Sample size requirement for digital image analysis of collagenproportionate area in cirrhotic livers

Andrew Rennie Hall,1 Emmanuel Tsochatzis,2 Richard Morris,3 Andrew Kenneth Burroughs2 &

Amar Paul Dhillon1

1Department of Cellular Pathology, UCL Medical School, Royal Free Campus, London, UK, 2The Royal Free Sheila Sherlock

Liver Centre and University Department of Surgery, UCL, Royal Free Hospital, London, UK, and 3Division of Population

Health, UCL, London, UK

Date of submission 21 March 2012Accepted for publication 30 July 2012

Hall A R, Tsochatzis E, Morris R, Burroughs A K & Dhillon A P

(2013) Histopathology 62, 421–430

Sample size requirement for digital image analysis of collagen proportionate area in cirrhoticlivers

Aims: The requirements for adequate cirrhotic liverbiopsy size have not been established for quantitativefibrosis measurements (collagen proportionate area:CPA). We evaluated the CPA of virtual biopsies incirrhosis to elucidate (i) the amount of tissue requiredto achieve reliable CPA measurements and (ii) the effectof aetiology on sample size requirements.Method and results: A total of 120 cirrhotic tissueblocks (six aetiologies) were studied. A representative100 mm2 region was selected from each block and areference CPA measured. Each image (n = 120) wasdivided into 100 · 1 mm2 images; CPA was measuredfor each 1 mm2 and virtual biopsies of different sizeswere created from the 1 mm2 components. For each

virtual biopsy size the probability that the virtualbiopsy CPA would be within 5% of the reference CPAwas calculated. There were 441 000 virtual biopsies.Biopsy size versus probability plots indicated that, for90% probability that the virtual biopsy CPA can beexpected to be within 5% of the reference CPA, 22–28 mm2 of analysable tissue is required depending onliver disease aetiology; and that a 75% probability levelrequires a biopsy with 12–15 mm2 of analysable tissue.Conclusion: The sample size required for a givenprobability level depends on the aetiology of cirrhosis,and this should be taken into account when judgingthe reliability of cirrhotic liver biopsy CPA.

Keywords: biopsy, cirrhosis, collagen proportionate area, digital image analysis, fibrosis

Introduction

Liver fibrosis is staged typically into numbered catego-ries by the identification of morphological features,rather than by quantitative analysis of collagen con-tent.1,2 Despite advances in radiological imaging andserum non-invasive markers, liver biopsies and theirhistological evaluation remain the best standard toassess the degree of liver fibrosis and to diagnosecirrhosis.3–6 Traditional scoring systems are not able

to substage cirrhosis. There is increasing interest inquantifying fibrosis in liver biopsies, and a validatedmethod is the use of digital image analysis to quantifyfibrosis as the proportion of the whole tissue area that iscollagen, namely the collagen proportionate area(CPA).7 CPA correlates well with hepatic venouspressure gradient (HVPG)8,9 and liver stiffness, asmeasured with transient elastography10 and, impor-tantly, is a better predictor of future decompensationthan Ishak score.11–13 Furthermore, unlike histologicalstaging, CPA provides a continuous numerical scalethat allows more accurate use of statistical methods(unlike stage categories, codified as numbers, used inhistological staging systems). It is not suggested thatCPA replaces the use of morphological categorical

Address for correspondence: Professor A P Dhillon, Department of

Cellular Pathology, UCL Medical School, Royal Free Campus, Row-

land Hill Street, London NW3 2PF, UK. e-mail: a.dhillon@ucl.ac.uk

� 2012 Blackwell Publishing Limited.

Histopathology 2013, 62, 421–430. DOI: 10.1111/his.12010

systems, as it does not represent the same information,but CPA provides an extra item of information. Thefuture potential to reverse fibrosis and possibly cirrho-sis,14,15 the interest in assessing progressive versusregressive cirrhosis,16 and the concept of subclassifyingcirrhosis8,17–20 have generated a need for an accurate,quantifiable method of measuring liver fibrosis on acontinuous numerical scale.7–11,13,19,21

The categorical assignment of liver disease stage isdifferent from the measurement of liver fibrosis.7,22,23

Establishing sample adequacy (minimum sample size)for the accurate measurement of liver fibrosis is also adifferent process to establishing sample adequacy forthe confident determination of liver disease stage.However, the stage of the disease is an importantvariable for determining the adequacy of a biopsysample; this is the case for both measurement of fibrosisusing methods such as CPA,24 as well as moreconventional stage evaluation.25 In addition, the min-imum sample size for reliable fibrosis measurement islikely to be affected by the distribution of fibrosisthroughout the liver, and consequently by diseaseaetiology.

Our aims in this study were to assess formally inpatients with cirrhosis: (i) the amount of tissue that isrequired to achieve reliable CPA results (and theprobability levels of smaller biopsies) using virtualbiopsy area and actual biopsy length obtained with16G and 19G needles; and (ii) to ascertain whetheraetiology of liver disease influences the minimumsample size requirements.

Material and methods

The study cohort has been described previously.19 Insummary, the cohort consisted of the 10 most recentexplanted cirrhotic liver cases from the Royal FreeHospital routine diagnostic histology archives (1999–2009) for each of the following selected commonaetiologies: alcoholic liver disease (ALD), primarybiliary cirrhosis (PBC), primary sclerosing cholangitis(PSC), autoimmune hepatitis (AIH), hepatitis C virus(HCV) and hepatitis B virus (HBV). Only cirrhotic, well-fixed tissue was selected. Cases with multiple aetiologiesor with malignancies were excluded, e.g. HCV plusALD. Cirrhosis was defined histologically as describedby Anthony et al.26: ‘a diffuse process characterized byfibrosis and the conversion of normal liver architectureinto structurally abnormal nodules’.

A total of 120 tissue blocks from 60 explanted livers(one block of tissue from the left lobe and another fromthe right lobe) were included, the CPA results of whichhave been detailed previously.19 All tissue blocks were

required to contain at least 1 cm2 of cirrhotic paren-chyma. Of the 60 explant cases, 30 were from male and30 from female patients; median age was 53.5 years,range 20–69 years. The clinical data for these patientshave been detailed previously.19 Sections were cut at3 lm from the archived paraffin blocks and werestained with picro-Sirius red.27,28

cpa measurements

Collagen proportionate area was measured as describedpreviously.8 In brief, a Canon Powershot A640 digitalcamera was used for image capture and Zeiss KS300software for the digital image analysis (DIA). Using theimage analysis software, a greyscale slider was used toselect the tissue area that is calculated subsequently asa total tissue area in pixels. A red, green, blue (RGB)threshold was then used to detect areas of stainedcollagen and the collagen mask was calculated as anarea in pixels. The ratio between the two areas (CPA)was expressed as a percentage. Measuring CPAinvolved capturing the whole image of the stainedliver section at low power with a 10 · 10 mm grati-cule placed onto the slide; tissue within 5 mm of thecapsule and large blood vessels were avoided. Adjacentsmaller areas were captured to achieve a total of100 mm2 if either capsule or large blood vessels(‡0.5 mm) were present within the 10 · 10 mmgraticule.

The resulting image ⁄ images were cropped into 100separate 1 · 1 mm squares and labelled 1–100. Eachindividual 1 mm2 subcomponent was analysed forCPA, performed in batches of 100, each slide in asingle session. The greyscale and RGB levels were keptconsistent throughout the analysis of each slide(100 · 1 mm2). Each slide, therefore, had 100 sepa-rate CPA measurements, one for each subcomponent1 mm2 field, in addition to the overall (reference) CPAfor the 100 mm2 area (Figure 1). Thus, a total of12 120 CPA measurements were made from the 120cirrhotic tissue samples. All the image analysis wasperformed by one observer (A.H.).

study strategy

The method adopted for determining the adequacy ofthe size of the liver sample involved measuring theprobability that the CPA of a virtual biopsy of specifiedsize would fall within 5% of the ‘true’ CPA (i.e. thereference CPA for each cirrhotic tissue sample, which istaken as the CPA of the whole 100 mm2 of cirrhoticparenchyma for that sample). This was measured asthe proportion of virtual biopsies that differ from the

422 A R Hall et al.

� 2012 Blackwell Publishing Ltd, Histopathology, 62, 421–430.

100 mm2 reference values by <5% CPA. The value of5% was chosen as it covers deviations caused by bothintra- and interobserver error in the CPA evaluation ofcirrhotic explanted livers.19

For each case a reference value (the total CPA for thewhole 100 mm2 area) was established. Selection of thecomponent parts of each virtual biopsy can be per-formed either by using random 1 mm2 subcomponentfields or by using contiguous (sequential) 1 mm2

subcomponent fields. For each virtual biopsy (randomsubcomponents) a random number generator (1–100without replacement; simple random selection withoutreplacement) was used to select a specific number ofrandom 1 mm2 subcomponent fields. Virtual biopsieswere constructed by calculating the CPA of thecombined 1 mm2 subcomponent fields. Using thismethod, for each of the 120 cirrhotic samples (slides),50 virtual biopsies were created for each of the virtualbiopsy sizes from 2 to 50 mm2, and 25 virtual biopsieswere created for each of the virtual biopsy sizes 51–99 mm2, totalling 3675 [(49 · 50) + (49 · 25)] vir-tual biopsies per slide, ranging in size from 2 to99 mm2. Virtual biopsies were constructed both byusing sequential contiguous 1 mm2 subcomponentfields and by using randomly selected 1 mm2 subcom-ponent fields, without replacement. The results ob-tained for each of these field selection procedures weresimilar. This study has focused on the data obtainedwith randomly selected 1 mm2 subcomponent fields, asrandom selection is considered essential for the use ofthe probability statements that are pivotal to our

findings. This criterion cannot be fulfilled by contigu-ous selection, which is deterministic once the first areahas been chosen. For comparison, the results for thecontiguous subcomponent selection method are avail-able as additional online material.

For each virtual biopsy the absolute deviation (AD)was calculated, i.e. the difference between the CPA ofthe virtual biopsy (combined CPA of all subcomponent1 mm2 fields) and the reference CPA (CPA of100 mm2). The probability level, expressed as apercentage, is the proportion of virtual biopsies of agiven size (2–99 mm2) that have a CPA within 5% ofthe reference value CPA for that slide. For example, ‘fora 90% probability level a biopsy size measuring22 mm2 is required’ indicates that 90% of 22 mm2

virtual biopsies have a CPA within 5% of the referenceCPA (AD < 5%) (Figure 1).

These values were plotted as line graphs of virtualbiopsy size against proportion of biopsies within 5% ofthe reference CPA. These graphs permit the estimationof biopsy size requirements for selected probabilitylevels (Figure 2).

relationship between virtual biopsy area

and actual liver biopsy length

15 percutaneous (16G Menghini needle) and 15transjugular (TJ) (19G Tru-Cut needle) biopsies wereassessed for the width of the tissue cores on thehistological slides. The biopsies were selected as thoseperformed at the Royal free Hospital that were the most

Whole area reference value CPA = 39%

1 mm2 images

Tissue masks

Collagen masks

Total tissue mask pixels: 168 703

Total collagen mask pixels: 61 118

10 mm virtual biopsy CPA:

Absolute deviation: 39%–36.2% = 2.8%

61 118168 703

×100 = 36.2%

Img 003

16 894Pixels:

5457 6635 12 554 5280 2231 6932 5651 5635 6112 4631Pixels:

16 850 16 892 16 900 16 828 16 880 16 900 16 900 16 759 16 900

Img 014 Img 029 Img 033 Img 038 Img 063 Img 073 Img 077 Img 094 Img 099

Figure 1. Virtual biopsy selection and absolute deviation explained. Right lobe of liver explant from a 64 year old man transplanted for cirrhosis

due to alcoholic liver disease. This diagram illustrates the concept of constructing a 10 mm2 virtual biopsy from 10 random 1 mm2 images

selected via random number tables and the method for calculating the absolute deviation (AD). The probability level is the proportion of

virtual biopsies that are within 5% CPA of the reference value, AD < 5%.

Sample requirement for CPA 423

� 2012 Blackwell Publishing Ltd, Histopathology, 62, 421–430.

proximate to November 2011. Biopsy width wasassessed microscopically by measuring each core atthree points and taking the median of these measure-ments. The three points were at 25, 50 and 75% alongthe length of the core. Median actual biopsy width forcores taken using the 16G Menghini needle (internaldiameter 1.4 mm) was 0.8 mm (range 0.5–1.1 mm),and for those taken using the 19G needle (internaldiameter 0.9 mm) was 0.6 mm (0.4–0.7 mm); thesefigures were used to convert the virtual biopsy areasinto actual biopsy lengths.

statistical analysis

Numerical variables were summarized as median (andrange) and nominal variables as frequencies or asdistributions. Differences between quantitative vari-ables were assessed using non-parametric tests. Corre-lations were assessed by Spearman’s rank correlation.Significance testing was two-sided, and the criticalalpha value was set to 0.05.

Results

In total, 441 000 virtual biopsies were obtained from120 sections. For each slide there were 3675 virtualbiopsies, 73 500 virtual biopsies per aetiology. Themedian of the reference CPA (100 mm2) of all thecirrhotic explant liver tissue blocks studied was 21.1%(range 8–47%). The median reference CPA according

to aetiology was: ALD 32.5% (17–47%), PBC 24.8%(15–38%), PSC 23.1% (8–36%), AIH 20% (8–35%),HCV 18% (12–34%) and HBV 17.5% (11–24%)(Table 1).

sample s ize requirements versus aetiology

Table 1 summarizes the minimum biopsy sizes requiredfor selected probability levels by aetiology of cirrhosis;these were calculated from plots as shown in Figure 2.

For a 75% probability that virtual biopsies will havea CPA measurement within 5% of the reference CPA, abiopsy size measuring 12 mm2 (this is equivalent to a15 mm specimen length with a 16G needle or a20 mm specimen length with a 19G needle) is requiredfor HCV (median AD = 2.8% CPA, range 0–16.7%)and PSC (2.7%, 0–14.5%); 13 mm2 (specimen length16G: 16 mm, 19G: 22 mm) for HBV (2.5%, 0–15.3%)and AIH (2.5%, 0–20%); and 15 mm2 (specimenlength 16G: 19 mm, 19G: 25 mm) for ALD (2.7%,0–16%) and PBC (2.7%, 0–14.4%) (Table 1, Figure 2).

For a 90% probability that virtual biopsies will havea CPA measurement within 5% of the reference CPA, abiopsy size measuring 22 mm2 (this is equivalent to a28 mm specimen length with a 16G needle: or a37 mm specimen length with a 19G needle) is requiredfor HBV (median AD = 2% CPA, range 0–10%);23 mm2 (specimen length 16G: 29 mm, 19G:38 mm) for HCV (2%, 0–11%), AIH (2.0%, 0–9.1%)and PSC (1.7%, 0–10%); 27 mm2 (specimen length

100

80

60

40

Pro

port

ion

of c

ases

with

in 5

% o

f ref

eren

ce C

PA (

%)

20

02 7 12 17 22 27 32 37 42 47 52

Bx size (mm2)

AIH

ALD

HBV

HCV

PBCPSC

90% of virtual biopsies within 5%CPA

57 62 67 72 77 82 87 92 97

Proportion of biopsies within 5% of reference CPA (probability level)plotted against virtual biopsy size

Figure 2. ALD = Alcoholic

Liver Disease; PBC = Primary

Biliary Cirrhosis; PSC = Prim-

ary Sclerosing Cholangitis;

AIH = Autoimmune Hepatitis;

HCV = Hepatitis C Virus;

HBV = Hepatitis B Virus. From

plots such as this a biopsy size

can be deduced for a specific

probability level, or vice versa.

424 A R Hall et al.

� 2012 Blackwell Publishing Ltd, Histopathology, 62, 421–430.

Tab

le1.

Sam

ple

size

requirem

ents

for

sele

cted

pro

bab

ility

leve

lsusi

ng

random

sele

ctio

nof

virt

ual

bio

psi

es

Aet

iolo

gy

Ref

eren

ceC

PA

+ra

nge

(%)

Pro

bab

ility

leve

lPro

port

ion

of

virt

ual

bio

psi

esw

ith

CPA

that

falls

within

5%

of

refe

rence

CPA

70%

75%

80%

90%

Min

sam

ple

size

(mm

2)

Equiv

.16G

⁄19G

bio

psy

length

(mm

)

Med

ian

AD

+ra

nge

(CPA

%)

Min

sam

ple

size

(mm

2)

Equiv

.16G

⁄19G

bio

psy

length

(mm

)

Med

ian

AD

+ra

nge

(CPA

%)

Min

sam

ple

size

(mm

2)

Equiv

.16G

⁄19G

bio

psy

length

(mm

)

Med

ian

AD

+ra

nge

(CPA

%)

Min

sam

ple

size

(mm

2)

Equiv

.16G

⁄19G

bio

psy

length

(mm

)

Med

ian

AD

+ra

nge

(CPA

%)

ALD

32.5

17–4

713

16

⁄22

3.1

0–1

6.6

15

19

⁄25

2.7

0–1

619

24

⁄32

2.4

0–1

3.9

28

35

⁄47

1.9

0–1

3

PBC

24.8

15–3

813

16

⁄22

3.2

0–1

7.9

15

19

⁄25

2.7

0–1

4.4

18

23

⁄30

2.6

0–1

6.6

27

34

⁄45

1.8

0–1

1.1

PSC

23.1

8–3

610

13

⁄17

3.1

0–1

912

15

⁄20

2.7

0–1

4.5

14

18

⁄23

2.5

0–1

2.8

23

29

⁄38

1.7

0–1

0

AIH

20

8–3

510

13

⁄17

3.3

0–2

013

16

⁄22

2.5

0–2

017

21

⁄28

2.2

0–1

523

29

⁄38

2.0

0–9

.1

HC

V18

12–3

410

13

⁄17

3.4

0–2

1.3

12

15

⁄20

2.8

0–1

6.7

15

19

⁄25

2.4

0–1

6.3

23

29

⁄38

2 0–1

1

HBV

17.5

11–2

410

13

⁄17

3.2

0–1

8.6

13

16

⁄22

2.5

0–1

5.3

16

20

⁄27

2.4

0–1

5.5

22

28

⁄37

2 0–1

0

ALD

,A

lcoholic

liver

dis

ease

;C

PA

,co

llagen

pro

port

ionat

ear

ea;PBC

,prim

ary

bili

ary

cirr

hosi

s;PSC

,prim

ary

scle

rosi

ng

chola

ngitis

;A

IH,au

toim

mune

hep

atitis

;H

CV

,hep

atitis

Cvi

rus;

HBV

,hep

atitis

Bvi

rus;

AD

,ab

solu

tedev

iation.

Bio

psy

length

bas

edon

med

ian

wid

thof

16G

and

19G

form

alin

-fixe

dbio

psy

his

tolo

gic

alpre

par

atio

ns

of

0.8

and

0.6

mm

,re

spec

tive

ly.

Sample requirement for CPA 425

� 2012 Blackwell Publishing Ltd, Histopathology, 62, 421–430.

16G: 34 mm, 19G: 45 mm) for PBC (1.8%, 0–11.1%);and 28 mm2 (specimen length 16G: 35 mm, 19G:47 mm) for ALD (1.9%, 0–13%) (Table 1, Figure 2).

The data obtained using the contiguous selectionmethod for virtual biopsies is similar to that provided bythe random selection method, and is available asadditional material online (Table S1 and Figure S1).

sample size requirements versus reference

cpa value

Taking all aetiologies together on a case-by-case basis(n = 120), a strong correlation was seen between theCPA of the whole tissue area and the minimum samplesize calculated from the virtual biopsies for the selectedprobability levels: 70% (R = 0.67; P < 0.0001), 75%(R = 0.64; P < 0.0001), 80% (R = 0.62; P < 0.0001)and 90% (R = 0.58; P < 0.0001) (Figure 3).

Discussion

Sampling error is an important issue when makingjudgements about the whole liver based on analysis of asmall fraction of the organ; this is the case for liverbiopsy, as it represents only 1 ⁄ 50 000th of the wholeorgan.29 Liver biopsy sampling variability has beenstudied infrequently. Previous studies have estimatedthe minimum size of biopsy required for accuratestaging of liver disease.24,25,30–33 The traditionalpractice of requiring biopsies with six (interlobular)portal tracts or biopsies �1–1.5 cm long (which wasconsidered previously to be the minimum sample sizerequired for histopathological assessment) has beenfound to be incorrect for the staging of liver disease.29

As this study is limited to investigating only sample sizerequirements within the stage category of cirrhosis(where interlobular portal tracts are replaced by septa),

30

25

20

15

10

00 10 20 30

Reference CPA (%)

Reference CPA (%)

40 50

5

Sam

ple

size

req

uire

d fo

r 70

% o

f vir

tual

biop

sies

to b

e w

ithin

5%

CPA

(m

m2 )

A scatter plot of the reference CPA of a case againstthe sample size required for 70% probability

A scatter plot of the reference CPA of a case againstthe sample size required for 75% probability

A scatter plot of the reference CPA of a case againstthe sample size required for 80% probability

A scatter plot of the reference CPA of a case againstthe sample size required for 90% probability

30

25

20

15

10

00 10 20 30

Reference CPA (%)40 50

5

Sam

ple

size

req

uire

d fo

r 75

% o

f vir

tual

biop

sies

to b

e w

ithin

5%

CPA

(m

m2 )

30

35

25

20

15

10

00 10 20 30 40 50

5

Reference CPA (%)

30

35

40

50

45

25

20

15

10

00 10 20 30 40 50

5

Sam

ple

size

req

uire

d fo

r 80

% o

f vir

tual

biop

sies

to b

e w

ithin

5%

CPA

(m

m2 )

Sam

ple

size

req

uire

d fo

r 90

% o

f vir

tual

biop

sies

to b

e w

ithin

5%

CPA

(m

m2 )

Figure 3. All aetiologies together: on a case by case basis (n = 120), a strong correlation is seen between the CPA of the reference tissue

area (100 mm2) and the minimum sample size calculated from the virtual biopsies for different probability levels: 70% (R = 0.67;

P < 0.0001), 75% (R = 0.64; P < 0.0001), 80% (R = 0.62; P < 0.0001) and 90% (R = 0.58; P < 0.0001).

426 A R Hall et al.

� 2012 Blackwell Publishing Ltd, Histopathology, 62, 421–430.

we can evaluate only the area of biopsy with regard tosample size adequacy rather than the number of portaltracts.

It should be noted that the internal diameter of abiopsy needle is only the theoretically maximumpossible fresh tissue sectional diameter, and the actualbiopsy diameter of formalin-fixed histological prepara-tions is considerably less. For a 14G needle (internaldiameter �1.6 mm), the actual average core width in ahistological preparation is 0.9 [standard deviation(SD) ± 0.3].34 As shown in this study, the actualaverage core width of a histological preparation from16G Menghini needle (internal diameter �1.4 mm)biopsies is 0.8 mm (range 0.5–1.1 mm), and for the19G needle (internal diameter �0.9 mm) actual aver-age core width is 0.6 mm (0.4–0.7 mm). The 19G Tru-Cut needle was also shown by Cholongitas et al.35 toproduce a biopsy width of 0.6 mm (range 0.5–0.8 mm)in a much larger cohort (n = 312). These figuresindicating shrinkage of tissue after paraffin processingare in agreement with Menghini, who showed origi-nally that a 15G Menghini needle (internal diameter�1.5 mm) gives an average core width of0.75 mm.36,37

According to Colloredo et al.,25 for a reliable (94%)evaluation of staging (and grading) a 2 cm long–1.4 mm wide sample is recommended to ensure therequired minimum number of portal tracts (11–15portal tracts); this is a tissue area of (20 · 1.4 mm)28 mm2, assuming that the fixed section width is1.4 mm, rather than this width being the internaldiameter of the needle. Thus, given the actual diameterof fixed specimens, using a 16G Menghini needle, thelength needs to be greater than that reported previ-ously.25,34–37 Using our data on biopsy widths offormalin-fixed histological preparations, and that fromother studies,34–37 the 28 mm2 (20 · 1.4 mm) tissuesample proposed by Colleredo et al.25 would require alength of 35 mm from a 16G needle or 47 mm from a19G needle biopsy. Similarly, in patients with cirrhosiscaused by HCV and HBV, at the time of transplant wefound that the proportion of 28 mm2 virtual biopsieswith a CPA that falls within 5% CPA of the referencevalue is 94 and 95%, respectively. The recommendedlength of liver biopsy required for reliable staging andgrading in chronic viral hepatitis is thus similar to thatrequired for CPA measurement of fibrosis in cirrhoticlivers caused by chronic viral hepatitis.

We used the same material here as in our earlierstudy of the distribution of fibrosis in cirrhotic livers.19

In our previous study of cirrhotic explanted livers at thetime of transplantation, aetiology was related to CPA,with ALD showing the highest CPA (median 30%)

followed by PBC (23.5%), PSC (22.5%), then AIH(18.5%), HCV (17%) and HBV (16.5%). In the presentstudy, using 100 mm2 of cirrhotic parenchymal tissuefrom the same tissue sections as before, ALD showedthe highest CPA (median 32.5%), followed by PBC(24.8%), PSC (23.1%), then AIH (20%), HCV (18%)and HBV (17.5%). The close similarity between CPAresults from each 1 cm2 area studied in the presentwork, and in the previous study where whole sectionalareas were measured (median 248 mm2), demon-strates the robustness and reproducibility of the CPAmethod.8 First, using 100 mm2 as the reference CPA inthe present work can itself be considered to representadequately the larger whole sectional CPA. Bedossaet al.24 also showed that for cirrhosis (METAVIR F4)there was the equivalent of more than 90% sensitivityfor virtual biopsies of more than 100 mm2 in compar-ison to their reference fibrosis area of 495 mm2

(estimated from their Figure 5a). In the study24 therange of fibrosis area for a correct F4 classificationwas ± 5.2%, which is similar to our probability levelcalculation. Secondly, the same author (A.H.) carriedout the CPA analysis for our previous19 and the presentstudies. The close similarity of whole-section CPA inthe previous work and the reference CPA in the presentstudy indicates low intraobserver error associated withour CPA measurement technique.

To date, most studies of the adequacy of liver biopsyspecimens have been based on chronic hepatitis (pre-dominantly HCV), evaluating histological scoring ofstage and grade.24,25,30–33 Only Bedossa et al.24 havepreviously explored sample size with regard to quan-tification of fibrosis employing DIA of the fibrosis area,the methodology of which is similar to our DIA of CPA,with picro-Sirius red staining of chronic HCV liverdisease samples (though no manual editing38 of struc-tural collagen, as in our technique, was performed byBedossa et al.). In their study there were 17 cases ofHCV (three with cirrhosis, METAVIR F4) with thefollowing reference values (mean fibrosis area ± stan-dard error of the mean): stage F0, 2 ± 0.14%; F1,3.4 ± 0.25%; F2, 5.8 ± 0.7%; F3, 14.7 ± 0.77%; andF4, 25.1 ± 1.44%.24 In previous work by our group,Calvaruso et al.8 reported that in TJ biopsies from post-orthotopic liver transplant HCV patients, the CPAvalues that correspond to Ishak stages 0, 1, 2, 3, 4, 5and 6 were 2.7, 3.7, 5.8, 6.6, 9.4, 14 and 22.6%,respectively. When comparing the METAVIR to Ishakstages, as proposed by Rozario et al.39 (F0 = Ishak 0,F1 = Ishak 1, F2 = Ishak 2, F3 = Ishak 4 ⁄ 5 andF4 = Ishak 6), there is a reassuring similarity betweenthe values presented by Bedossa et al.24 and the workby Calvaruso et al.8 (Table 2), suggesting that routine

Sample requirement for CPA 427

� 2012 Blackwell Publishing Ltd, Histopathology, 62, 421–430.

clinical biopsy material can be used to measure CPAreliably across the disease stage spectrum (at least ofHCV), and that the biopsy sample size adequacy criteriathat we applied in our previous work are satisfactory.The results of our present study using virtual biopsies forbiopsy adequacy calculations are concordant with thebiopsy sample size adequacy criteria that we applied toroutine clinical biopsy material in our previous study.

The detailed study by Bedossa et al.24 comprised10 659 virtual biopsies derived from 17 cases of HCVresection specimens. As calculated from Figure 5a ofthat study,24 it was shown that in order to assessfibrosis correctly, as scored by the METAVIR system,75% of virtual biopsies were staged correctly forcirrhosis, F4 with a 19 mm length virtual biopsy1 mm wide (an analysable tissue area of 19 mm2

equivalent to a 24 mm length specimen from a 16Gneedle or a 32 mm length specimen from a 19Gneedle). Cirrhosis F4 was determined as area of fibrosisrange 19.9–30.2%, i.e. a 75% probability that allvirtual biopsies were ±5.2% fibrosis area of thereference value. We found that in our explanted liverswith cirrhosis due to HCV, a sample size of 12 mm2 ofanalysable tissue (16G needle: 15 mm length speci-men, 19G needle: 20 mm length specimen) is requiredfor a 75% probability level that the measured CPA willbe within 5% of the reference CPA. The difference inminimum sample requirements for the comparable

probability level may be due to methodological differ-ences between our study and that of Bedossa et al.: (i)in our study we avoided large septal tracts, bloodvessels and capsule as part of the reference CPA areacriteria (this was carried out as it is edited from ourroutine CPA analysis via a manual editing step);8,38

and (ii) Bedossa et al.24 used contiguous 2.5 mm2 areasfor fibrosis area measurements, whereas in the presentstudy we used random sampling. The additionalmaterial, available online, shows the results and figuresfor contiguous selection of virtual biopsies; these aresimilar to those produced through random selection ofthe subcomponents of virtual biopsies. The resultspresented in the main body of this study (simplerandom selection without replacement of the virtualbiopsy subcomponents) represent the method of dataanalysis advised as the most appropriate statistically.

We found that adequate sample size differs betweenaetiologies of cirrhosis for each of the levels of proba-bility we evaluated (70%, 75%, 80%, 90%). For 90%confidence that the biopsy CPA will be within 5% ofreference CPA, a biopsy size measuring 22 mm2 ofanalysable tissue is required for HBV, 23 mm2 for HCV,AIH and PSC, 27 mm2 for PBC and 28 mm2 for ALD.This would require a 24–31 mm analysable length ofbiopsy on a formalin-fixed histological preparation froma 14G needle, 28–35 mm from a 16G needle, and 37–47 mm from a 19G needle, according to the biopsywidths of formalin-fixed histological preparations.34–37

The systematic review by Cholongitas et al.40 com-piled data from 32 studies, and found that the meanlength of percutaneous liver biopsies (n = 8615) was14.3 mm (SD ± 3.2 mm, mean area with a 16Gneedle = 11.4 mm2) for Tru-Cut needles (often pre-ferred by radiologists performing biopsies), and19.9 mm (SD ± 6.6 mm, mean area with a 16Gneedle = 15.9 mm2) for Menghini needles. Therefore,to obtain optimal samples, two passes would be needed,which is not standard clinical practice.40 Based onthese data, in cirrhotic livers at the time of transplan-tation, depending on the aetiology of disease, a singlepercutaneous biopsy of analysable tissue from a 16GTru-Cut needle gives a probability level of between 68and 74%, and from a 16G Menghini needle of between79 and 85%, that the CPA results will be adequatelyrepresentative.

According to previous studies35,41 the median com-bined length with three passes of a 19G TJ needle is22 mm35 (range 3–46 mm; median area 13.2 mm2,range 1.8–27.6 mm2) and with four passes 23 mm41

(range 2–46 mm, median area 13.8 mm2, range 1.2–27.6 mm2). In cirrhotic livers at the time of transplan-tation, depending on the aetiology of disease, four

Table 2. Comparison of previous studies of liver biopsycollagen proportionate area (CPA) and fibrosis area accordingto stage of hepatitis C virus chronic liver disease

Ishakgrade CPA mean (SD)

METAVIRgrade

Fibrosis areamean (SEM)

0 2.7% (±2) F0 2% (±0.14)

1 3.7% (±3) F1 3.4% (±0.25)

2 5.8% (±3.5) F2 5.8% (±0.7)

3 6.6% (±4.1) Noequivalent

Noequivalent

4 9.4% (±4.8) F3 14.7% (±0.77)

5 14% (±8.1) F3 14.7% (±0.77)

6 22.6% (±11.3) F4 25.1% (±1.44)

SD, Standard deviation; SEM, standard error of the mean.

Comparison of METAVIR to Ishak stage systems as sug-gested by Rozario et al.39

Collagen area of METAVIR stages versus CPA of Ishak stagein chronic HCV, taken from Bedossa et al.24 and Calvarusoet al.,8 respectively.

428 A R Hall et al.

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passes of a 19G TJ needle of analysable tissue gives aprobability level between 73 and 80% (three passes71–79%) that the CPA results will be adequatelyrepresentative.

Taking all 120 individual cases separately, there wasa significant linear correlation between reference CPAand minimum sample size for the 70%, 75%, 80% and90% probability levels, i.e. there is a relationshipbetween overall quantity of fibrosis (CPA) in eachcirrhotic liver at the time of transplantation and thesample size required for a given probability level.

In a theoretical liver that consists entirely of fibroustissue (100% CPA) the minimum sample size would bevery small (e.g. 1 mm2); a similar situation pertainswhen there is no fibrosis at all (0% CPA), as all the1 mm2 components of the virtual biopsy will haveidentical CPA measurements. The correlation we pres-ent here shows a positive linear distribution with anincrease in CPA related to an increase in the samplesize required. If we had cases with >50% CPA we mightexpect that the minimum sample size requirementwould return to a low size at very high CPA levels, asthe distribution of fibrosis should approach homogene-ity. This suggests that the sample size to reference CPArelationship, rather than being linear (as in the rangeof reference CPA represented in our present studycohort) over the full theoretical range of reference CPA(0–100%), is likely to be bell-shaped. In the presentstudy of cirrhotic livers at the time of transplantationthere were no cases with a reference CPA >50%, andmore variable intrasample CPAs with larger minimumsample sizes for adequacy were observed in the higherreference CPA cases.

Our findings are presented as probabilities that canbe related to biopsies of varying sizes rather than adiscrete cut-off point between adequate and inadequatesamples; in essence, quantifying the level of uncer-tainty. We believe this is more appropriate thandemanding artificial levels of probability. Differentlevels of confidence can thus be chosen that areappropriate to the clinical situation or study asrequired. It is important to note that the sizes ofbiopsies given in this study are referring to analysablelengths of cirrhotic tissue (not macroscopic biopsydescriptions) and the data should be understood withinthis context.

Conclusion

Increasing sample size will inevitably increase thereliability of CPA results. However, above 20–25 mm2

biopsy size (16G needle biopsy with 25–31 mm lengthof specimen, 19G: 33–42 mm length of specimen), the

incremental improvement in probability that the biopsyCPA will be within 5% of the actual (reference) CPAdiminishes. CPA measured in a properly performed TJbiopsy sample (four passes of a 19G needle, i.e. yielding13.8 mm2 of tissue from a median 23 mm specimenlength) taken from a cirrhotic liver at the time oftransplantation can be expected to be within 5% of theactual CPA in 73–80% of patients (depending on theaetiology of cirrhosis).

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Proportion of biopsies within 5% ofreference CPA plotted against virtual biopsy (contigu-ous selection) size.

Table S1. Sample size requirements for selected %proportions of biopsies that fall within 5% CPA usingcontiguous selection of virtual biopsies.

Please note: Wiley-Blackwell is not responsible forthe content or functionality of any supporting materi-als supplied by the authors. Any queries (other thanmissing material) should be directed to the correspond-ing author for the article.

430 A R Hall et al.

� 2012 Blackwell Publishing Ltd, Histopathology, 62, 421–430.