MAKERERE

UNIVERSITY

EVALUATION OF THE PERFORMANCE OF ALERE PIMA

CD4 TEST IN DETERMINING ART ELIGIBILITY AMONG
ADULTS IN UGANDA.

GODFREY P. MUJUZI, BBLT (MAK)

DISSERTATION SUBMITTED IN PARTIAL FULLFILLMENT OF THE

REQUIREMENT FOR THE AWARD OF MASTER OF SCIENCE IN CLINICAL
EPIDEMIOLOGY AND BIOSTATISTICS OF MAKERERE UNIVERSITY KAMPALA

July 2016

DECLARATION

I hereby declare that this work is a product of my own effort and does not contain unreferenced
content copied from somewhere else. To the best of my knowledge the research topic undertaken
has never been published or submitted to a university or tertiary institution for purposes of
academic degree or other awards in its current form.

Signed
..
Godfrey P. Mujuzi

Supervisors

Prof Charles Karamagi: MBChB, MMeD, PhD

Dr. Baveewo Steven MBChB, MSc CEB

ACKNOWLEDGEMENTS
I am grateful to the Almighty God for the immense grace and support accorded to me in the
accomplishment of this work.
The guidance and support from my supervisors especially Prof Charles Karamagi right from the
development of the proposal to the writing of the final dissertation is highly appreciated.
My gratitude goes to Ms Jane Ndibaza, Noor Nansubuga, Shem Mwebaza, Ivan Ssekidde and the
entire technical team in the Mildmay Uganda laboratory for helping in sample collection and
analysis. May the almighty God reward you abundantly.
The tireless efforts of lecturers in the clinical Epidemiology will be something I will forever
treasure in the progress of my career.
I do thank Mrs. Christine Kusasira Maholo for all the help accorded to me and also to my
classmates of CEU 2012-2014, Wekiya Enock, Moses Matovu Aidah Nakawunde, Esther Nambi,
Shamim Namukasa, Denis Opio Nixon and Steven Abwoye. May God Bless you all.
I am indebted to the clients seeking care at Mildmay Uganda who accepted to participate in the
study and to the entire Mildmay Uganda counseling team who provided counseling support to the
study participants.
My deepest gratitude goes to my family; wife Immaculate, daughters Patricia and Preita Angella,
sons Nicholas and Primo Victor for their unflagging love and support throughout my life and for
allowing borrow a lot of their time to complete this dissertation.

TABLE OF CONTENTS

DECLARATION................................................................................................................ i
ACKNOWLEDGEMENTS.................................................................................................. ii
Table of Contents.......................................................................................................... iii
LIST OF ABBREVIATIONS............................................................................................... vi
List of Appendices........................................................................................................ vii
ABSTRACT................................................................................................................... viii
CHAPTER ONE............................................................................................................... 1
INTRODUCTION............................................................................................................. 1
1.0 Background.......................................................................................................... 1
1.1 Problem statement............................................................................................... 4
1.3 Conceptual Framework......................................................................................... 6
1.4 Study objectives................................................................................................... 7
1.4.1 Overall objective:........................................................................................... 7
1.4.2 Specific objectives:........................................................................................ 7
CHAPTER TWO............................................................................................................... 8
LITERATURE REVIEW...................................................................................................... 8
2.0 HIV/AIDS burden................................................................................................... 8
2.1 HIV disease monitoring........................................................................................ 8
2.2 CD4+ Lymphocytes.............................................................................................. 9
2.2.1 Physiological variability in CD4 count............................................................9
2.2.2 CD4 as a prognostic marker for HIV progression.........................................10
2.3 Methods for enumerating CD4+ cells.................................................................11
2.4 Validation of CD4 testing Technologies...............................................................13
3

2.5 The Pima point of care....................................................................................... 15

CHAPTER THREE.......................................................................................................... 16
Methods and Materials................................................................................................ 16
3.1 Study site and design......................................................................................... 16
3.2 Population.......................................................................................................... 16
3.2.1 Target population:........................................................................................ 16
3.2.2 Accessible population..................................................................................16
3.2.2 Study population.......................................................................................... 17
Inclusion criteria....................................................................................................... 17
Exclusion criteria...................................................................................................... 17
3.2.3 Sampling procedure..................................................................................... 17
3.2.5 Sample size determination...........................................................................17
3.2.6 Study Variables............................................................................................ 18
3.5 Data collection and management......................................................................18
3.4 Laboratory Methods:.......................................................................................... 19
3.4.1 Sample collection......................................................................................... 19
3.4.2 Sample analysis........................................................................................... 19
3.6 Statistical analysis.............................................................................................. 20
3.6 Quality control.................................................................................................... 21
3.7 Ethical consideration.......................................................................................... 21
CHAPTER FOUR........................................................................................................... 22
Results......................................................................................................................... 22
CHAPTER FIVE............................................................................................................. 25
Discussion................................................................................................................... 25
5.1 Sensitivity and Specificity of PIMA......................................................................25
4

5.2 Positive and Negative predictive values of PIMA................................................25

5.3 Positive and Negative likelihood Ratios of PIMA.................................................26
5.3 Study Implications and Limitations....................................................................27
CHAPTER SIX............................................................................................................... 28
Conclusions and Recommendations............................................................................ 28
6.1 Conclusions........................................................................................................ 28
6.2 Recommendations.............................................................................................. 28
Reference List.............................................................................................................. 29
Appendices.................................................................................................................. 39

LIST OF ABBREVIATIONS

AIDS:

Acquired Immunodeficiency Syndromes

ART:

Antiretroviral therapy

CCD:

Charge Coupled Device

CD:

Cluster of differentiation

ELISA:

Enzyme Linked Immunosorbent Assay

FN:

False Negative.

FP:

False Positive

HAART:

Highly Active Anti Retroviral Therapy

HIMS:

Hospital Information Management System

HIV:

Human Immune Virus

LIMS:

Laboratory Information Management System

POC:

Point of Care

TLC:

Total Lymphocyte Count

LIST OF APPENDICES
Appendix A: Informed Consent
Appendix B: Data collection Tool

Appendix C: Collection of blood samples

Appendix D: Procedure for preparation of controls and samples for analysis using BD
FACSCalibur
Appendix E: Procedure for running samples and controls using BD FACSCalibur.

Appendix F: Procedure for running samples and controls using PIMA

ABSTRACT
Background Access to CD4 testing remains a major bottleneck to the scale up of HIV
antiretroviral treatment services and the attainment of the UNAIDS 90-90-90 targets.

Point-of-

care CD4 testing with properly defined performance characteristics can improve pre-ART HIV
care by reducing time to eligibility assessment by bringing CD4 testing close to the people in
peripheral sites.
Objective: This study assessed the efficiency of PIMA point of care CD4+ testing device in
identifying ART eligible subjects using the now recommended cut off of 500 CD4+ cells/l.
Methods: Duplicate analysis of samples was performed on the FACScallibur flow cytometer and
PIMA POC device. Using the results of the FACScallibur as a reference, the sensitivity,
specificity and positive/negative predictive values and likelihood ratios were calculated using
standard methods.
Results: At the threshold of 500 CD4 cells/L, PIMA capillary blood had a sensitivity of 90.8%
(95% CI, 85.3-94.8%), specificity of 84.4% (95% CI, 76.490.5%), NPV of 86.6% (95% CI, 78.892.3%), and PPV of 89.2% (95% CI, 83.4-93.5). The positive and negative likelihood ratios were
5.8 (95% CI, 3.78-8.89) and 0.11 (95% CI, 0.07-0.18) respectively.
Conclusions: The PIMA point of care can be useful in pre ART HIV/AIDS care as it correctly
classifies over 80% of pre ART subjects in regard to ART eligibility. The likelihood of being ART
eligible increases six-fold given a PIMA CD4 count of 500cells/l of blood positive test result.

CHAPTER ONE
INTRODUCTION
1.0 Background
Infection with the Human Immunodeficiency Virus type 1 (HIV-1) and type 2 (HIV-2) is the
primary cause of the Acquired Immunodeficiency Syndromes (AIDS). HIV-1 accounts for the
majority of cases worldwide (Clavel et al., 1986; Barre-Sinoussi et al., 1983; Broder & Gallo,
1984; Gallo et al., 1984). The course of an infection with HIV may be divided into four distinct
stages. The first phase is the incubation period, which is asymptomatic and may last 2-4 weeks.
This is followed by the acute phase of infection lasting an average of 28 days characterized by
high viral replication and high viral load, which may be accompanied by nonspecific symptoms
including lymphadenopathy, pharyngitis, skin rashes, myalgia and malaise(Soogoor & Daar,
2005). After the acute infection, there is a reduction in plasma viral load probably due to the
production of Anti HIV specific antibodies and T-cells leading to the latency phase that lasts for
several years (Lutalo et al., 2007; Van der Paal et al., 2007) . During this period there is
gradual destruction of the immune system especially as a result of HIV infecting and destroying
CD4+ cells including the T-helper cells. When the CD4+ cells fall below a critical number,
immunity is greatly compromised leading to a wide array of infections and cancers
characteristic of AIDS.

The suppression of HIV viral replication by effective highly active anti retroviral therapy
(HAART) results in considerable restoration of the immune function and prolongs the survival
1

of HIV infected individuals (Cooper, 1996; Palella, Jr. et al., 1998; Patel et al., 2008; Wendland
et al., 1999). Unfortunately this lifelong treatment is associated with a wide array of serious
and sometimes fatal side effects (Carr et al., 1998; Subbaraman, Chaguturu, Mayer, Flanigan, &
Kumarasamy, 2007). HAART may be delayed until the CD4+ cell count has fallen to a certain
point below which there is considerable risk of developing AIDS defining symptoms(Phillips,
Lepri, Lampe, Johnson, & Sabin, 2003) as very early initiation increases the risk of developing
drug resistant strains as a result of poor adherence , late initiation is associated with poor
treatment outcomes(Severe et al., 2010).

Sub-Saharan Africa bears the greatest global burden of human immunodeficiency virus (HIV)
infection constituting approximately 70% (25.0 million) of all persons living with HIV/AIDS.
Approximately 1.5 million of these are Ugandans (Joint United Nations Programme on
HIV/AIDS & Global report, 2013a). Measurement of CD4+ cells is the principal laboratorybased method for determining HAART eligibility. The world health organization (WHO) from
time to time, based on available clinical and epidemiological data provides guidelines for
initiation ART and countries adopt WHO recommendations for a certain value of CD4+ cell
count as a cut off below which HIV subjects are deemed HAART eligible. Uganda is
implementing the 2013 guidelines and moving towards the

current WHO test and treat

guidelines(Ministry of Health Uganda, 2013).

Flow cytometry, which is generally considered as the gold standard for CD4+ cell enumeration is
2

a very expensive technique that requires highly trained laboratory personnel and can only be
performed in well built, often urban or centralized laboratory facilities with a long turnaround
time for results(Peter et al., 2008). Decentralization of HIV treatment to lower health facilities is
vital in increasing access to HIV/AIDS care. The lack of CD4+ testing at lower health facilities
has therefore remained a hindrance to the access of comprehensive HIV care and HAART. Point
of care devices such as the PIMA (Inverness Medical Innovations) provide a suitable alternative
as they are cheap, easy to operate, require minimal training and can be performed at lower health
facilities because they do not need a purpose built laboratory facility or highly trained personnel
and runs on a rechargeable battery (Mtapuri-Zinyowera et al., 2010) .
Although the Ministry of Health introduced the PIMA point of care devices for CD4+ cell
enumeration at lower health facilities in order to increase access to HAART, there is limited
data regarding the performance of these devices compared to the standard of care flow
cytometers. Studies that have evaluated the performance of PIMA have used other thresholds
for ART eligibility and not 500 CD4+ cells/l (Wade et al., 2014; Rathunde, Kussen,
Beltrame, Dalla, & Raboni, 2014; Su et al., 2013; Morawski, Meya, & Boulware, 2013a). A
study conducted in Rakai showed differing performance characteristics of the PIMA POC
when different cutoffs [350 or 500 CD4+ cells/l] were used. Sensitivity and positive
predictive values were reported to have increased at a higher cutoff of 500 CD4+ cells/l.
(Galiwango et al., 2014a). In this study, sensitivity at a threshold of 350 cells/l was
reported as 88.6% and specificity as 87.5%. The sensitivity and positive predictive values
improved to 96.1% and 88.3% at a threshold of 500 cells/l(Galiwango et al., 2014a).
This study unfortunately included HAART experienced subjects making it difficult to
3

generalize the findings to an HAART naive population. The aim of this study was to define
performance characteristics of the PIMA POC in a properly selected study population.

1.1 Problem statement

There is an estimated 1.5 million Ugandans leaving with HIV/AIDS with only approximately a
half of the 1 million who need HAART receiving this life saving treatment(Joint United Nations
Programme on HIV/AIDS & Global report, 2013b). Uganda has adopted the WHO
recommendation to start HAART at CD4+ 500 cells/l in asymptomatic subjects aged 15
years and above except in some special cases(Uganda AIDS comission, 2015). In Uganda,
there are only few laboratories that are able to determine CD4+ cell counts using
flowcytometry. In order to increase access to CD4+ testing, the Ministry of Health has recently
adopted the use of PIMA point of care CD4+ analysers at lower public health care
facilities(Morawski, Meya, & Boulware, 2013b). There is however limited data regarding the
performance characteristics of the PIMA compared to the gold standard flowctometry. Although
individuals may be ART eligible if their CD4 count is tested using flow cytometry the use of
PIMA machines may determine them as ineligible, no study has been done in Uganda to assess
the efficiency of Pima point of care CD4 testing device in identifying ART eligible subjects
in an ART naiv e population using the 500 CD4+ cells/l threshold in Uganda.
Misclassification as a result of using the PIMA can result in considerable expenditure in
management of opportunistic infections due to late initiation of ART that would have been
averted if ART was initiated early.

1.2 Justification
CD4 counts remain the major determinant for to initiate Anti-retroviral therapy. Despite
this, there is limited access to CD4 testing due to complexity of methods, facilities and need
for highly trained personnel to perform these tests. The PIMA point of care device is an
alternative to the more sophisticated flow cytometry systems. PIMA CD4 is more affordable,
technically simple, can use either electricity or battery, is fully automated and thus, useful in
remote areas in order to reduce the turnaround time for initiating ART(Belec & Bonn, 2011).
Despite the advantages that PIMA point of care device provides, there is need to determine its
diagnostic characteristics to understand the nature and direction of bias/misclassification that may
accrue compared to the use of flow cytometry in determining HAART eligibility. This study
therefore determined the sensitivity, specificity, positive and negative predictive values and
likelihood ratios of the PIMA point of care device compared to flow cytometry in
identifying ART eligible subjects at CD4 500 cell/l.

Personnel
Training
Experience

1.3 Conceptual Framework

The conceptual framework (Figure 1) identifies potential factors that may affect the availability
and quality of CD4 results. These include the level of training and technical expertise required to
perform the test, type of equipment used, types and volume of samples required, the facilities
Availability of reliable CD4 testing Service.
needed to support equipment function, availability of reagents and cold chain storage as well as
equipment costs, complexity and availability of service engineers.

All these may affect the

availability and quality of CD4 testing and its use in HIV/AIDS care.

Sample
Types
Volumes

Figure 1: Conceptual Framework.

1.4 Study objectives

1.4.1 Overall objective:
To determine the diagnostic characteristics of PIMA compared to the flow cytometry
method in determining CD4 count for ART eligibility.
1.4.2 Specific objectives:
1.

To determine the sensitivity & Specificity of the PIMA in determining ART eligibility

among ART naive HIV positive subjects attending Mildmay Uganda clinic at Lweza.

2. To determine the positive and negative Predictive Values of the PIMA while determining
ART eligibility among ART naive HIV positive subjects attending Mildmay Uganda clinic at
Lweza.

3. To determine the positive and negative likelihood ratios of the PIMA while determining
ART eligibility among ART naive HIV positive subjects attending Mildmay Uganda clinic at
Lweza.

CHAPTER TWO
LITERATURE REVIEW
2.0 HIV/AIDS burden
The Human immunodeficiency virus (HIV) is the causative agent for the life threatening clinical
condition of acquired immunodeficiency syndrome (AIDS). The estimated number of people
living with HIV globally increased to 35.5 in 2013 from 34 million in 2012. Sub-Saharan Africa
bears the greatest global burden of human immunodeficiency virus (HIV) infection constituting
approximately 70% (25.0 million) of all persons living with HIV/AIDS. Approximately 1.5
million Ugandans are living with HIV/AIDS. Prevalence is higher (10.1%) in urban areas
compared to 5.7% found in rural areas and higher among women (7.5%) compared to men
(5.0%). Fortunately AIDS related deaths among people living with HIV have decreased by 30
percent due to increased access to antiretroviral treatment (HAART). (Joint United Nations
Programme on HIV/AIDS & Global report, 2013c).

There has been a significant effort to scale

up access to antiretroviral therapy (ART) in Uganda, alongside prevention, care, and social
support services. ART was formally introduced into the public health system in Uganda in
2003 and has since been progressively scaled up to reach even rural health care settings. By
November 2011, approximately 700,000 people were estimated to be clinically eligible for
8

ART, of which almost 43% (300,000) were accessing treatment.

2.1 HIV disease monitoring
HIV has particular tropism for CD4 cells and causes progressive destruction of CD4+ cells in an
HIV infected individual.

When the CD4+ cells fall below a critical number, immunity is

greatly compromised leading to a wide array of infections and cancers characteristic of the last
stage - AIDS. Measurement of CD4+ T lymphocytes in peripheral blood is therefore a critical
laboratory parameter for the evaluation and monitoring of patients with HIV infection and for
determining ART eligibility.
CD4 counts are used for determining the immunological stage of HIV infection, identifying when
to start antiretroviral therapy (ART), to identify patients likely to benefit from co
trimoxazole/Fluconazole prophylaxis and to recognize those most at risk of developing immune
reconstitution syndrome. In addition, CD4 may be used in evaluating the response to treatment
and for recognizing treatment failure.
2.2 CD4+ Lymphocytes
CD4+ Tlymphocytes, also known as the helper Tcells, are the coordinators of the immune
response which protects the body against microbial disease and some forms of cancer. The
destruction of CD4+ Tlymphocytes by HIV is the main cause of the progressive weakening of
the immune system in HIV infection, and ultimately leads to the acquired immune deficiency
syndrome, AIDS.

2.2.1 Physiological variability in CD4 count

Changes in CD4+ counts in HIV infected individuals are not exclusively due to HIV disease
progression. For example CD4 counts are typically lower in the morning than in the evening
with variations as high as 60% in HIV negative subjects and about 15% in HIV
positive subjects(Fei, Paxton, & Chen, 1993; Malone et al., 1990).
Weektoweek and seasonal variation may occur, and changes in CD4 count may also be seen
related to exercise, smoking, concurrent infection, and some medications other than ART (e.g.
corticosteroids). Changes in total white cell count and lymphocyte count may contribute more to
the fluctuations in absolute CD4 count seen in response to these biological factors than does the
CD4% (Giorgi, 1993a; Malone et al., 1990). These physiological intra-subject variations in
CD4 count may account for as much as, if not more than, technical variability of CD4
measurement (Raboud et al., 1995; Mandigo, Raboud, Haley, & Montaner, 1995).

2.2.2 CD4 as a prognostic marker for HIV progression

CD4 count is a strong predictor of HIV progression to AIDS and death, and is by consensus
considered the best laboratory marker for deciding when to initiate antiretroviral treatment
(ART) (Giorgi, 1993b; Sterling, Chaisson, & Moore, 2001). There is much heterogeneity in rate
of CD4 decline, with a minimum period between seroconversion and AIDS of about 2 years, and
a median of 11 years (Giorgi, 1993b). In addition, the rate of decline in CD4 count for an
individual patient may be highly variable over time(Wolbers et al., 2010), making it difficult
to predict at higher CD4 counts the likely time course of an individuals progression to
advanced disease.
10

The use of clinical staging alone to determine ART initiation is limited by the uncertain
prognostic value of asymptomatic or mild disease. In a Malawian setting, 56% of 1663 patients
with WHO clinical stage 1 or 2 disease had CD4 counts of <350 cells/l, and 36% had CD4
counts <250 cells/l(Tayler-Smith et al., 2010).

Regular monitoring of CD4 counts, in

conjunction with clinical criteria, is therefore recommended for monitoring HIV progression,
with treatment initiation indicated when CD4 drops to below a defined threshold. From time to
time, the World Health Organization issues updated guidelines for the management and
treatment of HIV/AIDS. Uganda is still implementing the 2013, WHO guidelines that provided
500 CD4+cells/l as the ART initiation threshold as plans to rolling out the current WHO test
and treat guidelines for all populations.

2.3 Methods for enumerating CD4+ cells.

Several manual and automated systems are available for the enumeration of CD4+ lymphocytes.
The automated systems comprise flow cytometry platforms that can be used for several flow
cytometery

applications

including

CD4+

T-

cell

enumeration( B r o w n &

Wittwer, 2000; Mengarelli, Fryga, & Barberi, 2013)

and the dedicated

cytometers that are designed for the enumeration of limited range of cells usually CD4+ and
CD8+ cells(Strauss et al., 1984).

The manual methods require the use of a microscope or

ELISA to estimate the number of circulating CD4/CD8+ cells(Carriere et al., 1999; Nouanthong,
Pata, Sirisanthana, & Kasinrerk, 2006a). New manual technologies based on micro-arrays and
11

dipsticks are being developed but are not yet available commercially for clinical use(Wu et al.,
2007).

The gold standard technology for CD4+ Tcell counting is flow cytometry, a technology that
allows a single cell to be measured for a variety of characteristics. Flow cytometers are used
in cell counting or cell sorting applications (Nunez, 2001). For CD4+ T cell enumeration, cells
are stained with fluorescence tagged antibodies to cell surface markers. The standard
methodology for determining the percentage of CD4+ cells is by staining for CD45/CD3/CD4.
By first selecting the lymphocytes (CD45), the CD3/CD4 cytogram can be used to enumerate
CD4+ cells.
Generally flow cytometers give relative percentages of CD4+ or CD8+ cells present in
the sample analysed. Absolute counts are then determined using several approaches e.g. the
volumetric approach in which a known volume of sample is analysed or the bead based systems
in which a fixed volume of sample is mixed with a known number of fluorescent beads. Since
the fluorescent beads can easily be differentiated from cells by the flow cytometer, the counted
beads of the sample analysed permits volume determination and hence the concentration of cells.
The volumetric approach or systems using fluorescent beads are called single platform
approaches and are commercially available as TruCount (Becton Dickinson, USA), FlowCount
beads (Beckton Coulter) and Perfect Count (Cytognos) that can be used on practically anyflow
cytometer.

12

Another approach used to calculate absolute CD4+ counts is to use a haematology analyser. The
haematology analyser measures the total white cell count. The relative percentages of each of the
types of lymphocytes is then determined from the total lymphocyte count. This dual platform
approach introduces variability in absolute CD4+ counts due to combining results of two
platforms into a single calculation. Recent developments in staining panels and data analysis
(Panleucogating) offer better results over traditional dual platform methodology, increasing the
performance of the dual platform approach up to the single platform level(Stevens et al., 2008).

The high demand for CD4 enumeration in HIV infected individuals has resulted in the
development of dedicated CD4 platforms often called closed systems in addition to the
traditional open system flow cytometers. Dedicated single platforms in use in Uganda include
the FACScount (Becton Dickinson), Guava easy CD4 (Guava techonologies) and Partec Cyflow
Counter (Partec). The dedicated platforms in comparison to open system flow cytometers have
decreased applicability but allow CD4 + T-cell counts with reduced technical complexity. The
manual methods use microscopes, haemocytometers and beads that become distinctly visible by
microscopy when in contact with CD4+ T-cells allowing cells to be identified and
counted(Nouanthong, Pata, Sirisanthana, & Kasinrerk, 2006b; Srithanaviboonchai et al., 2008).
Manual methods available commercially include Dynal T4 Quant and Coulter Manual CD4
Kit(Carella, Moss, Provost, & Quinn, 1995; Lutwama et al., 2008)
2.4 Validation of CD4 testing Technologies.
Method validation ensures that the analytical procedures in question are suitable for the intended
use.

In H I V /AIDS care, CD4 counts are the primary determinants of when to initiate
13

ART. WHO and Ministry of health guidelines regularly revise cut offs for initiation of ART
under particular conditions. Validation of new technologies should therefore ensure that results
of the new technology can correctly identify individuals due for ART at the current cut-offs as
per national and international (WHO) guidelines.

Although flow cytometry is considered the gold standard for the enumeration of CD4+ cells,
flow cytometers use different approaches in determining absolute CD4 counts. Flow cytometric
CD4 assays are generally divided into dual platform (DP) and single platform (SP) approaches.
DP methods rely on measurements from two independent instruments, a flow cytometric
platform (cytometer) and a haematology analyzer. Cell counts obtained from a haematology
analyser and the relative percentages from the flow cytometer are used to determine the
absolute CD4 counts(Glencross, Scott, Jani, Barnett, & Janossy, 2002; Mandy, Bergeron, &
Minkus, 1997a).

SP systems, including bead-based systems(Stewart & Steinkamp, 1982; Valet, 1984), and
volumetric approaches (Connelly et al., 1995; Mercolino et al., 1995) including traditional
lymphocyte reference testing(Reimann et al., 2000; Schnizlein-Bick, Spritzler, Wilkening,
Nicholson, & O'Gorman, 2000), primary CD4 gating(Janossy, Jani, & Gohde, 2000) and
PanLeucogating ( G l e n c r o s s e t a l . , 2 0 0 8 ; S t o r i e e t a l . , 2 0 0 3 ) , all rely on
measurements from the flow cytometer alone and have been shown to have significantly
improved precision over traditional DP methodologies (Barnett, Granger, Whitby, Storie, &
Reilly, 1999; Gratama, Kraan, Keeney, Granger, & Barnett, 2002). However, the precision of
14

the PanLeucogating DP method(Mandy, Bergeron, & Minkus, 1997b), which does not use the
TLC, is comparable to SP methods.

Given this variability in CD4 technologies, variability in results produced is expected and
validation is essential before new technologies are taken on. Several studies have compared
results of one flow cytometer with another or with the results of a new CD4 testing technology.
Comparisons based on correlation coefficients, that are reported in many studies ( D i d i e r e t
al.,

2001;

Kalva

Borato,

Carraro,

We b e r

Ribas,

Kalva-Filho,

&

R e b u g l i o Vel l o s a , 2 0 1 2 ) a r e inappropriate as large levels misclassification can

happen despite excellent correlations.

Some studies have reported misclassification

probabilities or data from which this more clinically significant measure of accuracy could be
calculated(Karcher, Bohning, Downing, Mashate, & Harms, 2006; Thakar, Kumar, Mahajan,
Mehendale, & Paranjape, 2006).
2.5 The Pima point of care.
The Pima CD4 is a small bench top automated analyser that uses disposable Pima test cartridge
containing dried reagents to determine CD4 counts. The Pima analyser is equipped with
miniaturized low-cost multi-colour fluorescence imaging optics. Fluorescence signals are
detected by a charge coupled device (CCD) board camera and analysed using preinstalled
software. A multicenter study conducted in Belgium and Tanzania using both capillary and
venous blood showed a relative bias of 4.1 and -9 for capillary and venous blood
respectively at Dareslam in Tanzania while at Antwerp, Belgium it was -9.4 and -9.5 for
venous and capillary blood respectively. At a threshold of 350 cells/l, PIMA
15

misclassification rates were reported as 13% (Wade et al., 2014). A study conducted in
Brazil reported sensitivity of the PIMA at 94% and specificity at 93% with negative and
positive predictive values of 86% and 97% respectively at a threshold of 200 cell/l
(Rathunde et al., 2014). A recent study in Uganda has reported sensitivity at a threshold of
350 cells/l to be 88.6% and specificity at 87.5%. The sensitivity and positive predictive
values improved to 96.1% and 88.3% at a threshold of 500 cells/l (Galiwango et al.,
2014a). These results together with findings in other areas indicate that the PIMA point of
care is a potential alternative to the flow cytometry systems but its performance may differ
depending on whether venous or capillary blood is used, testing site and that the sensitivity
improves at higher CD4 count thresholds.

CHAPTER THREE
METHODS AND MATERIALS
3.1 Study site and design
This cross sectional study aimed at evaluating the performance of the PIMA point of care CD4
analyzer was conducted at the Mildmay Uganda clinics at Lweza Entebbe road Uganda.
Mildmay Uganda is a specialist HIV/AIDS care, prevention and training centre in Uganda.
Currently Mildmay Uganda provides care to over 16,000 HIV infected clients with close to
8,000 currently receiving various HAART combinations. The voluntary HIV counselling and
16

testing program (VCT) performs about 3000 HIV tests a month with an average of 25%
positives. HIV positive individuals are recruited into care while the HIV negative individuals are
counseled about retesting and keeping negative.
3.2 Population
3.2.1 Target population:
All HIV positive clients seeking care at Mildmay Uganda and its affiliated clinics within a radius
of 60km from the main site at Lweza.
3.2.2 Accessible population
All HIV positive clients who sought care at Mildmay Uganda main site at Lweza during the
month of March 2014.

17

3.2.2 Study population

Inclusion criteria
All HIV positive ART naive a d u l t clients who sought c a r e at Mildmay Uganda and
r e q u i r e d a CD4 test for the purposes of determining ART eligibility during the study period
and gave informed consent to participate.
Exclusion criteria
All individuals who were unable to provide suitable samples for analysis on both methods
and whose results were invalid for at least one of the methods were excluded.
3.2.3 Sampling procedure
Consecutive sampling method was used during the study until the required sample size
was achieved.
3.2.5 Sample size determination
The sample size was derived from Buderers formula basing on both specificity and sensitivity
as shown below (Buderer, 1996) .

N (sn) =

= 287 and

N (sp) =

271
Where N (sn) and N (sp) were the required sample sizes based on sensitivity and specificity
respectively,

= margin of error at 5% (standard value of 0.05) and p = the proportion of

people eligible for ART among ART nave subjects visiting Mildmay Uganda and

confidence level at 95% (standard value of 1.96). It is estimated that 20% of the subjects

18

recruited at Mildmay have a CD4 count 500 cells/ l. Using the reported sensitivity and
specificity at a threshold of 500 CD4+ cells/l of 96.1 and specificity of 83 %
respectively(Galiwango et al., 2014a), we used a sample size of 287.
3.2.6 Study Variables.
The variables measured were CD4 counts, age and sex. ART eligibility was defined as a CD4
cell count of 500 cells/l of blood.
3.5 Data collection and management
Demographic data was obtained from the hospital information management system (HIMS) by
pre-trained research assistants using pretested study tools after which it was delinked from the
subject by assigning a unique study ID. Results from samples analysis from the FACScallibur
were extracted from the hospital laboratory information system by the researcher. PIMA results
were recorded in a laboratory note book by pre-trained research assistants and verified against
instrument printouts by the researcher. Results of the PIMA and FACScallibur were linked
using a unique identifier and entered into EPI data V 3.1 and exported to excel. The excel files
were imported to STATA software for analysis.

3.4 Laboratory Methods:

19

3.4.1 Sample collection

Two blood samples (3) were collected from each subject using EDTA evacuated tubes and EDTA
capillary tubes for venous and capillary blood respectively. Samples were collected using
Mildmay Uganda standard practice as detailed in the Mildmay Uganda sample collection
manual. Details of sample collection procedures are given in appendix D [extract from the
Mildmay Uganda sample collection manual].
3.4.2 Sample analysis.
Venous blood was analyzed using the BD FACScallibur and the capillary blood analyzed using
the PIMA CD4 test (Inverness Medical Innovations) yielding paired results for each subjects.
We did not analyze venous blood on the PIMA or capillary blood on the FACScalibur as this was
outside the scope of this study.
3.4.2.1 Sample Analysis using the BD FACSCalibur.
Blood was analyzed on the BD FACSCalibur following the Mildmay Uganda standard operating
procedure for the use of the BD FACSCalibur for the determination of CD4+ cell counts. Extract
on preparation and use of BD FACSCalibur is given in Appendix E and F. Briefly, 50l of EDTA
anti-coagulated blood was stained with 20l of BD MultiTest CD3/CD8/CD45/CD4 fluorescence
labeled antibody mixture in BD true count tubes for 15 minutes in the dark at room temperature
and then lysed with BD lysing solution. Analysis was done on the BD FACSCalibur using BD
MultiTest software and results directly exported into LIMS.

20

3.4.2.2 Sample Analysis using the PIMA CD4 Test

Blood was analyzed on the PIMA following the Mildmay Uganda standard operating procedure
for the use of the PIMA point of care CD4 testing device. Extract on the procedure for use of the
PIMA is given in Appendix G. Briefly, 25l capillary blood was drawn into the sample collector
by holding the cartridge at 45 until the cartridge was filled. The filled cartridge was capped and
inserted into the Alere Pima Analyzer. The Analyzer automatically starts analyzing the sample
once the cartridge is inserted and displays absolute CD4 counts after 20 minutes. The displayed
results were printed, filled and entered into the laboratory information system (LIMS). On each
day that samples were tested, equipment performance was checked using a valid Pima Bead
Standards whose results were recorded in the LIMS. Samples were tested only if the result of the
Pima Bead Standards had passed.
3.6 Statistical analysis

To determine the reliability of PIMA POC in predicting ART eligibility, subjects were
divided into those who were eligible for ART ( 500 CD4 cell/mm

3)

and those who were

3
not eligible for ART (> 500 CD4 cells/mm ) using results of FACScallibur.
classification based on the results of the FACScallibur

as the

With the

reference, subjects

were

classified as True Positive (TP), True Negative (TN), False Positive (FP) and False
Negatives (FN). W e c a l c u l a t e d p e r formance characteristics as s e n s i t i v i t y (TP
(TP+ FN) 100), specificity (TN(TN+FP) 100) and diagnostic efficiency (TP+TN)
(TP+TN+FN+FP) 100. Positive and negative likelihood ratios were determined using standard
methods(John Attia, 2003). The upward and downward misclassification probabilities were
calculated for the cutoff of 500 cells/l. The upward misclassification probability was

21

defined as one minus the sensitivity (1-Sn) and the downward misclassification probability was
defined as one minus the specificity (1-Sp) of the PIMA CD4 analyzer.
3.6 Quality control
All persons involved in the study had experience in handling human samples for diagnostic
purposes and were appropriately trained on specific study procedures. Competence
assessments were done for all staff prior to the start of the study to ensure that they had the
skills necessary for performing the tests and were familiar with the study tools. The BD
FACScallibur was calibrated using the BS calbrite beads (BD Biosciences). The quality of
reagents and the specimen processing procedures were controlled using low and normal
controls (BD Biosciences). The reference cartridge for the Pima was used to check for the
performance characteristics of the PIMA CD4 analyzer. A vial of reference material at two
different concentrations Low and Normal was run on both Pima CD4 and BD FACSCalibur as
per the manufacturers instructions.
3.7 Ethical consideration
Approval to carry out the research was obtained from the Clinical Epidemiology Unit,
Mildmay Uganda Research and Ethical Committee

and the School of Medicine

Research and Ethics Committee. Informed consent was obtained from all potential study
subjects and only those who gave informed consent were included. Information was coded and
stored in password protected data bases and /or lockable drawers accessible by only authorized
personnel.

22

CHAPTER FOUR
RESULTS
Study Subjects: A total of three hundred and two (302) subjects were enrolled into this study.
Twenty four (24) subjects were excluded from the analysis due to incomplete data or failure to
produce valid CD4 test results on both methods (Fig 2). Two hundred and seventy eight subjects
aged 20-74 (Median age = 40, IQR = 32-48) years, who produced valid results on both PIMA
and FACScallibur were include in the final analysis.

302 Participants

24 excluded due
to incomplete
data or invalid

278 study
participants

Figure 2: Study Profile.

The study population consisted of 181 (65%) female and 97 (35%) males. Although the mean
CD4 count using PIMA (478) was lower than that of the FACScallibur (481), this difference was
statistically not significant (p = 0.5358). The prevalence of ART eligible subjects at a CD4 count
threshold of 500 cells/l of blood was 58.6% (Table 1).

23

Table 1: Demographic and clinical characteristics of the study participants

Overall population

FACS Calibur

n =278

FACS Calibur

500 CD4 cells/l

>500 CD4 cells/l

n= 163

n=115

Gender
Female n (%)

181(65%)

94(58%)

87(76%)

Male n (%)

97 (35%)

69(42%)

28(24%)

Age (years), median (IQR)

40 (32-48)

40(31-49)

39(34-47)

FACScallibur
( Flow Cytometry)

CD4+
(SD)
478
322(125)
Performance

PIMA
POC

PIMA, Mean
(239)
684(207)

500cells/L

>500cells/L

500cells/L

148

18

166

>500cells/L

15

97

112

163

115

278

Characteristics of the PIMA: One hundred and sixty six (166) subjects were classified as
positive i.e. ART eligible by the PIMA and one hundred and twelve (112) as negative i.e. ART
Ineligible by the PIMA. Using results from the FACScallibur as the reference, there was 15 false
negative 18 false positives (Table 2).
Table 2: Contingency table showing classification of subjects according to ART eligibility
using the PIMA and FACScallibur.

24

At the 500 CD4 cells/l threshold, the performance characteristics of PIMA were sensitivity
90.8 %, specificity 84.4%, positive and negative predictive

values were 89.16 and 86.61

respectively (Table.3).
Table 3: PIMA Performance characteristics at the 500 CD4 cells/l threshold.
Sensitivity %, (95% CI)
Specificity %, (95% CI)
PPV %, (95% CI)
NPV (95% CI)
LHR+ (95% CI)
LHR-, (95% CI)
Prevalence, (95% CI)

90.80 (85.28-94.76)
84.35 (76.4 90.45)
89.16 (83.40-93.45)
86.61 (78.87-92.31)
5.80 (3.78-8.89)
0.11 (0.07-0.18)
58.63 (52.60-64.48)

25

CHAPTER FIVE
DISCUSSION
This study showed performance characteristics of the PIMA CD4 analyzer using capillary blood
compared with venous blood CD4 values determined using flow cytometry in a Ugandan ART
naive population. At the threshold of 500 CD4cells/L, PIMA capillary blood had a sensitivity of
90.8% (95%CI 85.3-94.8%), specificity of 84.4% (95%CI 76.490.5%), NPV of 86.6% (95% CI
78.8-92.3%), and PPV of 89.2% (95% CI 83.4-93.5). The positive and negative likelihood ratios
were 5.8 (95% CI 3.78-8.89) and 0.11 (0.07-0.18) respectively.
5.1 Sensitivity and Specificity of PIMA
The sensitivity of PIMA at the 500 CD4 cells/l threshold has been reported as 96 % (CI 95.2 96.9 %) from a meta-analysis and 96.1% (94.497.8%) from a study in Uganda (Scott et al.,
2015; Galiwango et al., 2014b). The low sensitivity 90.80% (85.28-94.76) in our study could be
attributed differences in the type of samples and study subjects as previous studies included ART
experienced subjects and used venous blood for comparison.
5.2 Positive and Negative predictive values of PIMA
As indicated by the NPV of 86.6 %, the probability of being ART ineligible given that the PIMA
CD4 count is above 500 cells/l of blood is 0.866. This is however lower than the previously
reported NPV of 94.1% from a Ugandan population that included patients on ART (Galiwango et
al., 2014c). Results of this study indicate that in areas with a similar prevalence, 86.6% of
individuals classified as ART ineligible by the PIMA will actually have a CD4 count above 500
cells/l of blood. The remaining 13.4 % of subjects will incorrectly be classified as ART
ineligible by the PIMA.

26

The PIMA PPV in our study population was 89.2 % which is higher than that previously reported
from a study in Uganda. The PIMA positive test (indicating ART eligibility) in our study
population was 89.2% likely to come from a person with a CD4 count less than 500 cells/l of
blood. In areas with a similar prevalence, 10.8 % of tests indicating ART eligibility will be from
persons who are ART ineligible.

Positive and negative predictive values are influenced by prevalence of the disease and therefore
are not interchangeable between geographical and time points with deferring prevalence rates.
This might partly explain the differences between the predictive values in this study and those
reported elsewhere.

Sensitivity, specificity, positive and negative predictive values do not

provide the most important information for making clinical decisions i.e. what are the odds of
having the disease given the result. In this case what is the likelihood or odds of being ART
eligible given the classification of the PIMA point of care CD4 analyzer as being ART eligible or
ineligible? All studies reviewed did not provide this piece of information and therefore the
interpretation on how well the PIMA performs in regards to identification of people eligible for
ART could have been influenced by the prevalence of ART eligible subjects in the study
populations.
5.3 Positive and Negative likelihood Ratios of PIMA

In this study we report positive likelihood ratio of 5.8 for the 500 cells threshold. This means
that the likelihood of a patient being eligible for ART increases six-fold given a CD4 count of
<500 cells/l of blood. Converting the likelihood ratios to odds using Bayes theorem gives
posttest odds as 8.2 and posterior probability as 89% given a positive test. The Negative

27

likelihood ratio at the same threshold was 0.11 which translates to post test odds of having a
disease given a negative test of 0.2 and posterior probability of 13%. The reporting of likelihood
ratios with derived odds of having a disease given test result allows for comparison of
performance of a diagnostic assay performance at varying disease prevalence time points or
geographical areas.
This study demonstrates the potential of the PIMA to facilitate scale up of access to HIV/AIDS
care and treatment services by providing access to CD4 testing in remote areas where
conventional testing is not possible as indicated by good correlation with the conventional
FACScallibur coupled with high positive and low negative likelihood ratios.

5.3 Study Implications and Limitations.

ART ineligible subjects misclassified by the PIMA as ART eligible (10.8 %) will have ART
initiated at a CD4 count above 500 cells/l of blood. Given the current WHO test and treat
guidelines that have largely not been implemented due to costs, only the 13.4% misclassified by
the PIMA as ART ineligible will be disadvantaged by delayed ART initiation.

The study was conducted in an urban setting with well-trained laboratory technologists under
ideal laboratory conditions.

Results obtained therefore may not represent performance

characteristics in rural settings which may not have the facilities and skilled labor yet this is
where the use point of care devices is needed mostly. The absence of published likelihood ratios
limited our ability to compare our findings with findings elsewhere.

28

CHAPTER SIX
CONCLUSIONS AND RECOMMENDATIONS
6.1 Conclusions
The PIMA point of care can be useful in pre ART HIV/AIDS care as it correctly classifies over
80% of pre ART subjects correctly in regard to ART eligibility. Frequent retesting may be
necessary for borderline CD4 counts to improve ability to identify ART eligible subjects.
6.2 Recommendations
We recommend the reporting likelihood ratios for all PIMA evaluations to allow comparison of
performance of diagnostic tests with varying prevalence rates. In light of the impending rollout
of the test and treat guidelines in Uganda, which will render CD4 testing not necessary for the
determination of ART eligibility and the varying performance characteristics at different
thresholds, the performance characteristics of the PIMA CD4 test at thresholds for determining
or assessing risk of developing opportunistic infections should be studied such that the test is
used for assessing risk of developing opportunistic infections.

29

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APPENDICES

Appendix A: Informed Consent

MAKERERE UNIVERSITY COLLEGE OF HEALTH SCIENCES
SCHOOL OF MEDICINE RESEARCH AND ETHICS COMMITTEE (SOM-REC)
INFORMED CONSENT

Title of the proposed study: Evaluation of the performance of Alere Pima CD4 test in
determining ART eligibility among adults in Uganda
Investigators:
Godfrey Pizaroh Mujuzi, Mildmay Uganda
Background and rationale for the study:
Currently the best practical way of assessing for who needs ARV (ART) is by determining ones
CD4 count. The currents methods are very expensive and require highly skilled people and
therefore not possible in rural areas. The Alere Pima CD4 test is a simple inexpensive method for
determining CD4 counts. We need to understand if the results obtained from the same device
give information as those from the currently used method (flow cytometry).

Procedures:
We will collect about 4mls of blood from your arm using a blood collection system. The will be
used to determine your CD4 using the usual methods and the results will be compared with those
obtained from your using the Pima machine.
Who will participate in the study :

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All HIV/AIDS patients on ART attending Mildmay Uganda clinic have been asked to participate
in this study. A total 210 patient will be required for the study and study will need you for only
30 minutes.

Risks/Discomforts:
There should not be any risks or discomfort in this survey, except for a minor pain or bruise at
the site of needle stick in your arm which is common in every blood draw.
Benefits:
There are no direct benefits to you. The information from this study will inform doctors about the
suitability of the machine for use in determining the time to start HIV drugs (ARV).
Confidentiality:
Your name, telephone number and address will not be recorded in reports that come from this
survey. The survey will only report results. Your name will also not be used on the blood
collection tubes and blood results. All the information that we collect will be kept confidential
and anonymous. Its only the principal investigator and research assistants who will have access
to your information

Alternatives:
Taking part in this study is entirely voluntary. Your decision not to participate will not affect the
way you receive services at Mildmay Uganda.
Cost:
Participation in the is entirely free and any addition cost incurred during the study will be met by
the principal investigator
Compensation for participation in the study:
It is extremely unlikely that something will go wrong during this study. However, you should
know that the University has procedures in place for reporting, investigating, recording and
handling adverse events and complaints from study volunteers. The university is insured for its
staff and students to carry out research involving people. The University knows about this

42

research project and has approved it. Any complaint should be made, in the first instance, to the
chief investigator identified for this particular study. Any complaint you make will be treated
seriously and reported to the appropriate authority.
Reimbursement:
There will not be any reimbursement given to participants.
Questions:
Participant can reach the principal investigator on +256772443403 or +256312200210 for any
study related questions
Questions about participants rights:
All participants who have questions about their rights as research participant will be addressed
by the principal investigators

Statement of voluntariness:
Taking part in this study is entirely voluntary. Your decision not to participate will not affect the
way you receive services at Mildmay Uganda.
Consent:
STATEMENT OF CONSENT/ASSENT
........................................................................... has described to me what is going to be done, the
risks, the benefits involved and my rights regarding this study. I understand that my decision to
participate in this study will not alter my usual medical care. In the use of this information, my
identity will be concealed. I am aware that I may withdraw at anytime. I understand that by
signing this form, I do not waive any of my legal rights but merely indicate that I have been
informed about the research study in which I am voluntarily agreeing to participate. A copy of
this form will be provided to me.
Name Signature of participant Date ...

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Name Signature of parent/guardian for minors (If applicable)Date ..

Name .Signature of interviewerDate .

Appendix B: Data collection Tool

Client Code |___|___|___|___|

Note: Please print all written entries in BLOCK CAPITAL LETTERS and avoid writing outside
the space provided. Ensure that all copies are readable. Please make sure that you answer all
questions. If the section is not applicable, indicate N/A.
Section 1: Patient Demographics
1.

Date of visit (DD/MM/YYYY) |___|___|/|___|___|/|___|___|___|

2.

Sex (M=Male, F= Female) |___|

3.

Date of birth(DD/MM/YYYY) |___|___|/|___|___|/|___|___|___|

4.

MO Number|____|__|__|__|__|__|__|

5.

Time of sample collection (HH:MM) |___|___|:|___|___|

Laboratory Results.
Test

Results

CD4 test

BD Facs Calibur Machine

Alere Pima Machine

Sample ID

cells/mm3

cells/mm3

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Technician Initials ________Signature _________________Date____/_____/____

Appendix C: Collection of blood samples

10.1.1 Venipuncture
i)

If necessary, have the participant roll up the sleeve. Place the tourniquet above elbow and

tighten enough to find the appropriate vein.

ii)

Palpate the ante cubit al fossa area and locate the desired vein. Loosen tourniquet.

iii)

Starting from the center and moving outward clean area with 70% isopropyl alcohol in a

circular motion, use a dry cotton swab to dry the area or allow it to air dry. Do not re-palpate
disinfected site.
iv)

Choose the proper needle depending on the size of the selected vein and the age of the

participant.
10.1.2 Vacutainer Technique
i)

Open needle package but do not remove the needle shield. Thread the needle into the

holder until secured. Apply tourniquet. Do not re-palpate disinfected site.

ii)

Have the participant make a fist and straighten the arm.

iii)

Remove the needle cover and inspect the needle to ensure that it is not damaged.

iv)

Position needle with bevel up, parallel to and over the top of the vein. Insert the needle

quickly under the skin and then into the vein.

v)

After entry into the vein, push the tube all the way into the holder and allow the blood to

fill the tube. Collect the correct amount of blood as stated out by the protocol.
vi)

When blood starts to flow into the tube, release tourniquet and have participant release

fist. If multiple specimens are needed, release the tourniquet after the first tube is collected. To
fill other tubes, remove the full tube and insert new tubes until all required tubes are filled.
vii)

Tubes which contain additives must be mixed gently and correctly with the sample

before placed in a rack while the samples collected in clot tubes should be put vertically standing
in a rack.
viii)

If no blood flows into the tube or blood ceases to flow before an adequate specimen is

collected, the following steps are suggested to complete a satisfactory collection.

45

ix)

Push tube forward until tube stopper is penetrated. If necessary, hold in place to ensure

complete vacuum draw.

x)

Confirm correct position of needle in vein.

xi)

Tubes may have bad vacuum. Remove tube and replace with new tube.

xii)

If second tube does not draw, remove needle, discard and repeat procedure.

xiii)

Upon completion of the venipuncture, remove the needle from the participants arms and

apply pressure to the site, using a sterile piece of cotton wool. Instruct the participant to elevate
the arm slightly and continue to applying pressure for 2-3 minutes.
xiv)

Immediately dispose off needle into sharp container. If an accident needle stick occurs,

contact the laboratory manager immediately. Wash the area with soap and water until leaving for
follow up treatment. (See safety manual for needle stick injury)
xv)

Place labels on the tubes and place tubes in appropriate rack/rocker for laboratory testing.

xvi)

Labels the specimens adequately with the futures as above

xvii)

For urgent samples, the phlebotomist shall transport the sample immediately to the

laboratory and instruct the data entrant who shall record the client details for tracking in the
emergency record book.
Note: The data entrant shall be responsible for monitoring the completion of the testing of the
emergency request and ensures that the result is delivered to the requester.
xviii) When the venipuncture site has stopped bleeding, place a bandage over the site.
When bleeding does not stop immediately, maintain pressure on the site for minimum of five
minutes, place a pressure dressing on the site and have the subject apply more direct pressure to
the site for another 15 minutes.
xix)

After bleeding inform the participant of the next clinical procedure as you let him or her

out
xx)

If bleeding does not stop, a clinical officer will be notified immediately.

xxi)

Fill in the information required in the laboratory specimen collection register. State the

correct amount of blood collected, time and date of collection including phlebotomists staff
initial.
Capillary Puncture of the Finger
The best site for a finger puncture is just off the center of the finger pad of the 3rd (middle) or

46

4th (ring) finger of the hand. The sides and the tip of the finger should be avoided.
Select the proper lancet: The BD Microtainer Contact-Activated Lancet 1.5 mm (lavender) is
used for glumeters and the BD Microtainer Contact-Activated Lancet 2.0 mm (blue) is used any
time you need more than a drop or two of blood.
Prepare the finger by cleaning it with a Chlorhexadine wipe. Allow it to air dry.
Grasp the finger, and using a sterile lancet, press firmly against the finger to make a puncture.
The first drop contains excess tissue fluid and must be wiped away. Collect the drops of blood
into the collection device by gently massaging the finger.
Avoid excessive pressure that may squeeze tissue fluid into the drop of blood or cause bruising.
When full, cap and then gently invert the collection device 5-10 times to mix the blood.
Hold a gauze pad over the puncture site for a short time to stop the bleeding.
Dispose of the contaminated materials and lancet in the appropriate waste containers.
Place a band-aid on the patients finger or have someone continue to hold gauze on the finger.
Label the specimens immediately.
Appendix D: Procedure for preparation of controls and samples for analysis using BD
FACSCalibur
Label all sample tubes and corresponding Trucount tubes with the patient identity number
obtained from respective patient forms, referred in dispatch logs and control samples with the
manufacturer given ID on the control sample
Note: Inspect all Trucount tubes to confirm presence of lypholysed pellets below the perforated
metal plate.
Add 20l of the monoclonal antibody to each labeled Trucount tube. Pipette just above the
stainless steel retainer. Do not touch the pellet.
Pipette 50l of well mixed sample tubes into their respective tubes. STRICTLY USING
REsVERSES PIPETTING.
Vortex and incubate in the dark for 15 minutes
Prepare a 1:10 lysing solution in DI water. Add 450l into each tube. The prepared solution is
stable for 1 month when stored in a glass container at room temperature
Incubate in the dark for 15 minutes. It is ready to run. Vortex for at least 2 seconds before
running

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Appendix E: Procedure for running samples and controls using BD FACSCalibur.

5.1.0 Running controls and patient samples with loader using single platform

5.1.1 Launch Worklist Manager from the menu task bar

5.1.2 Sign in with full names of operator, laboratory director (as principle laboratory director)
and institution name (Mildmay Uganda laboratory) then click accept.
5.1.3 In the next window, change Assay type from Cell quest to MultiSET
5.1.4 For automatic saving option ensure data file, Laboratory report, and Physician report are
ticked).
5.1.5 Click Accept. If you have already run calibration, click on Skip FACSComp.
5.1.6 The work list manager opens and, enter sample/control details (Name, sample ID,
laboratory number as Case number).
NB The sequence of forms and samples from reception must be maintained during entry.
5.1.7 Before the work list is pasted onto worklist manager, the names of clients and their patient
IDs entered in an EXCEL sheet in the sequence of the samples and forms.
5.1.8 Sequencial laboratory IDs are also given by the technologist or technician with specific
codes that are assigned by the HOS. The codes must be displayed for any laboratory personnel to
see.
5.1.9 New sequence for laboratory numbers shall be generated weekly or a period that shall be
defined for particular sites, explained and displayed for other laboratory personnel to understand.
5.1.10 The list is coped from the EXCEL sheet to the work list manager to create a work list for
running samples.
5.1.11 A file(identified by year) must be created at the desk top to save the work lists every
month.

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5.1.12 Select the panel name Mildmay 4 Colour

5.1.13 Click Assign Rack, choose the rack ID from the drop down arrow and click ok. If Assign
rack is not active it means the details of work list entry are not complete.
5.1.14 Load your prepared samples in their assigned positions on the rack.
5.1.15 On the machine press HI and Run buttons and pressurize the machine.
5.1.16 Click the Run button on the computer monitor
5.1.17 Run tests. Save tests per date.
Note: When running controls, ensure that all values pass.

Appendix F: Procedure for running samples and controls using PIMA

The Pima test consists of a disposable Pima test cartridge, containing dried reagents, and the
Pima Analyzer. Pima test cartridges are supplied in boxes containing a product insert detailing
instructions for sample collection as well as the test specific performance characteristics.
8.6.1

Before performing a Pima test, carefully read the Pima test cartridge guide for details

on sample application and cartridge handling.

8.6.2
8.6.3

Check correct local date and time before running a test.

To initiate a test run and open the cartridge slot door press when prompted by the

following screen.
8.6.4

The cartridge slot door opens and the Analyser prompts the Operator to insert a cartridge.

8.6.5

The cartridge slot door can be closed by pressing X returning the Operator to the Run

test window.
NOTE:

If no cartridge is inserted within 30 seconds, the cartridge slot door closes

automatically and the display returns to the Run test window.

8.6.6

Inserting the pima test cartridge; Insert cartridge into the Pima Analyser in the direction

indicated by the arrow on the cartridge.

NOTE:

The Pima Analyser is designed to be used in combination with the Pima test

cartridge only. No other cartridges, test strips or device should be inserted into the Analyser.

Do not discard the cartridge pouch until the Pima Analyser has successfully started the

49

analysis. In case of a barcode error, the information printed on the cartridge pouch can be used to
enter the barcode manually (refer to appedix for Barcode Error message).
8.6.7

Upon inserting the Pima test cartridge a sensor in the Analyser recognizes that a cartridge

has been inserted and automatically starts the analysis process by fully drawing the cartridge into
the Analyser and closing the cartridge slot door.
8.6.6

The display screen changes briefly to Reading cartridge at this moment.

8.6.7

Once the Pima Analyser has accepted the cartridge it will prompt the Operator to enter

both the Operator and Sample ID. During data entry the test analysis continues in the
background.
8.6.8

After entering an Operator, the Enter Sample window is displayed on the screen. A

Sample ID of up to 20 characters may be entered using the keypad. Enter Sample ID and
confirm.
8.6.9

After Operator and Sample ID are successfully entered, the Analysis in Progress

window appears. The ID of the sample currently analysed and an estimated time to completion
are shown.
8.6.10 After the analysis has been successfully completed, the Analysis done. Remove
cartridge window appears and prompts the Operator to remove the cartridge from the Analyser.
8.6.11 Once the cartridge is removed, the Pima Analyser automatically displays the first of four
result windows, showing the Sample ID and test result. Additional test related parameters can be
viewed using and .
8.6.12 A Pima Test Report may be printed but this must be transcribed carefully into patient
files since the result is printed on thermal paper which fades out after sometime.

50