Transcript A Serum Index for Methaemalbumin: the M Index

A SERUM INDEX FOR
METHAEMALBUMIN: THE M-INDEX
GRD Jones, M Roser, B Zworestine
Department of Chemical Pathology,
St Vincents Hospital, Sydney, Australia
MHA APCCB 2004 1
Introduction
• Serum indices for haemolysis (H), icterus (I) and lipaemia
(L) are established tools for routinely identifying the
presence of these interferences.
• Methaemalbumin may be formed after intravascular
haemolysis, haemorrhagic pancreatitis or a retro-peritoneal
bleed and gives the blood a distinct brown discolouration.
• Failure to recognise this cause of haemolysis may lead to
misinterpretation of biochemistry results and miss the first
indication of this clinical problem.
• Intravascular haemolysis samples also commonly contain
elevated bilirubin and oxyhaemoglobin.
• In this paper we demonstrate a serum index for
metahaemalbumin, the M index.
MHA APCCB 2004 2
Theory
• Methaemalbumin has a Soret absorbance peak at 408 nm.
• This peak overlaps the 415 nm wavelength available on
Hitachi analysers (figure 1).
• The H index on Hitachi analysers relies on measurement of
the gamma peak of oxyhaemoglobin at 570 nm (Figure 2).
• We propose that absorbance at 415 nm will allow detection
of all haem-containing molecules in a sample.
• Oxyhaemoglobin and other coloured interferences are
quantified by serum indices and their contribution to
absorbance at 415nm is calculated.
• The proposed M-index, for detection of methaemalbumin, is
the actual absorbance at 415 nm minus the the absorbance at
415 nm predicted from the other indices.
MHA APCCB 2004 3
415
408
450 480
570 600 650
700
Figure 1. Spectrophotometric scan of methaemalbumin with
primary wavelength of Soret absorbance peak of 408 nm. Also
shown are Hitachi wavelengths of 415 nm (for primary wavelength)
and candidate secondary wavelengths: 450, 480 (used for I index),
570, 600 (H index), 650 and 700 nm (L index).
MHA APCCB 2004 4
480
570 600
Oxyhaemoglobin: 570/600
450
Bilirubin: 450/480
Figure 2. Aborbance spectra and
wavelengths measurements for L,
H and I indices
Primary wavelength
650 700
Lipaemia (Intralipid): 660/700
Secondary wavelength
All wavelengths in nm
(primary/secondary)
MHA APCCB 2004 5
Methods
• Serum indices on the Modular <P> were attached to a userdefined test (the S-test, named after the Soret peak).
• The S-test was set up as follows:
– Single point reading at cycle 3.
– Primary wavelength 415 nm; 2° wavelength 480 nm.
– Sample volume 8uL; R1 normal saline 250 uL.
– Assay conditions the same as standard serum indices
• The 2° wavelength was chosen from those available to
minimise interference from L, H and I (figures 1 & 2)
• Methaemalbumin standard was prepared by the method of
Cowley and Powell1.
• Scanning spectrophotometry was performed on a Unicam
UV/VIS spectrophotometer.
MHA APCCB 2004 6
Experiment 1: Predicting S-test results
• L, H, I indices and the S-test were measured on dilutions of
fresh haemolysate, Intralipid™, and high bilirubin.
• Data from these measurements were used to determine the
contribution of each of these substances to the S-Test.
Spredicted = 6 x L + 2.54 x H + 0.3 x I1
• S-Test results and Spredicted were compared for patient
samples with elevated L, H and I indices (figure 3).
• A good correlation was seen between S-test results and the
predicted value of S based on the other indices.
1 Serum indices set up with the following parameter values:
A - 25 (H in mg/dL); B - 12,200; C - 88.5 (L in SI units x 10);
D - 94 (I in umol/L); E - 19,000; F - 180,000.
Note: C value 1/10 of normal value to give required precision.
MHA APCCB 2004 7
800
S-Test
600
400
200
0
0
200
400
600
800
Spredicted
Figure 3. Plot of actual S-test result against predicted S-test results
for patient samples with varying degrees of Lipaemia (L index 0 –
341, Triglycerides (1 – 20 mmol/L), haemolysis (0 - 282 mg/dL),
and icterus (0 - 358 umol/L). Green line is line of identity.
Interpretation: In samples without methaemalbumin, the S-test
result can be predicted from the other indices.
1. See text for description of indices setup.
MHA APCCB 2004 8
Experiment 2: Detecting Methaemalbumin
• Methaemalbumin standard was added to 13 samples
(containing haemolysis, icterus and / or lipaemia) to give
final methaemalbumin concentrations of 25 and 50 mg/L.
• L, H, I and S-Test were measured and the Spredicted was
calculated for samples with and without added MHA.
• The Effect of MHA on the values of the S-test and Spredicted is
shown in figure 4.
• The M-index (S-Test - Spredicted) is shown in figure 5.
• The average values and standard deviations for the S-test,
Spredicted , and the M-index are shown in the table.
• It can be seen that there is appreciable variation in Spredicted ,
a possible limiting factor in the M-index sensitivity.
MHA APCCB 2004 9
800
S-Test
600
400
Patient serum
Serum + 25 mg/L MHA
Serum + 50 mg/L MHA
200
0
0
200
400
600
800
Spredicted
Figure 4. Actual S-Test results plotted against predicted S test
results for patient samples, to which some have added 25 or 50
mg/L methaemalbumin (MHA). Dashed lines are lines of best fit.
Interpretation: Addition of MHA increases the S-Test with only
minor changes in Spredicted.
MHA APCCB 2004 10
140
120
M-Index
100
80
25 mg/L MHA
60
50 mg/L MHA
40
20
0
0
200
400
600
800
Spredicted
Figure 5. M-Index results (S-Test - Spredicted) results plotted against
predicted S test results for patient samples, to which some have
added 25 or 50 mg/L methaemalbumin (MHA).
Interpretation: Addition of MHA reliably increases M-index, with
some scatter in the measurement.
MHA APCCB 2004 11
S-TEST
Spredicted
M-Index
Methaemalbumin
25 mg/L
50 mg/L
Average
SD
Average
SD
68.0
3.0
130.8
9.1
6.4
4.8
8.6
5.9
61.7
4.7
122.2
8.1
Table. Averages and SDs for S-test, Spredicted and M-index results
for samples with added 25 or 50 mg/L methaemalbumin (MHA).
Interpretation: The scatter in the M-index results is a combination
of the scatter in background estimation (Spredicted ) as well as MHA
measurement (M-index). Scatter in Spredicted may limit sensitivity of the
M index.
MHA APCCB 2004 12
Discussion
• In samples with icterus, haemolysis or lipaemia without
MHA, a strong correlation was seen between the
predicted and actual values of the S-test (figure 3).
• Addition of methaemalbumin to a range of samples
produced a reproducible increase in the measured S-index
without significant change in the other indices (figure 4).
• The sensitivity of the M-Index is may be limited by the
background variation in the Spredicted as well as
imprecision in the S-Test results (table). The smallest
amount of MHA which can be reliably detected depends
on this background noise.
• Adjustment of testing conditions may allow improvement
in this aspect of the M-Index performance.
MHA APCCB 2004 13
Conclusion
• The M-index on Hitachi analysers allows automatic
detection of samples containing non-oxyhaemoglobin haem.
• These haem molecules detected may include
methaemalbumin or methaemoglobin.
• The M-index is available at no additional cost on Hitachi
analysers and may allow early diagnosis of intravascular
haemolysis.
• Further optimisation of index conditions may improve MHA
detection.
REFERENCE
1. Cowley DM, Powell VR. Quantitative determination of serum methaemalbumin. Pathology. 1986; 18: 310-312
MHA APCCB 2004 14