Microcytic Red Cell Disorders in British Asian Children

Laboratory Investigation of Microcytic Red Cell Disorders in British Asian Children

Rod Hinchliffe
Biographical Information

This is an excerpt from Bloodline Reviews, Volume 1, Issue 2-R, 2001, Diagnostic Advances in Hematology.

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Iron deficiency is widespread in British Asian children. Both α and ß thalassemia are found in this group, and complicate the investigation of iron deficiency. This study was performed to compare laboratory methods for the detection of iron deficiency in this group of children. This investigation of microcytic disorders in young British Asian children suggests that scrutiny of the blood count, together with the percentage of both microcytic and hypochromic red cells, be used as the basis for further investigation.

The α-thalassemias are carried by about 10% of the population, and ß thalassemias are present in 3-5%. The overwhelming cause of microcytosis is iron lack, which is remarkably common according to several studies in the United Kingdom, which have shown an incidence of 25-50% in Asian children from eight to nine months of age through about ages 3-3.5 years. This iron lack is dietary in origin and is thought to be based, quite simply, on the fact that traditional practices of feeding children are continued. Factors that may contribute to the high incidence of iron lack in this population include prolonged breast-feeding, prolonged feeding with cow's milk, which contains very little absorbable iron, and perhaps also an inadequate amount of iron in the solid foods that these children receive.

Our particular study of microcytic red cell disorders in British Asian children at Sheffield Children's Hospital involved 227 children aged 4-43 months who were tested using the Technicon H1 analyzer. The study was conducted to compare laboratory methods for the detection of iron deficiency in this population. Reference ranges were based upon in-house data and published values from the literature, and were age-related.

The following tests were performed on all 227 children: automated FBC, assays of zinc protoporphyrin (ZPP), plasma ferritin, plasma iron, iron binding capacity, percent saturation of transferrin (% sat), and plasma soluble transferrin receptor (sTfR). Hemoglobin electrophoresis was performed when MCV was less than 70 fL (to detect HbE) and HbA2 was measured when MCV was less than 65 fL (to detect ß thalassemia trait). The α3.7 and α4.2 DNA deletions, which cause the common deletional forms of α thalassemia, were sought by polymerase chain reaction, and C-reactive protein (CRP) was measured as a marker of inflammation.

Based on laboratory results, the children were grouped into eight categories:

Group 1: Iron-Replete (all hematological and biochemical data within normal limits; N=32)
Group 2: Depleted Iron Stores (reduced plasma ferritin as the only abnormality; N=7)
Group 3: Iron-Deficient Erythropoiesis (normal Hb with hematological and/or biochemical evidence of iron lack; N=86)
Group 4: Iron Deficiency Anemia (anemia with hematological and/or biochemical evidence of iron lack; N=46)
Group 5: α+ Thalassemia Trait (presence of α3.7 or α4.2 mutations; in three cases the presence of reduced MCH in the presence of normal Hb and biochemical measures of iron status; N=22)
Group 6: α° Thalassemia Trait (homozygosity/double heterozygosity for α3.7 and/or α4.2 mutations; in 4 children the combination of normal/near-normal Hb, RBC >5.7 x 10 12 /L, and biochemical measures out of keeping with reduction in MCH, together with normal HbA 2 level, was used to place them in this group; N=9)
Group 7: ß Thalassemia Trait (HbA 2 α3.6%; included one carrier of HbE and one with HbE disease; N=20)
Group 8: Anemia of Chronic Disease (hypochromic anemia with normal levels of soluble transferrin receptor and raised CRP values; N=5)

The iron-depleted group (Group 2) was a small one, with ferritin levels of less than 10 µg/L in the presence of normal hematology and normal measures of iron status. The mean transferrin receptor value in this group was about 12% higher than in the normal group (Group 1), and transferrin saturation was about 15% lower. These data suggest that this group of children had, for all intents and purposes, no iron stores left in their bone marrow - just enough iron to maintain normal red cell production.

The largest group by far was the group with iron-deficient erythropoiesis (Group 3; N=86). One message here is that in any population where iron deficiency is common, just measuring hemoglobin is not enough. Just measuring hemoglobin in this study would have suggested that all the children in this group were normal, when in fact, all of them had some degree of iron deficiency. Within the group as a whole, there was a spectrum of iron deficiency with children who had just one or two trivial abnormalities of iron status to the other extreme where a number of children had very clear evidence of iron deficiency, but whose hemoglobins were just holding within normal limits. It was noted that in some children, it was possible to see ferritin levels down to 2 µg/L and still have normal hemoglobin.

Members of group four were children with iron-deficiency anemia (N=46). Again, within this group there was a spectrum of iron deficiency with some children having one or two trivial abnormalities of iron status and a hemoglobin that was barely subnormal, to children with hemoglobin values as low as 4 g/dL.

Group five contained those with definite or probable α+ thalassemia. In this group it was interesting that all the children had at least slight increases in the percentage of hypochromic red cells. It had not been expected that something as mild as a single α gene deletion would cause hypochromia, but it was a consistent finding, suggesting that one use of percent of hypochromic cells might be to detect α thalassemias.

Group six members were those with a α° thalassemia trait. In this group were four children who were, in fact, double heterozygous, with both α3.7 and α4.2 mutations. One child was homozygous with an α3.7 mutation. Also, there were two children in this group with mild anemia. It is worth noting that there was a large spread among children in this group in terms of results for percent of hypochromic cells, zinc protoporphyrin, and transferrin receptor. This may indicate that as we move to a more abnormal state of erythropoiesis we get large scatter of results, and that while we can detect fairly mild iron deficient erythropoiesis using some or all of the panel of normal tests, it becomes very difficult or impossible to use these values to say with any certainty at all that a given child does or does not have a mild degree of iron deficiency, because we do not know what the upper or lower limits of normal are in these children. What seems to be of special value in this group are the ferritin measures, because those results made it clear that although a child had, for example, a hemoglobin of 13 g/dL, there was impending iron deficiency.

The same message of impending iron deficiency comes across in children in Group 7 - those with ß thalassemia trait. In this Asian population, ß thalassemia trait is usually of the ß° form, and a great majority of these children, not just the ones involved in this study, but in studies of this population over many years, are mildly anemic. Hemoglobin values above 11 g/dL are quite rare and are probably only found in about 5% of the population at most. Looking at some of the other variables studied, it was found that zinc protoporphyrin was elevated in a great majority of children in this group, as were transferrin receptors. Seven of these 20 children had co-existing iron deficiency, as defined by ferritin levels.

Only five children fell into Group 8 - those with anemia of chronic disease, which is actually quite rare for children in the Western world. The key test in this group was the transferrin receptor, as most of these children were anemic, had a low MCH, increased hypochromia, increased ZPP, and ferritin within normal limits. These data suggest that microcytic anemia in these children was due more to chronic inflammatory disease and failure of iron release from the reticuloendothelial system, than to iron deficiency itself.

As the statistical analyses of this study have not yet been completed, it may be helpful to look, for now, a little more in depth at the largest group - those with iron deficient erythropoiesis. (Fig. 1)

Figure 1: Sensitivity of laboratory variables in the Group with iron-deficient erythropoiesis:

MCH Hypochromic RBC ZPP Ferritin Soluble transferrin receptor % sat

reduced in 39.5% increased in 67.1% increased in 55.8% reduced in 46.5% increased in 50.5% reduced in 46.3%

MCH was the least sensitive variable in this group, which may be due to the threshold being set too low.

Percent hypochromic red cells was the strongest variable, but it should be noted that this number may be inflated by a small number of subjects with non-deletional α+ thalassemia and normal MCH and iron status. This percent hypochromic red blood cells variable is also of particular interest because there are a number of advantages to using it: it is immediately available with a blood count at no extra cost, it is precise, and the same reference limit can be used at all ages. In this study a percent hypochromic RBC level of 2.5% was used, with anything above that point considered to be indicative of iron-deficient erythropoiesis. Percent hypochromic RBC is not affected by acute inflammatory illness because these are red cells circulating in the blood and hypochromia is not going to increase within a matter of hours or a few days because of infection. It can be used along with percent microcytic cells as a discriminant function to help differentiate iron-lack from ß thalassemia and α° thalassemia traits.

There are also some disadvantages to the use of percent hypochromic RBC, namely that it is spuriously raised in grossly over-anticoagulated samples, some aged samples, and in some subjects on intravenous fluids; it is raised in some subjects with α+ thalassemia and in virtually all subjects with α° and ß thalassemia traits; and it is less sensitive to iron lack in subjects with higher MCHC values.

Looking at both the advantages and disadvantages of percent hypochromic RBC testing in this population, we can see that because this variable is increased in most thalassemic traits, it is valuable in detecting thalassemias, but it also makes the measure nonspecific because it is not specific for iron deficiency. It does seem clear, however, that this measure is specific for defects in hemoglobin formation.

In conclusion, we come back to the point of this study: an approach to the laboratory investigation of the measures of iron status. Of course a blood count is necessary, and it has been shown that percent microcytic cells and percent hypochromic red blood cells are very important. If the facilities are available, by all means look for CHr. Examine the blood film, because there is often evidence of conditions such as hemoglobin E disease, for example marked numbers of target cells. Transferrin receptor measures have been found to be useful in assessing children with microcytic anemia secondary to infection, and finally, ferritin assays are important in subjects with α° and ß thalassemia traits because of the uncertainty of the cut-offs of the other variables in those disorders.

It bears repeating that reference ranges should be selected carefully, and that awareness of the causes of microcytosis and hypochromia in your population should be maintained. This enables the development of knowledge about the typical changes brought about by thalassaemia and iron deficiency in the local population, thus making it much easier to detect the atypical cases, such as instances of double heterozygosity.

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