Monoclonal Information and Courses from MediaLab, Inc.

After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry.An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity. The densitometer scan below depicts the separated bands from a serum sample electrophoresis. The SPIFE 3000, Helena Laboratories, electrophoresis splits the beta zone into two fractions for easier detection of small beta-migrating monoclonal gammopathies. The densitometer scan from this electrophoresis shows five bands with two peaks in the beta band. Recall the order of protein fractions from left to right is: Albumin, alpha 1, alpha 2, beta, and gamma.

As each cell makes contact with the laser beam, the instrument determines cellular intrinsic and extrinsic properties. These extrinsic characteristics are reflected by the presence of a fluorescent signal emitted by monoclonal antibodies that are bound to specific cellular antigens on the cell surface. The cell image below shows a cell stained with two different monoclonal antibodies, each with a different fluorochrome. Each fluorochrome will emit a different color when it intersects the laser.The second image below is an example of a histogram display. It represents the monoclonal antibody for CD5 labeled with FITC dye. When this dye intersects a 488 nm argon ion laser beam, it emits a fluorescent signal in the green portion of the spectrum (don't be confused by the red peaks; that is simply how this particular flow cytometer is programmed to display every color on a single color histogram).

Until now, the interpretation has been single color analysis. Ruling out a monoclonal B cell population does not guarantee that this is a normal peripheral blood. The T-cell lymphocyte population must also be evaluated. In order to do so, it is important to not only determine how many T cells are present, but also what subsets they represent. Dual color analysis can be used to distinguish C4 from C8 subsets in the C3 population.C2= 91%CD5= 90%CD7= 91%Image #1 CD3 is plotted with CD4. CD3/CD4 dual positive (+) cells = 55% Add Q1 and Q2 percentages together to get total CD3 = 88%Image #2 CD8 plotted with CD3. CD8/CD3 dual + cells = 33%Add Q1 and Q2 percentages together to get total CD3 = 88%Since this is the same sample and the same gated population, the total CD3 values should be very close from image to image. In the two images on the right, the total CD3 values are within one-tenth of each other (both round to 88%). C2= 91% CD5= 90% CD7= 91% CD3= 88% CD4= 55% CD8= 33%

Flow cytometry is used most often to analyze white blood cells. Some appropriate samples are: Peripheral blood Bone marrow Lymph nodes and other tissues Fluids (cerebrospinal fluid, peritoneal, pleural, etc.)Since size and granularity are utilized to help sort cell populations, it is imperative to keep the cells intact. Thus, flow cytometry is best conducted on viable cells. Dead cells can complicate result interpretation by: Non-specifically binding monoclonal antibodies and emitting false fluorescent signals. Falling in inappropriate portions of the light scatter graph and falsely elevating event/cell counts.

Sample preparationSamples for flow cytometric analysis are prepared based on the following procedural steps. Prepare the sample according to cell type. Bone marrow (BM) and peripheral blood (PB): Prepare a blood smear, stain with Wright's stain, and scan under the microscope to identify basic cell distribution and morphology. BM can contain spicules; these samples need to be filtered. Tissue: Mince and filter tissues in a sterile cell culture media to release cellular components from the solid tissue form. Fluid: Filter fluid, prepare a cytospin, and scan under a microscope to identify cellular components. Obtain a white blood cell (WBC) count. Unless red blood cells are the population of interest, they should be lysed with a mild agent that will preserve the integrity of the cells targeted for analysis. If the red blood cells are not lysed, they lead to false analytic results. Adjust the WBC count by concentrating the WBCs to optimize for ‘staining' with monoclonal antibodies (MoAbs). Incubate the prepared cell concentration with assorted monoclonal antibody concentrations to allow antigen-antibody complexes to form. Lyse red blood cells (RBCs) with ammonium chloride or equivalent that will preserve cellular viability and integrity. The purpose is to eliminate RBCs while maintaining WBC integrity. If RBCs remain in the sample, they will interfere with the cell scatter plot and skew the results. Centrifuge to precipitate cells. Pour off supernatant. Wash in phosphate buffered saline (PBS) or equivalent to eliminate cellular debris and unbound MoAbs, centrifuge, and decant. Resuspend cells in PBS. Analyze cells using flow cytometer.

Rapid detection of influenza was a key focus for method development for many years. Traditional viral culture methods require special transport mediums, appropriate cell culture lines, and staff well versed in the manipulation of these cultures. Although the introduction of shell vial cultures and monoclonal fluorescent staining provided some improvement, the availability of results did not always meet the clinical need.Direct fluorescent antibody (DFA) staining of specimen smears can provide more immediately available results; however the availability of trained staff to interpret these smears is an obstacle for many laboratories. Antigen detection kits employing enzyme immunoassay (EIA) or immunochromatographic membrane principles did provide easily performed alternatives that fit well in most laboratory settings and provided more immediate results. Despite the fact that published studies demonstrated less than desirable sensitivity, these assays had found a niche and remained in place, even as molecular methods began to target these viruses.

The most common laboratory tests for detection of C. difficile are enzyme immunoassays (EIA) for detection of C. difficile Toxin A and Toxin B. The immunoassays are simple to perform and provide rapid results. However the sensitivities of these tests are not as good as culture, CCNA, or molecular methods. Only liquid stool samples should be processed. Due to the fact that the colonization rate is high, a positive result with a normal stool sample proves that the patient is colonized with C. difficile but not necessarily infected. There are many test kits available commercially for detection of C. difficile toxins. Results are available in 15 minutes – 2 hours depending on assay. Initially Toxin A was thought to be the toxin responsible for the majority of the effects of C.difficile disease, so most early test kits only detected Toxin A (based on monoclonal anti-Toxin A antibodies) but with the realization that there are strains that produce aberrant or no Toxin A (A-) that are known to produce infection, and more recently Toxin B negative (B-) strains, it is now recommended that a kit is used that detects both toxins.

VirusMost Likely Means of Dissemination Primary Route of EntryGeneral Signs and SymptomsLaboratory TestingSmallpox: Image courtesy of CDCAs an aerosol Inhalation High fever with extreme lethargySevere headache, backache, and abdominal painRash that starts as red bumps but quickly develops into small, itchy blisters Consult local APHL prior to sample collectionShell vial and DFA Monoclonal IFAMolecular tests Viral Hemorrhagic Fevers (Ebola, Marburg, Lassa, and Argentine): SolidLiquidAerosol AbsorptionInhalationIngestion Vary by type of viral hemorrhagic fever (VHF), but initial signs and symptoms often include: Marked feverFatigueDizzinessMuscle aches, loss of strength, and exhaustionSevere cases of VHF often show signs of bleeding under the skin, within internal organs, or from body orifices like the mouth, eyes, or ears Enzyme-linked immunosorbent assay (ELISA)Polymerase chain reaction (PCR)Viral culture

Based on the results of the mini-panel, the laboratory concluded that only anti-D was present and that it was consistent with administration of RhIg at 28 weeks.Patient A.D. delivered a 5 lb 13 oz female by C. section with serologic test results on cord blood as follows. Well washed cord red cells were used for ABO and Rh(D) typing to remove possible Wharton's jelly.Before proceeding to the next page, evaluate if the infant's ABO and Rh(D) types are valid. You will be asked questions that assess basic knowledge of blood grouping practices and test results for newborns. ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D* 0 0 NT NT 3+ NT = not tested / * monoclonal IgM anti-D DAT Reagent DAT CC Polyspecific AHG w+ 2+ W+ = microscopic positiveAHG = antihuman globulin serum CC = IgG sensitized cells Note: It is the lab's policy to add IgG sensitized cells to weak antiglobulin test results.

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.