HEMATOLOGY SECTION - ORIGINAL ARTICLE
Year : 2008 | Volume
: 51 | Issue : 1 | Page : 105--107
Evaluation of iron status: Zinc protoporphyrin vis-a-vis bone marrow iron stores
Sheila Das, Kandathil Joseph Philip
Department Pathology, Christian Medical College, Ludhiana, India
Department of Pathology, Christian Medical College, Ludhiana - 141 008
Zinc protoporphyrin (ZPP) in the red cells is an indicator of iron status in the bone marrow (BM) and can be easily measured by Protofluor-Z Hematofluorometer from Helena Laboratories. It is well known that bone marrow iron is a gold standard for the diagnosis of iron deficiency anemia (IDA) even in the pre-latent phase. Hence, it was considered pertinent to evaluate the diagnostic utility of ZPP in comparison with bone marrow iron stores. 107 random BM were selected over a period of 2˝ years; in each case, RBC indices where recorded along with ZPP and Perls«SQ» Prussian blue reaction for BM iron stores. The specificity and sensitivity were found to be 77.8% and sensitivity 69.8%, respectively. However, the sensitivity increased up to 96.2% when Hb, RBC indices and ZPP were considered for the diagnosis of IDA.
|How to cite this article:|
Das S, Philip KJ. Evaluation of iron status: Zinc protoporphyrin vis-a-vis bone marrow iron stores.Indian J Pathol Microbiol 2008;51:105-107
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Das S, Philip KJ. Evaluation of iron status: Zinc protoporphyrin vis-a-vis bone marrow iron stores. Indian J Pathol Microbiol [serial online] 2008 [cited 2022 Dec 6 ];51:105-107
Available from: https://www.ijpmonline.org/text.asp?2008/51/1/105/40418
Heme is formed in the developing red cell by the insertion of iron into a pre-formed porphyrin ring. In the event of insufficient supply of iron or impaired iron utilization, zinc is substituted for iron into protoporphyrin IX as zinc protoporphyrin (ZPP).  This ZPP response occurs because iron and zinc interact as ferrochelatase substrates, with an increase in the utilization of zinc when the iron supply is diminished.  The resulting high zinc protoporphyrin/heme ratio (ZPP/H) in circulating erythrocytes reflects a state of relative iron-deficient erythropoiesis.  Thus, the level of ZPP in the red cell, is a functional indicator of iron utilization at the time of cell maturation. Zinc protoporphyrin can be measured in whole blood using a front-face fluorimeter. 
The evaluation of bone marrow (BM) iron is a sensitive and reliable means for the diagnosis of IDA even in its pre-latent phase.  Bone marrow iron within the macrophages is readily identified by Perls' Prussian blue reaction, in which ionic iron reacts with acid ferrocyanide to impart a blue color.  This small study attempts to find out the diagnostic utility of ZPP in the evaluation of iron status and comparing it with BM iron using the latter as the gold standard.
Materials and Methods
One hundred and seven cases were selected randomly over a period of two-and-a-half years, in whom BM aspiration was performed for PUO, pancytopenia or unexplained cytopenia, staging for NHL, suspicion of multiple myeloma, etc. Complete history and clinical features of every patient was noted at the time of admission. In each case, peripheral blood counts along with RBC indices, using Bayer's Auto blood cell counter Advia 120 were noted. Of these, 53 had reduced BM iron stores and 54 had normal BM iron stores.
Zinc protoporphyrin (ZPP) was performed in every case and the results were noted. The instrument used was the Protofluor-Z Hematofluorometer from Helena Laboratories. This is a front-face fluorimeter designed for the measurement of ZPP in whole blood. When blood sample is exposed to 415 nm light, ZPP is excited and emits light at 595 nm, and in 5 s, the value in µmol/mol heme is displayed. The normal range is 30-80 µmol/mol heme. Only one drop of anticoagulated blood is required for the test. Perls' Prussian blue reaction for iron was performed on every BM aspirate based on the method by Kass.  Next, the grading of iron stores was noted based on the criteria of Gale et al . 
All 107 bone marrows were divided into four groups based on BM iron stores and ZPP (1) "Normal," where BM iron stores were of normal score (2 + ) or increased score (>3 + ) and ZPP was normal: 30-80 µmol/mol heme; (2) Iron deficiency anemia (IDA), where BM iron stores were reduced: score zero and ZPP was increased (>80 µmol/mol heme); (3) Pre-latent Iron deficiency, where BM iron stores were reduced and ZPP was normal 30-80 µmol/mol heme; and (4) Anemia of chronic disorder (ACD), where BM iron stores were increased and ZPP also increased >80 µmol/mol heme.
The specificity, sensitivity, positive and negative predictive values of ZPP was determined using BM iron stores as the gold standard.
Of the 107 cases studied, Group (1) "Normal" where BM iron stores were normal score (2 + ) or increased score (>3 + ) and ZPP was normal: 30-80µmol/mol heme comprised 42 cases; Group (2) Iron deficiency anemia (IDA), where BM iron stores were reduced: score zero and ZPP was increased (>80 µmol/mol heme), comprised 37 cases; Group (3) Pre-latent iron deficiency, where BM iron stores were reduced and ZPP was normal (30-80 µmol/mol heme) comprised 16 cases; and Group (4) Anemia of chronic disorder (ACD), where BM iron stores were increased and ZPP also increased (>80 µmol/mol heme) comprised 12 cases.
Details of their peripheral blood counts, RBC indices and ZPP are tabulated in [Table 1]. In the IDA (Gr B) group, 13 patients had MCV >80 fl, and in 9, MCH >27 pg. This was possibly due to the association of megaloblastic anemia. Anemia was detected in 12 cases with pre-latent Iron deficiency (Group C) and 32 in Group A. This was probably due to the association of anemia of chronic disorder (ACD) such as multiple myeloma, lymphomas, CML, PUO and hypersplenism.
The comparison of ZPP with Iron stores is shown in [Figure 1]. Of the total 107 cases, 53 cases with reduced iron stores were detected, in which 16 (30.2%) were in the pre-latent phase of iron deficiency. Using ZPP as a single test for the detection of IDA in comparison with BM iron stores, which was taken as the gold standard, the specificity and sensitivity were found to be 77.8% 69.8%, respectively. However, in cases of microcytic hypochromic anemia (Hb ,,, However, the correlation decreases with the presence of inflammation or anemia of chronic disease.  In the present series, 12 cases (11.2%) with anemia of chronic disorder (ACD) were found, where ZPP was increased and BM iron stores were normal or increased. Similarly, in Group A and C, 32 and 12 cases were detected to have anemia of chronic disorder, where ZPP was normal. In Group C, 30.2% cases were detected to have normal ZPP and reduced iron stores. This is in agreement with the pre latent phase of iron deficiency. 
Bone marrow aspiration is an invasive procedure; hence, it is not feasible in every case of anemia unlike ZPP. The latter is a simple test that requires only a drop of blood. Hence, it is concluded that zinc protoporphyrin in conjunction with Hb and RBC indices are reliable in reflecting the BM iron status except in the pre-latent phase of iron deficiency.
We thank Dr. Rajesh Issac, Lecturer in Community Medicine, for expert help and guidance in the statistical correlation of the study. We also thank Mr. Avinash Singh, MSc, LT, for expert technical assistance.
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