Hemolytic assays
- 2. • Hemolytic assays have traditionally been used to assess the functional activity of the complement system. They provide insight into the integrity of the entire cascade reaction. • These tests are particularly useful in the investigation of suspected complement deficiencies.
- 3. • Serial dilutions of the sample to be analyzed are incubated with Ab-sensitized sheep erythrocytes at a defined temperature. • Hemolytic assays are performed either in tubes or in agar plates. • The results are usually expressed as reciprocal dilutions of the sample required to produce 50 or 100% lysis (CH50 or CH100, respectively)
- 4. • Tests evaluating the functional activity of the alternative pathway (AH50) use guinea pig, rabbit, or chicken erythrocytes as target cells.
- 5. • The total hemolytic complement assay (CH50) measures the ability of the classical pathway and the MAC to lyse sheep RBC to which antibodies has been attached. • This estimates the standard quantity of serum required as a complement source to produce lysis of 50% of standard quantity of sensitized sheep red cells. TOTAL HEMOLYTIC COMPLEMENT ASSAY(CH50)
- 6. • The CH50 is an random unit defined as the quantity of complement necessary for 50% lysis of red cells. • The CH₅₀ unit is determined under standardized conditions which depend upon: – Erythrocyte and antibody concentration; – Buffering concentration of medium; and – temperature
- 7. • As the complements are added to Ab-coated erythrocytes, an increasing proportion of the cells are lysed • Released hemoglobins in the supernatant can be tested by a photoelectric spectrophotometer. • The hemolysis level is related with total complement activity. PRINCIPLE
- 8. • When the hemolysis percentage is served as ordinate and the corresponding serum amount is served as abscissa, a sigmoid curve can be obtained. • The relation between the complement amount in the vicinity of 50% hemolysis and hemolysis level presents a straight line. • So the 50% hemolysis point is defined as an end point, named 50% complement hemolysis (i.e. CH50).
- 9. Von Krogh equation, which converts the S- shaped complement titration curve into nearly straight line X = K ( Y )1/n ( 1-Y ) where X = ml of diluted complement added Y = degree of percentage lysis K = constant n = 0.2 ± 10% under standard E and A conditions.
- 10. • Materials and equipments – Barbitone- buffered saline for complement tests – Sheep erythrocytes (SRBC) in Alsever’s solution – Serum : this should be either fresh, or guinea-pigs, serum preserved specially for complements fixation assays – Horse hemolytic serum ( source of anti-erythrocyte antibody) – spectrophotometer Standardization of erythrocytes
- 11. • METHOD 1. Dilute the barbitone-buffered saline to working strength. 2. Wash 4ml of the erythrocytes suspension 3 times in barbitone- buffered saline. 3. Resuspend the washed erythrocytes in 15 ml of barbitone-buffered saline.
- 12. 4. Mix 1ml of erythrocytes with 25ml ammonia solution to lyse the cells and read absorbance at 541 nm. For a 6% SRBC suspension, in a 1cm cuvette, absorbance should be 0.48-0.50 5. Mix 15ml barbitone-buffered saline, 0.1ml of horse hemolytic serum and 15ml of 6% SRBC. Strictly, the anti-erythrocyte serum should be titred until the highest dilution still giving full complement fixation is reached; for most purposes use a 1 : 150 dilution.
- 13. 6. Incubate at 37°C for 15 min. 7. Sensitized SRBC can be stored overnight at 4°C
- 14. • METHOD • Label a series of tubes in duplicate with 1:8, 1:16, 1:32, 1:64 and 1:128. • Prepare a series of two fold serial dilutions of control and test serum in BBS each in duplicate • Start at 1:4 (100μl serum + 300 μl BBS) and transfer 200 μl of sample into the next labelled tube. ESTIMATION OF CH₅₀ TUBE ASSAY
- 15. • Mix thoroughly between dilutions and transfer 200 μl to the next dilution with a fresh pipette tip. • Repeat until all five dilutions are made. • Discard 200 μl from the final 1:128 dilution. • Add 200 μl of suspended sensitised SRBC to all tubes.
- 16. • Label two separate tubes as BLANK and add 200 μl of sensitised SRBC + 200 μlBBS. These tubes will measure spontaneous lysis of the SRBC in the BBS. • Label another two separate tubes as TOTAL LYSIS and add 200 μl of sensitised SRBC + 200 μl distilled water. • Gently mix all tubes. • Incubate at 37°C for 30 minutes in a waterbath mixing after 15 minutes.
- 17. • Centrifuge the samples at 1,500 rpm for 5 minutes to sediment the RBCs. • Transfer 100 μl of supernatant from each tube to a well in a 96 well flat bottom plate. • Add 100 μl of distilled water to each well. • Read the absorbance of the samples at 540nm using a plate spectrophotometer.
- 19. • Calculations • Calculate the mean absorbance for each sample • Subtract the BLANK absorbance (spontaneous lysis) from all samples • Calculate the % lysis for each dilution using the following formula: % lysis = OD540 (test) – OD540(Blank) ×100 OD540(total lysis) – OD540(Blank) • Plot the percentage lysis (vertical axis) versus the serum dilution on the horizontal axis. • Calculate the dilution required for 50% haemolysis for the control and test serum
- 20. •To calculate the 50% lysis, a line is drawn from the 50% percent value until it intersects with the graph lines and then a vertical line is drawn down to the dilution
- 22. • This simple assay, which is analogous to single radial immunodiffusion, may be used on a routine basis with reference to a standard serum. • Antibody-sensitized red cells are incorporated into molten agarose and the mixture allowed to set. • Wells are cut in the agarose and filled with either the sera under test or dilutions of a standard serum. Estimation of CH100 by assay in agar
- 23. • The complement diffuses into the agarose and reacts with and binds to the antibody-coated red cells. • Circles of lysis appear, the size of which depend upon the complement content of the serum. • MATERIALS AND EQUIPMENT • Sensitized red cells (made up to 10% v/v) • Agarose, 2% w/v in barbitone buffer • Glass plates • Gel cutter
- 24. • METHOD 1. Warm 1.5 ml of the barbitone buffer to 56°C in a water bath. 2. Cool 1.2 ml of molten 2% agarose to 56°C and add to the barbitone buffer 3. Mix and cool to 45°C in a water bath. 4. Add 0.2 ml of sensitized red-cell suspension and mix gently. 5. Place the glass plate on a level surface 6. Pour the mixture quickly onto the plate to form a smooth, even surface.
- 25. 7. When set, place the plate in a box containing moist filter paper and chill to 4°C for a few minutes to harden the agarose. 8. Cut two rows of five wells, approximately 3 mm across using an gel cutter. 9. Dispense 8 µl samples of the sera under test into separate wells. Similarly, add four doubling dilutions of a standard serum to a series of wells. 10. Incubate the plate in a moist box overnight at 4°C.
- 26. 11. Warm the plate, still in the box, to 37°C for 2 h, to allow cell lysis to occur. 12. Measure two diameters at right angles across each well and calculate their mean. 13. Plot the value of the areas (pr2) of the standard serum dilutions (linear scale) against the log dilution. Determine the concentration of the unknown sera as a percentage of the standard by extrapolation from the standard curve.
- 27. • very useful screening tool to rule out or to verify suspected complement deficiency disease. • Disease associated with reduced serum complement concentration are, • Glomerular nephritis • Serum sickness • cirrhosis of liver • myasthenia gravis • allograft rejection APPLICATIONS
- 28. • Disease associated with elevated serum complement concentration are, • Gout • Obstructive jaundice • Acute rheumatic fever • Rheumatoid arthritis • Typhoid fever
- 29. • The complement alternate pathway (AH50) assay is a screening test for complement abnormalities in the alternative pathway. • The alternate pathway shares C3 and C5-C9 components, but has unique early complement components designated factors D, B, and P, as well as regulatory factors H and I. • This pathway is activated by microbial polysaccharides and does not require immune complex formation. AH50
- 30. • Patients with disseminated infections with pyogenic bacteria in the presence of a normal CH50 may have an absent AH50 due to hereditary or acquired deficiencies of the alternate pathway. • Patients with deficiencies in the alternate pathway factors (D, B, P, H, and I) or late complement components (C3, C5-C9) are unusually susceptible to recurrent Neisserial meningitis.
- 31. • Absent complement alternate pathway (AH50) in the presence of a normal total hemolytic complement (CH50) suggests an alternate pathway component deficiency. • Normal AH50 with absent CH50 suggests an early (C1, C2, C4) classic pathway deficiency.
- 32. • Absent AH50 and CH50 suggests a late (C3, C5, C6, C7, C8, C9) component deficiency or complement consumption. • Absent AH50 and CH50 in the presence of a normal C3 and C4 suggests a late (C5, C6, C7, C8, C9) component deficiency.
- 33. • Investigation of suspected alternative pathway complement deficiency, atypical hemolytic uremic syndrome, C3 glomerulonephritis, dense-deposit disease APLLICATIONS
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