title: Optimal conditions for the performance of a monocyte monolayer assay.
abstract: Various versions of the monocyte monolayer assay (MMA) have been used to assess clinical significance of red blood cell (RBC) alloantibodies in transfusion for more than 35 years. However, the optimal conditions, including anticoagulant used for whole blood samples, temperature and duration of storage, and optimal pH for assessing the response of monocytes to antibody-bound RBCs, have never been clearly delineated.
  
  
  
  Whole blood from healthy donors was collected in ACD, EDTA, or heparin and stored at room temperature (RT) versus 4°C for up to 2 days. pH was examined with and without buffers. Phagocytosis of anti-D-opsonized R2R2RBCs was used as the positive control for comparison studies. Whole blood was taken into ACD and kept at RT until testing, from patients with or without immune hemolytic anemia.
  
  
  
  No significant differences in the phagocytosis of the R2R2control RBCs were observed using ACD anticoagulant between freshly drawn or up to 36-hour-stored whole blood kept at RT, regardless of the donor. Physiologic pH during MMA was important for optimal monocyte interactions with antibody-opsonized RBCs. MMA results with patient samples, under optimal conditions, kept up to 30 hours in one instance of long-distance shipment, correlated with clinical hemolysis.
  
  
  
  MMA can be reliably performed on whole blood samples drawn into ACD and kept at RT for up to 36 hours and when physiologic pH is maintained during the assay. Future studies are required to confirm whether use of these conditions with patient monocytes can provide accurate determination of alloantibody significance in patients requiring blood transfusion.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, Canadian Blood Services, Toronto, Ontario.|Centre for Innovation, Canadian Blood Services.
authors.forename: Tik Nga
authors.initials: TN
authors.lastname: Tong
authors.affiliation: Centre for Innovation, Canadian Blood Services.
authors.forename: Emeralda
authors.initials: E
authors.lastname: Burke-Murphy
authors.affiliation: Centre for Innovation, Canadian Blood Services.
authors.forename: Darinka
authors.initials: D
authors.lastname: Sakac
authors.affiliation: University Health Network, Toronto, Ontario, Canada.
authors.forename: Jacob
authors.initials: J
authors.lastname: Pendergrast
authors.affiliation: University Health Network, Toronto, Ontario, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: Cserti-Gazdewich
authors.affiliation: CHU de Québec-Université Laval, Quebec City, Quebec, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Laroche
authors.affiliation: Department of Laboratory Medicine and Pathobiology, Canadian Blood Services, Toronto, Ontario.|Department of Medicine, University of Toronto.|Centre for Innovation, Canadian Blood Services.|University Health Network, Toronto, Ontario, Canada.
authors.forename: Donald R
authors.initials: DR
authors.lastname: Branch
mesh_terms: D000328:Adult; D000925:Anticoagulants; D001793:Blood Preservation / Q000379:methods; D001803:Blood Transfusion / Q000592:standards*; D004912:Erythrocytes / Q000276:immunology*; D005260:Female; D006461:Hemolysis / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007518:Isoantibodies / Q000097:blood*; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology*; D009895:Opsonin Proteins; D010587:Phagocytosis; D013696:Temperature; D013997:Time Factors; D065227:Transfusion Reaction; D055815:Young Adult
chemical_list: D000925:Anticoagulants; D007518:Isoantibodies; D009895:Opsonin Proteins