Background/Case Studies: Phenotyping needs three-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)Brown1988 method in AABB-TM20 supplement involves 6 washes, whereas 2)Sheladiya2016 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.
Study
Design/Methods: RBCs from 7-day-old-units were suspended in salines 0.20, 0.24, 0.26, 0.30, 0.32, 0.34, 0.36 & 0.40%, diluted glycerols 10-100% at increments of 10% (100%= 270mOsm) & Hct %s 0.5, 1, 1.5, 2, 2.5, 3%; ODs taken at 405, 450 & 620nm after: 0,1,5,10,15,20,25,30 min (&initially 12 hr) in flat well microplates with & without shaking. Samples from 6 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 & Rh mismatched mixture differential lysis were tested.
Results/Findings: Maximum lysis of PRBCs happen at < 5min especially at hcts < 1% then plateaus off. Above 1.5% Hct lysis curves segregate for reagents & incubation times (eg Fig 1A). At salinity >.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 reagent140 mOsm (approx half of original) showed behaves similar to 0.3% hypotonic saline. 0.3%S lysis worked in tube & gel card for Rh mismatch simulations but not ABO. Conclusions: Microplate reader screening of RBC-differential-lysis is cheap, simple and rapid. OD at 620 nm with shaking likely parallels unlyzed cells. Initial 5-min lysis is fast in all NaCl < 0.34%. Initial slight rise in OD in NaCl>0.34% possibly represent subcritical swelling. With lysis extracellular osmolality rises and slows/halts further lysis, thus the slow drop of OD with higher Hct/Salinity (1A & 1B). We show that Hct approx 2.0% could be used in optimized conditions as opposed to approx. 0.2% (1:400 of 80%) needed for single step washing. Lowering salinity, pH, glycerol increased lysis in graded manner but around 50% glycerol show dichotomous behavior (Fig 1C). Differential lysis works for DCEK phenotyping.
Importance of research: Thalassemics get frequent transfusions from a young age, and have perpetually mixed blood. In poor nations genotype is unavailable, and phenotyped only if >3 months transfusion free, thus TDTs receive blood Antigen-neg only for detected Antibodies instead of phenotype matched (DCcEeK) as recommended. Successful standardization of a cheap simple and rapid method will allow millions of thalassemia patients across the globe receive phenotype-matched blood and live a longer morbidity free life.
