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ABO and D type Antibody screen
D positivescreen negative
D negativescreen negative
*Sensitizing events,prophylactic anti-D
*Sensitizing events,prophylactic anti-D
*Sensitizing events,prophylactic anti-D
Cord samplefor D.typing
Cord sample for DAT and D typing if mother is D negative
Other clinicallysignificant antibodies
Identify
Anti-K
PartnerK negative
Repeat ABO/D type and antibody screen. Reassessment of antibody levels
D-negatuve women without immune anti-D receive routine antenatal anti-D prophylaxis
PartnerK positive/uncertain
PartnerK negative
D-negative women without immune anti-D receive routine antenatal anti-D prophylaxis
PartnerK positive/uncertain
Newly detected
anti-K
Newly detectedanti-D, -c
TEST AT4-WEEK
INTERWALS
TEST AT4-WEEK
INTERWALS
NORMALLY TEST AZ 2-WEEKS Interwals
Anti-D.-c
D negativescreen positive
D positivescreen positive
All D-negative mothers without immune anti-D, sample for assessment of feto-maternalhaemorrhage. Post delivre prohylactic anti-D for D-negative mothers of D-positive babies
Gestation
Ca. 12weeks
28weeks
34 weeks
BirthNo further
tests
Action chart *
Background
Anti-D is the most frequent antibody responsible for serious Haemolytic Disease of Fetus andNewborn (HDFN).The level of an antibody's concentration in the serum of a pregnant woman can indicate the sever-ity of HDFN.The following levels of anti-D have been used to guide the management of pregnancies:
* Less than 5 IU/ml: HDFN unlikely, continue to monitor.* Between 5 and 15 IU/ml: Risk of moderate HDFN, refer to specialist unit.* More than 15 IU/ml: Risk of severe HDFN, refer to specialist unit.
Monitoring the changes of the concentration helps identifying the fetuses threatened by HDNF.The recommended method for the quantification of anti-D is the Indirect Antiglobulin Test (IAT).
Under standard circumstances the quantity of the anti-D correlates to the strength of the anti-gen-antibody reaction.ACT analyser is the only instrument capable to ensure standard conditions for routine blood groupserological methods performed in tube. The measuring principle of the ACT is the analysis of the size of the agglutinated particles pro-duced in saline medium (photo-sedimentometry). The strength of the reaction is characterized by the sedimentation curve. As in the ACT analyser every step of the IAT is automatically standardized, it is a sensitive and reli-able instrument for antibody quantification, too.
Antibody /‘titration’/ quantificationResults of NISS IAT
Results of Serum anti-D in different dilutions
1:50 dilution (N=50) 1:100 dilution (N=100) 1:150 dilution (N=150)
Strength ofagglutination +++ ++ +reaction:
Result of Control anti-D
Control anti-D = 0,1 IU/mlStrength of agglutination reaction: +
Aims
The factors influencing the reproducibility of agglutination reactions had to be excluded. Firstly the strength of reaction was determined to which the sedimentation curve belonged thatshowed most sensitively the concentration of the antibody, then based on it the optimal condi-tions of the quantitative measurement was established.
Methods
It was found that the type of sedimentation curve belonging to the weak positive reaction (“+”)was the most suitable to follow the effects of the changing parameters. The varied parameters were:
* the medium, * the incubation time, * use of enhancement solutions, * the phenotype of the red blood cells,* the age of the red blood cells, * and the concentration of the cell suspensions. The test results were compared with known standards in International Units (IU/ml).
Reproducibility of weak reactions Serum anti-D in 1:600 dilution
Representing the results by the ACT analyser in the form of sedimentation curves and videopictures
Results:
Using various commercially available anti-D-s of known concentration, it was proven that thereaction strength and the corresponding sedimentation curve of “+” reaction equals to 0.1 IU/mlanti-D. As the critical interval of anti-D is 5-15 IU/ml, measuring two dilutions of the test sera (50 and 150times), a fair estimation can be reached to the severity of HDFN. (Both positive reactions meanhigh risk, both negative mean no risk.) The results got by 15 minutes incubation time while using LISS enhancement medium were notsignificantly different from those where the incubation time was 45 minutes and the medium wasNormal Ionic Strength Solution (NISS). The enhancement medium that had the strongest influence on sensitivity was Polyethylene Glycol (PEG). The most influential factor was the concentration of the red cell suspensions.
Conclusion:
In the laboratories where ACT analysers are used, it is possible to measure quantitatively the levelof the antibodies and to monitor the changes without the regular use of the international stan-dards except changing the test parameters. Performing the IAT in saline medium (NISS) and using 45 minute incubation time, the anti-D stan-dard of 0,1 IU/ml gave a weak (one cross) reaction.In case of anti-D, if the test serum is diluted (N-times) until it gives a “+” reaction, then the anti-D concentration of the serum is 0,1N IU/ml.
Any questions will be answered: [email protected]
I N S
CYTOTECH INSTRUMENTS LTD.H-1131 Budapest, Dolmány u. 18-20.tel/fax: (+36) 1-359-9360cytotech.hu * [email protected]
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Automated Quantification of Anti-D with Automatic Coombs Test (ACT) AnalyserI. Hoffer 1, A. Borbely 2, A. Friss 3, E. Benko 4, S. Tovolgyi 5 (Hungary, Budapest)
References
1. Addendum for guidelines for blood grouping and red-cell antibody testing during pregnancy. Transfusion Medicine, 1999. 9, 99.2. Bowell, P.J., Wainscot, J.S., Peto, T.E.A., Gunson, H.H. Maternal anti-D concentrations and outcome in Rhesus haemolytic disease of the newborn. British Medical Journal, 1982. 285, 327-329.3. Duguid, J. K. M. Antenatal serological testing and prevention of haemolytic disease of the newborn J Clin Pathol 1997: 50: 193-1964. Gooch, A., Parker, J., Wray, J., Qureshi, H. Guidelines for blood grouping and antibody testing in pregnancy. Transfusion Medicine, 2007. 17, 252-262.*5. ISBT Science Series, 3, 33-606. Judd, W.J., Steiner, E.A., Nugent, C.E. Appropriate serological testing in pregnancy. 1992. 63, 293-296.7. Mollison, P.L., Engelfriet, C.P., Contreras, M. Blood transfusion in clinical medicine. 1993. 9th ed. Blackwell, Oxford.8. Nicolaides, K.H., Rodeck, C.H. Maternal serum anti?D concentration and assessment of rhesus isoimmunisation. 1992. 304, 1155-1156.
Authors1 Saint János Hospital, 2 National Blood Service, 3,4 Cytotech Intruments Ltd., 5 Budapest University of Technology and Economics (Hungary, Budapest)
CYTOTECH INSTRUMENTS LTD.H-1131 Budapest, Dolmány u. 18-20.tel/fax: (+36) 1-359-9360cytotech.hu * [email protected]
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