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Flow cytometric immunophenotyping in the diagnosis and follow-up of immunodeficient children de Vries, E.; Noordzij, J.G.; Kuijpers, T.W.; van Dongen, J.J.M. DOI 10.1007/s004310100797 Publication date 2001

Published in European Journal of Pediatrics

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Citation for published version (APA): de Vries, E., Noordzij, J. G., Kuijpers, T. W., & van Dongen, J. J. M. (2001). Flow cytometric immunophenotyping in the diagnosis and follow-up of immunodeficient children. European Journal of Pediatrics, 160(10), 583-591. https://doi.org/10.1007/s004310100797

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Download date:25 Sep 2021 Eur J Pediatr -2001) 160: 583±591 DOI 10.1007/s004310100797

REVIEW

Esther de Vries á Jeroen G. Noordzij á Taco W. Kuijpers Jacques J.M. van Dongen Flow cytometric immunophenotyping in the diagnosis and follow-up of immunode®cient children

Received: 29 March 2001 / Accepted: 15 May 2001 / Published online: 19 July 2001 Ó Springer-Verlag 2001

Abstract From time to time, paediatricians are con- immunode®cient children according to the four main fronted with children who might suer from a primary clinical categories. immunode®ciency disease. For practical purposes, these children can be divided into four main clinical catego- Keywords Flow cytometry á Immunode®ciency á ries: -1) a relatively large group of childrenwith recur- Immunophenotyping rent ear-nose and throat and lower respiratory tract infections, in some cases caused by de®ciencies of anti- Abbreviations AID activation-induced cytidine bodies or complement; -2) children with failure to thrive, deaminase á BTK Bruton tyrosine kinase á intractable diarrhoea or an opportunistic infection CD40L CD40 ligand á ENT ear-nose and throat á which canbe caused by a T-lymphocyte or combined FSC forward scatter á HIV humanimmunode®ciency immunode®ciency; -3) children with infections with py- virus á IFN interferon á IL interleukin á LWB lysed ogenic bacteria or fungi as seen in case of granulocyte/ whole blood á McAb monoclonal antibody á monocyte function de®ciency; and -4) a small hetero- SCID severe combined immunode®ciency á SSC side geneous group of children with recurrence of particular scatter á TCR T-cell receptor á ICF immunode®ciency- infections. Also, acquired immunode®ciency becomes a centromeric instability-facial dysmorphism more commonproblem inpaediatric practice. Flow cytometric immunophenotyping of leucocytes appears to be an ecient and rapid tool in the diagnosis and follow- Introduction up of immunode®cient patients, supporting early recognition, before serious infections have compromised From time to time, paediatricians are confronted with the child's general condition. This technique can now be childrenwho might suer from a primary immuno- performed inmanyhospitals. Inthis review, we give de®ciency disease [21]. For practical purposes, these directions for the use of ¯ow cytometric immunophe- children can be divided into four main clinical catego- notyping of leucocytes in the diagnosis and follow-up of ries [7, 26, 28]. The ®rst clinical category comprises the relatively large group of childrenwith recurrent ear-nose and throat -ENT) and lower respiratory tract infections, only in some cases caused by an underlying E. de Vries -&) á J.G. Noordzij á J.J.M. vanDongen immunode®ciency, especially if encapsulated extracel- Department of Immunology, Erasmus University Rotterdam, lular bacteria are found as pathogens. In patients with PO Box 1738, Rotterdam, The Netherlands, hypo- or agammaglobulinaemia, de®cient opsonisation Tel.: +31-10-4088094 with antibodies leads to impaired phagocytosis of these Fax: +31-10-4089456 micro-organisms. Complement de®ciencies are rare and E. de Vries inmost types of complementde®ciencyaected chil- Department of Paediatrics, Bosch Medicentrum, dren present with collagen-vascular disease [15]. How- `s-Hertogenbosch, The Netherlands ever, some complement de®ciencies lead to severely T.W. Kuijpers impaired opsonisation with recurrent serious bacterial Department of Paediatrics, Academic Medical Centre, Amsterdam, The Netherlands infections resembling agammaglobulinaemia -C3 de®- ciency, factor D de®ciency and factor I de®ciency; to a T.W. Kuijpers Department of Experimental Immunohaematology, lesser extent C2 de®ciency). Most children with recur- Central Laboratory of the Netherlands Red Cross Blood rent, often viral, ENT and lower respiratory tract Transfusion Service, Amsterdam, The Netherlands infections do not have an antibody or complement 584 de®ciency, but are immunocompetent. Their problems of their light scatter characteristics, such as forward are due to other more commoncauses such as bron- scatter -FSC) as measure for size, and side scatter -SSC) chial hyperreactivity, adenoidal hypertrophy, or an as measure for cellular irregularity -Fig. 1) [12]. Leuco- allergic constitution, or they may suer from rare cyte count and dierential cell count can then be used to non-immunological diseases like cystic ®brosis or calculate absolute counts of the blood lymphocyte sub- immotile cilia syndrome. populations, which should be compared with age- The second clinical category comprises children with matched reference values [6]. failure to thrive, intractable diarrhoea or an opportu- Blood sample handling, cell separation methodology nistic infection, which can be caused by a T-lymphocyte and labelling techniques in¯uence the reliability of the or combined immunode®ciency with absent or func- results obtained by ¯ow cytometric immunopheno- tionally de®cient T-lymphocytes. Their defence against typing of leucocytes [12]. Dead cells show increased intracellular micro-organisms is inadequate. The third auto¯uorescence, potentially leading to incorrect inter- clinical category comprises children with super®cial and pretation of staining results. This can be minimised by systemic infections with pyogenic bacteria or fungi, who using freshly collected samples which are fully analysed apparently have problems with their ®rst line of within24 h. The immunophenotypingprocedures take defence against invading micro-organisms as in cases 4±5 h, implying that the sample should arrive in the of granulocyte/monocyte function de®ciency. Finally, laboratory in the morning. Cell separation via density the fourth clinical category comprises a small hetero- gradients may result in dierential loss of speci®c lym- geneous group of children with recurrence of particular phocyte subpopulations. This is avoided when the lysed infections such as recurrent neisserial infections in cases whole blood -LWB) technique is used. The currently of late complement component de®ciency, or recurrent available LWB techniques require fewer preparation mycobacterial infections in cases with a defect in the steps and less sample handling via direct incubation of interferon -IFN)-c receptor, interleukin -IL)-12 or the the anticoagulated blood with McAbs, in combination IL-12 receptor [19]. These last three clinical categories with red blood cell lysis. These LWB techniques also are only rarely encountered in general paediatric prac- reduce non-speci®c binding of McAbs, especially if the tice. applied McAbs are directly conjugated with ¯uoro- Most cases of acquired immunode®ciency are iatro- chromes. genic and can therefore be anticipated: as more and more children are being treated with immunosuppressive drugs, aggressive chemotherapy protocols, and stem cell transplantation, acquired immunode®ciency becomes a more commonproblem inpaediatric practice [4]. Also, the prevalence of paediatric human immunode®ciency virus -HIV) infection in Europe is increasing, especially in children born to parents who originate from HIV- endemic areas [8]. Laboratory studies for identi®cation and clinical follow-up of immunode®cient children can now be performed inmanyhospitals. Inthis review, we focus our discussion on the increasingly important role of ¯ow cytometric immunophenotyping of leucocytes in the diagnosis and follow-up of immunocompromised children inpaediatric practice [12]. Applicationof ¯ow cytometric techniques helps to speed up the diagnostic process, thereby supporting early recognition of primary immunode®ciencies before serious infections have compromised the child's general condition, reducing the Fig. 1 Flow cytometric immunophenotyping of lymphocytes in the chances of survival [21]. blood of a BTK-de®cient X-linked agammaglobulinaemia patient as compared to a healthy control. -A) Analysis of peripheral blood of a healthy control. Dot plot with FSC and SSC showing ``lympho-gate'', ``mono-gate'', and ``granulo-gate'' -left). The Flow cytometric immunophenotyping B-lymphocytes inthe left upper quadrantexpress CD19 ontheir cell surface -middle). The T-lymphocytes inthe right lower quadrant express CD3 on their cell surface -middle and right). Flow cytometric immunophenotyping allows precise The NK-cells in the left upper quadrant are de®ned as CD3- assessment of relative frequencies of leucocyte subpop- negative and CD16- and/or CD56-positive -right). -B) Analysis of ulations by detection of cell surface and intracellular peripheral blood of a BTK-de®cient X-linked agammaglobulina- markers with ¯uorochrome-labelled monoclonal anti- emia patient. This 3-year-old boy presented at the age of 2 years with skin infections. Dot plot with FSC and SSC showing bodies -McAb). Immunophenotyping can be focussed ``lympho-gate'' -left). Complete absence of CD19+ B-lymphocytes on lymphocytes, monocytes, or granulocytes, because -middle), with CD3+ T-lymphocytes and CD16+ and/or CD56+/ these leucocyte populations can be de®ned on the basis CD3) NK-cells normally present -right) 585 hyper-IgM syndrome can be made via mutation analysis Diagnosing children with primary immunode®ciency of the CD40L gene. The autosomal recessive form of hyper-IgM syndrome is caused by mutations in the ac- Children with recurrent ENT and lower respiratory tivation-induced cytidine deaminase -AID) gene. AID tract infections proteins are speci®cally expressed in germinal centre B- cells in secondary lymphoid organs and are required for In some children with recurrent ENT and lower respi- immunoglobulin isotype switching as well [23]. ratory tract infections, a relatively mild antibody de®- ciency like IgA de®ciency, IgG subclass de®ciency, or anti-polysaccharide antibody de®ciency is found [21]. Childrenwith failure to thrive, intractable diarrhoea, Lymphocyte number and immunophenotype are com- or with an opportunistic infection pletely normal in these children -Table 1). It is useful to perform immunophenotyping of lym- Many children with T-lymphocyte or combined immu- phocytes according to the ``limited'' protocol -Table 2), node®ciency do not present with recurrent infections but when a rare severe antibody de®ciency with hypo- or with failure to thrive or intractable diarrhoea [26]. The agammaglobulinaemia is found. Determination of T- dierential diagnosis is extensive in these cases. The lymphocyte counts generally rules out severe combined combination of lymphopenia and agammaglobulina- immunode®ciency -SCID) and determination of B-lym- emia candirect the search towards animmunode®cien- phocyte counts supports the discrimination between the cy, but these are not invariably present. Opportunistic various causes of severe antibody de®ciency -Table 1) infections almost always indicate an immunode®ciency, [21]. If immunophenotyping of lymphocytes shows that either acquired -see below) or congenital. B-lymphocytes are virtually absent in the peripheral The ``limited'' immunophenotyping protocol as blood compartment -Fig.1), this strongly suggests an showninTable 2 caneasily detect severe combined early stop inB-lymphocyte development.This occurs in immunode®ciency disease. Generally, T-lymphocytes X-linked agammaglobulinaemia with Bruton tyrosine will be absent or very low in number [21]. However, kinase -BTK) de®ciency as well as in rare autosomal transplacental transfer of maternal T-lymphocytes in recessive forms of agammaglobulinaemia, such as Igl SCID patients or remaining -oligoclonal) T-lymphocyte heavy chain de®ciency. Using a McAb directed against production in Omenn syndrome -Table 1) might coin- BTK, it is now possible to detect BTK de®ciency in most cidentally lead to seemingly normal T-lymphocyte patients by analysing the monocytes with ¯ow cytome- counts. The presence or absence of B-lymphocytes dif- try; normal monocytes also express BTK [14]. Further- ferentiates between B+ SCID and B- SCID, as found in more, the type of B-cell dierentiation arrest in bone X-linked common c-chainde®ciencyor autosomal re- marrow canbe assessed with a special research protocol cessive Janus-associated kinase-3 -JAK3) de®ciency and -Fig. 2) and can direct the diagnostic analysis at the in recombination activating genes -RAG)-1 or -2 de®- molecular level -Fig. 3). ciency, respectively -Fig. 5). Major changes in either the If B-lymphocytes are present in the peripheral blood CD4+/CD3+ helper T-lymphocyte count or the CD8+/ compartment of patients with a severe antibody de®- CD3+ suppressor/cytotoxic T-lymphocyte count can be ciency, common variable immunode®ciency or hyper- suggestive for the diagnosis of MHC class II de®ciency IgM syndrome are more likely diagnoses. In common with low CD4+/CD3+ counts [5] or a diagnosis of 70 variable immunode®ciency, progressive hypogamma- kD zeta-associated protein -ZAP70) tyrosine kinase de- globulinaemia with combinations of IgA de®ciency, IgG ®ciency with low CD8+/CD3+ counts, respectively subclass de®ciency, and/or anti-polysaccharide antibody -Fig. 3) -Table 1). However, inmanycases of combined de®ciency can be found. Immunophenotyping of blood immunode®ciency, immunophenotyping results are less lymphocytes can show accompanying T-lymphocytope- aberrant with lymphopenia developing over time. Ad- nia [25]. In X-linked hyper-IgM syndrome with a defect ditional tests are then required to obtain a diagnosis. inCD40 ligand-CD40L), severe hypogammaglobuli- Lymphopenia develops within months after birth in naemia with normal or increased IgM is found. Immu- adenosine deaminase or purine nucleotide phosphory- nophenotyping of activated blood T-lymphocytes can lase de®ciency due to metabolic poisoning of the cells. show absence or decreased expression of CD40L -Fig. 4) Lymphopenia also gradually develops in the ®rst years -Table 2) [16]. Insome patientsCD40L is expressed but after birth inWiskott-Aldrich syndromeandtowards not functional; tests for CD40 binding can identify these adolescence or even adulthood in ataxia teleangiectasia patients. The defect in CD40L disrupts the CD40L- -Table 1) [10]. CD40 mediated T-B cell communication, which is in- If immunode®ciency is highly suspected but the dispensable for isotype switching from IgM to IgG, IgA ``limited'' immunophenotyping protocol shows no ab- or IgE. Consequently, X-linked hyper-IgM syndrome is normalities, an ``extended'' immunophenotyping proto- not a pure antibody de®ciency but should be considered col as shown in Table 2 can identify additional children as a T-lymphocyte disorder, which probably explains with speci®c subtypes of T-lymphocyte or combined why aected childrenoftenpresentwith anopportu- immunode®ciency that are not accompanied by major nistic infection. The de®nitive diagnosis of X-linked changes in the main lymphocyte populations -Table 1). 586

Table 1 Primary immunode®ciency diseases Diseases Pathogenesis Immunophenotyping

Mild Antibody De®ciencies -recurrent ENT and lower respiratory tract infections, lambliasis, sometimes asymptomatic) IgA de®ciency Pathogenesis unknown; in Generally not useful Normal immunophenotyping IgG-subclass de®ciency anti-polysaccharide antibody results. Focus onserum Ig -sub) Anti-polysaccharide antibody de®ciency, B-lymphocytes are classes and antibody reactivity de®ciency unable to produce antibodies against polysaccharide antigens, despite adequate responses against protein antigens Severe Antibody De®ciencies -recurrent respiratory tract and ENT infections, life-threateningbacterial infections, enteroviral CNS infections, lambliasis) BTK enzyme de®ciency Early arrest inB-cell ``Limited'' protocol Absent blood B-lymphocytes. -X-linked agammaglobulinaemia) development -bone marrow protocol) Dierentiation arrest re¯ected in Igl heavy chainde®ciency precursor B-cell populationin -autosomal recessive) bone marrow -useful for directing Several other rare forms DNA diagnostics in non-X-linked -autosomal recessive) agammaglobulinaemia patients) X-linked hyper IgM syndrome CD40L de®ciency leads to absent `Èxtended'' protocol Absent or decreased CD40L with CD40L de®ciency isotype switch of B-lymphocytes -CD40L) expressiononactivated and associated T-lymphocyte T-lymphocytes inX-linkedform; defect due to absent CD40 blood B-lymphocytes present stimulation Autosomal recessive hyper Mutations in the AID gene ``Limited'' protocol Blood B-lymphocytes present IgM syndrome disrupt isotype switching and somatic hypermutationin germinal centre B-lymphocytes Commonvariable Probably defects inB-cell ``Limited'' protocol Blood B-lymphocytes present; immunode®ciency maturationandhelper sometimes T-lymphocytopenia T-lymphocyte function Combined Immunode®ciency Diseases -failure to thrive, intractable diarrhoea, eczema, and other chronic problems, or opportunistic infections) Adenosine deaminase de®ciency Accumulationof toxic purine ``Limited'' protocol Increasing T-lymphocytopenia in -autosomal recessive) metabolites due to enzyme the ®rst months after birth; also Purine nucleotide phosphorylase de®ciency B-lymphocytopenia in adenosine de®ciency -autosomal recessive) deaminase de®cient SCID MHC class I expressionde®ciency Impaired antigen presentation `Èxtended'' protocol De®cient MHC class I expression -autosomal recessive) -b2 microglobulin) MHC class II expression Impaired antigen presentation `Èxtended'' protocol De®cient MHC class II de®ciency -autosomal recessive) -HLA-DR) expression; low numbers of CD4+ T-lymphocytes Wiskott Aldrich syndrome Impaired cytoskeletal ``Limited'' protocol Increasing T-lymphocytopenia -X-linked) -patients also suer reorganisation upon activation of with time; -impaired CD43 from eczema, thrombocytopenia, platelets and T-lymphocytes expression) and malignancy) Severe Combined Immunode®ciencies -failure to thrive, intractable diarrhoea, eczema, and other chronic problems, or opportunistic infections) X-linked SCID -common Defect inT-lymphocyte and ``Limited'' protocol T-lymphocytes absent, NK cells c-chainde®ciency) NK cell development low, and B-lymphocytes present JAK3 de®ciency -autosomal recessive) RAG1 or RAG2 -autosomal Early arrest incommon ``Limited'' protocol T- and B-lymphocytes absent, recessive) lymphocyte development NK cells present Omenn syndrome -autosomal Defective T-lymphocyte ``Limited'' protocol Oligoclonal T-lymphocytes, very recessive; partial RAG development with B-lymphocyte low or absent B-lymphocytes de®ciency) -patients present de®ciency with severe eczema) ZAP70 proteintyrosine Defect inT-lymphocyte ``Limited'' protocol T-lymphocytes, especially CD8+ kinase de®ciency development decreased; B-lymphocytes present -autosomal recessive) CD3 c-or-chainde®ciency Defect inT-lymphocyte function ``Limited'' protocol T-lymphocytes decreased, CD3 -autosomal recessive) expressiondecreased; B-lymphocytes present IL-7 receptor de®ciency Defect in the a-chainof the IL-7 ``Limited'' protocol T-lymphocytes absent, NK cells receptor, leading to a defect in and B-lymphocytes present T-lymphocyte development Reticular dysgenesis -patients Stem cell defect leading to ``Limited'' protocol Pancytopenia present with pancytopenia) defective maturationof T- and B-cells and myeloid cells 587

Table 1 -Contd.) Diseases Pathogenesis Immunophenotyping

Milder Combined Immunode®ciencies DiGeorge -CATCH-22) syndrome Disturbance of embryonic `Èxtended'' protocol Sometimes TCRab+ -cardiac malformations, development; 22q11 deletion -TCRab, TCRcd) T-lymphocytopenia with normal hypoparathyroidism, thymic TCRcd+ T-lymphocyte counts insuciency) Ataxia teleangiectasia Mutationinthe ATM gene; ``Limited'' protocol Sometimes increasing -autosomal recessive) exact pathogenesis unknown T-lymphocytopenia with time Chromosomal breakage Mutationinrespectively the ``Limited'' protocol Immunophenotyping mostly syndromes -Nijmegen breakage NBS, BLM,andICF genes; normal, but disturbances may be syndrome, Blooms syndrome, exact pathogenesis unknown present ICF syndrome) -mental retardation, photosensitivity, abnormal physionomy) Auto-immune Defect inapoptosis `Èxtended''protocol Absence of CD95 -FAS) on blood lymphoproliferative syndrome -CD95) leucocytes insome patients, presence of CD3+/CD4±/CD8± T-lymphocytes [24] De®ciency of IL12, IFNc Disturbed communication `Èxtended'' protocol Absence of IL-12 receptor on receptor, or IL12 receptor betweenT-lymphocytes/NK cells -IL12, IFNcR, IL12R) T-lymphocytes and NK cells, or -recurrent mycobacterial and monocytes; failure of absence of IFN-c receptor or infections, salmonellosis) monocyte activation by intracellular IL-12 in monocytes T-lymphocytes/NK cells Granulocyte/monocyte De®ciencies -super®cial and systemic infections with pyogenic bacteria and fungi) Chronic granulomatous disease Defective microbial killing due Generally not used; Normal immunophenotyping -X-linked or autosomal recessive) to a defect inoneof the ®ve `èxtended'' results; focus ondefective NADPH oxidase components protocol -b558) microbial killing in granulocyte function tests; -in patients with X-linked chronic granulomatous disease the expressionof cytochrome b558 may be defective) Leucocyte adhesionde®ciency Defective leucocyte adhesiondue `Èxtended'' protocol Absence of CD18 expression, type 1 -autosomal recessive) to a defect inCD18, the integrin -CD18) otherwise normal b2 chain Leucocyte adhesionde®ciency Defective leucocyte adhesiondue `Èxtended'' protocol Absence of CD15s expression, type 2 -autosomal recessive) to a defect inCD15s, -CD15s) otherwise normal sialyl-Lewis-X Complement De®ciencies -neisserial infections, serious bacterial infections, collagen-vascular diseases, sometimes asymptomatic) C2 de®ciency -autosomal Impaired opsonisation due to Not useful Normal immunophenotyping recessive) classical pathway defect results; focus ondeterminationof sometimes leading to serious serum complement activity bacteraemic infections C3 de®ciency, factor I de®ciency Seriously impaired opsonisation Not useful Normal immunophenotyping -autosomal recessive), factor D leading to a phenotype similar results; focus ondeterminationof de®ciency to that seenin serum complement activity agammaglobulinaemia C6, C7, C8, C9 de®ciency Impaired terminal complement Not useful Normal immunophenotyping -autosomal recessive), pathway leading to predisposi- results; focus ondeterminationof properdin de®ciency -X-linked) tion to neisserial infections; serum complement activity exact pathogenesis not clear

For example, in MHC class II de®ciency the absence of cellular cytokines, such as IL2 and IFN-c -T-helper 1 HLA-DR surface expressiongives the clue to the diag- pro®le), and IL4, IL5, and IL10 -T-helper 2 pro®le). nosis [5]; CATCH-22 -DiGeorge) syndrome is some- These tests are generally not performed in routine lab- times accompanied by severe T-lymphocytopenia with oratories and will not be further discussed here. low to absent -TCR)-ab+ T-lymphocytes and normal TCRcd+ T-lymphocyte counts -J.J.M. van Dongen, unpublished observation). In unclassi®able cases, ex- Children with super®cial and systemic infections tensive specialised immunophenotyping protocols may with pyogenic bacteria or fungi help to unravel the immunode®ciency [12, 13, 21]. These protocols may include markers of maturation, such as Most children with super®cial and systemic infections CD45RA -``naive'') and CD45RO -``memory'') on T- with pyogenic bacteria or fungi suer from neutropenia lymphocytes, markers of activation and/or proliferation, due to haematological disorders or iatrogenic causes -see co-stimulatory and/or signalling molecules, and intra- below). Flow cytometric immunophenotyping of leuco- 588

Table 2 Flow cytometric immunophenotyping protocol for the diagnosis of primary immunode®ciency diseases in paediatric practice ``Limited'' protocol -performed in many hospitals; use absolute counts and age-matched reference values) [6] T-lymphocytes CD3+ Total T-lymphocytes CD4+/CD3+ Helper subset CD8+/CD3+ Suppressor/cytotoxic subset B-lymphocytes CD19+ or CD20+ Total B-lymphocytes NK cells De®ned as CD3± as well as CD16+ and/or CD56+ population Quality control The ``lymphosum'' should equal 92%±98% -=% T+% B+% NK) ``Extended'' protocol -performed in specialised laboratories only; select relevant items based on the information presented in Table 1; always perform the ``limited'' protocol as well; compare with normal age-matched controls run in parallel, if no reference values are available) Analysis of the arrest in B-cell development in the bone marrow and of the intracellular expression of BTK in monocytes of patients with agammaglobulinaemia with absent B-lymphocytes Common c-chainexpression-CD132) onlymphocytes inSCID patientswith B-lymphocytes CD40L expression -CD154) on activated T-lymphocytes in hypogammaglobulinaemia with normal or high IgM; CD40L function is assessed by CD40 binding assays HLA-DR -class-II) and b2 microglobulin -class I) expression on leucocytes in children suspected of severe combined immunode®ciency with normal lymphocyte numbers and phenotype on initial screening TCRab- and TCRcd-expression on T-lymphocytes in children with neonatal hypocalcaemia and congenital heart disease Extensive specialised protocols in unclassi®able cases of combined immunode®ciency with stainings for `naive' -CD45RA) and `memory' -CD45RO) T-lymphocytes, for markers of activation and proliferation, for co-stimulatory and signalling molecules, and for intracellular cytokines -T-helper 1 and T-helper 2 pro®les) Intracellular cytochrome b558 expression in phagocytes if chronic granulomatous disease is suspected in children with phagocyte function de®ciency CD18 and CD15s expression on leucocytes if leucocyte adhesion de®ciency type 1 or type 2 is suspected in children with phagocyte function de®ciency and extreme leucocytosis Analysis of the IL-12 receptor b1 chain on T-lymphocytes/NK cells and of intracellular IL-12 and the IFN-c receptor chains on monocytes in children with recurrent mycobacterial infections cytes plays only a minor role in the diagnostic process in these children. A defect in granulocyte/monocyte function, such as in chronic granulomatous disease or leucocyte adhesion de®ciency, is rare. In chronic granulomatous disease, granulocytes show de®cient microbial killing due to a defect in one of the ®ve NADPH oxidase complex components [21]. This can be investi- gated by function tests of granulocytes. Flow cytometric immunophenotyping with McAb directed against cytochrome b558 can show absence of this compound in many but not all patients with the most frequent -65%) X-linked form of chronic granulomatous disease -``extended'' protocol in Table 2) [3]. In leucocyte adhesion de®ciency type 1 -CD18 de®ciency) and type 2 -CD15s de®ciency), leucocyte adhesion is impaired. This leads to leucocytosis, which is evenmore pronounced Fig. 2 Flow cytometric immunophenotyping of lymphocytes in the bone marrow of a BTK-de®cient X-linked agammaglobulinaemia during infections [21]. CD18 and CD15s expression can patient as compared to a healthy control. -A) Analysis of bone be analysed with immunophenotyping -Table 2) -Fig. 6) marrow of a healthy child -bone marrow donor). Dot plot with [1, 11]. FSC and SSC showing ``lympho-gate'' -left). CD3+ T-lymphocytes from contaminating peripheral blood are present in the right lower quadrant -middle). CD19+ B-lineage cells are present in the left upper quadrant -middle). CD19 is present on all B-lineage cells Children with recurrent particular infections from the pre-B-I cell stage onwards. CD10 is expressed on immature B-lineage cells and CD20 is expressed on mature De®ciencies in one of the components of the terminal B-lymphocytes. Immature CD10+/CD20)/CD19+ precursor-B- cells as well as mature CD10)/CD20+/CD19+ B-lymphocytes are complement pathway result in recurrent neisserial, present in the right lower and left upper regions, respectively mainly meningococcal, infections -C6 de®ciency, C7 -right). -B) Analysis of bone marrow of a BTK-de®cient X-linked de®ciency, C8 de®ciency, and C9 de®ciency; also prop- agammaglobulinaemia patient -same patient as in Fig. 1). Dot plot erdin de®ciency) [15]. Lymphocyte number and im- with FSC and SSC showing ``lympho-gate'' -left). Few CD19+ munophenotype are normal in these children -Table 1). B-lineage cells are present -middle), which are mostly immature CD10+/CD20)/CD19+ precursor-B-cells -right) De®ciencies in the IFN-c receptor, IL-12 or the IL-12 receptor lead to recurrent mycobacterial infections [19]. monocytes by ¯ow cytometry. Information about the The IL-12 receptor canbe detected onT-lymphocytes or expressionpatternsof these molecules candirect the NK-cells and the IFN-c receptor canbe detected on diagnostic analysis at the molecular level, but it is im- 589

Fig. 3 Scheme of lymphocyte dierentiation in the bone marrow portant to realise that protein expression does not nec- and thymus. Relevant cell surface and intracellular markers are essarily imply intact protein function. indicated. Dotted lines show the assumed positions of the dierentiation arrests in various primary immunode®ciency diseases. -Cy cytoplasmic, Sm surface membrane) Follow-up of children with acquired immunode®ciency

During follow-up of children treated with immunosuppressive drugs or chemotherapy, the degree of neutropenia is easily assessed with a leucocyte count and dierential. The slow reconstitution of lymphocytes after bone marrow transplantation [2] can be monitored by a simple immunophenotyping protocol to determine the numbers of the main lymphocyte populations -``limited'' protocol inTable 2). It should be kept in mind, however, that functional restoration of the lymphoid system takes more time thanis needed for the normalisation of absolute counts [22]. HIV infection is generally diagnosed by serology or polymerase chain reaction. Immunophenotyping has a role inthe follow-up of HIV-infectedchildrenwith the determination of decreasing CD4+/CD3+ helper T- Fig. 4 Flow cytometric analysis of CD40L expression on activated T-lymphocytes inthe peripheral blood of anX-linkedhyper-IgM lymphocyte counts as a prognostic marker for the syndrome patient. -A) Analysis of stimulated peripheral blood of a development of AIDS: long-term risk of mortality healthy control -PMA and Ca-ionophore for 5 h). Unstimulated increases from 33% to 97% with baseline CD4+ per- + CD3 T-lymphocytes do not express CD40 ligand -left). Expres- centage decreasing from ³35% to <5% [18]. It also has sionof the early activationmarker CD69 - middle) and CD40L + a role, together with HIV RNA quanti®cation, for the -right) uponactivated CD3 T-lymphocytes. -B) Analysis of + stimulated peripheral blood of an X-linked hyper-IgM syndrome timing of antiretroviral therapy. The CD4 helper patient. This boy presented at the age of 8 months with chronic T-lymphocyte count is used for the classi®cation of the diarrhoea and was bone marrow transplanted at the age of 5 years. degree of immune suppression: ³1500/llunder12 CD3+ T-lymphocytes are normally present -left) and show normal expressionof CD69 - middle) but absent expression of CD40 ligand months of age, ³1000/ll from 1±5 years of age, and -right) uponactivation.-Courtesy of Dr. E.G. vanLochem, ³500/ll from 6±12 years of age indicate the absence of Department of Immunology, Erasmus University Rotterdam) immune suppression, whereas <750/ll under 12 months 590

Fig. 6 Flow cytometric detectionof CD18 molecules inthe diagnosis of leucocyte adhesion de®ciency type 1. In the healthy control -left panel), virtually all leucocytes express the CD18 Fig. 5 Flow cytometric immunophenotyping of blood lympho- molecule whereas those of the leucocyte adhesionde®ciencytype 1 cytes inSCID patients.- A) Analysis of peripheral blood of a B± patient are de®cient for CD18 expression -right panel) SCID patient with a mutation in the RAG2 gene. This girl presented at the age of 12 months with recurrent otitis media and respiratory infections, and chronic diarrhoea. Dot plot with FSC laboratories -``extended'' protocol in Table 2). This and SSC shows the ``lympho-gate'' -left). Withinthe ``lympho- + identi®es children with immunode®ciencies that are not gate'', virtually no CD19 B-lymphocytes were detected -middle), accompanied by major changes in the main lymphocyte and strongly reduced CD3+ T-lymphocyte counts were found -right), whereas the NK-cell counts -CD3)/CD56+/CD16+) were populations. However, it should be noted that not all within the normal range for age -right). -B) Analysis of peripheral childrenwith animmunode®ciencyshow alterationsin + blood of a B X-linked SCID patient with a mutation in the cc immunophenotyping results -Table 1). Also, genetic gene. This boy presented at the age of 4 months with pneumonia, mutations do not always lead to absence of protein ex- diarrhoea and sepsis. Dot plot with FSC and SSC shows the ``lympho-gate'' -left). Withinthe ``lympho-gate'' CD19 + B-lym- pression, even if protein function is severely hampered. phocytes were detected -middle), but CD3+ T-lymphocytes were More extensive immunophenotyping protocols used absent -right), and the NK-cell counts -CD3)/CD56+/CD16+) inspecialised laboratories or research settingshelp to were decreased -right) recognise new immunode®ciency diseases by identifying as yet undescribed staining patterns. Also, pathogenesis of known defects can be further elucidated: the absence of age, <500/ll from 1±5 years of age, and <200/ll of speci®c cell surface markers [16, 21], the loss of spe- from 6±12 years of age indicate severe suppression [27]. ci®c epitopes de®ned by McAb staining patterns [9, 17], + During antiretroviral therapy, monitoring of CD4 or the determination of the B-cell dierentiation arrest helper T-lymphocyte counts is useful for evaluating the inbonemarrow cansupport the precise characterisation treatment eectiveness [20] in addition to HIV RNA of the defect. Therefore, it is important to use these more quanti®cation [27]. For further guidelines, the reader is extensive immunophenotyping protocols in patients referred to the literature [27]. with an established immunode®ciency disease, as well as More elaborate immunophenotyping protocols can in patients with probable immunode®ciency disease that be reserved for research purposes, e.g. for comparisonof has not been proven with other methods. immune reconstitution after bone marrow transplanta- Due to the rapid developments in the ®eld of ¯ow tion with normal ontogeny. Such studies are important cytometry and antibody production on the one hand for understanding the regeneration processes, but have and the recognition of new immunode®ciency diseases no direct clinical relevance as yet. onthe other, it is likely that the ``limited'' and``extended'' immunophenotyping protocols de®ned here will need to be updated in a few years time. Conclusion

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