1. MHA APCCB 20041 A SERUM INDEX FOR METHAEMALBUMIN: THE M-INDEX GRD Jones, M Roser, B Zworestine Department of Chemical Pathology, St Vincents Hospital, Sydney, Australia

2. MHA APCCB 20042 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.

3. MHA APCCB 20043 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.

4. MHA APCCB 20044 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). 415 450 480 570 600 650 700 408

5. MHA APCCB 20045 Oxyhaemoglobin: 570/600Bilirubin: 450/480 Lipaemia (Intralipid): 660/700 Primary wavelength Secondary wavelength All wavelengths in nm (primary/secondary) Figure 2. Aborbance spectra and wavelengths measurements for L, H and I indices 650 700 570 600 450 480

6. MHA APCCB 20046 Methods Serum indices on the Modular were attached to a user- defined 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 Powell 1. Scanning spectrophotometry was performed on a Unicam UV/VIS spectrophotometer.

7. MHA APCCB 20047 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. S predicted = 6 x L + 2.54 x H + 0.3 x I 1 S-Test results and S predicted 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.

8. MHA APCCB 20048 Figure 3. Plot of actual S-test result against predicted S-test results for patient samples with varying degrees of Lipaemia (L index 0 – 34 1, 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. S-Test S predicted

9. MHA APCCB 20049 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 S predicted was calculated for samples with and without added MHA. The Effect of MHA on the values of the S-test and S predicted is shown in figure 4. The M-index (S-Test - S predicted ) is shown in figure 5. The average values and standard deviations for the S-test, S predicted, and the M-index are shown in the table. It can be seen that there is appreciable variation in S predicted, a possible limiting factor in the M-index sensitivity.

10. MHA APCCB 200410 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 S predicted. S predicted S-Test

11. MHA APCCB 200411 Figure 5. M-Index results (S-Test - S predicted ) 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. M-Index S predicted

12. MHA APCCB 200412 Table. Averages and SDs for S-test, S predicted 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 (S predicted ) as well as MHA measurement (M-index). Scatter in S predicted may limit sensitivity of the M index.

13. MHA APCCB 200413 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 S predicted 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.

14. MHA APCCB 200414 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