P‐IG‐37 | Repurposing NESTROFT/Pink Reagents by Optical Microplate Standardization for Optimal Differential Lysis Phenotyping of Transfused Thalassemia & Simulation Mixes

Phenotyping needs 3-month transfusion-free window never available in Transfusion dependent Thalassemics (TDTs). Thus, TDTs rarely receive phenotype matched (DCcEeK) blood as recommended. Isolation of reticulocytes by centrifugation or differential lysis of allogeneic cells are two economic approaches for phenotyping such patients. There are two hypotonic (0.3% saline) lysis methods: (1) Brown (1988) method in AABB-TM20 supplement involves six washes, whereas (2) Sheladiya (2016) needs less wash but 1:400 dilution, if starting amount small, has little left to phenotype. NESTROFT (0.36% saline) is a field screen for Thalassemia and Pink Test (acidified glycerol) is a spherocytosis screen, both capable of differential lysis. We repurposed Pink/NESTROFT type reagents for differential lysis and standardize discriminatory window of hematocrit, salinity, glycerol and incubation-time combinations by mapping the OD-time-profile of donor RBC-suspensions and thalassemic samples in optical microwells. Thalassemic cells are osmotically resistant and lysis is the dominant mechanism of OD drop under shaking. RBCs from 7-day-old-units were suspended in salines 0.20%, 0.24%, 0.26%, 0.30%, 0.32%, 0.34%, 0.36%, and 0.40%, diluted glycerols 10-100% at increments of 10% (100% = 270mOsm) and Hct %s 0.5%, 1%, 1.5%, 2%, 2.5%, and 3%; ODs taken at 405, 450, and 620 nm after: 0, 1, 5, 10, 15, 20, 25, and 30 min (&initially 12 h) in flat well microplates with and without shaking. Samples from six TDT patients transfused in 1 month were treated pink reagents of 286mOsm, 167mOsm, 148mOsm, 111mOsm, 85mOsm with pH 6.6 and compared with 0.3% saline (Fig 1D). ABO and Rh mismatched mixture differential lysis were tested. Maximum lysis of PRBCs happen at <5 min especially at hcts <1% then plateaus off. Above 1.5% Hct lysis curves segregate for reagents and incubation times (e.g., Figure 1A). At salinity >0.36% (1A) or glycerol >50% (1C), ODs higher due to low lysis. NS eventually causes slow lysis. Higher OD due to settling can be offset by shaking. Diluted pink reagent 140mOsm (approx half of original) showed behaves similar to 0.3% hypotonic saline. 0.3%S lysis worked in tube and gel card for Rh mismatch simulations but not ABO. FIGURE. Effect of different grades of salinity, sample dilution/hematocrit, and glycerol on differential lysis of 7 day old PRBCs and Thalassemia samples.