Conditions associated with extreme hyperferritinaemia (>3000 µg/L) in adults.

BACKGROUND: The clinical significance of extreme hyperferritinaemia has come under scrutiny with the increasing recognition of haemophagocytic lymphohistiocytosis (HLH) in adults. Most studies of hyperferritinaemia have focused on serum ferritin greater than 1000 µg/L, often in ambulatory patients. The conditions associated with more extreme hyperferritinaemia are poorly understood. AIMS: To examine conditions associated with extreme hyperferritinaemia greater than 3000 µg/L in acutely ill adults at a quaternary care hospital. METHODS: Patients with serum ferritin greater than 3000 µg/L at Vancouver General Hospital between 1 August 2011 and 1 August 2012 were identified. Those over 18 years of age and with clinical data available were included in the study. RESULTS: Eighty-three subjects were identified. Twenty-one cases (25%) were due to transfusional iron overload, 16 (19%) due to liver disease and 15 (18%) due to mixed factors. Haemophagocytic lymphohistiocytosis (HLH) was diagnosed in six of 83 patients (7%) with ferritin greater than 3000 µg/L, but six of eight patients (75%) with ferritin greater than 20 000 µg/L. CONCLUSIONS: Extreme hyperferritinaemia greater than 3000 µg/L is uncommon in adult patients. The highest serum ferritin values are seen in HLH, but the differential diagnosis for serum ferritin greater than 3000 µg/L remains broad with iron overload and liver disease being the most common causes.

Screening tests for porphobilinogen are insensitive. The problem and its solution.

Standard screening tests for porphobilinogen (PBG) do not turn positive until the concentration of PBG exceeds 10-20 times the upper limit of normal. The authors have developed a screening procedure that uses a new ion-exchange resin to separate PBG from interfering substances in urine. On addition of Ehrlich's reagent, the color is more intense than that produced in the Watson-Schwartz test, and its spectrum more closely resembles that of pure PBG. By measuring the absorbance of this solution at 555 nm, it is possible to discriminate between urine samples with 9 mumol/L (2 mg/L) of PBG and those with no detectable PBG. The new screening test had positive results in two patients with latent acute intermittent porphyria; the Watson-Schwartz test had negative results in both cases. This procedure is easy to perform, has much greater sensitivity than the Watson-Schwartz test, and uses objective spectrophotometric data to separate positive from negative results.

Triiodothyronine--binding immunoglobulin in a euthyroid patient.

We describe a clinically euthyroid 60-year old woman with a past history of 1311 therapy for treatment of possible toxic nodular goitre. She had an elevated thyroxine level of 188 microgram/L (normal range 50-125 microgram/L) following her therapy, and her TSH was within normal limits at 4.7 mU/L. However her T3 level, as determined by an RIA technique employing charcoal to separate bound and free T3, was not measurable. T3 added to the patient's serum could not be recovered, therefore the presence of an unusual protein capable of binding T3 was suspected. To avoid this interference, T3 analysis was performed on an ethanol extract of the patient's serum and was found to be 17 microgram/L (normal range 0.8-1.8 microgram/L). At this time her thyroid microsomal antibody titre was 1:100,000. A protein, capable of binding more than 70% of the patient's T3, was demonstrated in the gamma globulin fraction by agarose gel electrophoresis. This protein did not bind T4. Scatchard analysis for T3 binding revealed a protein, presumably IGG, with a binding affinity of 2 x 109 L/mole and binding capacity of 50 microgram/L. This case exemplifies the caution that must be taken in interpreting thyroid function tests. When thyroid hormone levels are inappropriate to the clinical status of the patient the presence of circulating antibodies which can bind the thyroid hormones should be considered.

Antibody interference with biochemical tests and its clinical significance.

Antibody interference with routine biochemical tests is becoming increasingly recognized as a cause of spurious results. It is important to recognize these interferences because inappropriate investigation may be instituted in response to the abnormal test result. The present review deals with the various biochemical tests in which antibody interference occurs. The tests include measurement of enzymes, hormones, immunoglobulins, vitamin B12, and a variety of other molecules and ions.

Digoxin-like immunoreactivity, displacement of ouabain and inhibition of Na+/K+ ATPase by four steroids known to be increased in essential hypertension.

An endogenous digoxin-like immunoreactive substance(s) (DLIS, "endoxin") may be of significance in the etiology of essential hypertension (EH). Progesterone, dehydroepiandrosterone sulphate (DHEA-S), 11-deoxycortisol and 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), four steroids known to be increased in essential hypertension, were found to have digoxin-like immunoreactivity at levels 1,000 times higher than physiological concentrations. Of these steroids, progesterone and 18-OH-DOC were the most efficient in displacing 3H-ouabain from canine kidney Na+/K+ ATPase whereas progesterone and 11-deoxycortisol were the most potent inhibitors of this enzyme's activity. Although 18-OH-DOC and DHEA-S cross-reacted with digoxin-specific antibodies, their ability to inhibit Na+/K+ ATPase activity was minimal. Although it is concluded that these steroids may contribute to DLIS as isolated from hypertensive patients, it is unlikely that they would be of physiological significance in the etiology of EH unless they were to accumulate and act synergistically within vascular wall smooth muscle tissues.

Low concentration galactose determination in plasma adapted to the Cobas-Bio.

Galactose elimination at blood concentrations lower than 2.22 mmol/L has been advocated as a measure of functional liver blood flow. We have adapted an assay employing galactose dehydrogenase (EC 1.1.1.48) to the Cobas-Bio to measure low galactose concentrations in plasma. The collection of blood in sodium fluoride/potassium oxalate anticoagulant tubes eliminated the necessity for the plasma deproteinization step required in similar, manual methods. The between run CV's for plasma samples spiked with galactose to concentrations of 0.13-0.5 mmol/L were 3.6% and 3.2%, respectively. Our automated assay was more precise and had a greater range of linearity than a manual galactose oxidase (EC 1.1.3.9) method set up in our laboratory (0.04-1.10 mmol/L as compared to 0.06-0.56 mmol/L). The total assay time was 20 min.

Artefactual decrease in total protein concentration in patients with monoclonal gammopathies: a method-dependent error.

We found total protein estimates in patients with monoclonal gammopathies to be erroneously low when using the Cobas-Bio centrifugal analyzer. This problem occurred only when a serum-water blank was used. This probably results from the precipitation of these proteins under conditions of low ionic strength resulting in high blank readings. The problem can be avoided if a serum-saline blank is used.

Beta 2-microglobulin levels in cerebrospinal fluid of children with leukemia and lymphoma.

Beta 2-microglobulin levels were determined in the cerebrospinal fluid (CSF) of 119 children (ages 1 1/2 to 18 years) with malignant conditions; 87 with acute lymphoblastic leukemia, 9 with acute myeloblastic leukemia, 15 with lymphoma, and 8 with solid tumours. A total of 491 CSF specimens and 202 serum specimens were analyzed over a 12-month period. The mean CSF beta 2-microglobulin and serum beta 2-microglobulin were 1.11 +/- 0.58 mg/L and 1.5 +/- 0.64 mg/L respectively and were not different from the mean CSF (1.20 +/- 0.45 mg/L) and serum beta 2-microglobulin levels (1.70 +/- 0.45 mg/L) found in control patients. Meningeal leukemia was diagnosed on the basis of cytology in 7 patients. No elevation of CSF beta 2-microglobulin was found in any specimen at the time of CNS disease. Eleven other patients showed a transient rise in CSF beta 2-microglobulin above the reference range (greater than 2.1 mg/L). No evidence of CNS involvement was found in any of these patients. Five of these patients had received a combination of intrathecal methotrexate and irradiation therapy within the previous 4 months. A transient rise in CSF beta 2-microglobulin (2-3-fold increase over baseline CSF levels), which did not exceed the upper limit of the reference range was seen in 5 of 7 other children receiving the above therapy. Our study fails to demonstrate the usefulness of CSF beta 2-microglobulin for the diagnosis of CNS metastases but suggests that a transient elevation of CSF beta 2-microglobulin may occur after intrathecal methotrexate and irradiation therapy.

Minimizing analytical interferences from digoxin-like immunoreactive substances (DLIS) in cases of digoxin toxicity.

Recently, the value of therapeutic drug monitoring for digoxin has been called into question by the finding of endogenous digoxin-like immunoreactive substances (DLIS) in the serum of individuals, especially premature and full-term neonates, not being treated with digoxin. In some cases, values have been as high as 10 micrograms/L. Levels as high as 20 micrograms/L and 80 micrograms/g can be found in bile and meconium. Because of the magnitude of this interference, it is essential that methods be developed for measuring digoxin in the presence of DLIS. This is particularly important when such analyses are required in forensic science cases of suspected digoxin toxicity. This report outlines the high performance liquid chromatographic (HPLC) and radioimmunoassay (RIA) methods that we used in assessing the relative contribution made by digoxin, its metabolites, and DLIS to serum and tissue digoxin concentrations obtained by RIA in a forensic pediatric case of suspected digoxin toxicity.

A possible alternate pathway of bacteriochlorophyll biosynthesis in a mutant of Rhodopseudomonas sphaeroides.

A previously uncharacterized bacteriochlorophyll-less mutant (mutant 8) of Rhodopseudomonas sphaeroides has been found to excrete a tetrapyrrole-protein complex into the incubation medium. The structure of the major pigment of the complex was characterized as 2-desacetyl-2-vinylbacteriopheophorbide. The corresponding magnesium derivative does not fit into the currently proposed biosynthetic pathway for bacteriochlorophyll, and thus may indicate the existence of an alternate pathway of bacteriochlorophyll synthesis in this organism. Such an alternate pathway would be possible if reduction from the chlorin to the tetrahydroporphyrin stage can occur either before or after hydration of the 2-vinyl substituent of chlorophyllide a to an alpha-hydroxyethyl group.

Falsely negative laboratory diagnosis for myocardial infarction owing to the concurrent presence of macro creatine kinase and macro lactate dehydrogenase.

Macro creatine kinase (CK, EC 2.7.3.2) and macro lactate dehydrogenase (LD, EC 1.1.1.27) were both present in the serum of a 70-year-old woman with myocardial infarction. This interfered with the interpretation of the CK and LD isoenzyme analyses. Gel filtration and immunoprecipitation showed that the macro CK consisted of IgG and CK and the macro LD of IgG and LD. The IgG in this patient bound both MB and BB isoenzymes of CK, resulting in a macro CK complex that co-migrated with CK-MM on cellulose acetate electrophoresis. This situation led to a falsely negative laboratory diagnosis for myocardial infarction.

Glycosylated hemoglobins: a review.

The glycosylated hemoglobins are becoming accepted as a measure of longterm glycemic control in diabetes mellitus. The present review discusses the biochemistry and clinical studies in diabetes which relate glycosylated hemoglobin to screening, control and development of longterm complications. Other causes of increased glycosylated hemoglobin are also discussed.

Quantitative fluorometric screening test for fecal porphyrins.

We describe a fluorometric method for screening and quantifying porphyrins in stool. A small sample of stool is extracted with concentrated HCI and is diluted 200-fold in 3 mol/L HCI before analysis. An excitation scan is done from 350 to 450 nm, monitoring emission at 603 nm. Total porphyrin is estimated at the isosbestic point for coproporphyrin and protoporphyrin (402.5 nm). Monitoring emission at 603 nm eliminates interference from chlorophyll, obviating the need for extraction with ether. The position of the excitation peak gives some indication of the nature of the porphyrins in the stool. The acid extract can be injected directly into an HPLC system for fractionation studies. Our method correlates well with the spectrophotometric method developed by Lockwood et al. (Clin Chem 1985;31:1163-7). However, in our method, the sample is easier to process and the assay has higher sensitivity than their assay. The reference interval for porphyrin in healthy individuals by the fluorometric method is less than 300 nmol/g dry weight. We can detect as little as 1 nmol of porphyrin per gram (dry weight) of stool. Results of the method vary linearly with stool porphyrin concentrations as great as 4000 nmol/g dry weight. The within-run imprecision of the method is 3%.

Decreased anion gap associated with hypoalbuminemia and polyclonal gammopathy.

Anion gaps were determined in 82 patients with hypoalbuminemia; 24 of these patients had a polyclonal increase in gamma-globulin levels (designated group 5). The 58 patients without the polyclonal increase in gamma-globulin levels were subdivided according to the origin of their serum albumin loss as follows: group 1, renal group 2, gastrointestinal; group 3, skin; and group 4, malignant neoplasms. All groups had a statistically significant reduction in their mean anion gaps when compared with normal control subjects. The greatest decrease was when hypoalbuminemia was accompanied by a polyclonal increase in gamma-globulin levels. Hypoalbuminemia with or without a polyclonal gammopathy is a cause of a low anion gap. No statistically significant correlation was found between the anion gaps and individual serum albumin concentrations.

Effect of assay conditions on cross reactivity of digoxin-like immunoreactive substance(s) with radioimmunoassay kits.

One or more digoxin-like immunoreactive substances (DLIS), most frequently present in serum of premature and full-term neonates. cross react to various extents with different digoxin immunoassay kit reagents. Mostly, this variation is attributed to the relative cross reactivity of DLIS with the antiserum in each kit. However, modification of standard assay procedures for digoxin can also greatly alter the relative cross reactivity of DLIS. Using sequential RIA kit methods for digoxin by 20 to 60% relative to the standard equilibrium RIA mode. Cross reactivity was decreased still more if the concentrations of antiserum (binding-site concentration) and tracer (125 l-labeled digoxin) were decreased, and conversely. Serum samples containing only digoxin, analyzed by the modified method, consistently yielded results well comparable with those obtained with the manufacturers' recommended procedures. We describe use of the different responses to digoxin and DLIS of standard and sequential radioimmunoassays and use of simultaneous equation to calculate the concentration of DLIS (in digoxin equivalents) in digoxin-containing samples.