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A 42-year-old male received 6 units of RBCs during open heart surgery 6 months ago. His antibody screen was negative at that time. He has returned for a follow up surgery and his antibody screen is now positive with both screen cells at the AHG phase.Reactions are occurring at AHG phase, which indicates a possible clinically significant antibody, Jka showing dosage. Refer to Case Study 1 panel below to see reactions of antibody panel.IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not doneCase study 1 conclusion:Patient's previous transfusion 6 months ago exposed him to the Jka antigen, causing the formation of this antibody, which is known for showing dosage.

Immune antibodies occur in the serum of individuals who become sensitized to foreign antigens through pregnancy or transfusion. IgM predominates in the primary response, IgG in the secondary response. Most react at 37°C and are considered clinically significant. Examples include antibodies in the Kell, Rh, Duffy, and Kidd systems. Immune antibodies can be classified as alloantibodies or autoantibodies.Alloantibodies Produced by exposure to foreign red cell antigens which are non-self antigens but are of the same species. They react only with allogenic cells. Exposure occurs through pregnancy or transfusion. Examples include anti-K and anti-E. Autoantibodies Produced in an autoimmune process and directed against one's own red cell antigens. React with patient's own cells and all cells tested. Can possibly mask the presence of other significant antibodies. It is very important to make sure that no underlying significant antibodies are present if an autoantibody is suspected. A positive direct antiglobulin test (DAT) or auto control could indicate the presence of an autoantibody. Examples include cold auto (P or I) or warm auto (Rh specificity).

Autoimmune diseases are a group of disorders where the body's immune system malfunctions and attacks its own tissues. One aspect of these diseases is the formation of antibodies that are directed to self-antigens (autoantibodies). Autoimmune diseases can be divided into two general groups: Organ specific, where the autoantibodies attack a specific organ, and Non-organ specific (or systemic), where the autoantibodies attack multiple organ systems. An example of an organ specific autoimmune disease is Hashimoto thyroiditis where autoantibodies damage the thyroid gland. An example of a systemic autoimmune disease is systemic lupus erythematosus (SLE) where the autoantibodies may attack any organ in the body.

The antinuclear antibody test (ANA) is a test used to screen for the presence of autoantibodies that are directed toward components in the nucleus of the cell. Clinicians use the ANA test to assess the likelihood that a given patient has a SARD. The results of the ANA test alone are not diagnostic for the SARD. The patient must also have clinical evidence of the disease as well. Because the early clinical presentation for many of the SARDs are nonspecific, the results of the ANA test and subsequent follow-up testing are key pieces to making the correct diagnosis.Rheumatoid arthritis (RA) is the most prevalent disease in this group; however, the ANA assay is not the primary laboratory test for RA. Instead, the test for RA looks for the presence of rheumatoid factor (RF) or more recently, cyclic citrullinated peptide antibodies (anti-CCP).For the other diseases in the SARD group, especially SLE and SSc, the results of the ANA test can be useful in determining a correct diagnosis. The utility of the ANA test is to detect the antibodies early in the disease process.The ANA results in conjunction with clinical presentation give the clinician solid evidence to intervene with an appropriate treatment. Studies have shown that once treatment is started, the formation of new antibodies slows or even halts.(Ref3)Currently there are no cures for the SARDs. Treatments primarily focus on keeping the patient comfortable and the immune response in check. Treatments can vary from non-steroidal anti-inflammatory drugs, to immuno-suppressive drugs, to stem cell transplants. Individual treatment is often dependent on the severity of the disease and the response to the selected drug regimen.

Beavers C, Kern W, Blick K. Isolated acute thrombocytopenia in a 21-year-old caucasian male. Lab Med. June 2009;40(6):337-339.Bromberg MB. Immune thrombocytopenic purpura, the changing therapeutic landscape. N Engl J Med. 2006; 355:1643-1645. Glassy EF. ed. Color Atlas of Hematology. Northfield, IL: College of American Pathologists; 1998.Kwon JY, Shin JC, Lee JW. Predictor of idiopathic thrombocytopenic purpura in pregnant women presenting with thrombocytopenia. Int J Gynacol Obstet. 2007;85-88. Taghizadeh, M. An update on immune-mediated thrombocytopenia. Lab Med. 2008;39(1):51-54.Tarr PI, Gordon CA, Chandler WE. Shiga like toxin producing Escherichia coli and hemolytic uremic syndrome. Lancet. 2005;365:1073-86.Woelke C , Eichler P. Washington G, etal. Post transfusion purpura in a patient with HPA-1a and GP1a/11a antibodies. Transfus Med 2006;16:69-72. Wyrick-Glatzel J.Thrombotic thrombocytopenic purpura and ADAMTS-13: New insights into pathogenesis, diagnosis and therapy. Lab Med. 2004;35(12):733-737.

Transplacental ITP may occur in newborn infants who are born to mothers with ITP. If the mother has had one baby born with thrombocytopenia, it is usually an indication that all subsequent infants will also be born with thrombocytopenia. A very small percentage of babies born with ITP will have severe thrombocytopenia. Neonatal alloimmune thrombocytopenia (NAIT) is caused by platelet destruction that is the result of alloantibodies stimulated by foreign antigens during pregnancy or blood transfusions. Platelet destruction by alloantibodies may occur in neonates if the mother lacks the platelet-specific antigen but the baby has inherited the antigen from its father. When maternal IgG antiplatelet antibodies cross the placenta, immune destruction of the neonate's platelets occurs. The major concern with both of these conditions is intracranial bleeding if the neonate's platelet count is less than 50 X 109/L. NAIT has a high mortality rate due to bleeding into the central nervous system. Prompt diagnosis of the condition and treatment is critical. The thrombocytopenia lasts on average 3 - 4 weeks postnatal until the maternal antibodies have cleared the newborn's system.

Notice the wide variety of bacterial types that are present in this smear. This variety reflects the diversity of organisms that make up normal bowel flora.When the bowel is torn or punctured, an incredibly large amount of bacteria and foreign matter is released into the peritoneal space. The body's immune responses draw many white blood cells to the area to remove this material. Since there is such a large amount of bacteria to remove, many of the white cells are overwhelmed by the amount of debris that they ingest.

The macrophage is the final stage of development in the monocyte lineage. It is a phagocyte whose roles include the removal of dead and dying tissue and the destruction and ingestion of invading organisms. Macrophages (histiocytes) act as immune modulators as they will present antigens from ingested pathogens to helper T-cells.Their primary role in the bone marrow is the removal of cellular debris, including old red blood cells (RBCs). As a result, they become a source of iron for maturing RBC precursors. A histiocyte is a less phagocytic form of a macrophage with fewer lysosomal granules. Histiocytes may form clusters, or even fuse together into mulitnucleated giant cells. These giant cells are particularly evident on bone marrow biopsy from a patient with a marrow granuloma.The top image on the right shows the early transformation of a monocyte into a macrophage (see red arrow). Notice the increase in the amount of cytoplasm present as the cell begins to ingest debris in the bone marrow. This is demonstrated by the increasing vacuolization present in the cytoplasm. The larger the debris ingested, the larger the vacuoles will be.The lower image on the right shows a macrophage with large vacuoles (red arrow) adjacent to an RBC cluster (blue arrow). This is a common placement, since the macrophage is the iron source for these developing RBCs in the bone marrow.

Rocourt J. Jacquet C. Reilly A.: Epidemiology of human listeriosis and seafoods. International Journal of Food Microbiology. 62:197-209, 2000 While rarely diagnosed prior to 1960, more than 10,000 cases of listeriosis were recorded in the medical literature between 1960 and 1982, and thousands more have been reported annually world-wide. This widespread increase in reporting is most likely due to demographic trends and changes in food production, processing and storage, especially the extended cold food chain and the ability of Listeria monocytogenes to grow at low temperatures L. monocytogenes is a bacterium responsible for opportunistic infections, preferentially affecting individuals whose immune system is perturbed, including pregnant women, newborns, people over 65 years, immunocompromised patients, such as cancer victims, transplant recipients, people on hemodialysis and AIDS patients. Thus, the increasing lifespan and medical progress allowing immunodeficient individuals to survive, partially explains the increasing incidence of listeriosis. Moreover, L. monocytogenes is ubiquitous and can grow at temperatures as low as 0 degrees C. At this temperature growth is very slow. The expansion of the agro-food industry, the widespread use of systems of cold storage and changes in consumers demands have led to a large increase in the pool of Listeria that can cause food-borne infections.

The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria. Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dL. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.

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.

Free radicals are well known to occur in biological systems. A free radical is an atom or small molecule with unpaired electrons. These unpaired electrons make the atom or molecule highly reactive and unstable. Free radicals are produced constantly via metabolic processes. They are also released by immune cells. Immune cells can undergo 'oxidative bursts' (also called respiratory bursts) to help fight pathogens. Oxidative bursts can help degrade pathogens phagocytosed by immune cells and therefore free radicals have an important role in immune system function.However, free radicals also have detrimental effects on surrounding cells. When LDL is co-localized with cells or tissues that are releasing free radicals (such as in an inflamed vessel wall) the free radicals can chemically modify the phospholipids and other components of the lipoprotein. The LDL becomes oxidized and the modification makes the LDL more atherogenic.

Before beginning the course take some time to review and think about what you already know about HDFN. For example, jot down brief notes to answer the following questions: Which antibody causes the most severe HDFN? Antibodies in which blood group system are the most common cause of positive direct antiglobulin tests (DATs) in newborns but rarely cause clinically significant hemolysis? Should DATs be performed on all newborns regardless of maternal ABO and Rh blood groups? What is Rh immune globulin (RhIg), its source, constituents, purpose, and mechanism of action? Which tests are used to determine postnatal RhIg dosage? Which type of D variant can produce anti-D? What follow-up tests are typically indicated if a pregnant female has a positive antibody screen when initially tested? Which laboratory findings would suggest that an infant may have ABO HDFN? How can the clinical status of fetuses at risk for HDFN be monitored? What are the characteristics of red cells suitable for intravenous transfusion to fetuses suffering from severe HDFN due to anti-D?

Since anti-D produces the most severe HDFN and was once relatively common, let's begin by reviewing how anti-D is produced.Immunization to D may occur when Rh-negative individuals are exposed to the D antigen, but developing anti-D varies greatly from person to person. Some individuals produce anti-D after being exposed to a small volume of D-positive red cells (e.g., 0.1 mL). For others, a relatively large volume of D-positive cells is required. Yet other persons will never produce anti-D, regardless of exposure.

To understand the history of HDFN due to anti-D, it is useful to review the immune response. A primary (1°) immune response is the response that occurs following the first exposure to a foreign antigen. A secondary (2°)/anamnestic immune response occurs following subsequent exposures. The main differentiating features as related to producing anti-D during pregnancy are shown in the table and figure. 1o immune response 2o immune response 1. Following the first exposure to the D antigen, a lag phase occurs in which no anti-D is produced, but activated B cells differentiate into plasma cells. The lag phase can be as short as several days, but often is longer. 1. When exposure to D occurs in subsequent pregnancies, the lag phase is short (3–7 days) due to the presence of memory B cells that quickly differentiate into antibody-secreting plasma cells. 2. Depending on the antibody detection method, it often takes 5–15 weeks before anti-D is detectable in serologic tests. 2. An increase in anti-D is usually detectable within days. 3. The amount of anti-D produced is relatively low. 3. The amount of anti-D rises to a higher level. 4. Anti-D titers decline fairly rapidly and may become undetectable. 4. Anti-D titers tend to remain higher for longer but eventually decline. 5. The first anti-D produced is mainly IgM (although small amounts of IgG are usually also produced). 5. The main type of anti-D produced is IgG (although small amounts of IgM may be produced).

Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking, assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k.

As noted, policies for administering RhIg to mothers with a variant of D vary among countries and within some countries. An Rh(D) red blood cell phenotype with a weak or variant expression of the D antigen occurs in 0.2% to 1% of whites and is slightly more common in African Americans. The phenotype is routinely called weak D, although several variants exist. A simple model includes these D variants: 1. Weak DMultiple weak D variants exist. Red cells have fewer D antigens/red cell (quantitative difference) and only minor variations in D antigen proteins. Some, but not all, weak D phenotypes are detected by today's Rh typing sera and may be classified as Rh positive or Rh negative by routine testing but will be positive when a weak D test (IAT with anti-D) is done. An extreme form of weak D is the Del phenotype, in which the D antigen is so weakly expressed that it may be demonstrated only by adsorption and elution of anti-D. Weak D individuals do NOT produce anti-D and can be considered to be Rh positive for transfusion and RhIg purposes.2. Partial DPartial D variants have altered Rh(D) proteins that differ sufficiently from normal D antigens (qualitative difference) to allow anti-D production. Partial D red cells may react with some but not all anti-D typing reagents. There are many categories of partial D antigens (e.g., DIIIa, DVI, DAR), each with a unique genetic basis.Some persons with partial D have weakly expressed D epitopes and are designated "partial weak D."In practice, partial D and weak partial D can be considered similarly, i.e., ideally they should be transfused with Rh negative RBC and are candidates to receive perinatal Rh immune globulin depending on the policy in their location.

Laboratories use different protocols to confirm anti-D from RhIg administration and simultaneously exclude other antibodies in pregnant females at delivery.The following protocols are examples only and assume that the patient has: Received RhIg (this needs to be confirmed); That the antibody screen is positive (2+ or less); Antibody reacts only in the IAT phase and only with D+ screen cells.In other words, the following protocols assume that the antibody looks like a relatively weak IgG anti-D consistent with RhIg administration.Antibody Exclusion Protocols1. Mini-panelProbably the most common protocol is to perform a mini-panel to exclude other antibodies and report "probable passive anti-D due to RhIg administration;" "passive anti-D consistent with recent RhIg administration" or similar.Some commercial panels indicate which panel cells are useful to rule out other antibodies in the presence of anti-D. 2. Full panelSome labs do a full antibody identification panel to confirm anti-D and exclude other antibodies. This protocol is acceptable, but may be overkill, given that the same results can be achieved with fewer red cells.Passive versus ImmuneUnfortunately, there is no definitive test to determine if anti-D is passive or immune. Some labs perform a titration with the assumption that an anti-D titer greater than 4 likelyrepresents active immunization. While a high titer can exclude passive anti-D, a low titer cannot.This issue is discussed in detail in Rh Negative Female with Anti-D at Delivery: A Case Study on Dealing with the Issues, a case study that complements this course.

The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology 2006;22(4):188–195. (scroll to article)Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice. Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP TODAY. May 2008. Accessed January 18, 2011.*Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010. Accessed January 18, 2011.

The mucicarmine staining procedure is very specific in its detection of mucins of epithelial origin and is used to identify adenocarcinomas. It is particularly useful in detecting adenocarcinomas originating in the gastrointestinal tract. This procedure can also successfully stain and detect the capsule of a fungal organism known as Cryptococcus neoformans . This fungus is usually identified in the lungs and in nervous tissues. C. neoformans is more likely to infect immunocompromised patients than persons with active immune systems.

Genetic mutations in HIV are well known and are very likely, considering the presence of two RNA molecules per virus. Either or both RNA molecules can mutate. These mutations potentially lead to drug resistance or encourage the virus to evade the body's immune response. Mutations have created three major groups of HIV - M, N, and O. M is found in 99% of all the HIV cases in the world. N and O are primarily found in West African countries. N, though, infects only a very small number of individuals. The M group has subgroups lettered A to J. Subgroup B predominates in North America.

Genetic mutations in HIV are well known and are very likely, considering the presence of two RNA molecules per virus. Either or both RNA molecules can mutate. These mutations potentially lead to drug resistance or encourage the virus to evade the body's immune response. Mutations have created three major groups of HIV - M, N, and O. M is found in 99% of all the HIV cases in the world. N and O are primarily found in West African countries. N, though, infects only a very small number of individuals. The M group has subgroups lettered A to J. Subgroup B predominates in North America.

HR-HPV type viral infections, if left untreated, can convert normal cervical lining to cervical intraepithelial neoplasia (CIN). With a good immune response, the hyperplasia regresses and no carcinoma develops. This regression ocassionally occurs with CIN 2, but rarely occurs with CIN 3. If the CIN does not regress, high-grade squamous intraepithelial lesions (HSIL) develop. HSIL may progress to invasive carcinoma, typically in older individuals.

Important Flow Cytometry DefinitionsCluster designation or cluster differentiation (CD): A group of antibodies that recognize the same antigen.Fluorochrome (fluorophore): Fluorescent marker that is excited by light of one wavelength (generated by the laser of the flow cytometer) and emits light of a different wavelength (fluorescent light). Gate: A boundary that is set up around a subset of data points to segregate those events for analysis, or to exclude other events from the analysis.Immunofluorescence: Fluorescent expression of immune reactions between antigens and antibodies.Monoclonal antibody (MoAb): An antibody that is produced from a single clone of cells and therefore has high purity and reproducibility. A monoclonal antibody that is used in flow cytometry is directed against a single antigen. For example, the CD2 MoAb will bind to the CD2 antigen on the cell surface of a T cell.

The predominant immunoglobulin class for the B antibodies produced by individuals with group A phenotype and the A antibodies produced by individuals with group B phenotype is IgM. Small quantities of IgG and IgA may also be present.The ABO antibodies found in the serum of group O individuals include anti-A and anti-B. An antibody designated anti-A,B is also present. Anti-A,B in group O individuals tends to be predominantly IgG, although IgM and IgA components are also present.Infants of group O mothers are at higher risk for hemolytic disease of the fetus and newborn (HDFN) than those born to mothers with group A or B because IgG immunoglobulins readily cross the placenta. IgM molecules do not cross the placenta because of their larger size. However, the HDFN that results is usually mild and often subclinical. Infants generally survive with little or no intervention.It is important to note that immune antibodies are usually IgG. Both naturally occurring and immune ABO antibodies are critically important in transfusion since both sensitize, and usually hemolyze, red cells with the corresponding antigen.

Adipose tissue is no longer considered just a storage area for fat for future energy needs or a provider of body insulation. Recent discovery that adipose tissue secretes a large number of bioactive peptides known as adipokines classifies adipose tissue as an endocrine system. Besides adipocytes (fat cells), adipose tissue also contains connective tissue, nerve tissue, stromovascular cells, and immune cells. Each adipocyte can synthesize and secrete into systemic circulation a large number of adipokines. Additionally, the nonadipocyte fraction of adipose tissue secretes proteins.

IL-6 responds to tissue injury. IL-6 is synthesized and secreted by many different cells in addition to adipocytes including immune cells, fibroblasts, endothelial cells and skeletal muscle. IL-6 is increased in obesity and insulin resistance and those with elevated levels are at higher risk for type 2 diabetes and myocardial infarction. Similar to TNF-a, IL-6 increases NEFA release and reduces adiponectin secretion. IL-6 increases insulin resistance by inhibiting insulin receptor signal transduction in liver cells. It also increases other inflammatory cytokines, interleukin-1 (IL-1) and TNF-a, and stimulates the liver to produce C-reactive protein (CRP), an important protein marker of inflammation.

Another organism that has more recently become problematic is Clostridium difficile. Usually, normal gut flora resist overgrowth and colonization by this organism. However, antibiotic use that suppresses the normal gut flora, allows proliferation of C. difficile. The organism releases toxins that cause inflammation and damage to the mucosal lining of the colon, leading to severe diarrhea. An antibiotic-resistant strain has developed that can result in colitis, sepsis, and death. Elderly patients, patients with severe underlying illness, and patients undergoing immunosuppressive therapy are at higher risk of becoming infected since their immune response to the bacteria and its toxins is diminished.

As mentioned in the course introduction, MRSA infections fall into two general types:Healthcare-associated MRSA (HA-MRSA) Infections that occur in people who are, or have recently been, hospitalized. Community-acquired MRSA (CA-MRSA) Infections that are apparently acquired in the community There are a number of factors that distinguish HA-MRSA from CA-MRSA isolates. These factors are summarized in the table below.FactorHA-MRSA CA-MRSAOrigin of strainsNosocomial infectionsFive isolates associated with healthcare settings: USA100, -200, -500, -600, -800USA100 is the predominant isolate while USA 200 is the second most common isolate. USA700 has been isolated in both healthcare and community settings.Evolved from endemic methicillin-susceptible S. aureus (MSSA) strains Two clones, USA300 and USA400, are associated with the majority of CA-MRSA infections in the United States. USA300 has emerged as the most prominent clone and is not found among hospital strains.Genetic lineageIsolates usually carry large SCCmec types I, II or III (34-67 kb)The larger size of SCCmecII and III permits the inclusion of other non-beta-lactam resistance genes so that HA-MRSA strains tend to be multi-drug resistantIsolates carry a smaller SCCmec variant, predominantly type IV (24 kb), less often type V or variant VT. SCCmecIV (except for mecA) does not permit the inclusion of other non-beta lactam resistance genes so that CA-MRSA isolates exhibit resistance to only methicillin and erythromycin and are more often susceptible to other non-beta lactam antibiotics (eg., trimethoprim/sulfamethoxazole (SXT) and clindamycin). Affected populationLargely affects older adults and people with weakened immune systems; those who have undergone surgical procedures are at increased risk. Healthy persons in the general population without established risk factors for MRSA acquisitionClinical syndromesFound at multiple sites, most commonly bloodstream infections, urinary tract infections (UTI) and respiratory tract infectionsPredominantly skin and soft tissue infections (SSTIs), such as abscesses, cellulitis, folliculitis and impetigo and a serious form of pneumoniaGenes for Panton-Valentine leukocidin (PVL) are associated with SCCmecIV; the clinical spectrum of infections caused by CA-MRSA is directly related to the presence of PVL genes, coding for the production of a cytotoxin that causes tissue necrosis and leukocyte destruction.

Clostridial toxins are among the largest bacterial toxins reported to date and C. difficile produces two potent toxins: Toxin A ((TcdA), an enterotoxin and Toxin B (TcdB), a cytotoxin. It is the production of these toxins in the gastrointestinal tract that ultimately leads to disease. There is a relationship between toxin levels, the development of pseudomembranous colitis (PMC), and the duration of diarrhea. Levels of Immunoglobulin G against TcdA correlate directly with protection from disease following colonization, suggesting that a robust immune response is sufficient for protection from C. difficle-associated diarrhea (CDAD). The role of TcdB is not as well understood. Naturally occurring Toxin A negative/Toxin B positive (TcdA-TcdB+) strains have been identified from clinical isolates, which are capable of causing disease, even extensive PMC, suggesting a role for TcdB in CDAD. Toxin A had always been regarded as more important than Toxin B in infection. However, recent work utilizing mutant C. difficile, strains which did not, or could not produce Toxin A, and which were capable of producing very serious disease has led researchers to completely rethink the roles of Toxin A and Toxin B in CDAD. Toxin B was found to be responsible for the more serious damage to intestinal cells. In addition to the primary virulence factors (Toxin A and Toxin B ), Clostridium difficile also produces a third toxin, binary toxin (CDT). The prevalence of CDT in clinical isolates varies widely and its clinical relevance and role in pathogenicity are still not well defined.

Lymphocytes are primarily involved in the body's immune response mechanism. This involves complex phenomena which end in the development of humoral and cellular immunity. Humoral ImmunityHumoral immunity involves the production of antibodies (immunoglobulins), and is brought about by lymphocytes which we call B-cells. B-cells are bone-marrow derived lymphocytes. After B-cells are stimulated by an antigen, they proliferate and transform into plasma cells which produce specific antibodies. Cellular ImmunityCellular immunity includes delayed hypersentivity reactions, graft rejection, graft-versus-host reactions, defense against intracellular organisms, and probably defense against neoplasms. Cellular immunity is mediated by lymphocytes which we call T-cells. T-cells are so named because they are dependent on the thymus for their production and development. The majority of T-cells are long-lived with an average lifespan of 4.4 years, but it is known that some survive for as long as 20 years or more. T-cells are capable of leaving and re-entering the circulation many times during their long life. T and B cells cannot be differentiated when viewing blood films. They are identified through the use of immunologic cell markers.

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.

Days to weeks after exposure, the patient may begin to complain of fever, headache, and fatigue. This may also be accompanied by a rash.For the first several months after the infection, the exposed individual may be HIV-antibody negative and the disease may not be detected. However, the individual is still infective and can transmit the disease during this period.The disease may remain silent in the patient for months to years, even with no treatment.When the immune system is weakened enough, the patient will develop opportunistic infections and be classified as having acquired immunodeficiency syndrome (AIDS).

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

Examples of conditions in which spherocytes can be seen include hereditary spherocytosis and immune hemolytic anemias (i.e., ABO incompatibility). Spherocytes can also form in conditions where there has been a direct physical or chemical injury to the cells. An example would be a smear from an individual who has suffered severe burns. In hereditary spherocytosis, a condition where spherocytes are numerous, the MCHC value will be at the upper limits of normal, or about 36. The identification of spherocytes on the smear of a patient with hereditary spherocytosis can aid significantly in the diagnosis of the disorder. Artifactual spherocytes can appear when blood is stored for a prolonged period of time.

This case concerns a common scenario in the transfusion service (TS) laboratory, the detection of anti-D at delivery in a female who has received Rh immune globulin (RhIg) during pregnancy.Distinguishing between passive and immune anti-D is important clinically: If passive anti-D is misinterpreted as immune, RhIg prophylaxis may be omitted leading to D sensitization. If immune anti-D is misinterpreted as passive, appropriate follow-up of the antibody may be curtailed putting the fetus at risk.Unfortunately, differentiating between immune and passive anti-D is often impossible. This case study presents an opportunity to review perinatal testing programs and the crucial role of RhIg in preventing hemolytic disease of the fetus and newborn (HDFN) due to anti-D. The case also examines practical aspects of routine serologic testing involving neonates and women who have received RhIg during pregnancy. The case is a companion to "Hemolytic Disease of the Fetus and Newborn" and complements its content.In brief, the case will: Guide participants through laboratory findings that need to be interpreted and resolved; Examine current best practices in perinatal testing programs; Review the characteristics of RhIg and its use in pregnancy; Review and investigate key issues associated with detection of anti-D in women who have received antenatal RhIg; Discuss crossmatch and LIS policies related to RhIg-derived passive anti-D.

In cases like those of Patient A.D. two main serologic questions need to be answered:Are the reactions due to passive anti-D from RhIg or due to active (immune) anti-D?Are there other antibodies that need to be excluded Unfortunately, since there is no way to definitively differentiate immune from passive anti-D, certain assumptions are usually made (more later). Most laboratories first confirm that, as expected, only anti-D is being detected.

An issue related to reaction strength of RhIg in serologic tests is how long passive anti-D from RhIg can be detected post-injection. The half-life of IgG is 23 to 26 days. Following injection of RhIg, serologically detectable levels of anti-D peak within hours (IV injection) or days (IM injection).Although the half-life of passive anti-D from RhIg is approximately 3 weeks, it may be detectable by serologic tests for approximately 8 weeks by the indirect antiglobulin test (IAT) and up to 12 weeks or more by continuous flow analyzers used to quantify anti-D. Levels of passive anti-D will decrease over time.Immune anti-D becomes detectable later (e.g., ~4weeks after exposure to D+ red cells), and generally reaches a peak after 6–8 weeks. Levels of immune anti-D will remain constant for longer and will increase following exposure to another immunizing dose of fetal D+ cells. Depending on the many variables that can affect reaction strength (mentioned earlier), as detected serologically, passive anti-D from RhIg can be detected for about 8 weeks or longer by routine, sensitive antibody detection methods.Since RhIg is injected at about 28 weeks, it is routinely detected at delivery, which could occur well before the ~40 weeks considered to be normal gestation (37–42 weeks by the World Health Organization).

Unfortunately, there is no definitive test to determine if anti-D is passive or active. Tests can be done that will suggest whether the anti-D is probably passive or active. However, many laboratories do not perform such testing routinely since it's both safe and efficient to consider a post-RhIg anti-D to be passive without further testing, thereby triggering a post-natal RhIg injection. Further testing would be done if serologic test results suggest an immune anti-D, e.g., 4+ reactions with D+ red cells.One test that may be used to try to interpret if anti-D is passive or active is antibody titration.

Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k.

Delayed HTR result from a secondary (anamnestic) immune response causing a weak, undetectable antibody to become stronger.Upon re-stimulation by donor RBC positive for the antigen corresponding to the patient's antibody:* Patient's memory B cells differentiate into antibody-producing plasma cells.* As new IgG antibody is produced, it sensitizes antigen-positive transfused donor red blood cells.* The IgG-sensitized donor red blood cells are then removed by extravascular hemolysis (EVH) mainly in the spleen.

Susceptibility to an influenza virus strain is dependent on the immune status of the human host, since our bodies become immune only to the strains that we have been exposed to previously (either naturally or by vaccination). The lack of previous exposure to this subtype resulted in the rapid spread and increased number of infections, especially in the younger population. Respiratory infections occurred, even among those individuals who had received the seasonal flu vaccine because that vaccine was not formulated to protect against the influenza A 2009 H1N1 subtype.

Most patients who have suspected or confirmed cases of H1N1 infection have a mild, uncomplicated, self-limited illness that may not require antiviral treatment. If infected individuals have a normal immune system, they should be able to recover from the infection with symptomatic treatment only and without antiviral therapy. However, it is the decision of the patient's physician whether to treat or not to treat. The CDC provides this decision tree as a guideline if the illness is mild and uncomplicated:The CDC suggests that patients with suspected or confirmed influenza should be treated if: They are hospitalized as a result of the illness They are at risk for severe disease including these patients: Patients that have certain medical conditions, such as asthma, diabetes, heart disease, or patients with weakened immune systems that may exacerbate the infection. Children younger than 2 years old Adults 65 years or older Pregnant women or women up to 2 weeks post-partum They have a progressive or complicated illness characterized by signs of: lower respiratory tract disease such as hypoxia or abnormal chest x-ray CNS complications such as encephalitis Complications of low blood pressure including shock or organ failure Myocarditis Invasive secondary bacterial infection The treatment options indicated for the 2009 H1N1 infection include oseltamivir (brand name Tamiflu®), an oral tablet, and zanamivir (brand name Relenza®), an inhaled antiviral agent.Reference: Centers for Disease Control and Prevention. Updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season. December 7, 2009. Available at: http://www.cdc.gov/h1n1flu/recommendations.htm. Accessed January 18, 2010.

Adverse complications of transfusions can be classified into several categories: Immune-mediated transfusion reactions are those that trigger a response from the patient's immune system. Many transfusion reactions are mediated by the recipient's immune system. These reactions occur as a result of antigen-antibody interactions. Antibodies involved include those with specificity towards antigens on red cells, white cells, or platelets. In general, the immune responses occur in three stages: the immune system detects foreign material (antigen) the immune system processes the antigen the immune system mounts a response to remove the antigen from the body Non-immune mediated hemolytic transfusion reactions are caused by the physical or chemical destruction of transfused RBCs, bacterial contamination, circulatory overload, or citrate toxicity. Acute reactions are those that occur during or within 24 hours after the transfusion. There is usually a rapid onset of symptoms and these reactions may be fatal. Delayed reactions occur weeks or months after the transfusion of blood or blood components.

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

TB infection is usually followed by an immune response and latency after exposure. In about 5-10% of cases, the latent period progresses to an active infection.Infection occurs when a susceptible person inhales droplet nuclei containing Mycobacterium tuberculosis and the organism reaches the alveoli of the lungs. The minimal infectious inoculum may be as low as one viable organism.About 2-12 weeks after infection, the immune system limits multiplication of additional bacteria and the immunological test becomes positive.Latent tuberculosis infection (LTBI) is the stage when the viable organism remains in the body; the individual has no symptoms and is noninfectious.Most persons infected with M. tuberculosis do not experience clinical illness and are noninfectious. About 5-10% of persons who are infected and are not treated will develop active TB during their lifetime. The risk for progression is highest during the first several years after infection.Most often, M. tuberculosis infects the lungs. However, it can infect almost any organ in the body, including bones and joints. Tuberculosis meningitis is a TB infection that occurs outside the lungs with devastating consequences, most often in young children and patients with AIDS.

If an initial skin test is classified as negative, a second skin test should be administered 1 - 3 weeks after the first result was read.If the second test is positive, it probably represents a boosted reaction from a past infection. Response to tuberculin decreases over time. The initial TST stimulates the immune system, so that there is an immune response to a subsequent TST.If the second test is negative, the person is classified as not infected.Two-step testing eliminates the false-negative test results due to a weakened immune system.The two-step skin testing is not used in contact investigations or in other circumstances in which ongoing transmission of M. tuberculosis is suspected.

Plasma cells are uncommonly observed in the peripheral blood smear. They are normal constituents of lymph nodes, spleen, connective tissue and bone marrow. The presence of plasma cells in the peripheral blood is indicative of a large number of conditions, mostly related to infections , immune disorders, malignancies, toxic exposures, hypersensitivity reactions and their responses.Although mature plasma cells have a distinct appearance, they still may be confused morphologically with immature plasma cells and other cells with inclusions, reactive changes or nucleated red bloods cell with altered identities. In the image to the right, a plasma cell is present. The plasma cell has an eccentric immature nucleus with a muddy chromatin pattern. Note also clumping and stacking of the erythrocytes, typical of rouleaux formation, implicating an increase in plasma gamma globulin. Further studies are in order, including a bone marrow examination, where at least 30% of bone marrow cells should be variations of mature and immature plasma cells. Serum protein electrophoresis will reveal a monoclonal globulin spike, and light chains in excess of 1.0 gm/24 hours may be seen in the urine. The presence of lytic bone lesions is a convincing clinical clue. With these findings in combination, a diagnosis of myeloma can be made with assurance.