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Abstract

Sulphated haemoglobin (SulfHb) is a rare entity caused by irreversible sulphation of the haem moiety in haemoglobin, which leads to a similar clinical presentation to methylated haemoglobin (MetHb). The diagnosis of SulfHb is challenging. The presence of a SulfHb was suspected in a 73-year-old lady with low oxygen saturation (SaO2 ~75%), central cyanosis and normal arterial oxygen partial pressure (pO2 ~12kPa). Repeated arterial blood gas analysis on different systems returned error messages for MetHb quantification. There was improvement in oxygen saturation and cyanosis after exchange transfusion when the patient represented with atrial fibrillation and dyspnoea. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF MS) was undertaken on whole blood samples from the patient pre- and post-transfusion, alongside normal controls. These demonstrated the presence of a SulfHb in the patient’s blood, specifically identifying sulphur, sulphur monoxide and sulphur dioxide bound to the haem moiety; levels fell after exchange transfusion.
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Sulphated haemoglobin (SulfHb) is a rare entity caused by irreversible sulphation of the
haem moiety in haemoglobin, which leads to a similar clinical presentation to methylated
haemoglobin (MetHb). The diagnosis of SulfHb is challenging; it is often difficult to
distinguish from MetHb in the arterial blood gas analysers in common use in the UK due
to overlap in the absorption spectra of the two species. The presence of a SulfHb was
suspected in a 73-year-old lady with low oxygen saturation (SaO2~75%), central cyanosis
and normal arterial oxygen partial pressure (pO2 ~12kPa). Repeated arterial blood gas
analysis on different systems returned error messages for MetHb quantification. There was
improvement in oxygen saturation and cyanosis after exchange transfusion when the
patient represented with atrial fibrillation and dyspnoea. Spectrophotometry of the
patient’s blood was suggestive of the presence of SulfHb, with peak absorption at 620nm.
Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF
MS) was undertaken on whole blood samples from the patient pre- and post-transfusion,
alongside normal controls. These demonstrated the presence of a SulfHb in the patient’s
blood, specifically identifying sulphur, sulphur monoxide and sulphur dioxide bound to
the haem moiety; levels fell after exchange transfusion. MALDI-ToF MS represents a
new, rapid, sensitive and specific diagnostic test for the rare haematological syndrome of
SulfHb. In addition, it can identify different sulphur compounds bound to haem, providing
useful diagnostic clues as to the source of SulfHb where this is unclear.
Introduction
A 73-year-old lady presented with a short history of cyanosis. She was found to have
peripheral oxygen saturations of around 75% and cyanosis of her lips, tongue and fingers,
with otherwise normal examination findings. The patient underwent extensive
investigation by the local Cardiology and Respiratory services, with ultimate exclusion of
primary cardiac or respiratory pathology. Her oxygen saturation remained low on repeated
arterial blood gas sampling, despite normal PaO2levels (around 12kPa). Methylated
haemoglobin was suspected, but repeated error messages for MetHb quantification were
returned by arterial blood gas machines made by different manufacturers in three separate
hospitals. Haemoglobin high performance liquid chromatography (HPLC) and capillary
electrophoresis results were normal. Analysis of the patient’s haemoglobin by a National
Reference Laboratory returned normal results, including sequencing of her globin genes.
Screening for paroxysmal nocturnal haemoglobinuria and glucose-6-phosphate
dehydrogenase deficiency were negative. After a subsequent presentation with fast atrial
fibrillation with dyspnoea, the patient underwent an exchange red cell transfusion, with
improvement in her symptoms, cyanosis and oxygen saturation. The blood venesected
from the patient was noted to be dark brown in colour (Figure 1). A literature review
identified SulfHb as another potential cause of the patient’s
clinical signs. She gave a history of rheumatoid arthritis,
migraines and severe constipation, and potential sources of
sulphur identified among her medications were the migraine
drug rizatriptan, and a compound containing Epsom salts
(magnesium sulphate) that was prescribed for constipation.
A clinical diagnostic biotechnology startup company, MAP
Sciences, was approached to undertake analysis of the patient’s
blood to establish if SulfHb was detectable.
Methods
Visible light absorption spectral analysis of patient whole blood
20µl of whole blood samples were lysed in 2ml of ddH2O to causes cell lysis but also
release of haem moieties from the globin proteins; at a dilution of 1/100 the main visible
colour is due to the abundant haem moieties. Visible spectral absorption was measured at
10nm intervals between 500 and 700nm, blanking at each wavelength against ddH2O.
Spectra absorption was normalised against peak absorption at 530nm to correct for
variation in total Hb concentration of individual samples.
MALDI-ToF MS of blood for haem adducts
Control blood samples, along with the patient’s blood samples at presentation,
immediately prior to exchange transfusion, and post-exchange transfusion were all
analysed. 1µl of whole blood, or dried blood spots soaked in ddH2O, at a dilution of 1 in
2000 were subjected to MALDI-ToF mass spectrometry. MALDI ToF plates were pre-
coated with 1µl of alpha-cyano-4-hydroxycinnamic acid (CHCA) (Sigma Aldrich) at a
concentration of 20mg/ml, dissolved in 1:2 0.1% trifluoroacetic acid and acetonitrile,
which was allowed to dry to crystals. 1µl of the diluted patient sample was added and,
before completely drying, another 1µl of CHCA was added. After the sample matrix mix
had completely dried to crystals, this sample was analysed on the Shimadzu 8020 Clinical
Linear MALDI ToF mass spectrometer, which was optimised for analysis at 500 700
m/z. MALDI was internally calibrated with singly and doubly charged Bradykinin 1-7
fragment (Sigma Aldrich) protein standard.
Conclusion
MATRIX-ASSISTED LASER DESORPTION IONISATION TIME-OF-FLIGHT MASS SPECTROMETRY AS A
NOVEL TECHNIQUE TO DETECT SULPHATED HAEMOGLOBIN
Suzanne Docherty1, Ray Iles2, Raminta Zmuidinaite2, James Coulson3, Martin Besser4
1Haematology Department, Norfolk & Norwich University Hospital, Norwich, 2MAP Sciences , Bedford, 3Pharmacology
Department, Cardiff University, Cardiff, 4Haematology Department, Cambridge University Hospitals, Cambridge, United Kingdom
Figure 2: Absorbance across visible light spectrum of patient’s
blood on first admission (red line), pre-transfusion (orange) and
post-transfusion (blue) compared with normal control (yellow)
Figure 3: MALDI-ToF MS spectrum on patient blood
showing peaks for haem moiety at 618Da, with
additional peaks for bound sulphur (+32Da), sulphur
monoxide (+48Da) and sulphur dioxide (+64Da)
The addition of whole blood to ddH2O at a dilution of 1 in 2000 causes red cell lysis, and
also dissociation of haemoglobin into free haem and free alpha & beta globins. As these
are the most abundant proteins in lysed whole blood by several orders of magnitude, all
others molecules are effectively diluted out. Sinapinic acid as the matrix preferentially
ionises large proteins and was optimised for globin analysis as previously described (Iles et
al. 2013). Lysed whole blood was examined by UV visible spectroscopy. A control sample
was also analysed (one male with normal Hb). Three of the patient’s samples at
presentation, immediately prior to transfusion, and post transfusion, were analysed. The
expected absorption pattern is two major peaks at 550nm (OxyHb) and 670-680nm
(DeoxyHb). Comparison of theadmission sample against the control show a large increase
in absorption from 600nm to 700nm, with a minor peak at 620nm (arrowed) (Figure 2).
Comparison of the admission, start of transfusion and post transfusion samples show a
significant decrease in the absorption at 600-700nm post transfusion. However, a peak at
620nm is still visible in the post transfusion sample. This has decreased from 32% of the
normal Hb signal at 530nm to 12%, but this is still double that found in the control
samples (5-6%). MALDI-ToF mass spectrometric analysis of patient whole blood on the
admission sample identified peaks for the expected haem moiety at 618Da, with further
peaks at +32Da, +48Da and +64Da, correlating with sulphur, sulphur monoxide and
sulphur dioxide bound to haem (Figure 3).
SulfHb was identified in the blood of a patient who presented with persistently low oxygen
saturation on pulse oximetry, normal oxygen partial pressure on arterial blood gas sampling,
and repeated error messages returned by arterial blood gas machines from two manufacturers
for haemoglobin species present. It illustrates the diagnostic difficulties encountered where
the rarely encountered SulfHb is present, and the potential for confusion with MetHb
(reviewed by Lu et al., 1998). MALDI-ToF MS successfully identified the presence of
sulphur, sulphur monoxide and sulphur dioxide bound to haem moieties.
SulfHb is a rare complication of exposure of haem groups to sulphur. It causes irreversible
binding of sulphur to the porphyrin ring of the haem moiety, with resultant cyanosis as
SulfHb does not bind oxygen. The clinical presentation is similar to that of MetHb, but it does
not respond to treatment with methylene blue or ascorbic acid. However, the presence of
SulfHb decreases oxygen affinity of unaffected haemoglobin, with left-shift of the oxygen
dissociation curve and improved oxygen delivery to tissues; the converse is true of MetHb.
SulfHb thus tends to be associated with milder clinical symptoms than MetHb. Where
treatment is felt to be required, exchange transfusion is the intervention of choice as it can
reduce the proportion of SulfHb; once binding of sulphur to haemoglobin has occurred, it will
last for the 120-day lifespan of the erythrocyte.
To the authors’ knowledge, this is the first report using MALDI-ToF MS in the detection of
SulfHb. Many previous cases of SulfHb described in the literature are thought to relate to
hydrogen sulphide causing SulfHb, often in the context of bacterial overgrowth in the
gastrointestinal tract. MALDI-ToF MS enabled the specific identification of sulphur dioxide
rather than hydrogen sulphide bound to haem in this patient’s case, establishing the most
likely source of SulfHb in this patient as drugs containing sulphur groups; this patient was
taking an undeclared Epsom Salts compound, and the problem resolved rapidly when it was
ceased. MALDI-ToF MS is a highly specific and rapid methodology for the investigation of
dyshaemoglobin molecules; it should be considered alongside gas chromatography for the
definitive diagnosis of SulfHb.
References
Iles RK Iles JK, Abban T, Docherty SM, Naase, M. (2013) METHOD FOR DETECTING ABNORMALITIES IN
HEMOGLOBIN WO Patent App. PCT/GB2015/052,491
Lu HC, Shih RD, Marcus S, Ruck B, Jennis T. Pseudomethemoglobinemia: A Case Report and Review of Sulfhemoglobinemia.
Arch. Pediatr. Adolesc. Med. 1998;152(8):803805
Abstract Results
Figure 1: The patient’s
venesected blood (left)
with a red cell unit for
transfusion (right)
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Patent
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(EN)The method describes rapid screening of whole blood samples, pin prick and blood spot cards, subjected to MALDI – ToF Mass spectrometry. The spectra is generated and compared to those from normal healthy controls. Characteristic spectra are indicative of the presence of a hemoglobinopathy and the method can be used to screen/diagnose all sickle cell diseases, alpha and beta Thalassemias. (FR)L'invention concerne le criblage rapide d'échantillons de sang entier, par piqûre d'épingle et tests de Guthrie, soumis à la spectrométrie de masse MALDI-ToF. Les spectres sont générés et comparés à ceux de témoins normaux en bonne santé. Des spectres caractéristiques indiquent la présence d'une hémoglobinopathie et le procédé peut être utilisé pour cribler/diagnostiquer toutes les drépanocytoses, et les thalassémies alpha et bêta.
To see if methemoglobin could potentially be misdiagnosed and the limitation of present cooximeters. A 17-year-old girl who overingested a combination of cimetidine, acetaminophen, ibuprofen, and naproxen in a suicide attempt. Use of pulse co-oximeters to aid in the diagnosis of suspected sulfhemoglobinemia. Diagnosis of sulfhemoglobinemia achieved with final confirmation made with gas chromatography. Patient steadily improved with supportive care. There is a potential for the diagnosis of methemoglobin with some of the limitations of present co-oximeters. The laboratory diagnosis of sulfhemoglobinemia can be difficult to make.