Washing Erythrocytes to Remove Interferents in Measurements of Zinc Protoporphyrin by Front-Face Hematofluorometry

Abstract

Zinc protoporphyrin (ZPP) is determined by hematofluorometry of whole blood to detect iron deficiency in blood donors. In hospitalized patients, ZPP did not correlate with established markers of iron status. We performed 4500 ZPP measurements with the Aviv front-face hematofluorometer in samples from 475 patients and measured ferritin, transferrin saturation, hemoglobin, and erythrocyte indices. We found that the fluorometric determination is affected by substances dissolved in plasma but that this interference can be eliminated by using washed erythrocytes. In validation tests the within-day variation was < 3.5%; the day-to-day variation was < 6.8%. In 130 healthy persons without iron deficiency, ZPP was < or = 40 mumol/mol heme, which we consider a normal value. Mean ZPP in 46 iron-deficient patients was 256 (SD 105) mumol/mol heme (correlation with ferritin: -0.73; with hemoglobin: -0.85; P < 0.001). When washed erythrocytes are used, the hematofluorometric determination of ZPP is sensitive and specific for detecting iron deficiency in otherwise healthy individuals and hospitalized patients.

Observations on the measurement of zinc protoporphyrin in both whole blood and washed red cells in young children

“…The only previous report of the plasma fluorophore contribution to whole blood ZPP values in healthy subjects indicated a constant level equivalent to about 20 μmol/mol haem in adults. 1 In contrast, our findings in nonanaemic infants and young children, some of whom had mild iron deficiency and/or mild infection, 2 were quite different, in that, in most the plasma fluorophore component contributed <20 μmol/mol haem to whole blood values, whilst, importantly, there was a wide range of values, equivalent to 0-45 μmol/mol haem. In this situation, there is the risk of both falsepositive and false-negative values occurring.…”

High prevalence of subclinical iron deficiency in whole blood donors not deferred for low hemoglobin

“…ZPP levels were measured with a hematofluorometer (Model 206D, Aviv Biomedical, Lakewood, NJ) on the next day in the samples collected in lithium heparin. Before ZPP measurement RBCs were washed with phosphate‐buffered saline to minimize the possible influence of other fluorescent elements in plasma …”

Oxidative stress may modify zinc protoporphyrin/heme ratio in hematofluorometry

“…Plasma interference may potentially give false increased values, primarily because of bilirubin (Lamola, Eisinger & Blumberg, 1979;Schifman & Finley, 1981). Elimination of plasma interference requires washing the erythrocytes by saline (Hastka et al, 1992). Other less common analytical pitfalls that have been cited include incomplete hemoglobin oxygenation (Luoro & Tutor, 1994) as well as the practise of considering zinc protoporhyrin and erythrocyte protoporphyrin as equivalent (Labbe, Vreman & Stevenson, 1999).…”