ABSTRACT: In quantitative electromyography (EMG), a sample size of 20motor unit potentials (MUPs) is standard. The effect of increase in thenumber of MUPs above 20 is not known, although advanced MUP analysistechniques make such samples practical. In the present study, using multi-MUP analysis, pools of 3,720 neuropathic and 2,526 control MUPs wereobtained from external anal sphincter muscles. From each pool, 10,000random samples of 5, 10, 15, 20, 30, 40, 50, and 100 MUPs were obtainedby a computer. For each sample size, 95% normative limits for mean values,SDs, and “outliers,” and sensitivities were calculated for eight MUP param-eters. As the magnitude of MUP samples increased, normative limits nar-rowed and sensitivities increased (at 5: 20–30%; at 20: 30–55%; at 100:80–100%) for all statistics of all MUP parameters. Our results demonstrateda substantial increase in sensitivity by increasing the MUP sample to morethan 20. This option deserves consideration in an attempt to improve theusefulness of quantitative EMG.

Muscle Nerve 27: 196–201, 2003

SIZE OF MOTOR UNIT POTENTIAL SAMPLE

SIMON PODNAR, MD, DSc,1 and MICO MRKAIC PhD2

1 Institute of Clinical Neurophysiology, Division of Neurology, University Medical Center,SI–1525 Ljubljana, Slovenia2 The Fuqua School of Business, Duke University, Durham, North Carolina, USA

Accepted 21 October 2002

Electromyography (EMG) is most useful clinicallyin diagnosing patients with mild and clinically equiv-ocal neuromuscular disease. In this situation, sensi-tivity and specificity of the motor unit potential(MUP) analysis are still low.8 Although sensitivity canbe increased by application of additional diagnosticcriteria (it was 13–30% for individual parameters,51% on using a combination of eight MUP parame-ters, and 60% when an “outlier” criterion was addedto mean values in our earlier study8), this decreasesspecificity of analysis, which is more difficult to esti-mate.8 Thus, in order to improve diagnostic power ofMUP analysis, other approaches than cumulative di-agnostic criteria are needed.

It is well known from statistical theory thatincrease in magnitude of a sample reduces itsvariance. Thus, by increasing the number of MUPssampled from an individual muscle, the distinc-tion between normal and pathological musclesmight be significantly improved. It has been dem-

onstrated previously that an increase in MUP sam-ple size from 5 to 20 in an individual musclesignificantly improved the discrimination betweennormal and myopathic muscles.3 The current stan-dard is the compilation of 20 MUPs from an indi-vidual muscle.

In the early days of quantitative EMG, it requiredabout 1 h to compile a sample of 20–40 MUPs froman individual muscle and to measure values of MUPparameters from paper prints.1 Using a “single-MUP” analysis,2,10 which employs a trigger and delayunit, it required about 20 min to sample and quan-tify 20 MUPs. Nowadays, using computer-assisted,template-operated “multi-MUP” analysis, only 3–5min are needed to obtain and quantify a sample of20 MUPs.9,10,13 Thus, samples larger than 20 MUPshave now become practical. However, it has not beendemonstrated how useful it would be to increase theMUP sample obtained from individual muscle above20 MUPs in order to improve the diagnostic powerof MUP analysis. The aim of the present study was toexplore this.

MATERIALS AND METHODS

We studied a group of 56 patients with cauda equinaor conus medullaris injury8,10 and a control group of64 subjects,9,10 both of which have been described in

Abbreviations: EAS, the external anal sphincter; EMG, electromyography;MUPs, motor unit potentialsKey words: external anal sphincter muscle; motor unit potentials; needleelectromyography; sample size; sensitivityCorrespondence to: S. Podnar; e-mail: simon.podnar@kclj.si

© 2002 Wiley Periodicals, Inc.

196 Size of MUP Sample MUSCLE & NERVE February 2003

our previous studies. The National Ethics Commit-tee of Slovenia approved the study, and all subjectsgave informed consent.

MUPs were sampled from the external analsphincter (EAS) muscle by a standard concentricEMG needle (37-mm long; No. 22583; Oxford Med-ical, Old Woking, UK). An advanced EMG system(Keypoint, Medtronic Functional Diagnostics, Skov-lunde, Denmark), with standard filter settings (5Hz–10 kHz) and the template-operated multi-MUPanalysis program were used.6,10,13 In multi-MUP anal-ysis, the operator initiates the computerized acquisi-tion of the previous 4.8-s of EMG activity, from whichMUPs are then automatically extracted, quantita-tively described, and sorted into up to six classes,each representing consecutive discharges of a partic-ular MUP.10,13 Eight MUP parameters [amplitude,duration, area, number of phases, number of turns,thickness (� area/amplitude),4 size index (�2*ln(amplitude) � area/amplitude),11 and spike du-ration] were evaluated.

Control and patient MUP pools were formedfrom all MUPs obtained in these two populations.Using the matlab statistical system (The Math-Works, Natick, Massachusetts) “a bootstrap” tech-nique was applied, in which the computer ran-domly sampled 10,000 times samples of 5, 10, 15,20, 30, 40, 50, and 100 MUPs from the controlpool. For each sample, the mean value, SD, andthe lower and the upper 20th percentile values ofall evaluated MUP parameters were determined.Nonparametric statistics were used by setting the95% limits for these values as normative limits(specificity 95%) for mean values, SDs, and “out-lier” limits, respectively.9,10,12 Normative limits forMUP samples of the eight sizes indicated werecalculated in this way.

From the patient pool, 10,000 MUP samples ofthe same size (5, 10, 15, 20, 30, 40, 50, and 100MUPs) were sampled. Mean values, SDs, and theoutlier limits were determined. The obtained valueswere then compared to the normative limits forMUP samples of the same size. For each MUP pa-rameter, the sensitivities of the mean value, SD, andoutliers criteria were calculated for each sample sizeby dividing the number of patient samples of thatparticular size outside the normative limits by thenumber of all patient MUP samples of the same size(10,000).

RESULTS

In controls, 2,526 MUPs from 112 EAS muscles(mean, 23 MUPs/muscle),9,10 and, in patients, 3,720

MUPs from 138 EAS muscles (mean, 27 MUPs/muscle),5,8,10 were obtained. Statistically significantdifferences were found on comparison of MUPsfrom the patient and control groups for all evaluatedMUP parameters.5,8,10

Normative Limits. As the magnitude of the MUPsamples increased, normative limits for mean values,SDs, and outlier limits of all evaluated MUP param-eters progressively narrowed. This trend also contin-ued for MUP samples above 20, with “saturation”above 40–50 MUPs (Table 1, Figs. 1 and 2). A similarnarrowing of the 5–95% limits was observed in pa-tients (Table 1, Figs. 1 and 2).

Sensitivity. The sensitivity for mean values, SDs,and outlier limits progressively increased as the num-ber of MUPs within the sample increased (Table 1).This relation also continued for MUP samples ofmore than 20 MUPs, and was common to all evalu-ated MUP parameters (Fig. 3). The sensitivity washighest for MUP area and duration, followed by sizeindex, number of turns, spike duration, amplitude,number of phases, and thickness. The relations be-tween MUP parameters were fairly consistent overthe whole range of sample sizes (Fig. 3). For someMUP parameters at some sample sizes, sensitivitieswere the highest for mean values, whereas for theothers, they were highest for outliers. Sensitivities ofSDs were somewhat lower (Table 1).

DISCUSSION

To obtain a valid estimate of the skeletal muscle con-dition in quantitative EMG, individual MUPs do notsuffice, and a MUP sample is needed. This is due tovariability of MUPs caused both by muscle nonhomo-geneity, and signal recording characteristics. In thecalculation of normative limits, a number of musclesobtained from subjects without known neuromusculardisease are examined. By increasing the number ofMUPs generated by different motor units, variance ofthe sample decreases (as the atypical outlier MUPs are“averaged out”), and the MUP sample becomes in-creasingly representative of the whole muscle, as dem-onstrated in Figure 1, where 5th and 95th percentilesof MUP area converge to their actual population val-ues. This resulted in narrowing of the normative limitsand improvement in sensitivity of MUP analysis (Table1, Figs. 1 and 3), as reported previously for samples of5 to 20 MUPs in a small number of myopathic mus-cles.3 In the present study, we extended previous find-ings also to neuropathic condition and to samples ofmore than 20 MUPs.

Size of MUP Sample MUSCLE & NERVE February 2003 197

Our findings are not surprising as it is well knownfrom statistical theory that increase in sample sizereduces its variance, which is expected to narrownormative limits and increase sensitivity and specific-ity of distinction between different samples. How-ever, for most practical purposes, samples of up to 50are small, and asymptotic results cannot be used forthem. In order to uncover statistical distributions of

small samples, we resorted to random resampling,i.e., the bootstrap technique.

Although the same MUP sample was analyzed inboth our present and our previous studies, the norma-tive limits obtained here were narrower, and sensitivi-ties were twice as high.8,10 The main reason for theobserved differences was the random resampling (withthe bootstrap technique) of MUPs from the wholepatient and control MUP pools performed in thisstudy. MUP sampling from individual muscles used inprevious studies (and in EMG practice) is not random,because each patient’s and control subject’s musclepresents an individual MUP pool. Increase in MUPsample size thus cannot reduce interindividual (mus-cle/subject) variance. This is assessed by SDs of meanvalues from individual muscles/MUP samples, andwas much lower using the bootstrap techniquecompared to MUP sampling directly from individ-ual muscles (for MUP area and samples of 20MUPs: 79 �Vms and 135 �Vms, respectively8). Incontrast, with increase of MUP samples, both ap-proaches reduce intraindividual (muscle/subject)variance, which is assessed by mean values of SDfor individual muscles/MUP samples. This wassimilar using these two approaches (for MUP areaand samples of 20 MUPs: 319 �Vms and 331 �Vms,respectively).

Table 1. Effect of size of the MUP sample on the 5th and 95th percentile limits and sensitivity for mean values,SDs, and outliers for MUP area, duration, and number of turns.*

Statistics

MUPparameters

(samplesize)

Area (�Vms) Duration (ms) No. of turns

SensitivityControlrange

Patientrange Sensitivity

Controlrange

Patientrange Sensitivity

Controlrange

Patientrange

Mean 10 0.38 184–541 258–982 0.38 4.0–7.0 4.7–9.0 0.35 2.0–3.8 2.4–4.820 0.57 219–468 310–838 0.56 4.4–6.6 5.3–8.3 0.49 2.2–3.5 2.7–4.530 0.69 235–443 342–782 0.68 4.5–6.3 5.5–8.0 0.62 2.3–3.4 2.8–4.240 0.78 247–427 358–740 0.79 4.6–6.2 5.7–7.9 0.69 2.4–3.3 2.9–4.150 0.86 256–418 375–720 0.87 4.7–6.1 5.8–7.7 0.79 2.4–3.2 3.0–4.1

100 0.98 276–388 413–656 0.98 4.9–5.9 6.1–7.4 0.96 2.6–3.1 3.1–3.9SD 10 0.34 114–607 173–1,473 0.24 1.6–4.8 2.1–6.5 0.25 0.8–2.7 1.1–3.8

20 0.40 158–602 242–1,394 0.36 1.9–4.3 2.6–5.8 0.30 1.0–2.5 1.4–3.430 0.54 182–550 293–1,319 0.46 2.1–4.0 2.8–5.5 0.39 1.5–2.4 1.6–3.240 0.63 200–525 319–1,325 0.56 2.2–3.9 3.0–5.3 0.49 1.2–2.3 1.7–3.150 0.68 211–512 345–1,266 0.64 2.3–3.8 3.1–5.2 0.53 1.3–2.3 1.7–3.0

100 0.87 245–488 426–1,247 0.88 2.5–3.6 3.4–4.9 0.80 1.4–2.1 1.9–2.8Outliers 10 0.28 50–1,137 55–2,013 0.37 1.8–11.6 1.8–15.4 0.60 1–7 1–9

20 0.42 62–827 68–1,414 0.54 2.0–10.0 2.2–13.2 0.62 1–6 1–830 0.50 67–753 78–1,174 0.58 2.0–9.6 2.2–12.4 0.84 1–5 1–740 0.61 71–701 83–1,070 0.73 2.2–9.2 2.4–12.0 0.82 1–5 1–650 0.67 73–675 86–1,022 0.80 2.2–9.0 2.4–11.6 0.80 1–5 1–6

100 0.91 83–607 97–896 0.97 2.4–8.6 2.6–11.0 1.00 1–4 2–6

MUP, motor unit potential.*MUP samples of different sizes were obtained from the external anal sphincter (EAS) muscles by random sampling of control and patient MUP pools. Forbrevity, MUP samples of 5 and 15, and 5 evaluated MUP parameters5 were omitted.

FIGURE 1. Effect of the size of motor unit potential (MUP) sample(X-axis) on the 5th (circles) and 95th percentile limits (squares)for mean values (Y-axis) of controls (open symbols) and patients(solid symbols) with cauda equina or conus medullaris lesions.Results for MUP area are presented, but similar results were alsoobtained for other MUP parameters and other statistics (outliers,SD). At sample sizes above 40–50 MUPs, note “saturation” innarrowing of limits.

198 Size of MUP Sample MUSCLE & NERVE February 2003

As a consequence, the “normative data” obtainedin this study are not valid for clinical EMG practice,and sensitivities presented here should not be inter-preted in absolute terms. At the moment, normativelimits for larger MUP samples obtained by samplingfrom individual muscles (EAS or any other) are notavailable. Nevertheless, the normative limits and sen-sitivities obtained in this study are valid for compar-ison between different magnitudes of MUP samples,different statistical criteria of analysis (mean values,SDs, outliers) and different MUP parameters.

The demonstrated narrowing of normative limitswith increase in the MUP sample shows why, in EMGpractice, only normative limits obtained from sam-ples of the same or a smaller size than the evaluatedone can be used (Fig. 1). It is thus not appropriate touse normative limits for 20 MUPs when a sample ofonly 10 MUPs is evaluated using a mean-values cri-terion, because these normative limits will be toostringent. However, outlier analysis can be con-cluded when the number of MUPs outside the nor-mative range exceeds a certain number (determinedaccording to the sample size and percentile limits

FIGURE 2. Distribution of mean values for motor unit potential (MUP) parameter area of the control (C) and the patient (P) MUP samplesof increasing sizes (5, 10, 15, 20, 30 ,40, 50, and 100 MUPs). Similar results were obtained also for other evaluated MUP parametersand statistics (outliers, SD). Note different scales for MUP area (X-axis, �Vms) and for percentages of samples (Y-axis) in presentedhistograms. With increasing sample size, the spread and overlap of control and patient samples diminished.

FIGURE 3. Effect of the size of motor unit potential (MUP) sample(X-axis) on sensitivity of mean values criterion (Y-axis) to distin-guish between control and patient MUP samples for eight eval-uated MUP parameters. Similar results were also obtained fortwo other evaluated MUP statistics (outliers, SD).

Size of MUP Sample MUSCLE & NERVE February 2003 199

used). For example, with 10th percentile outlier lim-its obtained from samples of 20 MUPs, the musclecan be declared neuropathic and the analysis con-cluded when only 3 MUPs with values above theupper limit for any MUP parameter (area, duration,or number of turns)5 are obtained.12

Relations in sensitivities of different MUP param-eters and statistics were consistent over the wholerange of MUP sample sizes. Sensitivity was the high-est for MUP area and duration, followed by sizeindex, number of turns, spike duration, amplitude,number of phases, and thickness (Fig. 3), whichaccords with our previous results.8 MUP parametersof area, duration, and number of turns also “sur-vived” logistic and probit regression analyses in ourprevious study performed on the same data, andthus proved to be useful in MUP analysis.5 In thisstudy, they were three of the four most sensitiveMUP parameters in the whole range of sample sizes,which further supports their value.

It must be pointed out that the SD presentedhere evaluates variability within individual MUP sam-ples and not variability of their mean values. Thisresulted in much larger values of SD here than inour previous reports.9,10 Analysis of SD as used herehas not been applied previously either in our stud-ies8,10 or in reports by other authors. The reason forthe use of SD in the distinction between healthy anddiseased muscle is the increased MUP variability inpathological muscles.12 Pathological condition can(diffusely) change the entire population of MUPs toa similar degree or can affect only a proportion ofMUPs (focally). The mean-values criterion is sensi-tive for the first situation and the outlier,12 and SDcriteria for the second. In most human neuromus-cular diseases, both pathological processes (diffuseand focal involvement) occur in combination, so thecombined use of all criteria seems useful.10,12

Sensitivities for different statistics varied. Theywere highest for mean values of some MUP param-eters, and for outliers of the others (Table 1). In ourprevious studies, outliers were slightly more sensitivethan mean values.8,10 Although the sensitivity of SDwas lower, it was still reasonably good.

In the present study, neuropathic EAS muscleswere analyzed. Because our findings reflect a funda-mental statistical result (reduction in variance withincrease in sample size), they are independent ofparticular variables, their values, or distributions. Asa consequence, similar findings as in the EAS muscleare also expected for other skeletal muscles and fordifferent clinical conditions, such as myopathies.3

We assume that compilation of 40–50 MUPsfrom individual muscle would be optimal, as “satu-

ration” in narrowing of 5th and 95th limits was ob-served at larger sample sizes (Figs. 1 and 3). LargerMUP samples might also not be valid because, par-ticularly in small muscles such as the EAS, the num-ber of MUPs generated by different motor units islimited. However, with a systematic approach andthe use of advanced MUP analysis techniques (multi-MUP), it should be possible to obtain 40–50 validMUPs from individual muscle. Furthermore, due tothe similarity of MUPs from the subcutaneous anddeeper EAS muscles, MUPs obtained from these twomuscles on the same side may be pooled.7 In largelimb muscles, it should be relatively easy to obtain40–50 MUPs, and sampling of larger MUP sampleswould not be practical.

As explained earlier, in severely neuropathicmuscle, MUP analysis can be concluded using outliercriterion before even 20 MUPs have been obtained.Curiously, in our population, the mean number ofMUPs sampled from individual muscle was higher inpatients, perhaps due to their better motivation andto sensory loss leading to less pain on needle inser-tion, compared to controls.

We believe that our findings are important forEMG practice. They suggest that uncertainty in MUPanalysis can be reduced to a minimum simply byincreasing the number of MUPs sampled from theindividual muscle. Using the advanced multi-MUPtechnique, only an additional 3–5 min would berequired to sample 40–50, instead of 20–30 MUPs.Furthermore, sampling of additional MUPs wouldbe needed only for muscles with equivocal findingswhen 20 MUPs were sampled.

The authors thank Ewa Zalewska, PhD, and Prof. Janez Zidar forreview of the manuscript, and Dr. Dianne Jones for languagereview. The study was supported by the Ministry of Education,Science and Sport of the Republic of Slovenia, Grant No. J3 7899.

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