The frequency of detection of unexpected diabetes mellitus during haemoglobinopathy investigations

ABSTRACT Aims To establish the frequency of detection of previously undiagnosed diabetes mellitus as a result of detection of an increased glycated fraction of haemoglobin during high performance liquid chromatography (HPLC) for haemoglobinopathy diagnosis. Methods A prospective study was carried out over a 3-month period. During that period a total of 2094 patient samples were received for haemoglobinopathy investigation and were included in the study. Results Fifty samples were found to have an apparent increase in the glycated haemoglobin fraction and of these 38 were found to be from patients with known diabetes. Previously undiagnosed diabetes was discovered in 11 patients and it is likely that the twelfth patient also had diabetes. Conclusions The detection of evidence of undiagnosed diabetes during HPLC haemoglobinopathy investigations is not rare, there being four cases per month in this study. This incidental observation should be reported to clinical staff. A proportion of haemoglobin A undergoes posttranslational modification including glycosylation (non-enzymatic addition of glucose to the aminoterminal valine of the b chain) or glycation (less specifically, addition of carbohydrate to a protein). 1 Glycosylation occurs throughout the life-span of the red cell, at a rate determined by the ambient glucose concentration. Glycated haemoglobin is known as haemoglobin A 1 , of which 60e80% is glycosylated haemoglobin, haemoglobin A 1c . 1 In patients with diabetes mellitus, the proportion of haemoglobin that is haemoglobin A 1c has been found to be useful as an indication of the degree of hyperglycaemia during the preceding 3 months. In addition, since there are few other causes of an elevated haemoglobin A 1c , the finding of an elevated proportion can be useful in diagnosis. Glycated haemoglobin includes a labile fraction, which responds rapidly and transiently to raised blood glucose levels, and a stable fraction, to which the labile fraction is converted. 1 It is the stable fraction that is useful in judging long-term control of diabetes. Haemoglobin A 1c can be quantified by a variety of methods, of which high-performance liquid chromatography (HPLC) is increasingly used. When suspected haemoglobinopathies are investigated by cellulose acetate electrophoresis, the glycated fraction is not resolved and no specific abnormality is apparent in diabetic patients. However, when the technique used for such investigations is HPLC, the presence of a glycated fraction may be apparent. Our laboratory has previously drawn attention to the possibility that this may lead to the diagnosis of previously unsuspected diabetes mellitus. 2 Early diagnosis and good control of diabetes mellitus is important in reducing the end-organ damage that is characteristic of this disease, and it is therefore important for haematologists to alert clinical staff to the probability of this diagnosis when an increased proportion of glycated haemoglobin is observed. We therefore carried out a study to determine the frequency with which this is observed, and as a result of this study we developed a policy for notification of an increased glycated fraction to clinical staff. MATERIALS AND METHODS All tests were performed on peripheral blood samples anticoagulated with EDTA. HPLC for establishing relevant reference ranges and for haemoglobinopathy investigations was performed on a Bio-Rad Variant II instrument (Bio-Rad Laboratories, Hemel Hempstead, UK) using the b-Thalassaemia Short Program. Haemoglobin A 1c was quantified by HPLC using a Tosoh A1c 2.2 instrument. One hundred and two samples were obtained from fully informed young healthy volunteers of north European origin, in order to establish a reference range for haemoglobin A 2 . Data from the same samples were used to establish a reference range for peak 2 (P2), this being the peak with a retention time slightly longer than that of haemoglobin F, which we had previously noted to be increased in patients with an increased proportion of haemoglobin A 1c . The instruction manual of the Variant II states 'Diabetic specimens typically exhibit an elevated P2 peak'. In order to investigate the relationship between P2 on the Bio-Rad Variant II and haemoglobin A 1c on the Tosoh A1c 2.2, 93 samples from either healthy volunteers or patients with an elevated glycated fraction were studied in parallel on the two instruments. Results of all patients investigated by our haemoglobinopathy laboratory were surveyed over a 3-month period and when a sample was found to have a P2 fraction of 6% or greater, haemoglobin A 1c was measured and we investigated whether or not a diagnosis of diabetes mellitus had previously been established. If a review of request forms and laboratory records did not disclose this diagnosis, we contacted relevant clinical staff and requested review of clinical notes. If the patient had not been identified previously as suffering from diabetes mellitus, further tests were advised in order to confirm the diagnosis. Subsequently we followed-up the results of these confirmatory tests. Tests done were those usually carried out by the relevant clinical staff in order to confirm a diagnosis of diabetes

Diagnosis from the blood smear

A related slide show is available at www.nejm.org n examination of the blood smear (or film) may be requested by physicians or initiated by laboratory staff. With the development of sophisticated automated blood-cell analyzers, the proportion of blood-count samples that require a blood smear has steadily diminished and in many clinical settings is now 10 to 15 percent or less. Nevertheless, the blood smear remains a crucial diagnostic aid. The proportion of requests for a complete blood count that generate a blood smear is determined by local policies and sometimes by financial and regulatory as well as medical considerations. For maximal information to be derived from a blood smear, the examination should be performed by an experienced and skilled person, either a laboratory scientist or a medically qualified hematologist or pathologist. In Europe, only laboratory-trained staff members generally "read" a blood smear, whereas in the United States, physicians have often done this. Increasingly, regulatory controls limit the role of physicians who are not laboratory-certified. Nevertheless, it is important for physicians to know what pathologists or laboratory hematologists are looking for and should be looking for in a smear. In comparison with the procedure for an automated blood count, the examination of a blood smear is a labor-intensive and therefore relatively expensive investigation and must be used judiciously. A physician-initiated request for a blood smear is usually a response to perceived clinical features or to an abnormality shown in a previous complete blood count. A laboratory-initiated request for a blood smear is usually the result of an abnormality in the complete blood count or a response to "flags" produced by an automated instrument. Less often, it is a response to clinical details given with the request for a complete blood count when the physician has not specifically requested examination of a smear. For example, a laboratory might have a policy of always examining a blood smear if the clinical details indicate lymphadenopathy or splenomegaly. The International Society for Laboratory Hematology has published consensus criteria (available at www.islh.org) for the laboratory-initiated review of blood smears on the basis of the results of the automated blood count. The indications for smear review differ according to the age and sex of the patient, whether the request is an initial or a subsequent one, and whether there has been a clinically significant change from a previous validated result (referred to as a failed delta check). All laboratories should have a protocol for the examination of a laboratory-initiated blood smear, which can reasonably be based on the criteria of the International Society for Laboratory Hematology. Regulatory groups should permit the examination of a blood smear when such protocols indicate that it is necessary. There are numerous valid reasons for a clinician to request a blood smea

Erdheim‐Chester disease in bone marrow

No abstract available

Downloaded from www.nejm.org at UNIVERSITY OF WISCONSIN on

A related slide show is available at www.nejm.org n examination of the blood smear (or film) may be requested by physicians or initiated by laboratory staff. With the development of sophisticated automated blood-cell analyzers, the proportion of blood-count samples that require a blood smear has steadily diminished and in many clinical settings is now 10 to 15 percent or less. Nevertheless, the blood smear remains a crucial diagnostic aid. The proportion of requests for a complete blood count that generate a blood smear is determined by local policies and sometimes by financial and regulatory as well as medical considerations. For maximal information to be derived from a blood smear, the examination should be performed by an experienced and skilled person, either a laboratory scientist or a medically qualified hematologist or pathologist. In Europe, only laboratory-trained staff members generally "read" a blood smear, whereas in the United States, physicians have often done this. Increasingly, regulatory controls limit the role of physicians who are not laboratory-certified. Nevertheless, it is important for physicians to know what pathologists or laboratory hematologists are looking for and should be looking for in a smear. In comparison with the procedure for an automated blood count, the examination of a blood smear is a labor-intensive and therefore relatively expensive investigation and must be used judiciously. A physician-initiated request for a blood smear is usually a response to perceived clinical features or to an abnormality shown in a previous complete blood count. A laboratory-initiated request for a blood smear is usually the result of an abnormality in the complete blood count or a response to "flags" produced by an automated instrument. Less often, it is a response to clinical details given with the request for a complete blood count when the physician has not specifically requested examination of a smear. For example, a laboratory might have a policy of always examining a blood smear if the clinical details indicate lymphadenopathy or splenomegaly. The International Society for Laboratory Hematology has published consensus criteria (available at www.islh.org) for the laboratory-initiated review of blood smears on the basis of the results of the automated blood count. The indications for smear review differ according to the age and sex of the patient, whether the request is an initial or a subsequent one, and whether there has been a clinically significant change from a previous validated result (referred to as a failed delta check). All laboratories should have a protocol for the examination of a laboratory-initiated blood smear, which can reasonably be based on the criteria of the International Society for Laboratory Hematology. Regulatory groups should permit the examination of a blood smear when such protocols indicate that it is necessary. There are numerous valid reasons for a clinician to request a blood smea

Lower respiratory tract myeloid cells harbor SARS-CoV-2 and display an inflammatory phenotype

SARS-CoV-2 pneumonia may induce an aberrant immune response with brisk recruitment of myeloid cells into the airspaces. Although the clinical implications are unclear, others have suggested that infiltrating myeloid cells may contribute to morbidity and mortality during SARS-CoV-2 infection.1–3 However, few reports have characterized myeloid cells from the lower respiratory tract, which appears to be the primary site of viral-induced pathology, during severe SARS-CoV-2 pneumonia

Inhibitor of DNA Binding 3 Limits Development of Murine Slam-Associated Adaptor Protein-Dependent “Innate” γδ T cells

Id3 is a dominant antagonist of E protein transcription factor activity that is induced by signals emanating from the alphabeta and gammadelta T cell receptor (TCR). Mice lacking Id3 were previously shown to have subtle defects in positive and negative selection of TCRalphabeta+ T lymphocytes. More recently, Id3(-/-) mice on a C57BL/6 background were shown to have a dramatic expansion of gammadelta T cells.Here we report that mice lacking Id3 have reduced thymocyte numbers but increased production of gammadelta T cells that express a Vgamma1.1+Vdelta6.3+ receptor with restricted junctional diversity. These Vgamma1.1+Vdelta6.3+ T cells have multiple characteristics associated with "innate" lymphocytes such as natural killer T (NKT) cells including an activated phenotype, expression of the transcription factor PLZF, and rapid production of IFNg and interleukin-4. Moreover, like other "innate" lymphocyte populations, development of Id3(-/-) Vgamma1.1+Vdelta6.3+ T cells requires the signaling adapter protein SAP.Our data provide novel insight into the requirements for development of Vgamma1.1+Vdelta6.3+ T cells and indicate a role for Id3 in repressing the response of "innate" gammadelta T cells to SAP-mediated expansion or survival

Intraspecific Inversions Pose a Challenge for the trnH-psbA Plant DNA Barcode

BACKGROUND: The chloroplast trnH-psbA spacer region has been proposed as a prime candidate for use in DNA barcoding of plants because of its high substitution rate. However, frequent inversions associated with palindromic sequences within this region have been found in multiple lineages of Angiosperms and may complicate its use as a barcode, especially if they occur within species. METHODOLOGY/PRINCIPAL FINDINGS: Here, we evaluate the implications of intraspecific inversions in the trnH-psbA region for DNA barcoding efforts. We report polymorphic inversions within six species of Gentianaceae, all narrowly circumscribed morphologically: Gentiana algida, Gentiana fremontii, Gentianopsis crinita, Gentianopsis thermalis, Gentianopsis macrantha and Frasera speciosa. We analyze these sequences together with those from 15 other species of Gentianaceae and show that typical simple methods of sequence alignment can lead to misassignment of conspecifics and incorrect assessment of relationships. CONCLUSIONS/SIGNIFICANCE: Frequent inversions in the trnH-psbA region, if not recognized and aligned appropriately, may lead to large overestimates of the number of substitution events separating closely related lineages and to uniting more distantly related taxa that share the same form of the inversion. Thus, alignment of the trnH-psbA spacer region will need careful attention if it is used as a marker for DNA barcoding

Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/β-Catenin Signaling Pathway

While investigating the role of p38 MAPK in regulating melanogenesis, we found that pyridinyl imidazole inhibitors class compounds as well as the analog compound SB202474, which does not inhibit p38 MAPK, suppressed both α-MSH-induced melanogenesis and spontaneous melanin synthesis. In this study, we demonstrated that the inhibitory activity of the pyridinyl imidazoles correlates with inhibition of the canonical Wnt/β-catenin pathway activity. Imidazole-treated cells showed a reduction in the level of Tcf/Lef target genes involved in the β-catenin signaling network, including ubiquitous genes such as Axin2, Lef1, and Wisp1 as well as cell lineage-restricted genes such as microphthalmia-associated transcription factor and dopachrome tautomerase. Although over-expression of the Wnt signaling pathway effector β-catenin slightly restored the melanogenic program, the lack of complete reversion suggested that the imidazoles interfered with β-catenin-dependent transcriptional activity rather than with β-catenin expression. Accordingly, we did not observe any significant change in β-catenin protein expression. The independence of p38 MAPK activity from the repression of Wnt/β-catenin signaling pathway was confirmed by small interfering RNA knockdown of p38 MAPK expression, which by contrast, stimulated β-catenin-driven gene expression. Our data demonstrate that the small molecule pyridinyl imidazoles possess two distinct and opposite mechanisms that modulate β-catenin dependent transcription: a p38 inhibition-dependent effect that stimulates the Wnt pathway by increasing β-catenin protein expression and an off-target mechanism that inhibits the pathway by repressing β-catenin protein functionality. The p38-independent effect seems to be dominant and, at least in B16-F0 cells, results in a strong block of the Wnt/β-catenin signaling pathway