One-step enrichment of nucleated red blood cells

Journal oflmmunological Methods, 158 (1993) 277-280 © 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

277

J1M06620

Short communication

One-step enrichment of nucleated red blood cells A potential application in perinatal diagnosis Neelima M. Bhat, Marcia M. Bieber and Nelson N.H. Teng Department of Gynecology and Obstetrics, Stanford University, CA 94305, USA (Received 22 July 1992, revised received 14 October 1992, accepted 15 October 1992)

We describe a discontinuous triple density gradient to obtain a 25-fold enrichment of nucleated red blood cells from mononuclear cells, granulocytes, and mature red blood cells in a single centrifugation step. Key words: CD71; Nucleated red blood cell; Cell Isolation

Introduction

Various fetal cell types, nucleated red blood cells (erythroblasts), trophoblasts, and leukocytes cross the placenta and circulate within maternal blood (Schroder et al., 1975; Adinolfi et al., 1991). The use of these rare fetal cells circulating in maternal peripheral blood for perinatal diagnosis of genetic diseases is an attractive proposition. Unlike current methods, e.g., chorionic villus sampling, amniocentesis, and fetoscopy, this noninvasive technique would not increase infection or abortion risks. Several studies have reported the presence of fetal cells in maternal circulation by enzymatic amplification of gene sequences that are fetal specific (Bianchi et al., 1990; Camaschella et al.,

Correspondence to," N.N.H. Teng, Department of Gynecology and Obstetrics, Stanford University, CA 94305, USA. Tel.: 415-723-7334; Fax: 415-723-7737. Abbreviations: FL, fluorescein isothiocyanate; PE, phycoerythrin; TR, Texas red; FACS, fluorescent activated cell sorter; PBS, phosphate-buffered saline.

1990; Yeoh et al., 1991). Since the frequency of fetal cells in maternal circulation is low, enrichment/purification of these cells prior to amplification of specific genes is essential to obtain detectable signals. The method of choice is flow cytometric purification of cells tagged with fluorochrome-conjugated fetal-specific monoclonal antibodies (Bianchi et al., 1990; Camaschella et al., 1990; Yeoh et al., 1991). Flow cytometry, however, is an expensive and complicated method requiring highly skilled operators and expensive equipment. The quick and inexpensive triple gradient described in this paper allows a 25-fold enrichment of nucleated red blood ceils compared to the standard one-step method that is commonly used. This triple gradient is a modification of the double gradient that enriched mononuclear cells from erythroblasts in fetal liver (Bhat et al., 1990). The addition of the third gradient allows further separation of CD71 ÷ (transferrin receptor) erythroblasts from granulocytes and mature a n d / o r immature red blood cells that are CD71-. Since the frequency of erythroblasts in maternal peripheral blood is very low, we demonstrate this technique

278

using adult peripheral blood spiked with cord blood (30 : 1) as a source of nucleated red blood cells. Coupled with specific gene amplification to obtain detectable signals, this technique can be potentially applied to maternal peripheral blood for perinatal diagnosis. Studies in that direction are in progress.

Histopaque 1077 on top (Fig. 1). All Histopaque solutions were obtained from Sigma Diagnostics, St. Louis, MO. 7 ml of diluted cord blood cell suspension was very carefully layered on top of the gradient. The tubes were centrifuged at 600 x g for 30 min, cells at each interface were recovered, washed twice in PBS, and resuspended in Hanks' balanced salt solution with 3% fetal calf solution.

Materials and methods

Antibodies Sample preparation Heparinized adult peripheral blood and umbilical cord blood from normal deliveries at term (> 37 weeks) were obtained with the approval of the Committee for the Protection of Human Subjects at Stanford University. Specimens were diluted 1/3 in phosphate-buffered saline (PBS). 1 ml of diluted cord blood was mixed with 29 ml of diluted adult peripheral blood and separated on the two density gradients as decribed below.

Fluorescein isothiocyanate (FL)-conjugated anti-leukocyte HLe-1 (CD45, pan-leukocyte marker), phycoerythrin (PE)-conjugated anti-LeuM7 (CD13, pan-granulocyte and monocyte marker), and unconjugated anti-transferrin receptor (CD71) antibodies were obtained from Becton Dickinson Immunocytometry Systems (San Jose, CA). The anti-CD71 antibody was revealed with Texas red (TR)-labelled goat anti-mouse IgG (Caltag Laboratories, South San Francisco, CA).

The single gradient

Flow cytometry

9 ml of diluted blood samples were carefully layered onto a 4 ml of Histopaque-1077 in 15 ml sterile polystyrene tubes (Sarstedt, Germany). The tubes were centrifuged at 600 x g for 30 rain, cells at the interface were recovered, washed twice in PBS, and resuspended in Hanks' balanced salt solution with 3% fetal calf solution.

Four-color fluorescent activated cell sorter (FACS) analysis has been described in detail (Parks et al., 1986). In brief, 5 × 105 cells were incubated with 20/~1 of anti-CD71 antibody. After washing 25 ~1 of the revealing antibody, TRlabelled goat anti-mouse IgG (1:50) was added. Following another wash, the PE-anti-CD13 and FL-anti-CD45 were added. All incubations were performed for 15 min on ice. Following the last wash the cells were resuspended in 200 /~1 of staining medium and analysed on a highly modified dual-laser FACS II (Becton Dickinson, Mountain View, CA), interfaced with a VAX

The triple discontinous gradient A triple gradient was developed by layering 2.5 ml each of Histopaque-lll9 at the bottom, Histopaque 1107 (two parts Histopaque-1119 + one part Histopaque-1083) in the middle and

I

I~---Mononucleal cells

._~~

~-

Mononucleal cells ÷ Nucleated RB C' s

1077 1107 ....)~E~~

NucleatedRBC's

1077--4 Mature RBC's

Granulocytes

A

B

Fig. 1. A: single density gradient centrifugation. B: triple density gradient centrifugation. The major cell types recovered at each interface are shown with the arrows.

279 Triple Gradient

Single Gradient

Middle layer

Top layer

Bottom layer

too. I0-

~' 1

1O

1

1O0

I00

10

, -i 1

i I0

~ ] 100

i'

' ~ 1

, i' l0

'

J

100

Obtuse Scatter Fig. 2. The triple gradient allows enrichment of cells with granulocyte and erthrocyte-like scatter from mononuclear cells. The cells in the gates have scatter signals typical of mononuclear cells. The cells with small forward and obtuse scatter belong mainly to the erythrocyte lineage, and the cells with higher obtuse and forward scatter are monocytes a n d / o r granulocytes.

erythrocytes (low forward and obtuse scatter), lymphocytes (intermediate forward and obtuse scatter), and monocytes (high forward and obtuse scatter) are present (Fig. 2). In contrast to the single density separation, the top layer of the triple gradient contains predominantly lymphocytes and monocytes, and the middle and bottom fraction contain cells with scatter typical of granulocytes (high forward and obtuse scatter), and mature or immature red blood cells (Fig. 2). The expression of CD71 in fractions obtained from each interface shows that there is a considerable enrichment of CD71 ÷ cells in the middle layer of the triple gradient (Fig. 3A). Although

6300 computer (Digital Equipment, Maynard, MA) running FACS/desk software (Moore et al., 1986). Dead cells were gated out using propidium iodide. The signal for propidium iodide was collected in a fourth channel normally used to detect emission spectrum from allophycocyanin (650-670 nm).

Results and discussion

The forward and obtuse scatter profile of the fraction obtained at the interface of a single density gradient shows that all three cell types:

Triple Gradient

Single Gradient

Middle layer

Top layer

Bottom layer

100 -

100-

A CD71 SO

, - 50

3% ~

0

°t

._~

v,~_

_

B CD13 5O

,,..., I

10

100

h,,,,,. . . . . . . .

1

,,J'qllll 10

100

,,e,,

I'%t* qt%x o

t,

i

1

~ttt

,jr,, ""***'"

10

100

1

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Fig. 3. Enrichment of CD71 + cells in the middle layer of the triple gradient. A: histograms of CD71 expression of total cells at each interface. The arrow defines the cutoff used to calculate the percentage of CD71 + cells given in each box. B: histograms of CD13 expression of total cells at each interface.

280 TABLE I MAJORITY OF THE CD71 + CELLS FROM THE MIDDLE LAYER OF THE TRIPLE GRADIENT ARE CD45 AND CD13 NEGATIVE % of total ceils Single gradient

Triple gradient Top layer

Middle layer

Bottom layer

CD71 +

6

5

31

3

CD71 +/CD45 + CD71 +/CD45 -

5 1

5.5 0.5

8 24

0.5 2.5

CD71 +/CD13 + CD71+/CD13 -

3 3

3 2

4 27

2.5 0.5

6% of cells in the single density separation stained positively with CD71 ÷, 85% of these cells were CD45 +/CD71 +, suggesting they may be activated lymphocytes a n d / o r monocytes, which also express CD71 (Table 1). Only 1% cells of total cells and 15% of CD71 ÷ cells in the single density gradient were CD71 +/CD45-. In contrast about 24% of the total cells, and 75% of the CD71 ÷ cells in the middle layer of the triple gradient were CD45- (Table 1). Thus compared to the standard separation procedures most widely used, the triple gradient allowed a 25-fold enrichment of cells that were CD71 +, CD45-, and had scatter profiles typical of immature red blood cells. The separation of granulocytes in the gradient was traced using the CD13 antigen which is expressed on granulocytes and monocytes. The bright CD13 ÷ ceils in the single gradient interface and in the top layer of the triple gradient were monocytes as judged by scatter and reactivity to CD33 (data not shown). Although a large population of cells in the middle layer had a granulocyte/monocyte-like scatter (Fig. 2), majority of these cells were CD45 ÷ and negative/dim for CD13 expression (Fig. 3B). In contrast the cells with granulocyte-like scatter at the 1119 interface, of the triple gradient were brightly stained with the anti-CD13 antibody sug-

gesting these cells are mature granulocytes (Figs. 2 and 3B). Separation of granulocytes at this density was originally described by English et al. and is the popular method used to obtain one-step separation of mononuclear cells, granulocytes and mature red blood cells (English and Anderson, 1974). The triple gradient is a further modification of this technique to separate nucleated red blood cells of fetal origin from mononuclear cells, granulocytes and mature red blood cells which are of maternal origin.

References Adinolfi, M. (1991) On a non-invasive approach to prenatal diagnosis based on the detection of fetal nucleated cells in maternal blood samples. Prenatal Diagn. 11,799. Bhat, N.M., Bieber, M.M. and Teng, N.N.H. (1990) One step separation of human fetal lymphocytes from nucleated red blood cells. J. Immunol. Methods 131, 147. Bianchi, D.W., Flint, A.F., Pizzimenti, M.F., Knoll, J.H.M. and Latt, S.A. (1990) Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc. Natl. Acad. Sei. USA 87, 3279. Camaschella, C., Alfarano, A., Gottardi, E., Travi, M., Primignani, P., Caligaris Cappio, F. and Saglio, G. (1990) Prenatal diagnosis of fetal hemoglobin Lepore-Boston disease on maternal peripheral blood. Blood 75, 2102. English, D. and Anderson, B.R. (1974) Single-step separation of red blood cells. Granulocytes and mononuclear leucocytes on discontinous density gradient of Ficoll-Hypaque. J. Immunol. Methods 5, 249. Moore, W.A. and Kautz, R.A. (1986) Data analysis in flow cytometry. In: D.M. Weir, L.A. Herzenberg, C.A. Blackwell and L.A. Herzenberg (Eds.), The Handbook of Experimental Immunology, 4th edn. Blackwell Scientific Publishers, Edinburg, chapter 30. Parks, D.R., Lanier, L.L. and Herzenberg, L.A. (1986) Flow cytometry and fluorescence activated cells sorting (FACS). In: D.M. Weir, L.A. Herzenberg, C.A. Blackwell and L.A. Herzenberg (Eds.), The Handbook of Experimental Immunology, 4th edn. Blackwell Scientific Publishers, Edinburg, chapter 29. Sehroder, J. (1975) Transplaeental passage of blood cells. J. Med. Genet. 12, 230. Yeoh, S.C., Sargent, I.L., Redman, W.G., Wordsworth, B.P. and Thein, S.L. (1991) Detection of fetal cells in maternal blood. Prenatal Diagn. 11, 117.