Zinc protoporphyrin as screening test in female blood donors

¹ Laboratory of Hematology, Bosch Medicentrum, 5211 NL 's-Hertogenbosch, The Netherlands.

² Department of Clinical Chemistry, Akademisch Ziekenhuis Rotterdam, 3000 CA Rotterdam, The Netherlands.

³ Department of Hematology, Vrije Universiteit Amsterdam, 1007 MB Amsterdam, The Netherlands.
^a Address correspondence to this author at: Laboratory of Hematology, Bosch Medicentrum, Nieuwstraat 34 5211 NL 's-Hertogenbosch, The Netherlands. Fax 31-736162958; e-mail JLHART.EJLAST{at}inter.NL.net.

	Abstract

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Erythrocyte zinc protoporphyrin (ZPP) was measured in 102 women blood donors to evaluate its usefulness in screening for evolving iron deficiency anemia, a reason for the deferral of donors. The results were compared with serum ferritin determinations. Five women were deferred before their first donation and eight women were deferred after one or two donations. Women with increased ZPP values all had low serum ferritin concentrations, indicating iron-deficient erythropoiesis that was caused by iron depletion. The positive predictive value of an increased ZPP in predicting deferral of the donor after one or two donations was 75%, whereas a serum ferritin concentration 12 µg/L predicted deferral in 26% of the donors. The results indicate that the ZPP test can be recommended as a feasible and inexpensive predonation test to determine a subset of donors with iron-deficient erythropoiesis at risk of developing iron deficiency anemia.

	Introduction

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Blood donation causes a substantial loss of storage iron. Each unit of blood drawn from a man contains 236 mg of iron or 6% of the total iron stored in the body. Corresponding values for women are 213 mg of iron and 9% (1).

An inverse correlation exists between the size of body iron stores and absorbed iron (2)(3)(4). As body iron stores decrease, iron absorption increases. With continued iron loss, an individual either reaches equilibrium at a lower concentration of iron stores or becomes iron-depleted, eventually developing iron-deficient erythropoiesis and anemia.

Iron deficiency anemia is the major factor limiting the frequency of repeated blood donation.

In the majority of blood banks, hemoglobin (Hb) or hematocrit measurements are used as a screening test for the ability to donate blood. This approach has proven the most inexpensive method of protecting donors against the development of progressive iron deficiency anemia. However, there are important limitations to this method. The major limitations are that these measurements lack sensitivity and specificity, and the definition of a threshold value is very problematic (5).

Variations in Hb concentrations within one individual due to physiological variations in plasma volume and erythrocyte mass is another complicating factor. Moreover, Hb does not decline until stores are completely exhausted and iron-deficient erythropoiesis has developed. Obviously, assays of iron status that are capable of assessing iron deficiency before deferral because of low Hb are desirable for donor screening.

Serum ferritin determination is a reliable index of body iron stores. In healthy subjects, serum ferritin is directly proportional to body iron stores (6). Serum ferritin concentrations 12 µg/L reflect an iron-depleted state (7)(8). Serum ferritin measurement thus gives a good assessment of iron status. However, this test is expensive and takes several hours to perform. Moreover, a considerable number of regular donors will have abnormally low values but will not become anemic (9)(10)(11)(12)(13). Because current blood banking policy allows continued donation as long as the Hb concentration is maintained, it is clear that serum ferritin cannot be used as the only screen for potential donors.

When iron is in limited supply, Zn², instead of Fe², is incorporated into protoporphyrin IX in the last step of heme synthesis, causing an accumulation of zinc protoporphyrin (ZPP) in the erythrocytes (14)(15)(16).

Erythrocyte ZPP estimation is a sensitive test for diagnosing iron deficiency anemia (15). ZPP reflects the supply of iron to the erythrocyte relative to the rate of protoporphyrin formation during Hb synthesis. Therefore, increased ZPP values can occur not only in iron deficiency anemia but also in other diseases with iron-deficient erythropoiesis, such as anemia caused by chronic disease and hemolytic anemia. However, in healthy blood donors increased ZPP concentrations most likely will be caused by depleted iron stores and a deficit in the functional iron compartment. Therefore, erythrocyte ZPP measurement could be an attractive alternative as a predonation screening test. Subjects with iron-deficient erythropoiesis will show increased ZPP values before overt anemia has developed.

With the introduction of the hematofluorometer, designed to measure the ratio of ZPP to oxyhemoglobin in small volumes of blood, the measurement of ZPP has become a very simple, quick, and inexpensive procedure (15).

The present study evaluated the clinical usefulness of ZPP measurements as part of the screening procedure for the ability to donate blood.

	Subjects and Methods

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During six consecutive months, 102 female volunteer first-time blood donors were entered into the study. Each donor gave informed consent to participate. At the first visit, Hb, serum ferritin, and erythrocyte ZPP measurements were performed. At each successive predonation screening, Hb and ZPP were measured. In 38 women, serum ferritin measurements were repeated before the second blood donation. The donors were followed for a period of 12–18 months.

According to the Dutch rules for blood donation, donors were not allowed to donate when Hb values were <126 g/L. In these cases, either a new control or deferral followed (without further investigation of the cause of the anemia). Deferral for reasons other than anemia did not occur. A maximum of four donations a year was permitted.

Estimations of Hb and ZPP were performed in venous blood collected in EDTA. Hb was measured with an automated cell counter (NE 8000). ZPP concentrations were measured with a front-face hematofluorometer (AVIV Biomedical). To avoid the influence of plasma components, the erythrocytes were washed before determining ZPP (17). The ZPP, measured as the ZPP/Hb ratio, is expressed in µmol/mol heme. The reference interval for ZPP in our hospital is 35 ± 20 µmol/mol heme (mean ± 2 SD). Serum for ferritin assays was stored at -70 °C, and the analyses were performed in one assay. Ferritin determination was performed using an enzyme-linked assay (reference interval, 20–100 µg/L for women; IMX, Abbott).

	Results

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At the first visit, the mean age of the women was 29 years (range, 19–52 years of age). Seven women were older than 45 and three were older than 50 years of age; therefore, most of the donors were in the age of menstruation. The mean Hb concentration was 132 g/L (range, 98–151 g/L), and the mean ZPP was 40 µmol/mol heme (range, 10–150 µmol/mol heme). The mean serum ferritin concentration was 31 µg/L (range, 4–200 µg/L). Thirty-three of the 102 women (32%) had serum ferritin concentrations <20 µg/L, whereas 18 showed concentrations 12 µg/L.

Five women (5%) showed low Hb values and were deferred at their first-time donation. Two women with Hb values within the reference interval were not willing to donate, and the remaining 95 women donated 1–5 times (mean, 2.5 times) during a period of 12–18 months.

In 38 women, serum ferritin estimation was also performed before the second donation. Table 1 shows the mean serum ferritin concentration before and after the first donation and the mean percentage of the decrease in serum ferritin values. The time between two donations varied from 4 to 8 months.

A total of 13 donors were deferred during the period of the study. The data of these donors is shown in Table 2 .

The number of donors with iron depletion (serum ferritin 12 µg/L) and the number of donors with iron-deficient erythropoiesis (ZPP >55 µmol/mol heme), divided into deferred and nondeferred donors, are shown in Table 3 . The 10 women with serum ferritin concentrations 12 µg/L before their second donation had ferritin values varying from 14 to 27 µg/L before their first donation.

A total of 23 women donated despite serum ferritin values 12 µg/L; 5 were deferred after one donation and 1 after two donations, whereas 17 women donated a total of 36 times (mean, 2.1 times) without deferral. Thus, the positive predictive value of a low serum ferritin concentration in predicting deferral (after one or two donations) per donor was 26%. In 57 blood donors, serum ferritin estimations were not repeated. Assuming that these women showed the same decrease in serum ferritin as the donors mentioned in Table 1 , in ~15% of these women serum ferritin decreased to values 12 µg/L, but none of these donors was deferred. Therefore, the real positive predictive value in predicting deferral per donor would have been even lower.

The 17 donors who donated 2.1 times (on average) despite serum ferritin concentrations 12 µg/L probably had low serum ferritin concentrations before each successive donation.

In the total of 43 episodes where serum ferritin concentrations were 12 µg/L before donation, deferral followed donation 6 times; therefore, the positive predictive value of a serum ferritin value 12 µg/L in predicting deferral was 14%. Again, the 57 blood donors in whom serum ferritin estimations were not repeated were not taken into account.

Two women with increased ZPP concentrations at their first donation and two women with increased ZPP concentrations at successive donations were not deferred. The data of these donors are shown in Table 4 .

A total of eight donors donated in spite of ZPP concentrations >55 µmol/mol heme. Five were deferred after one donation and one after two donations, whereas two donors each donated three times. Thus, the positive predictive value of an increased ZPP concentration in predicting deferral (after one or two donations) per donor was 75%.

Of the 11 episodes where ZPP concentrations were increased before donation, deferral followed after donation 7 times; therefore, the positive predictive value of an increased ZPP value in predicting deferral was 66%.

	Discussion

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Regular blood donors who do not take supplementary iron have smaller body iron stores than do nondonors (1)(9)(18)(19)(20)(21). A substantial percentage of them, especially women at the age of menstruation and individuals who donate frequently, will develop varying degrees of iron deficiency. Because of the substantial variation in iron homeostasis among blood donors, one cannot predict which donor will become anemic, which donor will become iron-depleted, and which donor will reach an equilibrium at a lower concentration of iron stores.

To prescribe iron supplements to all donors does not seem advisable. Iron therapy frequently causes side-effects such as abdominal discomfort, and therapy compliance is low. However, it is important to prevent iron deficiency anemia in blood donors. Iron is a vitally important element in human metabolism. It has a central role in erythropoiesis but is also involved in many other intracellular processes in all the tissues of the body.

Erythrocyte ZPP concentrations will be increased in sideropenic donors without overt anemia, and the ZPP test could be of value in predonation screening.

In this study, we assessed whether an increased ZPP concentration is a good indicator of evolving iron deficiency anemia in women blood donors. We also compared the results with serum ferritin estimations.

A serum ferritin concentration of 12 µg/L is generally accepted as the point at which one defines the absence of storage iron. According to this definition, 12% of the first-time donors in this study were iron-depleted. Other authors reported percentages between 6% and 25% (1, 9, 10, 20–22).

We found a considerable decrease in the serum ferritin concentration after the first donation; this also has been reported by other authors (1)(10).

Schifman et al. reported that decreased serum ferritin concentrations have a low predictive value in predicting which donors will be deferred, a conclusion that is in agreement with our results (23).

Birgegård et al. found that, in female donors with a high prevalence of iron deficiency, a further increase in donation frequency was not necessarily accompanied by an increase in iron deficiency resulting in anemia (24). Strandberg Pedersen and Morling concluded that serum ferritin concentrations seem to be of little help in the selection of donors with a high risk of anemia (9). Thus, serum ferritin estimation is not useful as a predonation screening test.

The 14 donors with increased ZPP concentrations, either at their first-time donation or at later donations, all showed depleted or nearly depleted iron stores at the first-time donation, indicating that an increased ZPP concentration is evidence of iron-deficient erythropoiesis caused by iron deficiency in healthy blood donors. The specificity of an increased ZPP concentration in predicting low serum ferritin concentrations was 90% in a study of Morse et al. (22), and >80% in a study of Jensen et al. (25).

In contrast to our results, Schifman et al. found a rather low predictive value of an increased ZPP concentration in predicting donor deferral, but that study only evaluated the results obtained at the initial and the first follow-up visit (23).

Four of the 14 women with increased ZPP concentrations were not deferred (Table 4 ). The first and second donors showed a substantial increase in ZPP concentrations, and it is likely that these women would have developed anemia with further donations. In the third donor, the time between the first and second donations was 12 months, and the ZPP concentration returned to a value within the reference interval before the second donation. The fourth donor was iron-depleted and had mild iron-deficient erythropoiesis without a substantial increase in ZPP values after donating. She donated three times in 18 months without developing overt anemia.

Raftos et al. found that the increase in ZPP values depended on the frequency of donation (21). Our results show that the diagnostic value of erythrocyte ZPP estimation, used as a test to predict future anemia and deferral in blood donors, is satisfactory. The predictive value will probably increase in donors with a higher frequency of donation.

In our opinion, donors must be protected against the development of iron deficiency anemia, and erythrocyte ZPP estimation in conjunction with Hb before each blood donation could be of help in identifying donors who need iron supplementation.

The ZPP test is very easy to perform and takes less than one minute. In our laboratory, we wash the erythrocytes to remove plasma components that give rise to aspecific fluorescence. However, this is necessary only in some patients and not in healthy subjects. The test takes only a few drops of anticoagulated capillary or venous blood, and no further reagents are needed. The procedure could be performed in a bloodmobile. Hb measurement in combination with ZPP estimation is a technically feasible predonation screening procedure. We plan to perform a study in which donors with increased ZPP concentrations are randomized to receive either iron therapy or a placebo to compare the development of iron deficiency anemia and subsequent deferral in the two groups.

	References

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