Reference intervals for red cell variables and platelet counts in infants at 2, 5 and 13 months of age: a cohort study.

AIMS: To derive reference values for red cell variables and platelet counts from a cohort of infants sampled at precise ages during the first 13 months of life. METHODS: Blood counts, reticulocyte counts and zinc protoporphyrin concentrations were obtained from healthy term infants of North European ancestry at 2, 5 and 13 months of age. RESULTS: Mean cell volume (MCV) and mean cell haemoglobin (MCH) values did not differ significantly between 5 and 13 months and MCH concentration was unaffected by age. Values of all other variables at any one age differed significantly from those at the other two. Haemoglobin, mean cell haemoglobin, zinc protoporphyrin and platelet values (95% ranges) at 2 (n=119), 5 (n=97) and 13 months (n=42) were, respectively, 91-125, 101-129 and 105-133 g/L; 28.6-33.1, 24.5-28.7 and 24.3-28.7 pg; 36-116, 25-91 and 27-57 micromol/mol haem; and 216-658, 241-591 and 209-455×10(9)/L. At 2 and 5 months, respectively, 26.9% and 10.8% of subjects had platelet counts >500×10(9)/L. Reticulocyte counts at 2 months and MCV and MCH values at 5 months were significantly higher in girls. In boys, red cell distribution width values were significantly higher at 5 months, and zinc protoporphyrin values at both 2 and 5 months. CONCLUSIONS: These findings indicate the value of obtaining reference data at precise ages during infancy and confirm and extend earlier reports indicating a gender difference in laboratory measures used to assess iron status in early infancy.

Variable levels of carry over on platelet counts < or = 20 x 10(9)/l with the Bayer Advia 120.

Accurate platelet counts are essential for the safe management of severe thrombocytopenia (platelet counts < or = 20 x 10(9)/l). The effect of carry over on platelet counting in severe thrombocytopenia was investigated by performing counts before and after saline rinses on three Bayer Advia 120 automated blood counters. Counts were performed in both primary and manual closed tube system modes on two instruments and in manual open tube mode on a third. A total of 194 samples with platelet counts < or = 20 x 10(9)/l were studied. First counts were significantly higher in all groups. The magnitude of the difference varied both by analyser and counting mode. Carry over was minimal with one analyser in primary mode and second counts were on average only 5.5% lower; on a second analyser in manual closed tube system mode second counts were on average 37.7% lower. A first count of > or = 10 x 10(9)/l fell to <10 x 10(9)/l on the second count in 35 of 145 samples (24.1%). In five such samples, all tested on one analyser, the second count was <50% of the value of the first count. Two of 49 (4.1%) first counts of <10 x 10(9)/l increased to > or = 10 x 10(9)/l on repeat. These results show a variable and often potentially clinically important carry-over effect on severely thrombocytopenic samples using the Advia 120.

The clinical relevance of an isolated increase in the number of circulating hyperchromic red blood cells.

AIMS: To search for laboratory evidence of hereditary spherocytosis (HS) among apparently healthy children with the chance finding of an isolated increase in hyperchromic red cells (cells with intracellular haemoglobin concentration > 410 g/litre). METHODS: Blood and reticulocyte counts and Pink tests were performed on successive children found on routine counts to have > 4% hyperchromic red cells, and compared with age and mean cell haemoglobin concentration (MCHC) matched controls and children known to have HS. RESULTS: Thirty four patients with > 4% hyperchromic red cells had significantly higher absolute numbers of such cells (p < 0.0001) and higher reticulocyte counts (p < 0.01) than age matched controls, together with higher MCHC (p < 0.0001) and haemoglobin distribution width (p < 0.0001) values and lower mean cell volume (p < 0.02) values. Significant differences were also found among hyperchromic red blood cell, reticulocyte, and haemoglobin distribution width values when subjects were compared with MCHC matched controls. Pink test values were higher in children with increased hyperchromic red blood cells, but not significantly so. In patients with HS, most variables measured were significantly different both from those of children with > 4% hyperchromic cells and controls. Despite the differences found, few MCHC, HDW, reticulocyte, or Pink test values were outside of the normal limits, and only one child with increased hyperchromic cells had both a mild reticulocytosis and a slightly raised Pink test value. CONCLUSIONS: Subjects with an isolated increase in hyperchromic red blood cells have a profile of red blood cell changes similar to that of patients with HS, but to a lesser degree. They may carry a recessive form of the disease but lack the laboratory features of clinically manifest HS.

Total and differential leucocyte counts in infants at 2, 5 and 13 months of age.

Reference ranges for the total and differential leucocyte counts were determined from venous blood collected at 2, 5 and 13 months of age from a cohort of 112 healthy children of north European ancestry. At 2, 5 and 13 months, the ranges for neutrophils were found to be 0.7-4.7, 1.1-5.6 and 1.0-7.6 x 109/l, and for lymphocytes 3.3-10.5, 3.4-11.3 and 3.5-10.4 x 109/l, respectively. The upper limits for monocytes at each age were 1.2, 1.2 and 0.91 x 109/l, and for eosinophils 0.84, 1.0 and 0.88 x 109/l, respectively. Mean counts for all cell types, except monocytes, increased between 2 and 5 months of age. There was little change in mean counts between 5 and 13 months. Statistically significant correlations existed between the numbers of each cell type at 2 months of age, and were still present at 13 months between monocytes and each of the granulocyte series and between basophils and all other cell types. By comparison with older data these findings indicate a lower reference limit for neutrophils at 2 months of age, and a narrower range for this cell type at both 2 and 5 months of age. Reference ranges for lymphocytes and eosinophils are wider than indicated by some previous studies.

Measurement of haemoglobin using single drops of skin puncture blood: is precision acceptable?

The study aimed to investigate local concerns about clinically important discrepancies between repeat HemoCue haemoglobin measurements from single drops of blood. Two biomedical scientists and two health visitors each obtained a series of paired haemoglobin values by fingerprick sampling from healthy volunteers. Seven of 20 paired values obtained by health visitors and three of 20 obtained by scientists from the first drop of blood forming at the puncture site differed by > or = 10 g/l; 11 of 20 paired values obtained by health visitors and one of 20 by the scientists from the fourth drop of blood differed by > or = 10 g/l. After collecting and mixing a number of drops in EDTA tubes before analysis, seven of 40 paired values differed by > 5 g/l, and none by > 10 g/l. Pooling drops of blood before analysis improves precision of HemoCue haemoglobin measurement and allows users to achieve results comparable to those obtained by experienced laboratory staff. Measurement of haemoglobin from single drops of skin puncture blood should be discontinued.

Maintenance therapy with colloidal bismuth subcitrate reduces duodenal ulcer relapse.

AIM: To investigate the efficacy and safety of daily low-dose colloidal bismuth subcitrate in reducing duodenal ulcer relapse. DESIGN: Double-blind, double-dummy group comparative clinical trial with random allocation. Healing Phase: colloidal bismuth subcitrate 240 mg twice daily vs ranitidine 150 mg twice daily for up to 12 weeks. Maintenance Phase: nightly, colloidal bismuth subcitrate 120 mg vs ranitidine 150 mg vs placebo for up to 12 months (high-risk patients received active treatment only). ASSESSMENT: clinical, endoscopy, random blood bismuth levels (and rapid urease test for Helicobacter pylori in a subgroup). PATIENTS: 194 with active duodenal ulcer. OUTCOME: Cumulative healing at 12 weeks was 93% on colloidal bismuth subcitrate (of 92 patients) and 97% on ranitidine (of 102 patients). Relapse at 1 year was significantly less on active treatment as follows: placebo (50 patients) 60%; ranitidine (71 patients) 21%; colloidal bismuth subcitrate (64 patients) 33%. This was independent of the results of the rapid urease test which was positive in 78%, 88% and 76% of the patients respectively. Treatment was well tolerated. The highest median blood bismuth level (mcg/L) was 25 in the healing phase and fluctuated between 6 and 10 in the maintenance phase. CONCLUSIONS: Colloidal bismuth subcitrate, 120 mg nightly, is effective in reducing duodenal ulcer relapse and is well tolerated.

Mean cell haemoglobin concentration in subjects with haemoglobin C, D, E and S traits.

The Bayer H1 automated blood counter was used to assess the MCHC values of 40 nonanaemic patients with HbC trait, 21 with HbD trait, 23 with HbE trait and 69 with HbS trait. These were compared with values from controls with a normal Hb phenotype. Values were significantly higher in those with HbC, D and S traits and approached significance in those with HbE trait. In 45%, of subjects with HbC trait the MCHC value was > or = 35 g/dl. Such values may prove a useful marker for this abnormality. In a further 12 patients with HbC, D, E or S traits and coexisting iron deficiency anaemia, MCHC values were usually higher and the percentage of hypochromic cells (red cells with CHC <28 g/dl, directly measured by the H1) usually lower than values derived from controls with a normal Hb phenotype and iron deficiency anaemia of similar degree. In individuals with HbC, D, E or S traits, the MCHC and proportion of hypochromic cells are less sensitive indicators of iron lack than in subjects with a normal Hb phenotype.

Lymphocyte subset counts in skin puncture and venous blood compared.

To determine whether skin puncture blood can be used reliably for CD4 lymphocyte counts, the numbers of the major subsets of lymphocytes were assessed in paired venous and skin puncture blood samples from 22 children and 10 adults. Paired values were highly correlated, with skin puncture values being about 7% higher than venous values for each cell type. Differences were of borderline statistical significance for total lymphocytes and for each subset except CD3+ CD8+ T lymphocytes. Nevertheless, the magnitude of the differences was small and unlikely to be of clinical importance, and it seems that skin puncture samples may be preferable for CD4 counts in children or adults with difficult venous access.

Usefulness of red cell zinc protoporphyrin concentration in the investigation of microcytosis in children.

The clinical usefulness of the measurement of red cell zinc protoporphyrin (ZPP), an indicator of iron-deficient erythropoiesis, was assessed in a group of UK children undergoing investigation for red cell microcytosis. Of 213 children studied, 136 had increased ZPP values. Of these, 86 also had reduced iron stores as indicated by serum ferritin concentration. The 50 children with increased ZPP and normal ferritin values could be divided into two main groups. One group comprised 28 children who had evidence of coexistent infection or inflammatory disease. The other included 21 children who had beta-thalassemia trait (n = 19) or disease (n = 2). Among the 77 children with normal ZPP values, 22 had reduced serum ferritin concentrations and 45 did not, nor did they have evidence of beta-thalassemia. Microcytosis in some of these children could have been due to alpha-thalassemia trait. Measurement of ZPP is a simple, quick, and relatively cheap method of confirming the presence of iron-deficient erythropoiesis even when inflammation makes serum ferritin measurements unreliable. It is not as sensitive as the ferritin assay to the early stages of iron deficiency, and its specificity is reduced by the occurrence of raised values in most children with beta-thalassemia trait. Where there is microcytosis, normal values, together with normal hemoglobin A2 and serum ferritin concentrations, are likely to indicate alpha-thalassemia trait.

Frequency of coincident iron deficiency and beta-thalassaemia trait in British Asian children.

A study was carried out to determine the frequency of combined iron deficiency and beta-thalassaemia trait in a cohort of British Asian children to see whether the trait protects iron status. Of 470 consecutive children with red cell microcytosis, 77 had beta-thalassaemia trait and 26 (34%) of these also had evidence of iron deficiency. It was most common and profound in children under five years of age where the prevalence was 16 in 33 (48.5%). This suggests that iron deficiency is no less common in Asian children with beta-thalassaemia trait than in those without. It should not be presumed that the trait protects iron status or that the two are in any way mutually exclusive, at least in the early years.

Discriminant function using red cell indices to distinguish between HbC and HbE traits.

Values for MCV, MCH, MCHC and red cell distribution width (RDW) derived from subjects with HbC and HbE traits using a Technicon H*1 automated blood count analyser were compared. Significantly higher MCH, MCHC and RDW values were found in those with HbC trait. By use of a simple discriminant function (MCHC2 x RDW/1000), 19/20 subjects with HbC trait gave a value > or = 16.3, and 20/21 subjects with HbE trait gave a value < or = 16.2. The function described may be of value as an adjunct to electrophoresis in intralaboratory quality control.

Application of fluorescence in situ hybridisation to chromosome analysis of aged bone marrow smears.

AIMS: To evaluate the reliability of fluorescence in situ hybridisation (FISH) in the retrospective cytogenetic assessment of old bone marrow smears stored for periods of up to 20 years. METHODS: A series of bone marrow smears either Romanowsky stained, or frozen and unstained, and aged from one month to 20 years were hybridised with biotin labelled probes specific for the centromeric regions of human chromosomes X, 6, and 18. Sites of hybridisation were detected with fluoresceinated avidin. One hundred to 400 cells from each preparation were examined and the number of signals observed was recorded. RESULTS: All smears exhibited signals in most cells examined. In cytogenetically normal cases, an average 67.6% of cells (range 36%-90%) demonstrated the appropriate number of X centromere signals. In those samples known to contain extra chromosomes X, 6, or 18 the presence of cells with the abnormal copy number was clearly detected in each case. CONCLUSION: When applied in the way described, FISH can give consistent and accurate results with a variety of archival bone marrow smears, including aged prestained material. This will permit retrospective assessment of specific cytogenetic abnormalities in patients with leukaemia using their initial diagnostic slides even where these are several years old.

Errors in automated reticulocyte counts due to Heinz bodies.

Serial reticulocyte counts on a patient with an unstable haemoglobin haemolytic anaemia whose red cells contained Heinz bodies were obtained by two automated methods. One false low and several false high counts were obtained with standard threshold settings; each was accompanied by activation of the appropriate alarm. With the FACScan a bimodal red cell distribution was found, corresponding to the proportions of Heinz body positive and negative cells. This case illustrates the fact that Heinz bodies can interfere with automated reticulocyte counting methods, and to a degree that could be clinically important.