Reactive Information and Courses from MediaLab, Inc.

Rule-out (also referred to as exclusion or cross-out) is a process by which antibodies are identified as being unlikely in a given sample because of the absence of an expected antigen-antibody reaction. In other words, the absence of a reaction is noted with a cell that is positive for the corresponding antigen. Rule-out, while very useful, can lead to error. Ruling out an antibody should be combined with other supporting data to increase confidence in the solution; the more data collected, the higher the probability that the final solution is correct.Non-reactive cells are selected for rule-out. To be classified as non-reactive, a cell must NOT have reacted in any phase of testing in a given panel or screen. In the case of cold antibodies: if reactions are only occurring at immediate spin and are negative in the AHG phase, then that panel cell can be used as a rule out cell for IgG reactive antibodies but not for antibodies that react at immediate spin (IgM).If there is no reaction with a panel cell then it is possible that antibodies to the antigens on that cell are not present in the sample being tested.

There are a wide variety of solid tumors that can metastasize and spread into body fluids. As with cytospins positive for leukemia or lymphoma, any smear with tumor or suspected tumor should be sent for pathology or hematologist review.Body fluids tend to be a good growth medium for metastatic tumors. These tumor cells tend to be present in sheets and clumps. Frequently there will be reactive changes with increased mesothelial cells and macrophages associated with metastatic tumors as well.Tumor cells, in general, typically appear large with fine/open chromatin patterns, dismorphic or dysplastic nuclei and prominent nucleoli. They will have varying amounts of basophilic cytoplasm depending on the tissue of origin.

The macrophages in this image have multiple large phagocytic vacuoles. This sample is a reactive pleural effusion in a patient with widely metastatic rhabdomyosarcoma. These particular macrophages have been removing some of the red blood cells (RBCs) that have been released into the pleural space due to the spread of this patient's tumor. While there are no intact RBCs in these cells, the size of the vacuoles is good indication of what they have most likely been phagocytizing.

This cytospin is from a patient who presented in respiratory distress and was found to have a large mediastinal mass and large bilateral pleural effusions.The lymphoid cells in this image are large and immature in appearance. These lymphocytes were initially believed to be consistent with lymphoma cells but, after immunophenotyping, were found to be reactive T-cells instead of lymphoma cells.The three larger cells in the image look similar. The two larger cells on the left are just macrophages. The one larger cell on the right is actually the malignant cell (see arrow). The malignant cell has a larger nucleus with softer more open chromatoin and a slightly more promanent nucleoli. The cytoplasm is also more basophilic, and the vacuoles are atypical. They are not the typical round vacuoles seen in macrophages/histocytes; these vacuoles are more elongated.The diagnosis of ALCL was confirmed when the cytogenetics proved positive for the specific translocation, t(2;5), that defines this lymphoma.

Plasma cells are terminally differentiated B-lymphocytes that have developed a characteristic morphology while actively producing and releasing immunoglobulins. While plasma cells have their origins in the bone marrow as B-cells, they usually leave the bone marrow to develop and mature in the lymph nodes or spleen. Plasma cells begin to produce immunoglobulins after being stimulated by T-cells and exposed to processed antigens.Under normal circumstances, plasma cells are not a large percentage of the lymphoid cells found in a marrow. They are usually placed in a separate category in the differential, unlike viral/atypical lymphs. There can be a relative increase in plasma cells in reactive marrows, and both plasma cells and their early precursors will be markedly increased in plasma cell disorders.While mature plasma cells somewhat resemble lymphocytes, there are a few important differences. The size of the cell is usually larger with more abundant cytoplasm. The nucleus is eccentrically placed and the overall shape of the cell generally resembles a wedge or comet with the nucleus leading the cytoplasm. The chromatin is just as thick and clumpy as a lymphocyte's but is aligned in a more "spokey" or "clockwork" pattern. The cytoplasm is usually more basophilic than the cytoplasm of a normal lymphocyte and will have a well-defined perinuclear halo or noticeable clearing in the golgi area. Vacuoles may or may not be present.Notice the size of the single plasma cell in the top image (see red arrow). It is larger than the neutrophil precursors surrounding it and is almost rectangular in shape. Observe that the nucleus leads the cytoplasm, causing the wedge or comet shape. Notice the prominent perinuclear halo. Find the two plasma cells in the upper left corner of the second image. There is much more cytoplasm in these plasma cells compared to the occasional lymphocyte present in the field. Notice the eccentric nuclear placement as well as the characteristic clearing in the golgi area.

Smith KR, Fisher HC III, Hook, EW III: Prevalence of fluorescent monoclonal antibody-nonreactive Neisseria gonorrhoeae in five North American sexually transmitted disease clinics. J Clin Microbiol 34:1551-1552, 1996 We compared a direct fluorescent monoclonal antibody (DFA) test with alternative enzymatic and fermention tests for identifying presumptive gonococcal isolates in a systematic sample from patients attending five sexually transmitted disease clinics in five cities. Fourteen (2.5%) of 556 isolates from three clinics were nonreactive with the DFA confirmatory reagent and reactive by both the Quad-Ferm and Rapid NH tests. The prevalence of DFA-nonreactive Neisseria gonorrhoeae isolates varies geographically and is independent of local methods for the identification of possible gonococci. On the basis of our findings, we recommend that for use in medicolegal and other instances in which a diagnosis of gonorrhea has the potential to have far-reaching effects, it is appropriate to test DFA reagent-nonreactive, oxidase-positive, gram-negative diplococci by alternative methods of gonococcal confirmation. Although the prevalence of such isolates could change, the fluorescent monoclonal antibody confirmation reagents remain useful for many clinical situations. Their ease of use and ready applicability for screening large numbers of isolates make them useful for many laboratories.

A reactive pia arachnoid mesothelial cell as noted by the darker cytoplasm is present in this field. Reactive cells are a common finding in cytospin smears from spinal fluid samples and are sometimes difficult to distinguish from tumor cells. Mesothelial cells are usually interspersed among the other cells, rather than appearing in clumps. They have a single distinct nuclei that may be eccentric. The macrophages (histiocytes) are seen next to the mesothelial cell. Macrophages are distinguished from circulating monocytes by the irregular appearing cytoplasm. Bacteria, red cells or other debris can often be seen in the cytoplasm of macrophages.

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.

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

In 1900, a German scientist, Karl Landsteiner, discovered that blood groups differ from one individual to another. He took blood samples from five associates and himself, allowed them to clot, and then separated the serum from the cells. Landsteiner found that when he mixed the serum and red cells from different individuals, some samples clumped and some didn’t. Our present day classification of the ABO system is based on Landsteiner’s realization that agglutination occurred because of highly reactive antigens present on the red blood cell which corresponded to antibodies present in the serum. Landsteiner isolated and named the red cell antigens “A” and “B” and the corresponding antibodies “Anti-A” and “Anti-B.” If the red cells contained neither antigen, he called these cells “O,” representing zero antigens present. The fourth type of red cells, “AB” was discovered in 1902 by Von Decastello and Sturli, associates of Landsteiner. “AB” cells contained both A and B antigens on their surface.

Failure Mode, Effect, and Criticality Analysis uses the opposite approach of Root Cause Analysis. Ways FMECA and RCA differ: FMECA is proactive and RCA is reactive. FMECA occurs during development and RCA occurs after-the-fact. FMECA prevents errors and RCA satisfies patients or requirements. FMECA helps processes to work and RCA changes processes that do not work. FMECA encourages good outcomes and RCA changes bad outcomes.

Large lymphocytes have abundant pale blue transparent cytoplasm. If you imagine putting a printed page behind the cell, the cytoplasm looks as though you could see through it to read the words. Although there are usually no cytoplasmic granules present, a few large well-defined azurophilic granules (lysozomes) can occasionally be seen. In this case, the cells would be called large granular lymphocytes. A large lymphocyte can be found in the upper image to the right.Reactive, or atypical, lymphocytes are relatively fragile cells, and as a result can be squeezed out of shape by surrounding cells, giving them a scalloped appearance instead of a smooth cytoplasmic edge. The nucleus of the reactive lymphocyte is larger than that of the small lymphocyte, and is more irregular in shape. Sometimes it is rounded, oval or indented with a typical "stretched" appearance. A reactive lymphocyte can be found in the lower image to the right.

Lymphocytes "transform" in response to antigenic stimuli.Their nuclei becomes larger with more open chromatin and a greater degree of nuclear folding.The cytoplasm becomes abundant, the number of azurophilic granules may be increased and vacuoles may be present.The cytoplasmic membrane may be easily indented by surrounding red blood cells, resulting in a scalloped appearance of the cell's outer edge.These lymphocytes may also be referred to as reactive, activated or stimulated.

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.

It is important to recognize the presence of these morphology changes and to accurately identify them for several reasons: If the changes are pathological, their identification may aid the physician in diagnosing a specific condition. If the changes are not pathological, their identification alerts the physician to the fact that the changes are present, thus avoiding a possible misdiagnosis. If reactive, it indicates that although the cells are functioning normally, they are reacting to a stimulus. Indicating the presence of such cells may aid in determining the diagnosis or monitoring the course of disease once a diagnosis has been made.

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.