H. Robin

A new hybrid haemoglobin: haemoglobin Strumica/Beograd occurring in an individual with four haemoglobins.

Summary An investigation of the cord blood from full term twin infants revealed an additional haemoglobin F component due to an abnormal alpha chain. The father of the twins, whose blood picture was normal, was shown to have normal alpha and beta polypeptide chains together with variant alpha and beta polypeptide chains. Electrophoresis showed that he had four major haemoglobin components. Separation of the haemoglobin fractions by column chromatography, globin preparation, chain separation, tryptic and chymotryptic digestion and peptide map preparation led to the identification of haemoglobin A, haemoglobin Strumica (α2112 His ← Argβ2), haemoglobin D Beograd (α2β2121 Glu ← Val) and a hybrid haemoglobin Strumica D/Beograd (α2112 His ← Arg β2121 Glu ← Val)

Haemoglobin camperdown β104(G6) arginine → serine

Abstract Routine investigation of ante natal patients revealed a subtle change in the electrophoretic pattern on cellulose acetate of the proposita. Further investigations by isoelectric focussing in polyacrylamide gel suggested the presence of two major haemoglobin components. Using a modified cellulose acetate technique globin chain separation revealed an abnormal β-chain. Chain separation on a carboxymethylcellulose column provided a pure sample of the abnormal β-chain. After aminoethylation, tryptic digestion and peptide mapping, amino acid analysis of relevant peptides showed the abnormality in the β-chain to be a substitution of arginine by serine at the 104 position. The presence of a positively charged residue at this position would appear to be necessary for the stabilisation of the haemoglobin central cavity. The replacement by serine in this haemoglobin leads to slightly decreased stability but does not appear to affect the oxygen affinity.

Folate assays--an alternative to microbiological assays and commercial kits.

Summary A simple and reliable folate radioassay technique based on the principles of classic folate radioassays but incorporating several of the recently introduced technical innovations is presented as an alternative to the microbiological assays and commercial kits. Serum and red cell folate values from this method were compared with results from two commercial kits and a Lactobacillus casei (L. casei) assay. There was a good correlation with the L. casei assay for both serum and red cell folate (r = 0.95). the method uses N‐methyltetra‐hydrofolate standards diluted in folate free serum, bT‐lactoglobulin as a binder and an alkaline denaturation (‘no‐boil’) of endogenous folate binding proteins. Low hematocrit lysates were best performed at a 1 in 10 rather than 1 in 20 dilution. A comparison of the boil and ‘no‐boil’ kits from Diagnostic Products Corporation revealed that the ‘no‐boil’ kit produced results closer to those obtained from the L. casei assay than the boil kit.

Automated enumeration of reticulocytes using acridine orange.

&NA; Manual methods of counting reticulocytes using supravital stains, such as new methylene blue, have long been recognized to be subject to technical errors. Automated reticulocyte enumeration has recently become available with the development of an automated cell flow‐cytometer, the Ortho Spectrum III. In this method a fluorescent dye, acridine orange, which stains RNA in a manner similar to supravital stains, is used to distinguish reticulocytes from mature erythrocytes. We have evaluated this technique and found that it compares favourably with manual counting methods.

Immunoradiometric and electroimmuno assay of increased ferritin and apoferritin levels in serum.

Ferritin in serum from patients with increased serum ferritin levels has been studied both quantitatively and qualitatively. All techniques utilized in these studies are suitable to be used as routine screening tests for large numbers of patients. Electroimmuno assay (EIA) has been compared with the solid phase immunoradiometric (IRMA) assay as a technique to determine serum ferritin concentration (r = 0.99) and is suggested as a useful alternative when determining ferritin concentrations above 500 microgram/l. Iron stained EIA gels have been used to indicate the iron content of the ferritin molecule in sera. This simple screening test has demonstrated that apoferritin is found more often than iron-rich ferritin in the serum of patients with elevated serum ferritin levels. Immunoelectrophoresis precipitin bands suggest the heterogeneity of ferritin in serum from different patients.

EVALUATION OF THE TECHNICON H-1 HEMATOLOGY ANALYSER

&NA; The Technicon H‐1 is a new, random‐access hematology instrument performing a full blood count with leukocyte differential including eosinophils and basophils. Technical assessment showed good linearity and precision. Comparison with a Coulter S‐PLUS (II) showed close correlation for all full blood count parameters except MPV and MCHC on 149 unselected hospital inpatients over a wide clinical range. No significant carry‐over was detected in hemoglobin, red cell, white cell and platelet estimations. Differentials agreed closely with Technicon H6000 results for neutrophils, lymphocytes, monocytes and eosinophils. Poorer correlation with 100‐cell manual differentials for all cell types except neutrophils probably reflects the relative lack of precision in manual methods. Technological innovations on the H‐1 include a laser‐based optical system from which several new hematological parameters are derived. The contribution they make towards improved patient care awaits assessment.

A study of the true competitive protein binding of current vitamin B12 radioassays

&NA; The use of purified intrinsic factor in vitamin B12 radioassays has greatly reduced the misdiagnosis of pernicious anaemia in recent years, but anomalies still occur. Observations made on current vitamin B12 radioassays suggest that many methods fail to convert all serum cobalamins to cyanocobalamin and thus are not true competitive protein binding assays. No KCN in the extraction buffer resulted in an average 30% reduction in the vitamin B12 level in normal sera. High concentrations of KCN caused a significant increase in the vitamin B12 level in serum from most vitamin B12 deficient patients. This increase could have caused a misdiagnosis in 7 of 12 patients studied. The specificity of (57Co) cyano‐cobalamin binding to both transcobalamin II (Tcii) in a pool of normal sera and to intrinsic factor in a commercial kit binder reagent was 99%. We conclude that the KCN concentration in the extraction mixture should be between 2 and 5 mg/l.