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guideline

Guideline: the laboratory diagnosis of malaria

J. Wendi Bailey,1 John Williams,2 Barbara J. Bain,3 John Parker-Williams4 and Peter L. Chiodini5,6 for the General
Haematology Task Force of the British Committee for Standards in Haematology
1
Clinical Diagnostic Parasitology Laboratory, Liverpool School of Tropical Medicine, Liverpool, 2Diagnostic Parasitology Laboratory,
The London School of Hygiene & Tropical Medicine, 3Department of Haematology, St Marys Hospital, 4Department of Haematology,
St Georges Hospital, 5UK NEQAS for Parasitology, Hospital for Tropical Diseases, and 6The London School of Hygiene & Tropical
Medicine, London, UK

External Quality Assessment Scheme (NEQAS) surveys indi-


Summary
cate that there are continuing problems in malaria diagnosis:
UK National External Quality Assessment Service surveys inaccurate calculation of parasitaemia or failure to estimate it
indicate continuing problems in malaria diagnosis: inaccurate altogether, difficulty in distinguishing between Plasmodium
calculation of parasitaemia or failure to estimate it altogether, vivax and P. ovale, reporting the presence of malaria para-
difficulty distinguishing Plasmodium vivax from P. ovale, sites when none were present and misidentifying P. falcipa-
reporting malaria parasites when none were present and mis- rum as another species are still encountered. Furthermore,
identification of P. falciparum as another species still occur. P. knowlesi, normally a parasite of macaques, has now been
Therefore, the British Committee for Standards in Haematol- found to cause human malaria in almost all countries in
ogy Guidelines for the Laboratory Diagnosis of Malaria have South-East Asia, leading to illness as severe as that caused by
been revised. They are intended for use in the UK but may P. falciparum. It has been seen as an imported malaria in the
also prove useful in other non-endemic areas. Routine use of UK and is extremely difficult to distinguish morphologically
thick and thin films is advised for malaria diagnosis. Thick from P. malariae, which produces a much less severe illness.
films should be stained using Giemsa or Field stain. Thin For all these reasons, updating and revision of the guideline
films should be stained with Giemsa stain or Leishman stain. is considered essential. Accurate malaria diagnosis requires
Thick films should be examined by two observers, each view- adequate techniques and adequate training/experience. Both
ing a minimum of 200 high power fields. If thick films are are considered in this revised guideline. These Guidelines are
positive, the species should be determined by examination of intended for use in the UK and may also be applicable to
a thin film. In the case of P. falciparum or P. knowlesi infec- other non-endemic areas.
tion, the percentage of parasitized cells or the number of
parasites per microlitre (/ll) should be estimated and Recommended procedures
reported. Rapid diagnostic tests (RDTs) for malarial antigen
cannot replace microscopy but are indicated as a supplemen- Microscopy in the detection of malaria parasites and in
tary test when malaria diagnosis is performed by relatively the identification of species
inexperienced staff. Malaria RDTs are negative in babesiosis.
Basic procedures. High quality thick and thin films (Bain,
Keywords: malaria, diagnosis. 2006; Bain et al, 2011) should be prepared and examined in
all cases of suspected malaria, regardless of the results of
Since the publication of the first British Committee for Stan- immunochromatographic rapid diagnostic tests (RDTs) for
dards in Haematology (BCSH) guideline on malaria diagno- malarial antigen detection. The thick film should be used for
sis (The Malaria Working Party of the General Haematology the detection of malaria parasites and the thin film for iden-
Task Force of the British Committee for Standards in tification of species. It is useful to prepare four thick films
Haematology, 1997), the range of supplementary tests and four thin films so that two of each can be stained, leav-
available has altered considerably. In addition, UK National ing spare films to send to a reference centre (see Appendix
1) and for further study if there is diagnostic difficulty. Films
should be made without delay because morphological alter-
ation of parasites occurs with storage of K2ethylenediamine-
Correspondence: Professor Peter L. Chiodini, UK NEQAS for tetra-acetic acid (K2EDTA)-anticoagulated blood. Thin films
Parasitology, Hospital for Tropical Diseases, Mortimer Market, should be dried, exposed to acetone for 1 min and air-dried.
Capper Street, London WC1E 6JB, UK. A given laboratory should select one stain and become profi-
E-mail: peter.chiodini@uclh.nhs.uk cient at using it. If using Giemsa stain, the thin film should

2013 John Wiley & Sons Ltd First published online 8 October 2013
British Journal of Haematology, 2013, 163, 573580 doi:10.1111/bjh.12572
Guideline

then be fixed in methanol for 1 min prior to staining. As microlitre (/ll) of blood instead of percentage parasitaemia
Leishman stain is methanol-based the stain should be left on (World Health Organization, 2010). Parasite numbers/ll can
the slide for 1 min before adding buffered water pH 72 (see be calculated in relation to the number of white cells
Appendices 2 and 3). Thick films should be dried at 37C (Warhurst & Williams, 1996; Bowers et al, 2009) or from the
for 15 min or, if there is no urgency, for between 30 min percentage parasitaemia and the red cell count. Quantifica-
and 1 h at room temperature and should then be exposed to tion of parasites should be repeated daily until no parasites
acetone for 10 min in a Coplin jar prior to staining. Either (other than gametocytes) remain.
Giemsa stain or Field stain can be used. Some laboratories
use Field stain (see Appendices 2 and 3) for thick films Confirmation of diagnosis and species. All malaria films
because it is more rapid. Routine MayGr unwaldGiemsa should be examined by two trained observers. The second
(MGG), WrightGiemsa and Giemsa stains, including those observer may examine the film simultaneously or subse-
used in automated staining machines, are unlikely to be quently (e.g., next morning when the films have been exam-
satisfactory because the pH used is inappropriate. In the case ined on call). The second observer should have significant
of a gravely ill patient, it is useful to stain an extra fixed thin experience in the diagnosis of malaria and should keep his/
film with modified Field stain because this permits very her skills updated. The observer confirming the presence and
speedy diagnosis of P. falciparum infection. Giemsa or species of malaria parasites should also confirm that the
Leishman staining is still needed for precise identification of parasite count is of the correct order. However, it is not to
other species. be expected that a second parasite count will be exactly the
A minimum of 200 oil immersion fields (9100 objective) same as the first because the confidence limits of low counts
should be examined in the thick film; this will take about are fairly wide (Table I) and an amended count should only
510 min for an experienced observer but longer for those be issued if the first count is incorrect. If a patient who has
who do not often examine films containing malaria parasites. travelled in the Asia-Pacific region has parasites thought to
Some experienced microscopists prefer to scan with a950 oil be P. malariae, urgent referral should be made to the Malaria
immersion lens and use the 9100 objective to identify the Reference Laboratory for P. knowlesi polymerase chain reac-
species of suspected parasites. The 9100 should still be used tion (PCR) as these two species are very difficult to distin-
for counting parasites. Following the detection of malaria guish by morphology. P. knowlesi infection can progress very
parasites in a thick film, the thin film should be examined to rapidly as its erythrocytic cycle takes only 24 h, compared to
determine the species. If an observer is uncertain as to 72 h for P. malariae, so the clinical team should be informed
whether malaria parasites are present in a thick film, an that a diagnosis of P. knowlesi is being considered.
entire thin film should be examined with a9100 objective,
starting with the edges and the tail where parasitized cells
Table I. Ninety five percent and 99% confidence limits of parasite
may be more frequent. This is likely to take 2040 min. If counts if 1000 red cells are counted.
parasites are very rare, the co-ordinates of any parasites
detected should be recorded or an England Finder Graticule Observed 95% confidence 99% confidence
percentage limits limits
should be used, to permit later confirmation. It should be
noted that detection of P. falciparum gametocytes in the 0 000037 000053
absence of other stages of the life-cycle may be clinically 1 048184 035211
significant in an untreated patient as it may indicate 2 1231 1034
suppressed active infection (Warhurst & Williams, 1996). 3 2043 1847
4 2954 2659
Quantification of parasites. Whenever P. falciparum or 5 3765 3470
6 4677 4282
P. knowlesi is detected, the percentage of parasitized cells
7 5588 5193
should be quantified and reported promptly to the responsi-
8 6499 591045
ble clinical staff, as the severity of parasitaemia may affect 9 731095 68116
the choice of treatment. Quantification should be performed 10 82120 77127
using a thin film, with a minimum of 1000 red cells being 15 128174 122181
examined in different areas of the film. The use of an
The data in this table are derived from Diem and Lentner (1970). As
eyepiece with a graticule or grid, e.g., a Miller square or
an approximation, the confidence intervals can be calculated from
Index grid, facilitates quantification. In the case of a double
the formula p  [z.SE(p)] when SE(p) is the standard error of p and
infection, the quantification applies only to P. falciparum or
z is 195996 for 95% confidence intervals and 25758 for 99% confi-
P. knowlesi. Only asexual stage parasites should be counted dence intervals. SE(p) is calculated as [p(1 p)/n] when p is the
i.e. gametocytes of P. falciparum are excluded from the count observed proportion and n is the total number of cells counted. The
of positive cells. If the parasite count is <1 in 1000 cells, it is figures are predicted from probability theory and show the minimum
useful to quantify on a thick film. The World Health variability without taking account of technical or observational
Organization (WHO) uses the number of parasites per errors.

574 2013 John Wiley & Sons Ltd


British Journal of Haematology, 2013, 163, 573580
Guideline

Identification of the species when the thick film is positive and the results, laboratories may wish to consider adopting this
the thin film is negative. There are three possible ways to procedure.
determine the species when the thin film is negative. All may
be satisfactory, depending on the circumstances.
Supplementary tests
1 It is often possible for an experienced observer to deter-
mine the species on a thick film. A malaria RDT may also RDTs, also known as immunochromatographic tests, to detect
be performed. malarial antigen. RDTs are indicated to confirm the pres-
2 If only one or two ring forms are seen and it is not possi- ence or absence of P. falciparum assessed on a blood film,
ble to determine the species with certainty it is prudent particularly when there is a relatively inexperienced observer
for the patient to be treated as for P. falciparum infection. or when pressure of work out-of-hours prevents adequate
3 Films and a blood sample can be referred to a reference microscopic assessment (e.g. if on-call tests are being per-
laboratory (Table II). formed by a biomedical scientist who does not often examine
films for malaria parasites or who is simultaneously dealing
with a number of urgent requests) or in hospitals that exam-
Negative films despite a strong clinical suspicion of ine films for malaria parasites infrequently. In such a situa-
malaria. When the parasite count is very low, examining tion out of hours, a positive RDT result should be reported
1000 rather than 200 high power fields on a thick film will to the clinical team, adding that microscopic confirmation
increase the yield of positive results. When there is a strong and species identification will follow next morning. RDTs are
clinical suspicion of malaria but the initial films are negative, negative in Babesia sp. infection, so a negative RDT result on
repeat films should be made and examined after 1224 h a blood sample where falciparum parasitaemia is suspected
and again after a further 24 h. Laboratories should consider on morphological grounds should prompt urgent referral to
including a statement in every report that negative films do a reference laboratory. RDTs are not recommended for fol-
not exclude a diagnosis of malaria and that repeat films lowing the response to antimalarial treatment. They cannot
should be requested if clinically indicated. Relevant haemato- replace microscopy and therefore their use adds to the cost
logical abnormalities, such as thrombocytopenia or suspi- of malaria diagnosis. Furthermore their use in place of
cious findings with certain automated instruments (Sharma microscopy is not appropriate and may prove to be a false
et al, 2013), may strengthen a clinical suspicion of malaria economy. Recognized disadvantages of RDTs are (i) occa-
and be a further indication for repeat films. sional false positives occur (ii) they are less sensitive than ref-
erence microscopy (iii) persisting histidine-rich protein 2
High risk blood samples. It is not infrequent for malaria (HRP2) antigenaemia can give a positive test when no viable
diagnosis to be necessary on blood samples from patients parasites are present (iv) except in the case of P. falciparum
carrying the human immunodeficiency virus (HIV), hepatitis or P. vivax infection, the species cannot be determined (v)
B, hepatitis C or other blood-borne viruses. All malaria quantification is not possible (vi) operator misunderstanding
samples should therefore be regarded as potentially high risk. or misinterpretation of test line patterns may lead to appar-
If there is a suspicion of viral haemorrhagic fever, a clinical ent discrepancy between RDT and blood film results (vii)
assessment should be made and the relevant guidance (Advi- rarely, a prozone effect may occur with HRP2 based RDTs
sory Committee on Dangerous Pathogens, 2012) should be (Luchavez et al, 2011).
followed. For all other samples, standard laboratory proce- Currently available RDTs cannot specifically identify
dures, based on regarding all samples as potentially high-risk, P. knowlesi. HRP2-based test lines detect only P. falciparum.
should be followed (http://www.hse.gov.uk/index.htm). It is Plasmodium lactate dehydrogenase (pLDH) and aldolase-
possible that acetone may help inactivate enveloped viruses. based test lines may detect P. knowlesi as malaria parasites
As it is possible to introduce an acetone treatment step into but cannot identify the species. A hospital study in Malaysia
processing both thick films and thin films without impairing detected 74% of PCR-confirmed P. knowlesi using a

Table II. Procedure for referral to a reference laboratory.


Indications for referral (i) All positive results on blood film or rapid antigen detection test
(ii) Discrepancy between film and rapid antigen detection test
(iii) Negative cases with a strong clinical suspicion, after discussion with an infectious diseases/tropical medicine
specialist
Material to be referred (i) Two unstained thick films, two unstained methanol-fixed thin films and an aliquot of EDTA-anticoagulated
blood (to permit DNA analysis when necessary)
(ii) Patient report form (see Fig 1)
Method of transport (i) First class postal service, conforming to necessary regulations
(ii) Courier, packaging as for postal services

2013 John Wiley & Sons Ltd 575


British Journal of Haematology, 2013, 163, 573580
Guideline

PUBLIC HEALTH ENGLAND MRL No. _ _ /_ _ _ _


MALARIA REFERENCE LABORATORY
PATIENT REPORT FORM In confi dence

Family name:
All other names:

Home post code: Primary care trust:


Address in UK:

Date of birth: ___ / ___ / _____ Age: _____________ Gender: M / F

Country of birth: _____________________ Country of usual residence: _____________________

Malaria prophylaxis taken:


Ethnicity: (mark one) Reason for travel: (mark one)
(mark as relevant)
White Bri sh New entrant to UK NONE
Other White background Visi ng family in country of origin Mefloquine (Lariam)
Black African UK ci zen living abroad Malarone
Black Caribbean Civilian sea/air crew Doxycycline
Other Black background Bri sh armed forces Chloroquine (Nivaquine/Avloclor)
Indian Sub-Con nent Business/Professional travel Proguanil (Paludrine)
South-East Asian Foreign student studying in UK Unknown
Other Asian background Holiday travel to malarious country Other (please specify)
Mixed Ethnicity Foreign visitor ill while in UK
Other (please specify) Children visi ng parents living abroad Prophylaxis taken regularly? Y / N
Other (please specify) Con nued on return for _______ weeks

Date of onset of illness: ___ / ___ / _____ Date of star ng treatment: ___ / ___ / _____
Date of arrival in UK from malarious country ___ / ___ / _____ For India, please specify areas visited

Dura on of stay abroad : ____________________

Country(ies) where infec on acquired: _____________________________

G.P. Name & Address Name and contact details of person


comple ng this form if not G.P.

Tel. No.

Please complete other side


MRL pa ent report/referral form version 3 June 2013 - JW/DB

Fig 1. Public health England malaria reference laboratory patient report form.

576 2013 John Wiley & Sons Ltd


British Journal of Haematology, 2013, 163, 573580
Guideline

Hospital where diagnosis Date of diagnosis


made
____ /____ /______

Blood film Antigen test Please specify _________________


Method of diagnosis:
Clinical Other Please specify _________________

Species of malaria parasite: Was pa ent treated as: Outcome of illness:


P. falciparum Outpa ent Recovery
P. vivax Inpa ent
P. malariae Was pa ent: Death
P. ovale Pregnant Y / N _____ / 40
Species unknown Admi ed to ITU/HDU Y/N Unknown
No malaria parasites found Dura on of stay in hospital ______ days

Any other informa on relevant to this case:

If sending specimen(s) for referral please give the following informa on: Date of Sample
NHS/Hosp No. _____________________ Lab No. _____________________ ____ / ____ / ______

Type of specimen: Name and address for report:


Blood
Blood films
Other (please specify)

MALARIA IS A NOTIFIABLE DISEASE - PLEASE FILL IN A STATUTORY NOTIFICATION FORM AND FORWARD TO THE CCDC.

Please return this form to: MALARIA REFERENCE LAB - USE ONLY

PHE Malaria Reference Laboratory


London School of Hygiene and Tropical Medicine
Keppel Street (Gower Street)
London WC1E 7HT

Tel. No.: Surveillance 020 7927 2435


Laboratory 020 7927 2427
Fax 020 7637 0248

MRL pa ent report/referral form version 3 June 2013 - JW/DB

Fig 1. (Continued)

2013 John Wiley & Sons Ltd 577


British Journal of Haematology, 2013, 163, 573580
Guideline

pan-pLDH RDT test line and a pan-aldolase RDT test line Pathology Accreditation (UK) Ltd (CPA) accreditation for
detected 23%. The authors commented that neither the performance of these tests.
pLDH- nor aldolase-based RDT they tested demonstrated
sufficiently high overall sensitivity for P. knowlesi (Barber Use of a reference laboratory. For all positive cases, blood
et al, 2013). films and a 1 ml aliquot of the EDTA blood sample on which
The World Health Organization has produced detailed the diagnosis was made should be sent to the Malaria Refer-
performance assessment of commercially available RDTs (see ence Laboratory (MRL) for confirmation (Appendix 1 and
http://www.wpro.who.int/sites/rdt). Laboratory directors Fig 1). Cases in Scotland should be referred to the Scottish
should consult this site when selecting an RDT to ensure as Parasite Diagnostic Laboratory. If a reference centre other than
far as possible that it will meet their requirements. Whether the MRL is used to confirm the diagnosis, data should still be
or not a particular product is CE marked will also influence returned to the MRL. This is important as it provides surveil-
their choice. lance data that influence national policy decisions.

Quantitative Buffy Coat (QBC) blood parasite detection


Training, continuing education and maintenance of
method. Some laboratories use QBC as a screening test
expertise
backed up by thick/thin films on QBC-positive samples. Capi-
tal and test costs are high but, in busy haematology laborato- All laboratories must ensure that new members of staff are
ries where large numbers of malaria requests are received daily, adequately trained. In addition, laboratories that do not often
QBC screening may be a useful and time-saving technique. examine blood films for malaria parasites need to ensure that
staff maintain their skills. The following procedures are useful:
Polymerase chain reaction. PCR is at least 10-fold more 1 Sets of mixed positive and negative thick and thin films
sensitive than microscopy (Padley et al, 2003). It is also more should be available for examination during training and
reliable for determining the species in a mixed infection. for periodic practice; suitable films include NEQAS films
Whilst used largely as a reference test, nucleic acid detection and other films that have had the species confirmed by a
is likely to play an increasing role in primary diagnosis in reference laboratory. In addition, reference laboratories
well-resourced healthcare settings. Real time PCR or loop- can often provide spare films for training purposes.
mediated isothermal amplification (LAMP) nucleic acid 2 NEQAS films can be examined by all biomedical scientists
detection (Polley et al, 2010) can produce results within a and medical staff who carry out microscopy for malaria
clinically relevant time frame and real time PCR can assess diagnosis. This is a useful training exercise after the
the rapidity with which a malarial parasitaemia is reduced on correct diagnosis is known so that relevant features can
drug therapy (Beshir et al, 2010). be demonstrated immediately to any staff member who
fails to make the correct diagnosis.
Drug sensitivity. This is currently a research tool. It has a 3 High quality picutres of malaria parasites should be avail-
potential future role, particularly in determining prophylaxis able for reference (see http://www.who.int/en/ and search
policy. As more and more molecular markers become vali- malaria publications).
dated against clinical and in vitro drug sensitivity test results, Websites can be used for on-going training. The following
it will ultimately become possible to supply information on may be useful: www.dpd.cdc.gov/dpdx/HTML/Image_
likely antimalarial drug susceptibility to clinical teams. Library.htm (Centers for Disease Control and Prevention,
USA) http://www.rph.wa.gov.au/pubs.html (Royal Perth
Hospital, West Australia. Click on malaria information to
Quality control
access the teach and test yourself site) (Note: this is a use-
As part of internal quality control. ful resource but it should be noted that the films shown
may not have been stained at an optimal pH).
1 All malaria films should be examined by two observers
5 Training courses are available (see http://www.lshtm.ac.
2 All new batches of Giemsa or Leishman stain should be
uk/ or http://www.lstmliverpool.ac.uk/).
tested with a known P. vivax or P. ovale infection to ensure
that Schuffner dots and James dots are stained and that par-
asitized cells are decolourized. Blood films for this purpose
Acknowledgements
can be sealed in plastic slide boxes and frozen. Frozen films
must be brought to room temperature over 12 h before PLC wrote the manuscript. All authors met and discussed its
opening the box, to prevent condensation and red cell lysis. content in detail. All authors contributed material and
commented on the manuscript. PLC is supported by the
National Institute for Health Research University College
External quality assessment. All laboratories offering tests for
London Hospitals Biomedical Research Centre and by UK
malaria parasites should participate in one or both of the
NEQAS.
available NEQAS schemes. This is a requirement for Clinical

578 2013 John Wiley & Sons Ltd


British Journal of Haematology, 2013, 163, 573580
Guideline

References efficacy of anti-malarial drugs in vivo: quantita- targets increase sensitivity of malaria detection
tive PCR measurement of parasite clearance. using loop-mediated isothermal amplification.
Advisory Committee on Dangerous Pathogens Malaria Journal, 5, 312. Journal of Clinical Microbiology, 48, 28662871.
(2012) Management of Hazard Group 4 viral Bowers, K.M., Bell, D., Chiodini, P.L., Barnwell, J., Sharma, S., Sethi, N., Pujani, M., Kushwaha, S. &
haemorrhagic fevers and similar human infectious Incardona, S., Yen, S., Luchavez, J. & Watt, H. Sehgal, S. (2013) Abnormal WBC scattergram: a
diseases of high consequence. Crown Copyright. (2009) Inter-rate reliability of malaria parasite clue to the diagnosis of malaria. Hematology, 18,
Available at: http://webarchive.nationalarchives. counts and comparison of methods. Malaria 101105.
gov.uk/20130107105354/http://www.dh.gov.uk/ Journal, 8, 267. The Malaria Working Party of the General Haema-
health/files/2012/07/FINAL-VHF-guidance-for- Diem, K. & Lentner, C. (1970) Documenta Geigy tology Task Force of the British Committee for
publication.pdf [Accessed 18 July 2012]. Scientific Tables, 7th edn. J R Geigy S.A, Basle, Standards in Haematology (1997) The labora-
Bain, B.J. (2006) Cells, Blood: A Guide, Practical, Switzerland. tory diagnosis of malaria. Clinical and Labora-
4th edn. Science, Blackwell, Oxford. Luchavez, J., Baker, J., Alcantara, S., Belizario, V. tory Haematology, 19, 165170.
Bain, B.J., Bates, I., Laffan, M.A. & Lewis, S.M.(Eds) Jr, Cheng, Q., McCarthy, J.S. & Bell, D. (2011) Warhurst, D.C. & Williams, J.E. (1996) ACP
(2011) Dacie & Haematology, Lewis Practical, Laboratory demonstration of a prozone-like broadsheets No 148 laboratory diagnosis of
11th edn. Churchill Livingstone, Edinburgh. effect in HRP2-detecting malaria rapid diagnos- malaria. Journal of Clinical Pathology, 49,
Barber, B.E., William, T., Grigg, M.J., Piera, K., tic tests: implications for clinical management. 533538.
Yeo, T.W. & Anstey, N.M. (2013) Evaluation of Malaria Journal, 10, 286. World Health Organization (2010) Parasitological
the sensitivity of a pLDH-based and an aldolase- Padley, D., Moody, A.H., Chiodini, P.L. & Salda- confirmation of malaria diagnosis: WHO techni-
based rapid diagnostic test for diagnosis of nha, J. (2003) use of a rapid single-round, mul- cal consultation, Geneva, 68 October 2009.
uncomplicated and severe malaria caused by tiplex PCR to detect malarial parasites and Available at: http://whqlibdoc.who.int/publica-
PCR-confirmed Plasmodium knowlesi, Plasmo- identify the species present. Annals of Tropical tions/2010/9789241599412_eng.pdf [Accessed 9
dium falciparum, and Plasmodium vivax. Journal Medicine and Parasitology, 97, 131137. August 2013].
of Clinical Microbiology, 51, 11181123. Polley, S.D., Mori, Y., Watson, J., Perkins, M.D.,
Beshir, K.B., Hallett, R.L., Eziefula, A.C., Bailey, R., Gonzalez, I.J., Notomi, T., Chiodini, P.L. &
Watson, J., Wright, S.G., Chiodini, P.L., Polley, Sutherland, C.J. (2010) Mitochondrial DNA
S.D. & Sutherland, C.J. (2010) Measuring the

Appendix 1 Commercial stains (i) Field compound stain A (25 g) HD


Reference centres 1410 Field compound stain B (25 g) HD 1415 Leishman
(i) PHE Malaria Reference Laboratory, London School of stain (500 ml) HS400 Giemsa stain (500 ml) HS295.
Hygiene and Tropical Medicine, Keppel Street, London TCS Biosciences Ltd Botolph Claydon Buckingham MK18
WC1E 7HT www.malaria-reference.co.uk 2LR United Kingdom Phone: +44 (0)1296 714222 Fax: +44
(ii) Diagnostic Parasitology Laboratory Liverpool School of (0)1296 714806 Email: sales@tcsgroup.co.uk.
Tropical Medicine, Pembroke Place, Liverpool L3 5QA (ii) Gurr R66 Giemsa (500 ml) cat no 350864X VWR
http://www.lstmliverpool.ac.uk/ International Ltd (as above).
(iii) Scottish Parasite Diagnostic Laboratory Stobhill Hospi-
tal, Balornock Road, Glasgow G21 3UW http://www.
Appendix 3
spdl.scot.nhs.uk/
Methods
(i) Leishman stain for thin films
Stain recipe
Appendix 2
1 Add methanol-cleaned glass beads to 500 ml of methanol
Suppliers of kits and reagents
(AnalaR grade)
Immunochromatographic Kits
2 Add 15 g of Leishman powder
Given the large number of products available, potential
3 Shake well, leave on a rotary shaker during the day then
users of malaria rapid diagnostic tests are advised to consult
incubate at 37C overnight. There is no need to filter.
the WHO website (http://www.wpro.who.int/sites/rdt) to
inform their choice of product. Method
Suppliers: Quantitative Buffy Coat (QBCTM) 1 Make a thin film and air-dry rapidly.
Woodley Equipment Company Ltd Old Station Park 2 Place film in a staining rack and flood film with acetone;
Buildings, St. Johns Street, Horwich, Bolton, Lancashire, BL6 leave for 1 min.
7NY, United Kingdom Tel: +44 (0) 1204 669033 Fax: +44 3 Tip off acetone and allow the film to dry.
(0) 1204 669034 Email: sales@woodleyequipment.com 4 Flood the film with Leishman stain and leave for 30 s to
Buffered water 1 min to fix.
Buffer tablets (Gurr) pH 72 (50 tablets to make 100 ml 5 Add twice as much buffered distilled water (preferably
each) Cat. no. 33194 2F VWR International Ltd, Hunter from a plastic wash bottle as this allows better mixing of
Boulevard, MagnaPark, Lutterworth, Leicestershire, LE17 the solution), pH 72.
4XN TEL 0800 223344 FAX 01455 558586 6 Leave to stain for 15 min.

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British Journal of Haematology, 2013, 163, 573580
Guideline

7 Wash off the stain with tap water. 6 Rinse gently in tap water then drain and air-dry upright.
8 Dry film upright.
(iv) Giemsa stain for thick and thin films.
As an alternative, use commercially prepared Leishman Stain recipe
stain (HD Supplies, Aylesbury, Buckinghamshire, UK). Giemsa powder 38 g
(ii) Field stain for thick films. Methanol 250 ml (AnalaR grade) Glycerol 250 ml.
Stain recipe 1 Add stain and methanol-cleaned glass beads to amber
The same stain preparation method is followed for Field glass bottle.
stains A and B. 2 Add glycerol and methanol, shake vigorously and place at
1 Add 25 g of powdered compound stain to 80 ml of 37C for 24 h with further frequent shaking.
distilled water. 3 Remove from the incubator and shake again for 24 h; the
2 Mix well and filter before use. stain is then ready for use.
3 Change stains monthly. 4 Filter small amounts as required.
Method Method for Giemsa stain for thick film
1 Make a thick film and leave to air dry at room tempera- a Make a thick film and leave to air dry at room tempera-
ture for 30 min to 1 h or in a 37C incubator for ture for 30 min to 1 h or in a 37C incubator for 15 min.
15 min. b Place the slide in a Coplin jar or slide container and
2 Place the slide in a Coplin jar or slide container and cover cover with acetone; leave for 10 min.
with acetone; leave for 10 min, then air dry. c Tip off the acetone and leave to dry.
3 Stain with stain A for 5 s, then drain. d Dilute the stain 1:10 in buffered water, pH 72.
4 Rinse gently in tap water for 5 s, then drain.
Place the slide in a trough or stain upside down in a
5 Stain with stain B for 3 s, then drain.
staining plate; add stain, leave for 1040 min, depending on
6 Rinse gently in tap water then drain.
the specific stain used (Gurr R66 requires 3040 min to
7 Air-dry upright.
show all inclusions. Ready-made Giemsa stain [HD Supplies]
8 Examine the area where the nuclei of the white cells are
needs 2530 min. All laboratories should test every stain
stained purple.
against control slides to establish the correct time for the
(iii) Field stain for thin films. specific stain in use.)
Stain recipe e Pour off the stain and wash slide with tap water for a
1. Dilute 1 ml of Field stain B with 3 ml of buffered few minutes.
water (pH 72). f Dry upright.
Method
Method for Giemsa stain for thin film.
1 Make a thin film and air dry rapidly.
1 Make a thin film and air dry rapidly.
2 Place film in a staining rack and flood film with acetone;
2 Place film in a staining rack and flood film with acetone;
leave for 1 mi.
leave for 1 min.
3 Tip off acetone and allow the film to dry, fix with metha-
3 Tip off acetone and allow the film to dry.
nol for 1 min, tip off methanol and air dry.
4 Fix in methanol for 1 min then air dry the film.
4 Flood slide with the diluted Field stain B.
5 Proceed as for thick film.
5 Immediately add an equal volume of Field stain A, mix
thoroughly and leave for 1 min.

580 2013 John Wiley & Sons Ltd


British Journal of Haematology, 2013, 163, 573580