CYTOLOGICAL, CYTOCHEMICAL AND IMMUNOLOGICAL FINDINGS FROM TWO CHILDREN WITH CHEDIAK-HIGASHI SYNDROME

Cytological, cytochemical and immunological findings from two children with Chediak-Higashi


INTRODUCTION
Chediak-Higashi Syndrome (CHS) is an inherited autosomal recessive generalized cellular disorder characterized by partial albinism (depigmentation of eye, hair and skin), frequent pyogenic infections and abnormal large granules in leukocytes and other granule-containing cells.CHS patients frequently exhibit neutropenia, relative lymphocytosis, thrombocytopenia, nystagmus, peripheral neuropathy, fever of unknown origin and impairment of natural killer cells function.The disease evolves to an accelerated phase with pancytopenia and a diffuse mononuc1ear infiltrate '.This rapidly proliferative phase usual1y leads to death frorn infection or hemorrhage, and may be associated with a T -cell Iymphoma-, The identification of abnormally large cytoplasmic inclusions in circulating white blood cells and bone marrow is a very important morphologic markcr for CHS4,1l.These inc1usions are shown as multiple iregular greyto dark blue granules in neutrophils and one large azurophil granule in lymphocytes anel monocytes, when staineel with Wright's stain.ln neutrophils, cytochemical studies have shown that these granules were positive for peroxielase, acid phosphatase anel esterase.ln this report, we describe two unrelated patients with clinieal, morphologic and cytoehemical features of leukocytes similar to those from patients with CHS.One of them showed also lymphoeytosis.

Patient 1
Patient 1 is a 2.1-yr-old mulatto male who was a 7 pound, 8-ounee, 40-week-gestation produet of a.31-yr-old woman.Neonatal cyanosis during the period of 24 hours and nasal obstruction were noted.At age of 2 months the patient presented bronchopneumonia and at 5 months he was found to have hypopigmented skin and hair.
The family medical history reported a brother and a cousin who died aftcr presenting the same cutaneous picture.He has two normal brothers and two normal sisters.The parents are cousins.
The patient has no history of other serious infections, However, he did experienee repeated bouts of furunculosis.
The child was referred to the Instituto da Criança "Prof.Pedro de A1cântara", Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and the Chediak-Higash'i syndrome was diagnosed by the examination of the blood and bone marrow samples that revcaled large, abnormal granules in most granulocytes and in many lymphocytes and monoeytes.
At that time, the patient was found to have enlarged splecn and liver, moderate anacmia and otitis media.He was treated with ferrous sulphate, antibiotics and ascorbic acid (lg/day).After then, he showed a transient pcriod of lymphocytosis.

Patient 2
Patient 2 is a 2-yr-old mulatto girl who has no family history since her parents were anonym.Shc is adoptive daughtcr of a rich family.
The patient was referred to lhe "Instituto da Criança Prof, Pedro de Alcântar a, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo" due to derrnatolo gic problems.On admission the child presenteei partial albinism with dcpigrncntarion o f eye, hair anel skin, photophobia, nystagrnus, intermittcnt Icbr ile episodes anel pyogenic infcctions.Thc presence of abnormal larg e granules in lhe cytoplasm of peripheral leukccytes anel bone marrow finally led to lhe diagnosis of CHS.
.262 Nowadays, she is an a good health, with no signs of the accelerated phase of the disease.

Laboratorial Methods
Peripheral blood and bone marrow smears were proeessed by Leishman stain.The pcriphcral blood Ieukocytes were stained for acid phosphatase, alkalin phosphatase, peroxidase, mucopolysaecharydes (PAS), neutral fat (Sudan Black), nonspecific esterase using alpha-naphthyl butyrate as substrate!".The nitroblue-tetrazolium (NBT) reduction test, stimulated or not with lipopolysaccharide, was also perforrned!".All reagents uscd were from SIGMA Co., U. S. A Mononuclear cells from periphcral blood were isolated by ccntrifugation on a Iicoll-hypaque gradient.The number of T and B was determined by the rosettc method using shecp red blood cells and zymosan-complement complex '.
Dctcrrninations of the subset lymphocytes bearing the CD4 or CD8 marker were made by staining cells with a fluorescein conjugated monoclonal antibody (Leu 3a and Leu 2a, Becton-Dickinson, U. S. A), using a direct immunofluorescence assay '.
The lymphocytes of the patient 1 were stimulated with phytohernmaglutinin mitogen and aiso maintained in suspension culture for five weeks, without addition of external growth Iactors", The medium was changed weekly by a fresh one through centrifug ation of the cells in culture.The viability was determined by trypan blue exclusion tcchnique,

Cytologlcal study
The bone marrow smears showed abnormal granules in megakariocytes, promyelocytcs, myelocytes and leukocytes in both cases.

Light microscopic and cytochcmical studies
Irem pcripheral blood leukocytes showed the characteristic lysosomal granules, as previously dcscribcd in CHS.

Cytochemical Study
The results of the cytochemical reactivity of the granules in lhe leukocytes are summarized in Table 1. and also as a weak diffuse staining in the cytoplasm of neutrophils and monocytes.
The NBT histochemical stimulated test, using lipopolysaccharide from E.coZi was positive in 58% of neutrophils from patient 1 and 88% in neutrophils from patient 2.

Immunologlcal evaZuation
The results of the quantitative analysis of the blood cells are shown in Table 2.
The CRS patients showed a low neutrophils count.
The patient 1 exhibit leukocytosis and lymphocytosis during a period of the disease, when his T and B lymphocytes were diminished in percentage and increased in total number.The patient 2 displayed similar results of T and B lymphocytes in percentage but in absolute number there were normal.The lymphocytes subsets were found altered with a low number of T helper cells in patient 1 when compared with a control group.
The patient 1 was studied three times.In the second one he showed an episode of lymphoproliferation, returning to normal after two months.
The mononuclear cells obtained from patient 1 during the lymphoproliferative phase showed normal response to the PRA and remained alive in continuous culture for five weeks, with a viability ranging from 96 to 60%.The cclls death in the culture was due to fungal contamination.

DlSCUSSION
In this study two cases of CRS are presented.Both are mulatto children from the North East region of Brazyl where the consanguineous marriages are frequent, as referred in familial history of patient 1.
The clinical manifestations frequently reported in CRS were noted in these patients.Thcy showed skin hypopigmentation and silvery tint colo r of the hair '-".Patient 2 mentioned a susceptibility to sunlight and had also photophobia and rotatory nystagmus.Both patients referred repeated febrile episodes with reeurrent infectious diseases.
An interview with the mother of patient 1 revealed the death of another son and nephew with the same cutaneous picture.
The cutaneous hypopigmentation seen in these patients are specially related to giant melano somes present in melanocytes".
The genetic involvement in this disorder is determined by one recessive gene that is lethal in homozygous state!".The kariotype has been found normal in number of chromosome but with prevalent breakages in chromatide.'".Thc consanguineous marriages are frequent in these cases".
In both cases of this paper, the CRS was diagnosed by laboratorial pathognomonic finding of abnormal granules present in peripheral blood and bone marrow leukocytes, similar to those prcviously described in morphological and cytochemical tests+'! , 15. Variations in number, size, shape and color of the granules were displayed in polymorphonuclear leukocytes.The lysosomal nature of the granules was demonstrated by their acid phosphatase, peroxidase and esterase contento The amount of reaction product deposited in the abnormal granules and its distribution were extremely variable, even in the same cell, and this is probably associated with the presence of primary and secondary lysosomes.An explanation for the discrepancy in celllevels of lysosomal enzymes in CRS was previously dcmonstrated and revealed that most of the giant granules were lysosomes, loaded with substances derived from specific granules and cytoplasmic materials".
A1though in this study the majority of leukocytes showed large abnormal granules, the respiratory burst activity was normal, as demonstrated by a normal stimulated NBT test.Similar results were described in two patients with leukocytes granulation abnormalities associated with neurologic impairment, without other signs of CRS 8 .Abnormalities of cyclic nucleotide metabolism, disorders of microtubule assembly, impaired neutrophil and monocyte chemotaxis, and dclayed phagolysosomal fusion are related to the increased susccptibility of these patients to infection and justify the classification of this entity as a "phagocyte disorder"13,14,lS.
In order to correct the microtubule defect and membrane fluidity in both patients, mcgadoses of ascorbic acid was used 3 ,9.A transient reduction in the number of abnormal granules in Ieukocytes was observed in patient 1.On the othcr hand, after six months follow-up he dcveloped a marked leukocytosis and lymphocytosis.Despite of the increased number of lymphocytes, CD4+ lymphocytes were diminished in percentage.In addition to this, the mononuclear cells obtained from patient 1 at this time were capable of remaining alive for five weeks in continuous cell culture, without additional conditioned medium.
Several reports suggest that lymphoproliferative phase of the disease is a reaction to a viral infection atributed to Epstein-Barr virus, Cytomegalovirus, Herpes simplex virus or to Varicelazoster virus3•S,7.12.16; however, direct evidence for this generally lacking.The basis for the marked cellular proliferation is not understood.In the present study it was not possible to demonstrate virus-like particle in peripheral blood cells nor to perform serological tests.
Others observations do not exclude the possibility of a reactive process which coexist with a neoplasic one.So, lymphomas Hodgkin's and the T -cell type were described".Some patients experience rcpeated lymphoprol ifer ative phases interrupted by periods of remission.Our patient 1 presented rever, jaundice, hepatoesplenomegaly, lymphadenophathy and partcytopenia and at a peripheral level he presented a mixed population of lymphoid cells and not a homogcneous population of highly atypical cells.This is consis-tent with a benign reactive processo Continuous cell lines from peripheral blood lymphocytes were establishcd from one male patient with CHS (homozygous line) and frorn his father (heterozygous line), although no fungal, bacterial and viral material could be recovcredê.It's not known, whether viral agents are present "in vivo" in the cells obtained for culture or relcased from a latent stage upon being subjected to "in vitro" conditions.
Little is known about patient 2 since her parents are anonymous.Nevertheless, both patients have been followed, and abone .marrowtransplant is bcing evaluated as an approach to correct a sedes of abnormalities that involves hematopoietic progenitor cells.
. = not done.T helper cells = CD4+ lymphocytes.T suppressor cells = CD8+ lymphocytes.purple stained body (Figure lC), Patient 2 showed 81 % of neutrophils with 16 to 25 abnormal granules and 100% of eosinophils with a median of 40 inclusion bodies per cell.Forty percent of lymphocytes contained one large granule were around 40.
The reactivity of the neutrophil, eosinophil, lymphocyte and monocytc granules for acid phosphatase was positive (Figure lD) and nega-tive.Alkalin phosphatase reaction was negative in lymphocytes and monocytes and positive in neutrophils (Figure lE).Except in lyrnphocytes, all abnormal granules are strongly peroxidasepositive (Figure lF), Abnormal granulations in neutrophils were PAS negative, while the surrounding cytoplasm stained strongly pink (Figure lG).The giant granules of lymphocytes and monocytes gave positive PAS reaction (Figure IR).Therc were some variation in the intensity of the reactivity of the granules with Sudan-Black being strongly positive in neutrophils, eosinophils and monocytes (Figure 11), and negative in thc lyrnphocytes.Esterase activity of the granule was shown as a large, reddish brown aggregate (Figure lJ) resulting from a strong enzymatic activity A.; SANTOS-FORTUNA, E. de 10s & CARNEIRO-SAMPAIO, M. M. S. -Cytological, cytochemical and immunological findings from two childrcn with Chediak-Higashi syndrome.Rev. Inst.Adolfo Lutz,50(1/2): 261-267, 1990. .