discard the first 5 mL of blood before collecting the specimen. Patient is receiving heparin therapy. Heparin will prolong the aPTT If the patient is being evaluated for possible factor deficiencies or coagulation inhibitors, use a heparin digesting enzyme as a pretreatment before testing the PT or aPTT. .

A patient would generally need a level > 40% of each factor that is being detected by the test procedure to achieve a normal (non-prolonged) aPTT or PT test result. Therefore, a patient with an inadequate level, meaning less than 40%, of one or more coagulation factor will have a prolonged PT or aPTT test. In the mixing study, an aliquot of abnormal patient plasma is mixed with an equal amount of pooled normal plasma (PNP), which contains approximately 100% of all coagulation factors. The new mixed plasma sample contains at least a 40% level of each factor after the mix, including the factors that may have been present in very low levels in the original patient sample.For example, if a patient's sample contained 10% of the normal amount of factor VII, a prolonged PT test would occur. After adding an equal amount of the pooled normal plasma, with an approximate 100% of factor VII, the resulting quantity of factor VII in the sample would be 55%; enough to "correct" the PT test result.When this new mixture is retested for PT or aPTT, the results that were originally prolonged due to low coagulation factor concentrations will "correct". If an inhibitor is present that can counteract and nullify the newly added factors, there will be no correction in the assay, and the PT or aPTT will remain prolonged.

The specimen drawn for a mixing study must meet the following conditions for accurate testing: A properly filled 3.2% sodium citrate tube must be collected. Proper centrifugation to create platelet-poor plasma for analysis must occur; the presence of platelet phospholipids can interfere with the mixing study if an anti-phospholipid antibody is present. Testing must be performed within 4 hours of collection.

Step TwoThe next step is an immediate re-run of the PT and/or aPTT test with the newly created sample mixture. The results should be documented on a worksheet to compare to the original PT and/or aPTT tests.Step ThreeThe sample that has been made for the mixing study consisting of the pooled normal plasma and the patient plasma should also be incubated to rule-out any slow reacting inhibitors. To do this the "mixed" specimen is incubated at 37°C for 1 - 2 hours or as long as required by your laboratory's procedure. A set of control tubes should also be incubated at the same time as the mixed sample tube. A pure patient plasma sample and a pure pooled normal plasma sample will serve as the controls in this procedure. You may incubate all 3 tubes together in a water bath or heat block. Refer to the image to the right. The incubation of these controls will account for the heat-labile state of some coagulation factors which will be discussed again.After the incubation phase, the PT and/or aPTT tests should then be repeated once more. If any coagulation inhibitors were present in the patient sample, the incubation phase would have given the ideal temperature and time for the antibody-coagulation factor reaction to take place. This is especially helpful in the case of anti-factor VIII inhibitors since they are often slow acting or weak inhibitors.

A "corrected" mixing study result is defined as a PT or aPTT result that is now in normal range of the pooled normal plama control, or within approximately 10% of the normal range. A PT or aPTT test that was greatly prolonged at baseline, may not completely correct to within the normal reference interval on a 1:1 mix."No correction" would then mean that the patient sample/pooled normal plasma mixture had no significant decrease in clotting time for the PT or aPTT compared to the intial PT/aPTT results.

Various tools have been developed that identify whether a sample is "corrected" or "not corrected" by the addition of pooled normal plasma. One tool is the Rosner Index.The Rosner Index subtracts the clotting time of the pooled normal plasma (PNP) from the clotting time of the 1:1 mix. This result is then divided by the clotting time of the patient sample. The equation is as follows:Rosner Index = (1:1 mix clotting time result - PNP clotting time result) / initial prolonged clotting time of patient sampleWith this method, a high index value represents the possibility of an inhibitor. A low index value would represent a possible factor deficiency. For example, an index of 10 or lower indicates correction, 15 and above indicates no correction. If after the calculation is performed and a value of 10-15 is obtained, it is recommended that your test be repeated.Each laboratory must determine its own reference interval for the Rosner Index.

Another method used for the interpretation of mixing study results is the <70% correction method created by KM Devreese:% correction = [(aPTT patient plasma - aPTT 1:1mix)/(aPTT patient plasma - aPTT pooled normal plasma) x 100]A % correction >70% indicates correction; a % correction <70% indicates no correction.

Patients who have factor deficiencies may or may not require immediate treatment based upon their risk of bleeding. For example, a patient may only need therapeutic treatment if they are having an invasive surgery or a dental procedure. For the patients who do require treatment, some of the current options are: Transfusion of Fresh Frozen Plasma (FFP) Administration of factor concentrates

To quantify the actual amount of factor present in a factor-deficient patient sample, one can employ another test based on the PT or aPTT assays. This assay involves mixing various dilutions of a patient sample with the specific factor-deficient reagent plasma. The results of the various "mixtures" are then plotted against a standard curve that correlates with factor activity levels. When developing the standard curve, at least 3 points must be plotted to ensure accuracy when utilizing the curve for patient factor activity assays. Once the standard curve has been constructed and all points plotted, the approximate amount of factor contained in the patient sample can be interpreted. The patient sample must have two or more points plotted against the standard curve, which enhances accuracy and allows for the recognition of inhibitors in the patient sample. These points must fall within the acceptable range of the standard curve to be considered valid. Two or more assayed reference plasmas that validate the standard curve should also be tested at least every 8 hours of patient testing to ensure accuracy of patient results.

A 50-year-old male with a family history of diabetes visits his physician for routine physical. He reports that he feels his health is excellent. He exercises regularly, but often his diet is high in calories and fat.Physical Examination: Slightly overweight; blood pressure and pulse normal.A basic metabolic panel and PSA are ordered. All results are within reference range except the plasma glucose. The patient's physician orders a hemoglobin A1C (HbA1C) the following week.Laboratory results:Fasting plasma glucose (FPG)= 110 mg/dL (Reference interval 75 - 100 mg/dL)One Week Later:Hb A1C= 6.0% (Reference interval 4 - 6%)

In 1997, the ADA recommended significant changes in the diagnosis of diabetes. The poorly reproducible oral glucose tolerance test (OGTT) was replaced with easier to use and more patient-friendly diagnostic criteria. An elevated fasting plasma glucose (FPG) was the preferred test to document hyperglycemia according to the 1997 ADA Clinical Practice Recommendations. An elevated casual plasma glucose with symptoms of diabetes and 2-hour plasma glucose after an ingestion of 75 grams of dissolved glucose were also used for diagnosis. In 2010, the ADA affirmed the decision of an international expert committee's recommendation to use the HbA1c test to diagnose diabetes with a threshold > 6.5%.Any one of the four criteria can be used. The hyperglycemia should be demonstrated a second time by any of the four criteria unless the glucose level is significantly high and diabetes is unquestionable. The table below lists the diagnostic assays and criteria. Assay Description Criteria for Diabetes HbA1C Performed in laboratory by method NGSP certified and standardized to DCCT assay > 6.5 % Fasting plasma glucose At least 8 hour fast > 126 mg/dL Casual plasma glucose Symptoms of diabetes; Blood glucose measured at any time of day > 200 mg/dL Two-hour plasma glucose Following a glucose load of 75g anhydrous glucose dissolved in water > 200 mg/dL These criteria are reviewed regularly by ADA, WHO, and IDF.

Plasma Glucose Level Designation 75-100 mg/dL Reference range for Fasting Plasma Glucose < 100 mg/dL 2003 ADA Normal Fasting Plasma Glucose 100 mg/dL to 125 mg/dL ADA Impaired Plasma Glucose (IFG) 140 mg/dL to 199 mg/dL ADA and WHO Impaired Glucose Tolerance (IGT) HbA1C 4-6% ADA Recommended Due to limited A1C assay availability worldwide, WHO does not publish A1C recommended levels

These are the ranges that are recommended by the 2010 ADA Clinical Practice Guidelines for determining increased risk for diabetes: Glucose test Range indicating increased risk for diabetes Fasting plasma glucose 100 - 125 mg/dL 2-hour plasma glucose following 75g glucose load 140 - 199 mg/dL HbA1C 5.7 - 6.5%

Serum, plasma, and whole blood glucose levels are among the most common laboratory assays. Due to self-monitoring of blood glucose (SMBG), blood glucose is also the most common assay performed by patients themselves or their caretakers. Fasting, timed, and casual serum or plasma specimens are run in hospital laboratories for screening, diagnosis, and monitoring of patients.

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.html Accessed March 25, 2013.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, RochesterMN;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. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. 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.

Mixing studies may be ordered 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 on 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 orderProper patient identificationProper venipuncture procedure for blood collection Use of correct anticoagulant Collection of correct specimen type (eg - plasma, serum, whole blood)Order of drawProper storage Proper transportProcedures 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 RNA Bacteria Fungus Virus Antibody Antigen Detection techniques can include:Chemiluminescence Electrophoresis Enzyme Fluorescence Hybridization Radioactivity Amplification is not necessary in direct techniques.

Sickle cell anemia, in addition to being a hemoglobinopathy, is a hemolytic anemia. Hemolysis is both intravascular (about one-third) and extravascular (about two-thirds). Common markers of hemolysis include elevated LDH, bilirubin, and reticulocyte levels.The hemoglobin that is released into the plasma during intravascular hemolysis combines with nitric oxide (NO). The resulting decrease in NO availability contributes to the vaso-occlusive crisis by stimulating vasoconstriction, endothelial adhesiveness, and thrombosis.Hemolytic crisis also involves splenic sequestration, which occurs in an effort to remove damaged red blood cells. This can result in hypovolemia, which may lead to shock, especially in children. Children can also exhibit splenomegaly due to intrasplenic pooling of blood.Adults in hemolytic crisis may experience autosplenectomy. This occurs when the spleen has multiple infarctions, followed by fibrosis, which renders the spleen nonfunctional.

Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, for example, erythrocyte membrane defects or red cell enzyme deficiencies. The diagnosis of ABO HDFN in the laboratory differs from diagnosing Rh and other types of HDFN in which clinically significant antibodies must be identified. Diagnosis may be difficult, because the DAT on the newborn's red cells is unreliable. Indeed, many labs do not routinely do a DAT on infants born to Rh positive females, since many will be positive in the absence of clinically significant hemolysis. Cord blood is often retained (e.g., for 7 days) should the infant develop signs of HDFN and required testing.If ABO HDFN is possible, based on incompatible ABO blood groups and a positive DAT, and the mother's antibody screen is negative, many laboratories do not investigate the positive DAT as would be done for unexpected antibodies like anti-D or anti-K (the laboratory does not perform an elution on the newborn's red cells). Instead, the infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN.

Diagnosis of ABO HDFN is supported by these findings: ABO incompatibility between mother and child, with mother typically group O; Maternal antibody screen negative; Cord DAT weakly positive or negative; Newborn hyperbilirubinemia with jaundice occurring in first 24 hours; Increased spherocytes and reticulocytosis in the newborn; Presence of IgG anti-A or anti-B in cord plasma / serum.

The Red Blood Cells (RBC) that are chosen for exchange transfusion must meet these criteria: ABO-compatible with mother and infant (usually group O) and lack antigens to any maternal IgG antibodies; If mother has anti-D, RBCs are group O Rh negative; No greater than 7 days old; Reconstituted with AB Fresh Frozen Plasma (FFP) to obtain a prescribed hematocrit, e.g., 45–60% (0.45–0.60); CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from hypoxia-induced sickling; Irradiated with a minimum dose of 25 Gray (Gy) to prevent graft-versus-host disease.RBC are normally crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. Molecular methods exist for typing Rh (RHD and RHCE), Kell (K & k), Duffy (Fya & Fyb), and Kidd (Jka &Jkb) loci.In perinatal testing programs, molecular typing can determine the Rh type of the mother, father, and fetus and may be done if the mother has anti-D or another antibody known to cause HDFN. More specifically, if available, DNA methods are typically used in these circumstances: For women who type as weak D in serologic tests, to determine the Rh genotype of the mother to identify if she is partial D or weak D; For women who have made anti-D, to determine the Rh genotype of the father to see if fetal monitoring is needed; For women who have made anti-D, to determine the Rh type of the fetus if the father is heterozygous for RhD or unavailable for testing. Fetal blood typing can be done using fetal DNA from cells obtained by amniocentesis or by testing cell-free, fetal-derived DNA present in maternal plasma at 5 weeks gestation and later. Like all diagnostic methods, DNA typing has limitations and is not 100% sensitive and specific. For example: The blood group's molecular basis may be unknown; Not all alleles in ethnic populations are known; Rare mutations in the RHD and other genes may not be detected; Silencing changes (switching off of a gene) may affect antigen expression; Fetal typing using amniotic fluid may give false-negative results because of maternal cell contamination.

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.

Connective tissue is classified by cellular patterns and the composition of the matrix. There are 4 categories of connective tissue in adults:Connective tissue proper - The matrix is composed of collagen, elastin, and reticulin fibers as well as adipose and mast cells.Cartilage - The matrix is composed of firm, gel-like matrix is made up of water, collagen fibers and chondrocytes.Osseous tissue (bone) - Bone is comprised of a rigid, mineralized matrix of collagen fibers and osteocytes.Vascular tissue (blood) - The matrix is a liquid called plasma, which consists of red and white blood cells, leukocytes and platelets.

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.

Metabolic syndrome may vary in definition and diagnostic criteria depending on the organization that is consulted. Health-related organizations that have developed diagnostic criteria for metabolic syndrome include: National Heart, Lung, and Blood Institute (NHLBI)/American Heart Association (AHA) World Health Organization (WHO) American Association of Clinical Endocrinologists (AACE) International Diabetes Foundation (IDF) European Group for Study of Insulin Resistance (EGIR)Each organization's set of criteria is slightly different in its parameters and its details for diagnosis. All of the above organizations agree that defining glucose intolerance, obesity, hypertension, and dyslipidemia is important. However, there are varied opinions in how important each risk factor is in relation to the others. Varied criteria to determine obesity is utilized: waist to hip ratio, BMI, and waist circumference. One organization does not include a measurement of obesity. Glucose intolerance is determined by measuring plasma insulin and/or glucose levels. Lack of standardization in insulin measurement and assay availability makes criteria using insulin levels impractical.

Blood is composed of an isotonic fluid, called plasma, in which various peripheral blood cells (hemocytes) are suspended. There are three major groups of peripheral blood cells. The three major groups include:Red Blood Cells (Erythrocytes)White Blood Cells (Leukocytes)Platelets (Thrombocytes)

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

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 (H20) 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.

Red cell reaction strengths at delivery from an antenatal RhIg injection at 26–30 weeks (usually 28 weeks) are typically 2+ or less, although stronger reactions are possible depending on the detection method, time since injection, and other factors. Multiple variables can affect the reaction strength of passive anti-D seen post-RhIg injection: Amount of RhIg injected (the greater the number of IU of anti-D administered, the stronger reactions will be); Titers of anti-D in the plasma pool used to manufacture RhIg (occasionally a donor with an exceptionally strong anti-D may be in the pool); Maternal physical size and related blood volume (a larger volume of maternal plasma will dilute RhIg more); Time between RhIg administration and testing (passive antibody will decrease in strength over time); Sensitivity of antibody detection method (e.g., gel-IAT and PEG-IAT may give stronger reactions than LISS-IAT); Volume of FMH (amount of D-positive fetal RBC available in the mother to adsorb anti-D); Route of RhIg administration: Some RhIg products can be administered IM only, whereas others can be given both IM and IV (see later). Peak levels of RhIg are reached faster with IV compared to IM administration (within hours with IV administration compared to days with IM administration). Also, with IV administration, higher levels of IgG anti-D are achieved. Operator variability (technologist techniques vary in removing cell buttons when reading IATs). Because of these variables, many laboratories consider 2+ or less reaction strengths to be consistent with passive anti-D.

Below are the results of a mini-panel of red cells specifically chosen to exclude other clinically significant antibodies in the presence of anti-D. Besides an autocontrol, a positive control (Ror) was included to confirm that the mother's plasma containing the probable anti-D was reactive at the time of testing. Recall that the results of the initial antibody screen showed that the possible (unexcluded) antibodies were anti-C, D, E, K, Fyb, Jka, M, s, Leb(with anti-M less likely as a cause of HDFN and anti-Leb not a cause).Antibodies excluded by Screen Cell #3 included anti-c, e, Fya , Jkb, N, S, P1 and anti-Lea.Before proceeding to the next page, assess whether the unexcluded antibodies from the initial antibody screen have been excluded by the mini-panel below using the guidelines in the antibody exclusion protocol.Mini-Panel ResultsCellRhRhesusKellDuffyKiddMNSsPLewisResultsCDEceKkFyaFybJkaJkbMNSsP1LeaLebGel IAT*1rr000+++++0+00++0+S+002rr000++0+0++0++0++S+003r'r+00++0++00++0+00+004r'r+00+++++++++0+++0+05r"r00+++0+0+0+++0+++006r"r00+++0++++++++++0+07Ror0++++0++++++++++0+2+8Auto0* IAT = indirect antiglobulin test All panel cells are negative for low frequency antigens and positive for high frequency antigens unless noted otherwise. All cells are also negative for Cw, Kpa, and Lua.

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

Therapeutic drug monitoring helps to ensure that a dosing regimen is appropriate for a given patient. The blood plasma concentration of the drug is measured to determine the correct dose that will achieve a therapeutic level of the drug without overdosing into a toxic range. When a drug enters the body, it reaches a peak concentration that starts to fall as the drug is eliminated.The amount of time it takes for a drug's concentration in the body to decrease by 50% is called the drug's half-life. The longer a drug's half-life, the slower it is removed from the body. Most drugs are eliminated from the body in one to three days, but some drugs with longer half-lives can still be detected in the body weeks after the initial dose.The figure on the right illustrates a typical concentration pattern for a drug that is given orally (ingested).

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 irregularly-shaped cylinders of coarse, or fine, highly refractive particles. A granular cast containing coarse brown granules is indicated by the yellow arrow in the image on the right (brightfield, 400X magnification). A hyaline cast can be seen just to the left of the granular cast (blue arrow). The presence of an occasional granular cast is not considered pathologic.

Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include: Antibody Binding to Red Blood Cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of Complement The end result of complement activation is red cell lysis. Activation of Mononuclear Phagocytes and Cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of Coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobulinemia and hemglobinuria which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system.

A febrile non-hemolytic transfusion reactions (FNHTR) is defined as a temperature increase of 1oC over 37oC occurring during or after the transfusion of blood components. FNHTRs are more common in the transfusion of platelets. Multiply-transfused patients and multiparous women make up the largest populations experiencing this type of reaction. There are two mechanisms involved in the manifestation of a FNHTR. The first one involves the presence of a white cell antibody in the patient's plasma that interacts with the white cells in the blood product. These antibodies may be directed against granulocyte antigens or human leukocyte antigens (HLA). This interaction causes endotoxins to be released, which act on the hypothalamus and stimulate a fever. The second mechanism involves the generation of leukocyte cytokines during product storage. The production of cytokines usually occurs during storage in warmer temperatures, which is why non-leukoreduced platelets are commonly implicated.

Allergic reactions are grouped into three categories depending on severity: mild or uncomplicated moderate or anaphylactoid life-threatening or anaphylactic reactionsMild allergic reactions occur in about 1-3% of patients receiving blood products containing plasma. Symptoms are usually mild and include urticaria, erythema (skin redness), and itching. Hives can appear any where on the body and may vary in size. Symptoms usually occur within minutes after the start of the transfusion. They can often last for hours or even days. 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 that are bound to IgE on mast cells and causes the release of histamines. Allergen substances may be drugs or food consumed by the blood donor. Anaphylactoid and anaphylactic reactions have similar presentations. These reactions are rare but life-threatening. Anaphylactoid and anaphylactic reactions are severe systemic reactions with symptoms such as hypotension, dyspnea, nausea, vomiting, urticaria, and diarrhea. The most life-threatening symptoms include lower airway obstruction, laryngeal edema, cardiac arrhythmia, cardiac arrest, shock, and loss of consciousness. None of these reactions present with fever.

Diagnosis of allergic reactions is based on the recognition of a skin rash associated with itching. Treatment involves temporarily discontinuing the transfusion and administering an antihistamine. The rash will usually heal when the transfusion is stopped or when an antihistamine is given. Once symptoms have been alleviated, the transfusion may be resumed. If symptoms continue or progress, the transfusion must be stopped and a new donor unit obtained. Premedication will usually prevent urticarial reactions in patients with a history of allergic reactions. If premedication is unsuccessful, washed cellular products may prevent a reaction. Leukoreduction has no role in preventing an allergic reaction. Anaphylatic and anaphylactiod reactions should be recognized when patients develop symptoms described on the previous page. The transfusion must be stopped immediately. Differential diagnosis includes hypotensive reactions, transfusion-related acute lung injury (TRALI), myocaridal infarction, and pulmonary embolus. An IgA deficiency should be investigated and is confirmed by the presence of anti-IgA. Treatment includes timely administration of epinephrine in addition to other supportive care such as vasopressors and airway support. Prevention involves avoiding transfusion of IgA. Cellular products should be washed to remove residual plasma. Products may also be collected from donors who are known to be IgA deficient. Autologous donations are an alternative.

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 has made several recommendations for preventing TRALI including: Blood collection facilities should implement interventions to minimize the preparation of high-plasma-volume components from donors known to be leukocyte-alloimmunized or at increased risk for leukocyte alloimmunization. Blood transfusion facilities should work toward implementing appropriate evidence-based hemotherapy practices to minimize unnecessary transfusion. Blood collection and transfusion facilities should monitor the incidence of reported TRALI and TRALI-related mortality. Transfusion services should work with clinicians to educate providers about the risks of TRALI and about the need to work toward implementing evidence-based transfusion practices for all blood components, with special emphasis on high plasma-volume components. High-plasma-volume components include the following: FFP obtained from whole blood or apheresis Plasma frozen within 24 hours Cryoprecipitate-reduced plasma Apheresis platelets Whole bloodThere have been several other suggestions for preventing TRALI, which include: Screening of all donors for anti-neutrophil or anti-HLA antibodies. Once donors are identified, they are excluded from donating, or their blood is used for products that do not contain much plasma. This method would not prevent TRALI in recipients who have alloantibodies. Use of pre-storage leukoreduced blood. Use of younger blood products. Appropriate utilization of blood products. Using blood products only when clinically indicated may reduce the frequency of TRALI. Because TRALI can coexist with other transfusion reactions and with pulmonary complications unrelated to transfusion, the diagnosis of TRALI is difficult, but it is an important step in monitoring the effectiveness of TRALI risk-reduction strategies.

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.

Post-transfusion purpura (PTP) is a very rare complication of blood transfusion. It has been most commonly associated with the transfusion of red blood cells (RBCs) and whole blood, but has also been seen in platelet and plasma transfusions. It is characterized by a rapid onset of thrombocytopenia, or decreased platelet count, which results from the product of a platelet alloantibody. Platelet counts are usually less than 10,000/uL. Reactions occur around 7 to 14 days post-transfusion. Patients present with purpura, bleeding from the mucous membranes, gastrointesinal ,and/or urinary tract bleeding. Melena and vaginal bleeding have also been reported. The thrombocytopenia is usually self-limiting. Platelet counts and coagulation studies aid in the diagnosis. Patients can also be tested for platelet specific antibodies, human leukocyte antigen (HLA) antibodies and lymphocytotoxic antibodies. The differential diagnosis includes other causes of thrombocytopenia.