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In infants and children, whole blood ZnPP/H ratios measure iron-deficient erythropoiesis. Because immature erythrocytes are less dense than mature erythrocytes, we hypothesized that the sensitivity of ZnPP/H is improved if measured in the least dense cells. Blood was collected from control suckling, mild and severe iron-deficient (ID) suckling rats. Cord blood was collected after uncomplicated (control), diabetic (severe ID) and intermediate iron status pregnancies (mild ID). ZnPP/H was measured before and after density centrifugation. ZnPP/H in the lightest cells, the top fraction, was reproducible. The difference between whole and top fraction was defined as Δ ZnPP/H. In rats, although the whole blood or top ZnPP/H differed by postnatal age, P<0.0001, Δ ZnPP/H was greatest after the interval with least body iron accrual, P<0.0001. In mild ID rats, whole blood ZnPP/H was similar to, but Δ ZnPP/H was greater than controls, P<0.005. In humans with mild ID, whole blood ZnPP/H was similar to, but Δ ZnPP/H was relatively greater than controls, P<0.001. In rats and newborn humans, Δ ZnPP/H is more sensitive than whole blood ZnPP/H in identifying conditions associated with impaired erythrocyte iron delivery and may become a useful tool in measuring erythrocyte iron incorporation in early development.
There is need for a sensitive clinical index of iron-deficient erythropoiesis in growing premature infants. In premature infants, clinical practices, such a conservative transfusion criteria and therapeutic recombinant erythropoietin therapy may deplete tenuous iron stores (1–4). When iron supply is limited, zinc replaces iron in the protoporphyrin IX ring to form zinc protoporphyrin (ZnPP) (5). The ratio of whole blood ZnPP to heme (ZnPP/H) measures impaired erythrocyte iron incorporation (5, 6). In older infants, whole blood ZnPP/H is a more sensitive indicator of early, preanemic iron deficiency than hemoglobin or classical indices of storage iron (7). Whole blood ZnPP/H ratios from cord blood or ill newborns have also been shown to reflect iron status (4, 8). ZnPP/H ratios are higher in newborns born small for dates and offspring of mothers with insulin-treated diabetes, two conditions commonly exhibiting severely impaired tissue iron content (severe ID) at birth (4, 8). In premature infants, whole blood ZnPP/H ratios fall in response to iron therapy (9) or with indirect delivery of iron via transfusion (3). In premature infants, whole blood ratios are inversely related to gestation (8), necessitating gestation-based normal values for interpretation (4, 8–10).
In mild ID, measuring ZnPP/H in the most immature circulating erythrocytes, the reticulocytes, would improve the sensitivity of ZnPP/H to detect impaired iron incorporation. Previous work supports that measuring iron incorporation into reticulocytes (reticulocyte hemoglobin, or percent hypochromic reticulocytes) with clinical flow cytometry-based automated hematology analyzers is able to identify mild ID (1, 8, 11–15). Although powerful, sophisticated instrumentation to investigate these parameters is not universally available. However, immature erythrocytes can also be separated based on density centrifugation. After centrifugation, the lighter fraction contains greater relative numbers of reticulocytes than is seen in whole blood. Employing this low tech approach, we sought to examine whether reticulocyte enrichment via differential centrifugation (16) would allow us to examine whether a difference between whole blood and top fraction ZnPP/H (Δ ZnPP/H) is clinically useful.
We hypothesized that ZnPP/H ratios measured in the least dense erythrocytes are reproducible and that Δ ZnPP/H is more sensitive in detecting mild ID than whole blood ZnPP/H ratios during early rat and human development.
Sprague-Dawley dams (Charles River Laboratories, Wilmington, MA, U.S.A.) and pups were studied. UW Animal Care and Use Committee approved the study. Pregnant dams were delivered to University animal housing one week prior to delivery. Control pups with litter size of 10 were grown to specific ages to demonstrate normal developmental ZnPP/H ratios. In the developmental studies, rats were examined at postnatal day (P); P1 newborn, P4 suckling, P8 suckling, P12 suckling, P18 weanling and adults (10–15 weeks).
For treatment studies, a subset of rats underwent randomization to damfed or experimental manipulation with an artificial cow milk-based formula with insufficient iron to meet the needs of suckling rats. The formula provided 8 mg/L elemental iron using ferrous sulfate. Formula-fed rats were removed from the dam and artificially fed by gastrostomy in temperature-controlled pint-sized polypropylene deli cups as previously described (17, 18). Feeds were adjusted to maintain daily weight gain equal to that of dam fed rats with a litter size of 10. Artificial feeding began at P4 for severe ID with anemia or P8 for mild ID without anemia. An additional group, mild ID+Fe, included artificial feeding beginning at P8 supplemented with a once daily dose of 6 mg·kg−1 oral ferrous sulfate (Fer-In-Sol, Mead Johnson, Evansville, IN, U.S.A.). Formula groups were compared to dam fed control and sacrificed at P12.
Rats from both developmental and treatment groups were randomly selected for either blood analysis or body iron determination. In the blood group, after lethal isofluorane anesthesia, blood was drawn via cardiac puncture and placed into EDTA anticoagulated tubes. For rats assigned to body iron determination, no blood was drawn. Midline incision was performed and gastrointestinal tract was removed from the body to remove iron not yet absorbed, but residing within the gastrointestinal lumen. Blood lost during the gastrointestinal harvest was included with the body. The body without gastrointestinal tract was analyzed for iron content by atomic absorption after nitric acid/perchloric acid digestion. Briefly, the bodies were pre-digested for 24–48 hr at RT in 30 mL concentrated nitric acid and then transferred to Teflon® digest tubes with an additonal 10 mL of concentrated perchloric acid. The samples were digested at 90°C for 1.5 hr, then at 200°C until digests were clear (duration varied by size of rats). Digests were allowed to cool to room temperature and diluted to a constant volume with deionized water. Total Fe concentration of the digests was measured, with accompanying ferrous nitrate reference standards on a Perkin Elmer 2280 Atomic Absorption Spectrophotometer (Waltham, MA, U.S.A.).
Cord blood samples from three separate groups of newborns were analyzed. EDTA-anticoagulated cord blood is normally collected at all deliveries in the Birthing Center at Meriter Hospital, Madison, WI, U.S.A. and held for 7 days in the Blood Bank. We obtained the blood just before samples were to be discarded. Blood was obtained between June 2005 and December 2006. The term control group was randomly collected from normal term deliveries. The second group, offspring of term/near-term insulin-treated diabetes (IDM), was collected because of the marked risk for abnormal iron status (4, 8). IDM newborns admitted to the NICU are more severely affected by maternal illness. The intent of the third, at-risk group was to obtain cord blood from term ethnic minority and Caucasian mothers exhibiting intermediate iron status. From the at-risk group, we defined a mild ID subset with cord plasma ferritin values in the lowest quartile of our samples, but normal cord whole blood ZnPP/H (10). The fourth, premature group included cord blood from newborns with birth weight less than 1500 g, but without known impairment of iron status by maternal diabetes or growth restriction. Because cord ZnPP/H ratios in premature normally fall as gestation increases (4, 8), this group was included to determine whether Δ ZnPP/H is observed in premature newborns. The premature and IDM groups were identified from the Meriter Hospital Nursery Intensive Care Unit (NICU) admission log and the at-risk group from the healthy newborn admission log. No other data were collected from these mother-child dyads. Approvals from the UW-Madison Human Subjects Committee and Meriter Hospital Institutional Review Board were obtained to analyze deidentified samples.
ZnPP/H was determined before (whole blood) and after (top) the density centrifugation step. Samples were tested for clots, stored at 4°C, and hematology profile performed by a Coulter MD Coulter Counter, (Coulter-Beckman, Hialeah FL, U.S.A.). An aliquot of whole blood was centrifuged in a 500 µL microcentrifuge tube (Fisher Scientific, Hampton, NH, U.S.A.) at 800 × g in an Eppendorf, 5415D Microcentrifuge (Eppendorf AG, Hamburg, FRG) for 4 minutes. Plasma was removed. The cell pellet was rinsed in PBS three times to remove interfering pigments (19). After rinsing, packed red blood cells were diluted 1:1 with PBS and whole blood ZnPP/H determined with a front-face hematofluorometer (Aviv Biomedical, Lakewood, NJ, U.S.A.).
To obtain ZnPP/H from the top fraction, or least dense, immature erythrocytes, we employed the principle of density gradient centrifugation (11, 16, 20). We centrifuged an aliquot of whole blood for 30 minutes at 1500 × g (16) in a 400 µL 8 × 48 mm microcentrifuge tube (Fisher Scientific, Waltham, MA, U.S.A.) in an Eppendorf, 5415D Microcentrifuge. Plasma was removed with a Fisherbrand® gel loading pipet tip (Fisher Scientific) and set aside. The top 25% of packed cells was identified using a custom-made polyethylene Tube Reader (Figure 1). The top 25% of cells were transferred to a new 8 × 48 mm tube, an equal volume of plasma added, and centrifugation repeated. The top 25% of these cells were transferred to a new tube, resulting in the lightest 6.25% of cells from the original sample. After rinsing three times, the top fraction ZnPP/H was measured. Δ ZnPP/H was defined as the difference between the top fraction and the whole blood ZnPP/H.
The Tube Reader was custom-manufactured by UW-Madison Biomedical Engineering students as a team project. The Tube Reader was 4.7 × 2.4 × 2.0 cm (12 × 6 × 5 in), L × W × H manufactured in static dissipative ultra-high molecular weight polyethylene with place holders drilled for 8 × 48 mm microcentrifuge tubes. The laminated scale was placed behind a Plexiglas® window and adjusted to match each tube’s packed cell volume (see supplemental materials).
An aliquot of blood obtained before reticulocyte enrichment was stained for manual reticulocyte percentage with the supravital new methylene blue stain (Sigma, St. Louis, MO, U.S.A.). Reticulocyte percentage was also obtained on an aliquot of the top fraction after reconstitution with plasma. Blinded reviewers determined the reticulocyte percentage at 100× power.
Sample size was determined to be 8 per group for P12 control and mild ID rats assuming 80% power, P value <0.05, to measure a 50 µmol/mol rise in ZnPP/H with top fractionation. Sample size was determined to be 9 per group for at-risk and human IDM, assuming 80% power with P value <0.05 to measure a 25 µmol/mol rise in ZnPP/H with top fractionation. Reproducibility of top ZnPP/H from multiple aliquots of the same sample within-day and between-day was examined by repeated measures ANOVA. Additionally, paired and unpaired t testing, Kruskal-Wallis testing, or factorial ANOVA, with Fisher's post hoc testing was employed. Values reported are mean ± SEM.
The minimal initial sample volume necessary for reproducible top fraction ZnPP/H was 300 µL. Consistent with the literature (19, 21, 22), we previously showed that aliquots of whole blood ZnPP/H assayed within- or between-days for up to 10 subsequent days were similar (23). If stored at 4°C, aliquots assayed for top fraction ZnPP/H were also reproducible in human and rat samples within- or between-days for 10 days (Fig. 2A & B). Mean coefficient of variation on individual repeated samples was 9.6% (range 1.4–26%). We also determined whether sample processing artifactually elevated ZnPP/H by examining the bottom fraction ZnPP/H in P4 rats. P4 rats were selected because these rats exhibited the greatest Δ ZnPP/H (difference between whole blood ZnPP/H and top ZnPP/H). Mean ZnPP/H from the heaviest 75% (103.5 µmol/mol) was similar to whole blood ZnPP/H (94.0 µmol/mol), while the lightest 12.5% or top fraction (228 µmol/mol) was higher, P<0.002.
In the rat developmental study, whole blood or top fraction ZnPP/H differed by postnatal age, P<0.0001 (Fig. 3). The top fraction ZnPP/H was higher than whole blood ZnPP/H at all ages, except adults, P<0.0001. Δ ZnPP/H, body iron concentration (µg·g−1), body weight (g), daily body Fe increment (µg·d−1) and mean reticulocyte percentages (whole/top fraction) differed over development and are shown in the table of Fig 3. Suckling and weanling rats exhibited higher Δ ZnPP/H ratios than adult rats. However, Δ ZnPP/H ratios in suckling and weanling rats were similar to each other, except at P4, when higher ratios accompany the lowest daily body increment and greatest fall in body iron concentration (Fig. 3).
Whole blood ZnPP/H ratios were sufficiently sensitive to identify a longstanding, severe ID. In rats with severe ID, mean hemoglobin (85.3 ± 2.7 g/L) and mean body iron concentration (25.2 ± 1.5 µg·g−1) were both lower than controls (93.4 ± 2.6 g/L and 40.3 ± 2.5 µg·g−1), P<0.005. Figure 4 shows that whole blood ZnPP/H ratios in severe ID were higher than control, P<0.0001. The white box labeled “Severe ID” shows the mean ± 2 SD whole blood ZnPP/H ratios in severe ID. In mild ID and mild ID+Fe, mean hemoglobin values (99.0 ± 2.7 and 97.3 ± 2.8 g/L, respectively) were similar to controls. Whole blood ZnPP/H in mild ID and mild ID+Fe were also similar. However, Δ ZnPP/H was able to discriminate a difference between mild ID and controls or mild ID+Fe, P<0.001 (Fig. 4). Although mean rat weight was similar, body iron concentration (µg·g−1) differed by treatment (Fig 4). Mean reticulocyte percentages (whole/top fraction) are shown in Fig 4.
In humans, cord whole blood ZnPP/H from the severe ID (IDM) group was sufficiently sensitive to show a difference from control term newborns, P<0.0001. Mean ± 2 SD from severe ID is shown in white box on Fig. 5. In control newborns, whole blood ZnPP/H ratios in term controls were similar to mild ID, but Δ ZnPP/H in mild ID is higher than controls, P<0.0001 (Fig. 5). Mean reticulocyte percentages (whole/top fraction) are also shown in Fig 5. To investigate the specificity of Δ ZnPP/H, we examined a group with developmentally higher ZnPP/H ratios, but without evidence of iron disturbance. Although higher than controls, cord whole blood (153.7 ± 19.9 µmol/mol) and top fraction (165.2 ± 26.5 µmol/mol) ZnPP/H ratios from premature newborns were similar, with no appreciable Δ ZnPP/H.
This is the first study to show that measuring ZnPP/H in the least mature erythrocytes from newborn rats or humans improves the sensitivity of ZnPP/H to detect impaired iron delivery. Examining Δ ZnPP/H removes the overlap between normal and abnormal groups and lessens the developmental differences in values. Whole blood ZnPP/H was previously shown to measure erythrocyte iron incorporation in umbilical cord blood and NICU neonates (3, 4, 8). Erythrocytes circulate for up to 90 days in newborns (24). With maturation, erythrocytes progressively increase in density (25). Reticulocytes, the most immature erythrocytes, are identifiable for 2 days by supravital staining of retained cytoplasmic RNA, mitochondria, ribosomes, centriole and Golgi bodies (26). Methods of reticulocyte enrichment have been described for years, including Percoll-Renografin gradients, phthalate ester separation, silicone oil gradients, and employing high-speed/longer duration floor model centrifuges for density separation (11, 16, 20, 25). With these complex methods, the least dense cells immature cells can be concentrated up to 10 times, using reticulocyte percentages as indicators. We selected a simpler, lower speed centrifugation using universally accessible microcentrifuges to accommodate small (300 µL) sample volumes. Although other low density immature erythrocytes remain in the top fraction and contribute to the top fraction ZnPP/H, we measured reticulocytes as the marker of density separation (16, 20, 25) because of the ease of measurement and because percentages were sufficiently higher (doubled).
In the rat developmental study, ZnPP/H changes with postnatal age, but the highest ZnPP/H and largest Δ ZnPP/H were found in rats with virtually no net 4-day body iron accretion. This observation supports our previous published data supporting a limitation of erythrocyte iron delivery in normal rats at P4–P6 (27). The relatively lower ZnPP/H and Δ ZnPP/H at P8, P12 and P18 accompanied increased iron accretion. However, adult rats exhibited the lowest Δ ZnPP/H. Because of this, our data support that the relatively higher Δ ZnPP/H in normal developing rats compared to developing humans could be because rats exhibit a relative faster growth rate and require relatively greater iron for erythropoiesis, compared with humans. For rats in the treatment studies, hemoglobin and body iron levels confirm the presence of mild and severe ID. At P12, rats with severe ID exhibited relatively higher whole blood ZnPP/H, but ratios in dam fed control, mild ID or mild ID+Fe rats were similar. The advantage of Δ ZnPP/H is its ability to discriminate a difference between untreated mild ID from control and mild ID+Fe.
In cord blood from term newborns (controls), we anticipated and observed no appreciable Δ ZnPP/H, reflecting normal fetal erythropoiesis at term. The severe ID (IDM) group was included to demonstrate that Δ ZnPP/H is unnecessary when whole blood ZnPP/H ratios are markedly abnormal. In two-thirds of IDM, impaired transferrin receptor-mediated iron transport in placenta, fetal hypoxia, exaggerated growth, and exaggerated erythropoiesis (8, 23, 28) result in impaired tissue iron. In IDM, ZnPP/H (4,8) or erythrocyte protoporphyrin (29) were correlated with disturbed iron status, and to surrogates of maternal glycemic control (23, 29). From mothers at-risk for iron deficiency, we defined the mild ID group as the subset with the lowest quartile plasma ferritin levels, but normal whole blood ZnPP/H. In cord blood, using Δ ZnPP/H improved the sensitivity of ZnPP/H to detect mild ID. Previous work shows that whole blood ZnPP/H ratios are inversely related to gestation (4, 8). We examined premature infants to show that, although whole blood ZnPP/H ratios were higher than at term, absence of appreciable Δ ZnPP/H supports normal fetal erythropoiesis.
Other potential clinical indicators of iron status in newborns have been investigated. A soluble truncated transferrin receptor (sTfR), used in older patients, was more strongly correlated with erythropoiesis than iron indices in newborns (30–32). Reticulocyte indices (reticulocyte hemoglobin or percent hypochromic reticulocytes), available on newer clinical flow cytometer analyzers, are effective at identifing preanemic mild ID in infants (1, 8, 15). Although our method was designed for simple nonautomated hematofluorometers, a channel measuring reticulocyte ZnPP/H could be also developed for automated clinical flow cytometry analyzers, as suggested by Labbe’ and Dewanji (33).
Because reticulocyte indices are not universally available, whole blood ZnPP/H ratios are simple, cost-effective and require minimal sample volume when screening iron status of premature infants (9, 10). Ratios are directly measured by two commercially available hematofluorometers on drops of blood with minimal cost (34, 35). Changes in whole blood ZnPP/H ratios are relatively specific, with lead poisoning, unlikely in newborns and hospitalized premature infants, raising ratios (19). In newborns, jaundice or pigmented drugs may artifactually elevate ZnPP/H, but simple rinsing removes plasma pigments (19). We found that reticulocyte enrichment step was reproducible and Δ ZnPP/H relatively more sensitive than whole blood ZnPP/H in determining preanemic mild ID in newborn rats and humans. Four additional observations supported that Δ ZnPP/H was not artifact. First, the most common disturbance of red cell processing is hemolysis, but we observed that ZnPP/H falls with hemolysis (unpublished data). Secondly, we mathematically projected that the bottom fraction ZnPP/H would be unchanged after centrifugation and found that it was unchanged. Third, Δ ZnPP/H was unappreciable in normal term and preterm human samples and adult rats. Fourth, immature erythrocytes comprise nearly all cells in the top fraction. Previous reports describe that adult whole blood also contains a very small percentage (less than 0.1%) of low-density mature cells identified by resistance to shrinking with valinomycin (36). We treated neonatal cord blood with valinomycin to identify these mature low-density in the top fraction, but found scant numbers (data not shown).
Measuring whole blood and Δ ZnPP/H ratios may minimize the disadvantage of developmental differences in whole blood ZnPP/H in rats and humans, minimize the overlap between normal and abnormal values and improve sensitivity in identifying mild ID. We speculate that identifying mild ID by Δ ZnPP/H when treating the anemia of prematurity could be clinically useful. Miller, et al. showed that whole blood ZnPP/H may respond to iron therapy (9), but by employing whole and Δ ZnPP/H would potentially improve the senstitivity to observe an iron response, and when iron dose is titrated to ZnPP/H (9), avoid overtreatment. Future work in this field is necessary.
This work was supported by NIH M01 RR03186 from UW GCRC/CReFF (PJK), the University of Wisconsin Women in Science and Engineering Life Cycle Grant (PJK), Thrasher Research Fund (PJK), and NIH R01-HD-29421 (MKG). The authors would like to acknowledge experimental design support from John A. Widness, M.D and Richard Eisenstein, Ph.D., technical support by Elizabeth Goetz M.D., Debra Schneider B.S., Kelsey J. Kleven, and Aisha K. David. We also acknowledge engineering design from Undergraduates in Biomedical Engineering 201 students, Katy Reed, Sarajane Stevens, Christopher Westphal and Anita Zarebi, with advisor Kristyn Masters, Ph.D.