Download Anti HumanGlubulin

‫به نام خدا‬
‫‪‬‬
‫ارزيابی آنتی هيومن گلوبولين‬
‫دکتر کتايون خداورديان‬
‫اداره مديريت تضمين کيفيت‬
Anti HumanGlubulin
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AHG Reagents:
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a-polyspecific
AntiIgG,AntiC3c/Anti
C3d
b-Monospecific
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Appearance
Culture
Package
Information of Label
Information of Brochure
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3-Specificity and potency of anticomplement antibodies
A polyspecific reagent should contain
anti-C3c & anti C3d at control level to
avoid false positive reactions
anti C4d must be avoided
SAMPLE
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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)
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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
Recommended AHG test
procedure
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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
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2-Washed#4 with minimum of 3 ml saline per
wash
3-Add 2 volume AHG reagent,centrifuge
without delay
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RCF(g) 100 200-300 500 1000
Time(s) 60 25-30
15
8-10
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Quality Control:
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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.
False positive Reactions
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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
False Negative Results
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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