GAIN INDEPENDENT COMPENSATION
Opened in 2011, the Institute of Molecular Biology gGmbH (IMB) at Johannes Gutenberg University, Mainz, Germany is housed in a brand new building with state-of-the-art scientific facilities. The architect suggested that ultra-low temperature freezers housed in the Institute’s Core Facilities should be capable of being linked to a central water-cooling system, an integral part of the building’s modern infrastructure.
Centre of excellence
Described as ‘a centre of excellence in the life sciences in the heart of Europe’, IMB has been funded for an initial period of ten years by the Boehringer Ingelheim Foundation, a charity dedicated to promoting outstanding research in medicine, biology, chemistry and pharmaceutical science. As a non-profit entity, which operates like an innovative academic research centre, IMB focuses on key questions in developmental biology, epigenetics and DNA repair. The aim is to transform our understanding of how we develop, adapt to our environment, age and develop diseases such as cancer.
IMB’s core facilities
Researchers at IMB have access to cutting-edge technologies and the latest equipment provided centrally. This arrangement has significant benefits, as Dr Bernhard Korn, Director of IMB Core Facilities, explains: ‘The decision to set up centralised resources comprising cost-intensive instrumentation and high-level expertise enables even small research groups at IMB to run ambitious major projects’.
‘Secondly, it is much more economical for the Institute to run instruments and equipment, such as freezers, at the limit of their capacity by sharing resources among multiple users. In addition, by locating our ULT freezers together in a dedicated area we can make use of a centralised monitoring and alarm system which constantly checks the temperature, power supply and cooling water for all units.’
Selecting ULT freezers
IMB chose to equip its core facilities with 23 SANYO MDF-U74V upright -86ºC freezers and three of the company’s MDF-C2156VAN cryogenic freezers. All are equipped with the water-cooled condenser option, to meet a requirement to utilise the Institute’s water-cooling system. The space-saving -86ºC freezers are used to hold a collection of more than 25,000 different human protein-coding clones, while the -150ºC freezers store a large number of human cell lines, tumour cell lines and tissue samples. Later, patient specimens will also be stored prior to the extraction of nucleic acids and proteins.
According to Bernhard Korn, ‘Installing SANYO freezers was essentially a ‘plug-and-play’ experience for us. The freezers arrived, they were hooked up to the cooling water, switched on and they worked. They provide a very stable, constant environment with no temperature fluctuation – this is what we really love about them. The insulated inner doors on the MDF-U74 model reduce temperature change when the freezer is opened and there is enough space for large boxes. A wide choice of shelves provides the flexibility to accommodate the storage needs of our different research groups. These freezers are very easy to operate and we like the fact that the main power switch is on the side. This means there is no danger of a unit being turned off accidentally – a recognised hazard if this switch is placed on the front panel.’
Advantages of water-cooling
The architect of the IMB building suggested that wherever possible, equipment should be water-cooled, rather than relying on traditional air cooling. So, in addition to ULT freezers, there are centrifuges, laser systems and sterile work benches which are also connected to the building’s central water-cooling system.
As Dr Korn points out, there are various advantages to water-cooling freezers, ‘With less heat dissipated in the freezer room only ventilation is needed, rather than air-conditioning that not only requires energy but wastes heat. In fact with a total of over 100 instruments throughout the Institute hooked up to the water-cooling system, only a very few labs and measurement rooms, around five per cent of the total space, require active cooling.
‘Water is circulated at 18ºC and leaves the freezers at 23–24ºC. However, this heat energy is not wasted as the warmed water is used to contribute to the central heating for the whole building. Therefore the Institute is able to reduce energy costs and benefit the environment.’
SANYO’s water-cooled ULT freezers incorporate a double plate heat exchanger which maximises energy transfer from the refrigerant to a closed water circuit. As water is more efficient than air at removing heat, the compressor efficiency is improved. As a result, not only is energy consumption 15–20 per cent lower than for an equivalent air-cooled model, but temperature recovery after door opening and sample loading is faster, protecting samples.
Further developments at IMB
2012 will see IMB expanding to its full complement of around 12 groups, with the core facilities being extended to support the additional researchers. The success of the current water-cooled ULT freezers is underlined by plans to add five or six more of the same -150ºC freezers and up to ten of the -86ºC models, all with water-cooled condensers.
Dr Korn commented, ‘SANYO is well known not only for the quality and reliability of its ULT freezers, but also for building good customer relationships and providing excellent technical support. Another important factor for IMB is the high level of service and full guarantee provided by EWALD Innovationstechnik GmbH, the German distributor. Although initially more expensive than equivalent air-cooled freezers, choosing the water-cooled condenser option enables the Institute to use less energy and make significant savings in the medium and long term. We believe this is the way to go for the future.’
Institute of Molecular Biology GmbH
funded by the Boehringer Ingelheim Foundation
Ackermannweg 4
55128 Mainz
Tel: +49-6131-39-21501
www.imb-mainz.de
SANYO E&E Europe BV
Biomedical Division
Tel. +44 (0) 1509 265265
www.eu.sanyo.com/biomedical
Recently several clinical laboratories have reported antibody cocktails to perform leukocyte differentiation for routine screening. Distinct advantages over microscopic leukocyte differentiation are the large number of counted cells (tens of thousands) and objective immunological definition of the cell types. Here we review the published protocols and their usefulness for a routine setting.
by Dr G-J van de Geijn, Dr M. Beunis, Dr H. Janssen and Dr T. Njo
Differential white blood cell counting
Differential white blood cell count (dWBC) is an important and widely applied diagnostic test. The current generation of routine cell counters automatically produce a fast and reliable dWBC for most non-pathological samples. If dWBC results are aberrant or there are technical issues, the routine haematological analyser typically ‘flags’ a sample, and microscopic differentiation is mandatory. This dogma has been challenged by recent publications from independent groups. Although technically different, the approaches these groups have in common is that they each use a single flow cytometric tube for dWBC. This makes an implementation, which may be technically complicated and expensive, potentially feasible for clinical practice. Here we review the relative merits of the different flow cytometric approaches and attempt to position flow cytometric dWBC in clinical practice.
To appreciate the merits and disadvantages of the new flow cytometric approaches against the current microscopic practice, one must realise that leukocyte identification by flow cytometry is fundamentally different from microscopy. For example, microscopy can not differentiate lymphocyte subsets (B, T and NK cells), which are essentially defined immunologically. Flow cytometry can not replicate the microscopic classification of myeloid precursors because antigen expression in myeloid differentiation follows a different path from the microscopic phases. Although some of the dogmas for the interpretation of the microscopic leukocyte differentiation are more ‘practice-based’ than ‘evidence-based’, microscopy has the distinct advantage of a long history in clinical practice. A significant amount of training is required to ensure and maintain sufficient expertise among technicians to offer reliable round-the-clock service for microscopic dWBC. Due to the low number of cells counted (100-200) and the unequal distribution of cells on the slide, statistical variation and inter-observer differences are significant, well known disadvantages of microscopy [1,2].
Advantages of flow cytometry over microscopy are the large number of cells that are analysed (tens of thousands and more) and the objective immunological definitions of the different leukocyte types, using monoclonal antibodies defined by the international Human Leukocyte Differentiation Antigens (HLDA) classification system. This facilitates a more robust and evidence-based approach. In addition, different and more classes of leukocytes can be defined using flow cytometry compared to microscopy, providing growth potential for defining new cell populations for diagnosing and following up clinical diagnoses. Disadvantages of flow cytometry are increased costs of equipment, and that it is currently not used in many first-line haematology labs.
A new position for flow cytometry in routine clinical practice?
In the current clinical diagnostic setting, flow cytometry is almost exclusively performed in specialised laboratories during office hours, mainly as an established technique in leukaemia and lymphoma diagnostics. In routine haematological practice flow cytometry is not widely adopted. It is sometimes used as a reference method for quantifying leukocytes, erythroblasts and platelets during validation of a routine cell counter. Besides quantifying platelets and CD4 cell counts in the Celldyn-4000 and Sapphire routine cell counters, there is currently no widely adopted application in the routine laboratory. For leukocyte differentiation flow cytometry is mentioned in the CLSI guidelines as a candidate reference method for leukocyte differentiation. However the current reference method is still microscopic differentiation [3].
Flow cytometry
Flow cytometry uses specific monoclonal antibodies to detect cellular characteristics. These antibodies are labelled with fluorescent dyes emitting light at different specific wavelengths. Cell suspensions stained with a cocktail of antibodies can be analysed rapidly by flow cytometry which runs the cells past a laser. Light scatter and fluorescent signal are detected in different channels to give information on cell type, granularity and maturity of cells. Using combinations of these parameters the different cell types are detected in two-parameter dot-plots by so-called gates.
Flow cytometric differential white blood cell counting
In recent years several labs have reported antibody cocktails combined with acquisition protocols to perform flow cytometric dWBC [4-7]. The goal of these publications is to test if flow cytometric dWBC can be performed in a single tube as a screening tool for samples flagged for review by the haematology analyser [Figure 1]. Flagged samples are tested first by flow cytometry, which may reduce the number of microscopic differentiations required. These protocols have in common that they use a single tube approach requiring a small amount of blood and a cocktail of antibodies to determine an extended dWBC by using flow cytometry. Which leukocyte populations are defined, the number of leukocyte populations and the strategy used to define them differs [Table 1]. The main features of these protocols are discussed below.
Faucher and colleagues were the first to report their antibody cocktail, discriminating 12 different cell populations using a 6-marker/5-colours protocol [4]. This is the only cocktail using CD2, which enables identification of mature T-cells as well as T-blasts. This can be an advantage in detecting T-ALL with CD34- CD3- blasts. Another difference with the other cocktails is the use of CD294 to positively identify basophils, eosinophils and T-cells. The description of the lymphocyte subsets is incomplete as NK and T-cells cannot be discriminated. Although there is no general blast-marker to aid blast detection, blasts are detected and classified as T-lineage, B-lineage, monocytic or other blasts. NRBCs and plasma cells are not detected.
Using this antibody cocktail with a slightly adapted gating strategy, the first routine application, with flow cytometric dWBC integrated in the workflow of a haematology laboratory was published [8]. Samples flagged by the haematology analyser were analysed by flow cytometry before microscopy. Flow cytometer acquisition software that automatically adapts the gates to fit the different leukocyte populations and an automated pipetting station were used as technical aids. The authors show that this approach reduces the number of microscopic differentiations, manual hands-on time and turn-around-time. A group from Korea tested this cocktail with automatic gating software on a set of leukopenic samples, known to give problems with a reliable microscopic dWBC [9]. Both groups report that the gates were set correctly by the automatic software in >75% of the samples.
The cocktail reported by Bjornsson et al differentiates all nucleated cells in 11 categories using 6 markers and DRAQ5 staining with a 5-colour flow cytometer [5]. This protocol cannot discriminate between T and B-lymphocytes and uses CD203 to facilitate basophil detection. In contrast to the other cocktails, when the sample is diluted and re-measured using a low acquisition rate, CD36 can also be used to detect platelets.
Cherian and colleagues describe a 10 markers/8-colour cocktail including Hoechst staining to detect 12 leukocyte categories and NRBCs [6]. Strong points of their approach are the inclusion of CD34/117 for more robust blast detection, resulting in good correlations with microscopy. Furthermore CD33/64 is used for positive definition of monocytes and eosinophils, CD123 for basophils, CD38 for plasma cells and Hoechst to quantify NRBCs. No positive defining marker for T-cells is included.
Recently we reported our 10 marker/5-colour flow cytometric dWBC cocktail called Leukoflow [7]. Compared to the other cocktails, this cocktail uses the largest number of antibodies on a 5 colour machine. Although behind the scenes this requires a complex gating strategy to define the populations, the manual gating is not too difficult. Compared with the other methods, this assay is the most complete in defining lymphocyte subsets. Using CD3, CD19, CD16, CD56 and CD4 all lymphocyte subsets can be defined, including CD4-positive T-cells, except for the double positive CD3 and CD8 cells. CD138 is used to detect plasma cells. CD34 aids detection of blasts which can be further subdivided into blasts of the B-lymphoid, T-lymphoid or myeloid lineage. There is no positive marker for basophils. NRBCs can be quantified using a separate staining with DRAQ5 and antibodies.
Correlations between flow cytometry and cell counter/microscopy
The results of each of these reported flow cytometric protocols were compared with the results from haematology analysers and microscopy for sets of normal and abnormal blood samples. For normal, implicit, blood samples there are no real differences in the correlations between flow cytometry and microscopy for the different cocktails. In general, the correlations for neutrophils, lymphocytes and eosinophils are very good (>0.9) whereas the correlation for monocytes is lower (0.63-0.86) and the correlation for basophils is the poorest (0.29-0.70). To assess how these protocols compare when differentiating leukocytes in abnormal blood samples (e.g. containing plasma cells, blasts or immature granulocytes), these protocols should be compared on the same samples. This has not yet been reported in literature.
Additional clinical value of flow cytometric dWBC
Given the fact that different and more leukocyte populations can be identified with flow cytometric dWBC the question arises as to whether this additional information also has additional diagnostic value. Several examples of this have already been demonstrated. Roussel et al report efficient use of the ratio of T and B lymphocytes to discriminate B lymphoproliferative disorders in a random selected group of 349 with WBC >4×109/L [8]. This indicates that other flow cytometric dWBC methods that measure B- and T-lymphocytes, such as the ones reported by Cherian et al and our group can also use this [6,7]. Faucher et al demonstrated that in patients without known haematological disease flow cytometric dWBC can help to detect those with inflammatory syndrome (acute bacterial infection, heart failure, cancer, systemic disease) by their enhanced count of CD16-positive monocytes [4]. CD16-positive monocytes are found in nearly all inflammatory diseases [10]. CD16 positive monocytes are modulated during conditions such as atopic eczema, malaria infection and sepsis [11,12]. Information on CD16-positive monocytes can also be obtained with the other antibody cocktails using CD14, CD36 or CD33+CD64 to define monocytes.
The cocktail by Cherian et al contains CD64, which is reportedly upregulated on granulocytes during infection or sepsis [6,13]. Proper validation of the added clinical diagnostic value of all these parameters requires further investigations comparing patient cohorts homogeneous for the conditions mentioned above with the appropriate control patients.
Conclusion
All studies reported so far demonstrate that flow cytometric dWBC is technically feasible, and its results in general correlate well with the other known dWBC techniques. In order to compare the performance of these cocktails with each other it is crucial that they are compared on the same sample set. To our knowledge, such a comparison has not been published yet. Since all publications report good correlations between their flow cytometric dWBC and other methods for dWBC, we expect no big differences between the different cocktails for normal samples. For abnormal samples there will be differences due to the different composition of the cocktails. For implementation in a routine setting as a screening technique in between the haematology analyser and microscopic smear review, an automatic gating protocol is a significant advantage. This is only available for one of the reported methods so far. However, in order to make it a robust system suitable for use by a large group of technicians with 24/7 service, development of a flagging system that detects abnormalities/errors in the automated gating, as is present on haematology analysers, is a must. Unfortunately this is not available for any of the reported flow cytometric protocols yet, but it deserves significant attention to make this promising technique attractive for routine laboratories.
References
1. Pierre RV. Peripheral blood film review. The demise of the eyecount leukocyte differential. Clin Lab Med 2002;22(1):279-97.
2. Ruemke CL. The statistically expected variability in differential leukocytes counting In: Koepke JA, editor. Differential Leucocytes Counting. CAP Conference Aspen College of American Pathologist 1977; p 39-46.
3. Koepke JA, Van Assendelft OW, Brindza LJ, Davis BH, Fernandes BJ, Gewirtz AS, Rabinovitch A. Reference Leukocyte (WBC) Differential Count (Proportional) and Evaluation of Instrumental Methods; Approved Standard-Second Edition. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute 2007; 1-35 p.
4. Faucher JL, Lacronique-Gazaille C, Frebet E, Trimoreau F, Donnard M, Bordessoule D, Lacombe F, Feuillard J. ‘6 markers/5 colors’ extended white blood cell differential by flow cytometry. Cytometry A 2007;71(11):934-44.
5. Bjornsson S, Wahlstrom S, Norstrom E, Bernevi I, O’Neill U, Johansson E, Runstrom H, Simonsson P. Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer. Cytometry B Clin Cytom 2008;74(2):91-103.
6. Cherian S, Levin G, Lo WY, Mauck M, Kuhn D, Lee C, Wood BL. Evaluation of an 8-color flow cytometric reference method for white blood cell differential enumeration. Cytometry B Clin Cytom 2010;78(5):319-328.
7. van de Geijn GJ, van Rees V, van Pul-Bom N, Birnie E, Janssen H, Pegels H, Beunis M, Njo T. Leukoflow: multiparameter extended white blood cell differentiation for routine analysis by flow cytometry. Cytometry A 2011;79(9):694-706.
8. Roussel M, Benard C, Ly-Sunnaram B, Fest T. Refining the white blood cell differential: the first flow cytometry routine application. Cytometry A 2010;77(6):552-63.
9. Jo Y, Kim SH, Koh K, Park J, Shim YB, Lim J, Kim Y, Park YJ, Han K. Reliable, accurate determination of the leukocyte differential of leukopenic samples by using Hematoflow method. Korean J Lab Med 2011;31(3):131-7.
10. Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 2007;81(3):584-92.
11. Novak N, Allam P, Geiger E, Bieber T. Characterization of monocyte subtypes in the allergic form of atopic eczema/dermatitis syndrome. Allergy 2002;57(10):931-5.
12. Skrzeczynska J, Kobylarz K, Hartwich Z, Zembala M, Pryjma J. CD14+CD16+ monocytes in the course of sepsis in neonates and small children: monitoring and functional studies. Scand J Immunol 2002;55(6):629-38.
13. Davis BH, Olsen SH, Ahmad E, Bigelow NC. Neutrophil CD64 is an improved indicator of infection or sepsis in emergency department patients. Arch Pathol Lab Med 2006;130(5):654-61.
The authors
Dr Gert-Jan van de Geijn, Dr Marlène Beunis, Dr Hans Janssen and drs Tjin Njo, MD.
Department of Clinical Chemistry (KCHL),
Sint Franciscus Gasthuis,
Kleiweg 500,
3045 PM Rotterdam,
The Netherlands.
e-mail: g.vandegeijn@sfg.nl
The challenge of providing an effective allergy testing service in the face of the increased prevalence of the condition and a limited number of local allergists is being met by a centralised and specialised South African lab with the help of state-of-the-art equipment and facilities.
by Dr Cathy van Rooyen
Allergy is on the increase, especially in developing countries where industrialisation, lifestyle changes and socioeconomic factors lead to an increase in allergy prevalence. South Africa is no exception, as shown by data from prevalence studies of asthma in rural and urban populations [1]. The challenge in South Africa is to provide an adequate and comprehensive allergy service to a growing allergic population, in a healthcare system that has until recently been ill-prepared to meet allergy demands. South Africa has a dire shortage of trained allergists, as a postgraduate allergy diploma has only recently been introduced by the college of family medicine in South Africa and recognition of Allergy as a subspecialty of Paediatrics and Internal Medicine has only been introduced this year. There is no stand-alone postgraduate specialisation in Allergy or Immunology as seen in European countries.
The diagnostic laboratory not only faces the increased demands for allergy testing from the handful of clinicians with formal allergy training, but also the majority of requests from general practitioners without adequate allergy knowledge and who in conjunction with allergy testing, require in-depth interpretation of results, and advice on additional testing and patient management.
Allergy testing at AMPATH pathologists
AMPATH is one of the largest private pathology providers in South Africa and also provides the most extensive diagnostic service for allergy in South Africa. After consideration was given to the most effective model for the provision of this service, a model of centralisation of in vitro allergy testing was adopted. AMPATH built a National Reference Laboratory (NRL) for centralisation of allergy and other specialised tests, which has been operational since July 2011.
In addition to the centralised in vitro testing services offered, in vivo allergy diagnostics, including skin prick testing, intradermal testing and patch testing is also offered at selected centres. AMPATH also provides a diagnostic referral clinic for Allergy, where patients with complicated allergy can be referred for a detailed workup, which includes history, examination and appropriate testing. Patients are referred back to the clinician with a detailed report on the patient’s allergies and management advice. A separate service for allergen immunotherapy is also available at this clinic.
Centralisation of allergy tests in the AMPATH NRL
The AMPATH NRL is the main testing hub for specialised laboratory tests in AMPATH, South Africa. Centralisation has many advantages, which include cost-savings by increasing and optimising test volumes on a single platform and staff and resource savings, uniform test results, pooling of expertise in test performance and interpretation, etc. However, specimens do take longer to reach a centralised testing centre. The biggest challenge in such an environment is to provide a guaranteed and acceptable turnaround time (TAT), as many specimens are transported from various locations throughout the country.
The major factors that should be addressed to improve TAT are pre-analytical (easy registration of specimens, frequent, reliable and speedy transport, immediate specimen processing and sorting), analytical (24 hour continuous analysis of patient specimens on robust platforms with a large and high-speed processing capacity) and post-analytical (real-time result reporting and interpretation). It is also important to recognise that the perception of TAT for referred work from peripheral sites is largely influenced by intricate overnight transport options to the laboratory with same-night processing, ensuring patient result availability by 6h00 the following morning.
AMPATH rose to this challenge by providing logistical and pre-analytical solutions to transport specimens quickly to the AMPATH NRL using different transportation systems. Once the specimens reach the laboratory, processing is automated and specimens are sorted by high-speed sorters (Beckman automate 2550) [Figure 1] and transported regularly to the Serology Department, where allergy testing is performed. In the Serology department further sorting and aliquotting is performed by another high-speed sorter (Beckman automate 2550) down to test / analyser level. Specimens are processed continuously as soon as they reach their sorting targets.
From a logistic, quality, cost and time-management perspective, AMPATH adopted a policy of automation and consolidation of assays onto automated platforms where possible. A large autoline from Beckman Coulter was installed in the Autolab where various chemistry, immunochemistry and infectious disease testing was consolidated [Figure 2]. Allergy and autoimmune testing was not available on this system and after meticulous research a decision was made not to integrate third-party analysers onto the system. A separate system was therefore required to manage automation of allergy and autoimmune testing.
Previously allergy testing was done on one Phadia 1000 and two Phadia 250 instruments from ImmunoDiagnostics*. These systems were chosen for the quality of their assays, ease of use and reliability. We therefore looked to ImmunoDiagnostics for higher capacity instruments that would meet our needs. We were informed of the new Phadia 2500, which is an integrated and automated system which provides for high volume allergy and autoimmune testing on one platform. This would provide an ideal solution in terms of staff and space saving, easier workflow by consolidating tests on one instrument and increased processing capacity to accommodate increased test volumes due to centralisation. At the same time we would still have the same superior test quality that we require. Unfortunately the Phadia 2500 was not ready for installation when we moved into our new laboratory facility in July 2011, but ImmunoDiagnostics prioritised our request and our Phadia 2500 was installed in October 2011. After a full instrument validation, we have transferred all of our IgE mediated allergy testing as well as a significant portion on our autoimmune testing to the Phadia 2500 [Figure 3].
Performance of the new Phadia 2500 in the AMPATH NRL
We have been extremely happy with the performance of the Phadia 2500 in our laboratory. We have experienced the following advantages after switching to the system:
– Consolidation of allergy and autoimmune diagnostics onto one platform which can be operated by a single technologist, thereby saving one full time equivalent staff member.
– Space saving, as the footprint of the Phadia 2500 is significantly smaller than the three previous allergy and autoimmunity instruments (Phadia 1000 and two Phadia 250s)
– Continuous random access with continuous specimen processing.
– Large processing capacity, which eliminates bottlenecks over peak times.
AMPATH routinely tests for approximately 260 individual allergen-specific IgEs, but there are approximately 700 different allergens available which can be ordered when requested. ImmunoDiagnostics is also constantly adding new allergens and recombinant allergen components to its test menu. We are therefore able to offer the widest range of allergens available for allergy testing in South Africa.
Autoimmune testing in the AMPATH NRL
Patients are screened for connective tissue diseases using immunofluoresence on HEP-2 cells. In patients with positive screening tests, further testing is done by performing a connective tissue disease (CTD) screen for extractable nuclear antigens (ENA) on the Phadia 2500 instrument. This screen contains 16 Individual ENAs. Should the screen be positive, a breakdown of the individual autoimmune components is performed. The new Phadia 2500 streamlines this process through high sample throughput and software solutions which supports our workflow protocols, thereby ensuring optimal TAT.
Range of allergy tests performed in the AMPATH NRL
AMPATH’s main objective is to be the national leader in allergy diagnostics. This is not only beneficial for individual patients, but also leads to a competitive advantage in our local market. We spend a lot of time on research and development in allergy and also on implementing scientifically proven methods or tests in our testing repertoire.
In addition to IgE mediated allergy testing to crude allergen extracts, AMPATH is also placing emphasis on testing to different allergen components. This is either available as singular allergens on the Phadia 2500, or as part of a multiplex microchip array (ISAC or Immuno-Solid Array Chip) from ImmunoDiagnostics. The latest ISAC assay detects specific IgE to 112 recombinant allergens and can provide additional diagnostic insight by the prediction of cross-reactivity, prediction of risk for severe reactions, prediction of whether allergies will be outgrown and additional information on the heat-stability and bio-degradibility of certain allergens.
There has also been an increasing focus on cellular allergy (non-IgE mediated allergy) and AMPATH has expanded its test repertoire by offering Basophil Activation Testing (BAT) by flow-cytometry for foods and food additives, drugs, venoms and inhalants [Figure 4]. Our BAT was developed in-house, and commercially available allergens from Buhlman Laboratories are mostly used, except for drug allergies, where the suspect drug itself is used and tested in different concentrations in comparison to a non-allergic control patient. A similar testing strategy is followed for T-cell mediated allergies, where a modified T-cell proliferation assay (LTT) is used, mainly for drug allergies, metal allergies and occupational allergies. The demand for these services has increased substantially and cellular testing volumes are approaching a third of our total allergy testing requests.
A new philosophy in allergy testing – from bench to bedside
Experience has showed us that in vitro allergy testing only isn’t sufficient in our local environment.AMPATH has therefore instituted in vivo allergy testing at multiple allergy testing depots throughout the country. A range of in vivo allergy testing, such as skin prick testing (SPT), prick-prick testing, intradermal testing and patch testing may be performed at some of these depots.
Although AMPATH’s combined allergy testing services with careful interpretation by allergy consultant pathologists is usually adequate, there is a small minority of complicated patients that still require additional testing or for whom the most appropriate testing protocol cannot be identified. After becoming aware of this void, we decided to take allergy testing from the bench to the bedside by providing a diagnostic referral clinic for allergies and immunology. Clinicians can refer complicated patients to this clinic for an appropriate diagnostic service where the patient’s history and physical examination is considered before testing and management guidelines are given based on test result interpretation. Allergen immunotherapy is also provided where indicated [Figure 5].
Lessons learnt from centralisation and automation
Automation can lead to significant benefits in diagnostic pathology when applied appropriately. The full benefits of automation can only be reached when both pre-analytical as well as analytical steps are automated. Optimal workflow planning is essential to the success of the automation project. Automation works optimally in a simplified workflow environment – too many rules, exceptions and workarounds slow the process. Automation also works optimally if hardware, instruments and software are from the same supplier. Although third party analysers can easily be connected to an automated track, software may often not be fully compatible with middleware and can lead to suboptimal TAT.
The way forward
We want more automation, more integration and more consolidation. We envisage a smaller staff complement of highly trained staff. We are looking at more automation solutions, e.g. slide processing and other manual techniques performed in our serology department. We are also aiming to expand our cellular allergy/immunology department with further research and test development. We are also looking at software solutions to aid with allergy diagnostic interpretation, especially considering unique South African sensitisation patterns and cross-reactive allergens. Through these efforts and by dedication to our patients and clinicians, we aim to embrace the future of allergy diagnostics in South Africa.
References
1. Weinberg EG. Urbanisation and childhood asthma: An African perspective. JACI 2000; 105(2):224-231.
The author
Dr Cathy van Rooyen, MBChB, MMed Path, FRC Path
Ampath Laboratories, South Africa
*ImmunoDiagnostics (formerly Phadia) is part of Thermo Fisher Scientific
Hereditary spherocytosis is an inherited haemolytic anaemia due to fragile red cells. This article gives a brief overview of the pathophysiology of this red cell disorder, and presents the key points on the different screening tests.
by Dr May-Jean King
Membrane structure of human red blood cell and associated defects
The human red blood cell (RBC) is discoid or biconcave in shape. It deforms when navigating through blood vessels and capillaries. The integrity and elasticity of RBC are maintained and regulated by a series of interactions between two layers of proteins localised to the outer lipid bilayer and the cytoskeleton on the cytoplasmic side [Figure 1]. The resulting RBC membrane is a 3D structure composed of specific transmembrane proteins (the band 3 macro-complex, and the glycophoein C-protein 4.1R) and a 2D network of skeleton proteins spectrin, actin, protein 4.1R and other minor components [1]. A qualitative or quantitative abnormality in one of these membrane proteins will lead to fragile red cells and haemolytic anaemia. Hereditary spherocytosis is associated with defects in the vertical interaction of the band 3 macrocomplex (i.e., band 3, CD47, and Rh complex) with protein 4.2, and ankyrin to which β-spectrin binds directly [Figure 1]. Hereditary elliptocytosis has abnormalities in protein 4.1R or defective spectrin self-association [2]. A partial deficiency of protein 4.1R can affect its interaction with glycophorin C and P55 in a junctional complex, which is stabilised by a band 3-adducin-spectrin bridge [3]. The mutations located in the self-association site for spectrin αβ heterodimers can affect the formation of tetramers or higher oligomers that enable the extension of the spectrin-based cytoskeleton to cover the cytoplasmic side of the red cell membrane. HS and hereditary elliptocytosis are not single-gene diseases.
Hereditary spherocytosis
Hereditary spherocytosis (HS) is more prevalent among the Northern European populations (about 1 in 2000 to 5000 births) than in other ethnic groups. Where the cytoskeleton fails to attach to band 3 in the membrane via protein 4.2 and ankyrin, that area of membrane becomes detached and is pinched off from the intact RBC. This continuous loss of membrane lipid and integral membrane proteins reduces the RBC volume and transforms it into a spherocyte. Splenic sequestration of spherocytes reduces their lifespan in circulation to <120 days. Therefore a patient with HS presents a haemolytic anaemia with reticulocytosis, jaundice and possibly gallstones and/or splenomegaly [4]. The clinical phenotype of HS is heterogeneous, ranging from asymptomatic, mild, moderate to severe haemolysis requiring blood transfusion. HS is diagnosed in newborn or as late as in the fifth to seventh decade of life. A mild HS condition can be exacerbated by an infection (e.g., Parvovirus B19, CMV, Herpes 6, gastroenteritis), resulting in a severe haemolytic anaemia.
Laboratory testing for HS
Membranopathy is suspected when the cause of haemolytic anaemia remains unknown after the exclusion of enzymopathy, haemoglobinopathy and other extrinsic factors. Finding spherocytes in a blood smear indicates HS, but is not necessarily definitive. Exclusion of immune haemolytic anaemia (AIHA) is important because this condition also presents with spherocytosis [5]. Typical HS is expected to present almost all of the following features: evidence of a haemolytic process (e.g., raised bilirubin and LDH, low or no haptoglobin), low Hb, reduced mean cell volume, elevated mean cell haemoglobin concentration, and raised reticulocyte count [Figure 2]. The raised MCHC and increased % hyperdense RBCs are useful markers [6]. The diagnosis of dominant HS (75% of cases) is straightforward when family history of HS and the results for the red cell indices and blood chemistry are available. In the case of recessive HS, the proband may present a severe haemolytic anaemia with the blood smear showing anisocytosis and occasional cell fragments whereas the parents are apparently asymptomatic.
The majority of subjects with HS can be diagnosed by using a screening test without resorting to further investigation [Figure 2]. Two traditional screening tests for HS are still in use: the osmotic fragility test [7] and the acid glycerol lysis time test [8] [Table 1]. The cryohaemolysis test uses a change in temperature to effect red cell lysis [9]. The ektacytometer gives specific deformability profiles for a range of red cell disorders [Table 1]. However, this technique can give similar profiles for both HS and AIHA. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins is the confirmatory test because it detects all the membrane proteins known to be associated with HS [Figure 3, panel I]. Molecular analysis of membrane protein genes is usually performed by research laboratories. However, knowing the membrane protein defects and the associated protein gene mutation(s) does not influence the management of HS patients [12]. Unlike the aforementioned HS screening tests, the unusual feature of the EMA (eosin-5’-maleimide) Binding test [13] is the use of a flow cytometer, which analyses individual intact RBC in a sample. Confocal microscopy of EMA-labelled RBCs showed emission of both green and red fluorescence. RBCs of different sizes and shapes are labelled [Figure 3, panels II and III], [14]. The test is robust, only a low volume of patient specimen (5 µL packed RBC) and test reagent is required, and the test gives consistently reproducible results.
Conclusion
There is no screening test that has 100% sensitivity and 100% specificity for the diagnosis of HS. The adoption of the EMA Binding test is because it is easy to use and an abnormal result often indicates a membrane-associated red cell disorder. When this flow method is used in conjunction with the Osmotic Fragility test, differential diagnosis of HS and hereditary stomatocytosis can be made [described in 12].
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The author
May-Jean King
Membrane Biochemistry
NHS Blood and Transplant
North Bristol Park
Filton
Bristol BS34 7QH
UK
e-mail: may-jean.king@nhsbt.nhs.uk
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