Objective: Immunophenotyping of blood lymphocytes is an important tool in the diagnosis of hematologic and immunologic disorders. Because of maturation and expansion of the immune system in the first years of life, the relative and the absolute size of lymphocyte subpopulations vary during childhood. Therefore we wished to obtain reference values for the relative and the absolute size of all relevant blood lymphocyte subpopulations in childhood. Study design:We used the lysed whole blood method for analysis of lymphocyte subpopulations in 429 blood samples from neonates (n = 20), healthy children (n = 358), and adults (n = 51). The following age groups were used: I week to 2 months (n = 13), 2 to 5 months (n = 46), 5 to 9 months (n = 105), 9 to 15 months (n = 70), 15 to 24 months (n = 33), 2 to 5 years (n = 33), 5 to 10 years (n = 35), and 10 to 16 years (n = 23).Results: Our results show that the absolute number of CD19* B lymphocytes increases twofold immediately after birth, remains stable until 2 years of age, and subsequently gradually decreases 6.5-fold from 2 years to adult age. The CD3* T lymphocytes increase 1.5-fold immediately after birth and decrease threefold from 2 years to adult age. The absolute size of the CD3*/CD4* T-lymphocyte subpopulation follows the same pattern as the total CD3 + population, but the CD3+/CD8 * T lymphocytes remain stable from birth up to 2 years of age, followed by a gradual threefold decrease toward adult levels. In contrast to B and T lymphocytes, the absolute number of natural killer cells decreases almost threefold in the first 2 months of life and remains stable thereafter. Our study also showed that changes in the absolute size of lymphocyte subpopulations are not always consistent with changes in their relative size. This demonstrates that the relative counts of lymphocyte subsets do not reflect their actual size and are therefore of limited value. Conclusion:On the basis of this study we strongly recommend that immunophenotyping of blood lymphocytes for the diagnosis of hematologic and immunologic disorders be based on the absolute rather than on the relative size of lymphocyte subpopulations. Our data can be used as age-matched reference values for blood lymphocyte immunophenotyping. (J Pediatr 1997; 130:388-93)

Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during persistence. The relationship between isolates was analyzed by fingerprinting their chromosomal DNA. The fingerprints of eight strains (isolated from eight patients) with various MOMP compositions were different, whereas fingerprints of isolates with identical MOMP compositions were indistinguishable. In 12 patients, two or more strains with different MOMP compositions were found; one strain was isolated from the sputum samples of each of the six remaining patients. In seven of the 12 patients, strains with different MOMPs but with indistinguishable fingerprints were found. The differences were found in proteins b,c (five patients) and d (five patients). In patients with COPD, the MOMPs of H. influenzae are subject to changes that may enable this bacterium to escape immunological defense mechanisms.

To analyze whether exacerbations in chronic obstructive pulmonary disease (COPD) coincide with reinfection by Haemophilus influenzae, 16 COPD patients were studied longitudinally for 3 years. Exacerbations coincided with reinfection by H. influenzae, either endogenous, by a strain with a DNA fingerprint indistinguishable from the strain previously present but with another major outer membrane protein (MOMP) pattern (2 patients), or exogenous, by a strain with a different DNA fingerprint and MOMP pattern (3 patients). The other patients, remaining in an infectious state without clear exacerbations for longer periods, were persistently infected by a particular H. influenzae strain (median persistence time, 5.5 months; range, 2-23 months). Of 8 antibiotic-treated patients, 7 remained infected by H. influenzae with the same DNA fingerprint, although all strains were sensitive to the antibiotics prescribed. Results of the study suggested that exacerbations in COPD patients coincide with endogenous or exogenous reinfection by H. influenzae, persistently infected patients keep the same H. influenzae strain for longer periods, and antibiotic treatment was not effective in eradicating H. influenzae.

The titer and specificity of antibodies to the infecting Haemophilus influenzae was determined in sera and sputa from 27 patients with chronic obstructive pulmonary disease (COPD) to analyze the specific immune response. COPD patients had significantly higher serum IgG and IgA antibody titers than 13 healthy controls (mean IgG titers 12,302 and 5,623, respectively; mean IgA titers, 2,398 and 912; p less than 0.001). The mean IgM titers were comparable: 501 and 447, respectively. Specific IgA antibodies were also detectable in the sputum of the COPD patients (mean IgA antibody titer, 776). The local antibody production was determined by calculating the relative coefficient of excretion (RCE) to albumin. The mean RCE of 89.1 for IgA indicated statistically significant local production (p less than 0.02), in contrast to a nonsignificant increase for IgG (mean RCE of 3.6). Specific IgM was below the detection level. Immunoblotting experiments showed that the antibodies in sera from COPD patients and controls were directed against most of the outer membrane proteins of H. influenzae, with individual differences between IgG, IgA, and IgM. The IgA and IgG antibodies in serum had a similar specificity as those in sputum. The appearance or persistence of H. influenzae coincided with minor changes in antibody titer and specificity. From these results we conclude that COPD patients are infected with H. influenzae despite the presence of at least as many antibodies in sputum and serum as in controls and that these antibodies are directed against a variety of antigenic determinants of the infecting strain.

Differences in the major outer membrane protein b,c (molecular weight, 39,000 to 41,000) of related Haemophilus influenzae strains isolated from the sputum of patients with chronic obstructive pulmonary disease were analyzed biochemically and immunologically. Protein b,c was isolated from a total of six related H. influenzae strains from two chronic obstructive pulmonary disease patients. After CNBr digestion of the proteins, the differences in size appeared in the larger of the two fragments. Trypsin and chymotrypsin digests of proteins from related H. influenzae strains showed that proteins differed by only a few peptides or were very similar, in contrast to the peptide maps of proteins from nonrelated strains. Peptide analysis of b,c proteins from related H. influenzae strains by high-performance liquid chromatography after Staphylococcus aureus V8 protease digestion and amino acid analysis of corresponding fractions revealed highly comparable patterns, indicating only minor differences in the amino acid sequences of these proteins. Immunization of rabbits with intact bacteria of four related H. influenzae strains resulted in a strong anti-protein b,c antibody response in all animals. In three of four rabbits, antibodies specific for the b,c protein of the strain used for immunization were elicited, indicating that the changed proteins contained specific immunodominant epitopes. Anti-protein b,c antibodies promoted strain-specific, complement-dependent, bactericidal activity. From these results, we conclude that H. influenzae shows antigenic drift in immunodominant epitopes, caused by small changes in amino acid composition of the b,c protein. Antibodies to these epitopes promote complement-dependent bactericidal activity.