serology Agglutination Techniques and Blood Cell Identification

Transcription

1 Serology: Agglutination Techniques and Blood Cell Identification S erology is a branch of immunology dealing with techniques to identify and measure antigens, and to detect serum antibodies. Agglutination is a serological reaction in which antibodies react with antigens Jones the & Bartlett surface of Learning, particles and LLCcause the particles to clump together, NOT or agglutinate. FOR SALE This OR reaction DISTRIBUTION generally results in a visible mass that can be seen with the unaided eye. In the diagnostic laboratory, the agglutination reaction is a valuable aid in the identification of an unknown bacterium, because the unknown can be pinpointed by its agglutination with known antibodies. NOT Although FOR SALE serology OR has DISTRIBUTION become highly automated, this exercise NOT FOR illustrates SALE OR DISTRIBUTION how agglutination reactions can be studied without sophisticated or specialized equipment. In Part A, the slide agglutination technique will be performed to learn the identity of an unknown bacterium. In Part B, the tube agglutination technique will be performed to determine the amount of antibodies in a Jones & Bartlett solution Learning, of serum. In LLC Part C, the agglutination of Jones blood cells & Bartlett will be Learning, studied. LLC A. Slide Agglutination The slide agglutination Jones & technique Bartlett Learning, is used to determine LLC which of several PURPOSE: to identify a bacterial FOR species SALE using a OR poly-distribuvalent antiserum. bacteria agglutinates NOT FOR a preparation SALE OR of DISTRIBUTION selected antibodies. Emulsions of NOT unknown bacteria are mixed with drops of known antibodies on a microscope slide, and the mixture is observed for agglutination. This process is essentially a trial-and-error method because different combinations of bacteria Jones and antibodies & Bartlett are Learning, tried until LLC agglutination is observed. Jones In this & exercise, NOT Salmonella FOR SALE antibodies OR DISTRIBUTION will be combined with various NOT bacteria FOR SALE to OR DISTRIBUTION Bartlett Learning, LLC determine which, if any, is a Salmonella serotype. S pecial Materials Polyvalent Salmonella antiserum in dropper bottles Unknown broth cultures of various bacteria..5054

2 Wooden applicator sticks or toothpicks Beakers of disinfectant Saline solution in dropper bottles Nutrient broth culture of Salmonella Tube of nutrient broth NOT FOR P rocedure SALE OR DISTRIBUTION 1. Obtain a clean glass slide and, using a wax pencil, draw a circle for each of the unknown broth cultures available. Aseptically deposit two loopfuls of each broth culture into their respective circles. 2. Add one drop of the Salmonella antiserum NOT FOR (a solution SALE of OR Salmonella DISTRIBUTION antibodies) to each circle. Mix the bacteria and antiserum well, using a wooden applicator stick or toothpick. Use different sticks for each sample, and place the stick in the beaker of disinfectant after its use. 3. As a positive control, Jones take a clean & Bartlett glass slide, Learning, draw a circle, LLCand mix a drop of Salmonella antiserum NOT FOR with two SALE loopfuls OR DISTRIBUTION of a known broth culture of Salmonella. An agglutination reaction should be evident. As a negative control (to show the absence of agglutination), mix a drop of saline solution with two loopfuls of the known Salmonella culture in a second circle. Also as a negative control, mix a drop of antiserum with two loopfuls of sterile, Bartlett bacteria-free Learning, nutrient LLC broth in a third circle. Jones & 4. Observe the various unknowns and determine which agglutinated with the Salmonella antiserum by comparing the mixtures to the control reactions. Enter your results in Table 1 of the Results section, using ( ) to indicate agglutination and ( ) for nonagglutination. Discard the slide in the beaker Jones & Bartlett Learning, of disinfectant LLC after use. From your Jones data, determine & Bartlett the Learning, identity of the LLC unknown organism. If this is not possible, NOT FOR indicate SALE why. OR DISTRIBUTION B. Tube Agglutination PURPOSE: to determine an antibody titer in a serum sample. In the tube agglutination technique, the object is to use the agglutination reaction to determine the titer of antibodies in a serum sample. The titer is a measure of the amount of antibodies in the sample. It is determined by find- ing the most dilute concentration of antibodies that gives a detectable reaction FOR with SALE an antigen. OR DISTRIBUTION Various dilutions of antibodies NOT are prepared, FOR SALE after OR DISTRIBUT NOT which a standard amount of antigen is added. Following an incubation period, the titer is determined. This section will demonstrate the tube agglutination technique and the method for ascertaining the titer of antibodies. S pecial Materials Polyvalent Salmonella antiserum Salmonella antigen solution Saline solution (0.85% NaCI) Serological tubes with racks..5054

3 Mechanical pipetters Beakers of disinfectant 1-ml pipettes 5-ml pipettes P rocedure 1. Select ten NOT clean FOR serological SALE tubes, OR DISTRIBUTION number them, and set them up in a rack on the desk. Obtain samples of coded Salmonella antiserum, Salmonella antigen, and saline solution with which to work. 2. Using a mechanical pipetter and a sterile 5-ml pipette, place 0.9 ml of Jones saline & solution Bartlett in Learning, tube #1, and LLC ml of saline solution in tubes Jones #2 through & Bartlett Learning, LLC NOT FOR #10, as SALE indicated OR in DISTRIBUTION Figure With the mechanical pipetter and a sterile 1-ml pipette, transfer 0.1 ml of the antiserum solution from the stock supply to tube #1. This yields a 1:10 dilution of antiserum. Mix the contents by drawing them up and expelling Jones & Bartlett them Learning, from the LLC pipette, and then transfer Jones ml from & tube Bartlett #1 to Learning, the saline LLC NOT FOR SALE OR in DISTRIBUTION tube #2. This forms a 1: 20 dilution, as shown NOT FOR in Figure SALE 1. OR Mix DISTRIBUTION the contents of tube #2, and transfer ml to tube #3, as shown in figure. Mix and transfer ml to tube #4, and continue this process on through to tube #9. Remove ml from tube #9, and discard it into a beaker of disinfectant. Tube #10 will Jones be a control & Bartlett tube and Learning, will not receive LLC antiserum. Discard the pipette as directed by the instructor. 4. Using a mechanical pipetter and a sterile 5-ml pipette, transfer ml of Salmonella antigen solution to each tube. This doubles the dilutions to the final dilutions shown in Figure 1. Mix the contents, and set the rack aside to incubate at 37º C. A water bath is helpful for the incubation step. ml ml ml ml ml ml ml ml ml discard 0.1 ml antiserum Saline Jones (ml) & Bartlett 0.9 Learning, LLC Initial dilution 1:10 1:20 1:40 1:80 1:160 Jones & Bartlett Learning, LLC 1:320 1:640 1:1280 1:2560 Control Antigen (ml) Final dilution 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 FIGURE 1 Preparation of a dilution series for the tube agglutination test. Control..5054

4 5. After an hour or longer Jones at the instructor s & Bartlett suggestion, Learning, observe LLC the tubes for evidence of agglutination NOT FOR by tapping SALE the OR bottom DISTRIBUTION of the tube to suspend the sedimented material. Large masses should be observed in the lower dilutions (e.g., tubes #1 and #2), and the amount of agglutinated material should decrease as the dilution becomes higher. Eventually there should Jones & be Bartlett a tube that Learning, shows the LLC last evidence of agglutination. Jones The & dilution Bartlett of Learning, LL NOT FOR this SALE tube OR is the DISTRIBUTION titer of antibodies. Note that tube NOT #10, the FOR control SALE tube, OR DISTRIBUT should show no evidence of agglutination since it does not contain antiserum. Overnight refrigeration will enhance the results. 6. Enter your results in Table 2 of the Results section, using ( ) to indicate maximum agglutination, ( ) to indicate moderate agglutination, ( ) to indicate minimum agglutination, and ( ) to indicate no agglutination. Determine the titer of antibodies in the serum sample by noting the dilution of the tube where the last evidence of agglutination appeared. Write your answer in the Results section. C. Hemagglutination: Determination of Blood Type NOT Hemagglutination FOR SALE OR DISTRIBUTION is a type of agglutination that involves NOT FOR blood SALE cells. OR In DISTRIBUT microbiology there are many clinical uses for this reaction, including the diagnostic procedures for measles, mumps, and influenza. The hemagglutination reaction is also important in blood banking because it is used to determine the blood type of an individual prior to using the blood for transfusion. Learning, Most textbooks LLC contain accounts Jones of the importance & Bartlett Learning, of compatible LLC Jones & Bartlett NOT FOR SALE OR blood DISTRIBUTION types in transfusion. PURPOSE: to determine ABO and Rh blood types. In this exercise, the blood type will be determined by hemagglutination. The blood used in this exercise will not be taken from the student unless specified by the instructor. Instead, a human blood substitute will be Jones & Bartlett Learning, used. LLC This blood substitute is Jones commercially & Bartlett available Learning, and consists LLC of animal blood or microscopic beads NOT bonded FOR SALE to blood OR antigens. DISTRIBUTION It will give reactions identical to those of human blood. Four major blood types are recognized. Type A individuals possess A antigens on the red blood cells, while type B persons have B antigens. Both A Jones and B & antigens Bartlett occur Learning, type AB LLC individuals, and neither Jones is found & on Bartlett the red Learning, LL NOT blood FOR cells SALE of type OR O DISTRIBUTION persons. The antigens may be detected NOT by FOR reactions SALE of the OR DISTRIBUT blood with antisera containing A or B antibodies, as shown in this exercise. Another antigen, the Rh factor, is found in 85% of Caucasians, who are said to be Rh-positive. It is absent in the remaining 15%, who are Rh-negative. In Jones & Bartlett African-American Learning, LLCpopulations, the percentages Jones are & 93% Bartlett positive Learning, and 7% negative. This factor is an important consideration in hemolytic disease of the new- LLC born. Its presence also may be determined by a hemagglutination reaction. S pecial Materials 70% ethyl alcohol and gauze or cotton pads Anti-A and anti-b typing sera..5054

5 NOT FOR SALE Toothpicks OR DISTRIBUTION or applicator sticks Vials containing ml of 0.85% sterile saline solution Sterile disposable lancets Anti-Rh typing serum P rocedure 1. Wash and dry two glass slides. Divide one slide into sections A and B by marking the underside of the slide with a wax pencil or felt pen. The underside is used to prevent wax or ink from interfering with the agglutination reaction.! 2. Obtain the tube of blood to be used for this exercise. The instructor will When taking your own NOT FOR SALE OR blood, DISTRIBUTION be certain that the specify the source of the blood. The type of this blood will be determined. lancet is taken from a If the student s blood is to be used, the directions for securing the blood will sealed package and is sterile. be explained by the instructor. Jones & Bartlett 3. Place Learning, a drop LLC of blood in sections A and B Jones the first & Bartlett slide and Learning, a drop LLC the center of the second slide. Also, place a small drop in the vial of saline solution. 4. Place a drop of anti-a serum next to the blood in section A, and a drop of anti-b serum next to the blood in section B. These sera contain A and B antibodies, respectively. Place a drop of anti-rh serum next to the blood on the second slide. Anti-Rh serum (Rh antibodies) detects the D antigen, which causes the Rh-positive condition. 5. Mix the drops of blood and respective antibodies with clean toothpicks or applicator sticks, being sure to use a fresh one for each mixing. Agglutination with anti-a and anti-b sera is observed by the presence of large, grainy, dark clumps on the slide. Agglutination with anti-rh sera is determined by NOT FOR rocking SALE the slide OR DISTRIBUTION back and forth for approximately 2 minutes NOT FOR over SALE very OR DISTRIBUTION mild heat. A warming tray or warming box at 45º C may be used. Small, finer clumps will be seen on close examination. It should be pointed out that determination of the Rh factor by this method is questionable at best and that Jones & Bartlett further Learning, testing LLC is necessary for a definitive conclusion. Jones & Bartlett Learning, LLC NOT FOR SALE 6. OR Record DISTRIBUTION results in the Results section using NOT the FOR following: SALE OR DISTRIBUTION Agglutination with anti-a serum... type A blood Agglutination with anti-b serum... type B blood Agglutination Jones with both & Bartlett anti-a and Learning, anti-b sera LLC... type AB blood Agglutination NOT with FOR neither SALE anti-a OR DISTRIBUTION nor anti-b sera... type O blood Agglutination with anti-rh serum... Rh-positive No agglutination with anti-rh serum... Rh-negative 7. To verify your results, repeat the above process using blood from the Jones saline & Bartlett vial. The test Learning, should be LLC performed only for A and B Jones antigens, & since Bartlett Learning, LLC NOT FOR detection SALE of the OR Rh DISTRIBUTION antigen will not work by this method. NOT Cover FOR the bloodantiserum mixtures with coverslips, and observe the presence or absence SALE OR DISTRIBUTION of agglutination under the lower power (10x) objective of the microscope. Clumps of red blood cells indicate that agglutination occurred with the Jones & Bartlett antiserum. Learning, Unclumped, LLC free-floating cells Jones reflect nonagglutination. & Bartlett Learning, LLC..5054

6 D. Blood Smear A blood smear provides the opportunity to view red blood cells and various types & Bartlett of white Learning, blood cells LLC that function in body defense. Jones Noting & Bartlett which Learning, LL Jones NOT cells FOR are SALE present OR in DISTRIBUTION unusual numbers may provide insight NOT FOR to the SALE nature OR of DISTRIBUT the disease. For example, the number of abnormal lymphocytes is unusually high in patients with infectious mononucleosis, and the monocyte count is elevated in cases of listeriosis. In this exercise, the different blood Jones & Bartlett cells Learning, will be observed, LLC and their percentage Jones may & be Bartlett determined. Learning, LLC S pecial Materials 70% ethyl alcohol and gauze or cotton pads Sterile disposable lancets Wright s stain Dropper bottles of distilled water Buffer solution (Giordano) if available PURPOSE: to identify blood cell types and perform a differential white blood cell count from a blood smear. P rocedure Jones 1. & Clean Bartlett two glass Learning, slides and LLC dry them thoroughly. One slide Jones will & be Bartlett used for Learning, LL NOT FOR the SALE blood OR smear, DISTRIBUTION the second for spreading the blood. NOT Obtain FOR a large SALE drop OR of DISTRIBUT blood as specified by the instructor, and place it at the end of one slide. 2. Spread the drop of blood across the face of the slide, using the second slide as a spreading slide as follows: Place the spreading slide at a 45º angle, and Jones & Bartlett Learning, bring it LLC back into the drop as shown Jones in Figure & Bartlett 2A. Allow Learning, the blood LLC to spread out to the slide s edge, and then NOT drag FOR the blood SALE the OR length DISTRIBUTION of the slide one time only, as shown in Figure 2B. Lift the spreading slide at the end of the smear in order to feather the end. Repeat the process with a fresh drop of blood and a new slide if a successful smear is not obtained the first time. 3. Thoroughly air-dry the slide. Cover the smear with Wright s stain while counting the number of drops added. Permit the stain to remain for 2 minutes. Without washing the slide, add an equal number of drops of distilled water or buffer solution to the slide, and mix the stain and water through gentle rocking. Allow the mixture to remain 2 additional minutes. A A clean slide is drawn back into the drop of blood. FIGURE 2 Preparation of a blood smear. B The spreading slide is pushed across the face of the sample slide, thereby spreading out the blood

7 Jones & Bartlett 4. Gently Learning, wash the LLC slide with water for 30 seconds and blot it dry. Examine the NOT FOR SALE OR slide DISTRIBUTION under the oil immersion (100x) lens, NOT beginning FOR SALE at the feathered OR DISTRIBUTION end. 5. Note the presence of red blood cells, which should appear as pale orange disks if the residual stain has been washed free. Red blood cells (erythrocytes) do not stain with Wright s stain, but if the dye has not been removed, the Jones cells may & appear Bartlett pale Learning, to dark blue. LLC Try to locate an uncluttered area where NOT the FOR cells are SALE standing OR alone DISTRIBUTION and may be observed individually. Enter a representation of several red blood cells in the Results section. 6. Note the presence of white blood cells (leukocytes), which should appear larger than red blood cells. Stained white blood cells will exhibit a Jones blue & nucleus Bartlett in Learning, an unstained LLC or pale blue cytoplasm. Neutrophils Jones & Bartlett are Learning, LLC NOT FOR the most SALE common OR DISTRIBUTION white blood cells observed (Figure 3). NOT These FOR cells SALE have OR DISTRIBUTION Name Neutrophils: Pale pink granules Microscopic appearance Approximate percentage of white blood cells Description Most abundant WBCs; pale pink granules, multilobed blue nucleus Eosinophils: Red granules 2 4 Red granules, blue nucleus Basophils: Jones & Bartlett Learning, LLC Blue granules Blue granules, NOT <1 FOR SALE OR blue DISTRIBUTION nucleus Lymphocytes: Jones & Bartlett No Learning, LLC NOT FOR SALE granules OR DISTRIBUTION Large oval or Jones & Bartlett Learning, LLC spherical blue nucleus Monocytes: No granules 2 8 Largest cell; irregular blue nucleus Erythrocytes Pale pink disks Thrombocytes (platelets) Fragmentlike cells FIGURE 3 Formed elements of stained blood smears

8 FIGURE 4 Counting procedure for a differential white blood cell count. dark blue nuclei divided into two, three, or more lobes. Lymphocytes are the next most common white blood cell. These cells have large blue nuclei that take up almost the entire space of the cytoplasm and make the entire cell appear dark blue. Monocytes are relatively uncommon. They Jones & Bartlett Learning, are large LLC cells, each with a large blue Jones nucleus & characteristically Bartlett Learning, indented LLC on one side. Eosinophils and basophils NOT FOR are SALE the least OR common DISTRIBUTION white blood cells. They appear as multilobed, nucleated cells with distinctive red and blue granules, respectively. Small groups of cells also will be observed as clusters of blue fragmentlike bodies. These are the thrombocytes (platelets) used in blood Jones clotting. & Bartlett Enter representations Learning, LLCof the types of white blood cells and NOT the FOR thrombocytes SALE OR in the DISTRIBUTION Results section. 7. A differential white blood cell count may be obtained by counting 100 white blood cells and determining the percentage of each of the five types. To perform this step, begin at the feathered end and move the smear vertically Bartlett while Learning, recording LLC each type of white blood cell Jones as it is & observed. Bartlett Learning, LL Jones & NOT FOR When SALE the OR end DISTRIBUTION of the smear is reached, move horizontally NOT FOR to the SALE next plane OR DISTRIBUT (Figure 4) and once again move the smear vertically, continuing the tally. Determine the percentage of each type of white blood cell, and enter it into Table 3 of the Results section. The slide may be labeled and retained if necessary. Q uestions 1. Explain the importance of positive and negative control preparations in the slide agglutination procedure. Jones & Bartlett Learning, LLC 2. Why is ml of material NOT FOR removed SALE and discarded OR DISTRIBUTION from tube #9 in the tube agglutination procedure? 3. What is meant by titer, and why may the titer be a significant factor in the diagnosis of disease? NOT FOR 4. Consider SALE OR the DISTRIBUTION blood type you have determined in the NOT laboratory. FOR SALE What type OR DISTRIBUT or types of blood could successfully be transfused to this individual under emergency conditions? Explain carefully. 5. Name several factors that contribute to a successful blood smear and several that LLC contribute to a poorly executed Jones blood & smear. Bartlett Learning, LLC Jones & Bartlett Learning,..5054

9 Name Date Exercise Results Section Agglutination Techniques A. Slide Agglutination Jones & Bartlett Learning, Table LLC 1. Determination of Unknown Jones Organism & Bartlett Learning, LLC Organism Agglutination Identity of unknown NOT organism: FOR SALE OR DISTRIBUTION Jones B. Tube & Bartlett Agglutination Learning, LLC Table 2. Determination of Titer of Antibodies Tube number Dilution 1: 20 1: 40 1: 80 1: 160 1: 320 1: 640 1: : : 5120 Control Agglutination Titer of antibodies: Serum sample code: Observations and Conclusions:..5054

10 C. Hemagglutination: Determination of Blood Type A NOT FOR SALE Blood OR DISTRIBUTION type: B Rh D. Blood Smear Magnif.: Table 3. NOT Differential FOR SALE White OR Blood DISTRIBUTION Cell Count Type of WBC Neutrophil Basophil Eosinophil Lymphocyte Monocyte Jones Number & in Bartlett blood Learning, LLC Percentage in blood % % % % % Observations and Conclusions:..5054