Results

DEVELOPMENT OF MICROASSAY

We incubated 100, 40, 20, and 10 μl of blood with monoclonal antibodies. When using 20 μl of blood, at least 100 000 nucleated cells remained available after completion of the staining procedure in all samples tested, as determined by use of a cell counter (Coulter Counter; Hialeah, FL). However, when using 10 μl of blood, major and variable cell loss was observed. Relative frequencies of lymphocyte subpopulations determined in parallel with either 100 μl or 20 μl of blood per test tube gave fully comparable results in five separate neonatal blood samples. Therefore, 20 μl of blood incubated with 20 μl of each monoclonal antibody per test tube was used in all further experiments.

The determination of the relative frequencies of lymphocyte subpopulations by either double or triple immunostaining was compared in one adult and six cord blood samples—for example, triple CD4/CD8/CD3 vs double CD4/CD8, CD4/CD3 and CD8/CD3 immunostaining. When using triple immunostaining, monoclonal antibodies were either incubated consecutively, with each incubation followed by two washes, or all together. No significant differences were found between the relative frequencies of lymphocyte subpopulations, as determined by the different staining methods. Therefore, in all further experiments only triple immunostaining was used with incubation of all three fluorochrome conjugated monoclonal antibodies simultaneously to speed up the microassay and minimise cell loss due to multiple washes.

DETECTION OF ERYTHROID CELL CONTAMINATION

When a scatter gate is set around the lymphocyte population, it is generally not possible to exclude fully other contaminating cells without excluding lymphocytes. However, as long as the sum of the relative frequencies of B lymphocytes, T lymphocytes, and natural killer cells (the so-called “lympho-sum”) is at least 95% (or 0.95), the lympho-gate is often considered to be placed correctly. The remaining events within the lympho-gate are monocytes, granulocytes, and unidentified events, which generally consist of dead cells and debris. In our neonatal cord blood samples the lympho-sum rarely reached 95% of the lympho-gate (table 1). In preterm neonates, in particular, the lympho-sum seemed to be far too low. This was caused by a relatively high frequency of unidentified events (fig 1).

Normoblasts express CD71 and GpA, whereas erythrocytes express only GpA. In contrast, leucocytes express CD45, unlike normoblasts and erythrocytes. Therefore, CD71⁺/GpA⁺/CD45^- cells within the lympho-gate can be regarded as normoblasts, whereas unlysed erythrocytes have the CD71^-/GpA⁺/CD45^-immunophenotype.5 By triple immunostaining with CD71-FITC, GpA-PE, and CD45-PE-Cy5, these unidentified events could be identified as contamination by normoblasts and in some samples by unlysed erythrocytes as well (fig 2) (table 1). The FSC and SSC of these normoblasts and unlysed erythrocytes was checked (“back-gating”), and was found to be relatively low, as expected for these cells (figs2C and D).

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Figure 2

Triple staining for CD71, GpA, and CD45 identifies normoblasts and unlysed erythrocytes within the lympho-gate. (A) Lympho-gate setting. (B) CD71⁺/GpA⁺/CD45^- population of normoblasts and the CD71^-/GpA⁺/CD45^- population of unlysed erythrocytes within the CD45^- lympho-gate. (C) Back-gating of CD71⁺/GpA⁺/CD45^-normoblasts into the lympho-gate. (D) Back-gating of and CD71^-/GpA⁺/CD45^-unlysed erythrocytes into the lympho-gate.

A small percentage of events remained unidentified—that is, they were neither CD45⁺ leucocytes, nor CD71⁺/GpA⁺/CD45^- normoblasts, nor CD71^-/GpA⁺/CD45^- unlysed erythrocytes (left lower quadrant of dot plot in fig 2B). These events may represent dead cells and debris.

ERYTHROID CELL CONTAMINATION IN NEONATAL BLOOD

CD71/GpA/CD45 staining was used to determine the relative counts of normoblasts and unlysed erythrocytes and to calculate the relative counts of lymphocyte subpopulations. The differences in relative frequencies of lymphocyte subpopulations in neonatal cord blood with and without a correction for normoblasts and unlysed erythrocytes are shown in table 2 for two samples. Normoblasts were not found by manual differentiation or flow cytometric immunophenotyping in blood samples from the same neonates at 1 week of age nor in blood samples from adults, but unlysed erythrocytes were still present in some samples at 1 week of age (table 1).

Discussion

We have developed a microassay for flow cytometric determination of blood lymphocyte subpopulations in neonates and young infants by adapting the lysed whole blood technique, using one step triple immunostaining. When only double immunostaining is available, more blood is needed and less detailed results are obtained—for example, double positive CD45RA⁺/CD45RO⁺CD3⁺ T lymphocytes cannot be identified. This micro-assay needs only about 0.4 ml of blood to determine T lymphocyte subpopulations for monitoring HIV infected infants, and 0.5 to 0.8 ml of blood is sufficient for detailed evaluation of infants with suspected congenital immunodeficiencies. Extensive protocols for research purposes can also be performed, because 1.0 ml of blood is sufficient for more than 40 triple immunostainings. These are acceptable volumes in infants and even in preterm babies.

Normoblasts and unlysed erythrocytes can be present in considerable numbers in neonatal samples.4 The presence of normoblasts cannot be prevented by technical measures, but strict adherence to a rigorous lysing protocol can minimise the number of unlysed erythrocytes in a sample. Therefore, identification of both cell populations is of interest. Using triple immunostaining for CD71, GpA, and CD45, we were able to identify both normoblasts (CD71⁺/GpA⁺/CD45^- cells) and unlysed erythrocytes (CD71^-/ GpA⁺/CD45^- cells) within the lympho-gate, and to measure their relative frequencies. When only double immunostaining is available, GpA⁺/CD45^- cells can be used to measure the relative frequency of erythroid cells, but normoblasts and unlysed erythrocytes cannot be identified separately. The relative frequencies of erythroid cells can be used to calculate the correct relative frequencies of blood lymphocyte subpopulations. This is especially important in preterm neonatal and prenatal blood samples, where the relative frequencies of normoblasts and unlysed erythrocytes can be high (tables 1 and 2),6 and the frequencies of lymphocyte subpopulations can be underestimated. This can be confusing if a congenital immunodeficiency is suspected. The erythroid cell contamination of the lympho-gate rapidly disappears after birth and is virtually absent by 1 week of age (table1).

In conclusion, we have developed a fast and easy to use lysed whole blood microassay for immunophenotyping of neonatal and infant blood lymphocyte subpopulations. It requires only a small volume of blood (20 μl per test tube), and offers a method to correct for the presence of normoblasts and unlysed erythrocytes in neonatal samples. It would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies when genetic markers are not available.7