1. به نام خدا ارزيابی آنتی هيومن گلوبولين دکتر کتايون خداورديان اداره مديريت تضمين کيفيت

2. Anti HumanGlubulin AHG Reagents: a-polyspecific AntiIgG,AntiC3c/Anti C3d b-Monospecific

3. Appearance Culture Package Information of Label Information of Brochure

4. Quality Control 1-Specifity The reagent should only agglutinate red cells sensitized with Ab &/or coated with complement component 2-Potency of anti-IgG serological titration

5. 3-Specificity and potency of anti- complement antibodies A polyspecific reagent should contain anti-C3c & anti C3d at control level to avoid false positive reactions anti C4d must be avoided

6. SAMPLE Serum prefer (complement inactivated by EDTA) Whole blood at room Tem. up to 48 hour, & 7 days at 4 degree Patient Serum or plasma is best store at - 20 degree (specially for complement activity)

7. Potency: a-prepare cell sensitized with IgG: Diluted a weak IgG anti-D(0.8 iu/ml) 1/1…………1/16 Prepare sensitized O + cells (R1r Dce/dce) Incubation at 37 Washed#4 Tested with 1/1………1/8 AHG *The AHG reagent should not show prozone by immediate tests using 2 volume of AHG. prepared cell sensitized with strong anti D (Blend IgG&IgM)

8. Potency cell coated with complement cell sensitized in LISS: add 1-2 ml of fresh group O whole blood to20 ml of 100g/l sucrose (10%), 15’ incubation 37 washed#4 prepared 5% cells,tested with 1/1……..1/8 AHG

9. -False positive reactions: a-Test for excess anti-C3d, Incubate fresh serum with 6 ABO compatible cells donor unit segments(10-30 days old) -0.2ml fresh serum+0.1ml compatible cell → 45 min. 37 degree - Washed#3 with salin - Add 0.2 ml AHG - +1 macroscopic reaction →Rejection of reagent

10. b-Tests for contaminating red cell antibodies (against washed A1,B and O cells) 0.1 ml 0(+) cell,B(+)cell,A(+)cell 3% + 0.2 ml AHG incubation at 37 degree reaction must be negative

11. -Non specific reactions prepared 5% O neg(Du-) cell + Anti D Incubation at 37 Washed#4 Tested with AHG *No Reaction - stability Control

12. Recommended AHG test procedure 1-Sensitized red cells: NISS:4 volume serum+1 volume cells (3%) [ #3washed & suspension in PBS], 45’ incubation at 37 LISS: 2 volume serum+2 volume cells(1.5%) [2#washed PBS,1#washed LISS & suspension in LISS ], 15’ incubation at 37 2-Washed#4 with minimum of 3 ml saline per wash 3-Add 2 volume AHG reagent,centrifuge without delay

13. RCF(g) 100 200-300 500 1000 Time(s) 60 25-30 15 8-10 Quality Control: Positive control :An IgG anti-D diluted to give 1+or2+reactions with Rh D positive(R1r) cells. Negative control An inert group AB serum with the same RhD positive cells. Addition of sensitized cells to all negative tests.

14. False positive Reactions Chemical Conta. (Cu, Zn,Fe Present in Saline, detergent traces on glassware). Poor Quality of Saline (hypertonic, Bacterial Conta.) Colloidal Silica From glass bottle. AHG with additional Ab Specificity. Bacterial Cont. of Reagents, Test cells, and/or unknown serum. Cold Agglutinins (Cells store for long time at 4 ◦c ) Clots, Fibrin Particles Over Centrifuge Dirty Glassware

15. False Negative Results Inadequate washing of RBCs. Excessive packing of RBCs in washing procedure, this traps protein between the red cells neutralize the Reagent Contamination AHG with human Pro. Neut. Antiglobulin Ab In sufficcent incubation of cells & serum. Elution of Ab from RBCs after prolonged contact with AHG reagent. Delay washing (elution of weakly attached Ab) Serum/cell ratio too low Incubation at Tem. Other than optimal Improper centrifugation