Long-term stabilized blood samples as controls for flow cytometric HLA-B27 screening: a feasibility study.
BACKGROUND
Long-term stabilized blood samples are potentially useful as positive or negative procedure controls for flow cytometric HLA-B27 screening, and could serve as test samples in an external quality assessment (EQA) scheme. We evaluated long-term stabilized whole blood specimens as prepared for the UK NEQAS for Leucocyte Immunophenotyping EQA scheme (Sheffield, UK).


METHODS
Peripheral blood samples were obtained from nine blood bank donors with known HLA-B typing. Short-term stabilization with Trans-FIXtrade mark was performed before shipment to Sheffield. Thereafter, long-term stabilization was performed. Commercially available HLA-B27 mAb were tested periodically between 1 week and 12 months on (i) fresh, (ii) short-term stabilized, and (iii) long-term stabilized blood samples using a stain, lyse, and wash technique. We compared the forward scatter (FSC), sideward scatter (SSC), and fluorescence signals of lymphocytes as a function of time. Furthermore, a pilot send-out with stabilized blood samples of four blood bank donors was distributed among the participants to the Benelux EQA scheme for HLA-B27 screening, and results were compared with historical EQA data obtained using nonstabilized blood samples from the same donors.


RESULTS
There were no major effects on FSC and SSC characteristics of lymphocytes. Background fluorescence of stabilized samples increased and specific fluorescence of stabilized HLA-B27 positive samples decreased as compared with fresh samples. However, discrimination between the investigated HLA-B27 positive and HLA-B27 negative samples remained feasible poststabilization. In the pilot send-out, the results obtained with stabilized samples were less concordant than with the corresponding fresh samples due to variable quality of the stabilized samples.


CONCLUSIONS
Long-term stabilized whole blood samples are potentially useful as true HLA-B27 positive and true HLA-B27 negative control cells for daily and longitudinal quality control of flow cytometric HLA-B27 screening. In the same way, long-term stabilized samples may be used for EQA purposes. However, these samples are currently not feasible for reagent validation purposes. Extensive quality control of long-term stabilized samples is necessary before distribution in multicenter surveys.
Levering
Wilfried H B M
WH
Laboratory for Histocompatibility and Immunogenetics, Sanquin Blood Bank South West Region, Rotterdam, The Netherlands. w.levering@sanquin.nl
Wind
Henk
H
Granger
Vivian
V
Sintnicolaas
Kees
K
Hooijkaas
Herbert
H
Reilly
John T
JT
Gratama
Jan W
JW
Barnett
David
D
D000483:Alleles; D000911:Antibodies, Monoclonal; D001793:Blood Preservation; D003429:Cross Reactions; D005434:Flow Cytometry / Q000379:methods / Q000592:standards*; D015235:HLA-B Antigens / Q000097:blood / Q000235:genetics; D015796:HLA-B27 Antigen / Q000097:blood* / Q000276:immunology; D006331:Heart Diseases / Q000175:diagnosis / Q000276:immunology; D006650:Histocompatibility Testing / Q000379:methods / Q000592:standards; D006801:Humans; D016130:Immunophenotyping / Q000379:methods / Q000592:standards; D011786:Quality Control; D012015:Reference Standards; D025242:Spondylarthropathies / Q000175:diagnosis / Q000276:immunology
D000911:Antibodies, Monoclonal; D015235:HLA-B Antigens; D015796:HLA-B27 Antigen