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Previous Volume 330:398-401 February 10, 1994 Number 6 Next

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Post-transfusion graft-versus-host disease (GVHD) is generally thought to result from the engraftment of lymphocytes in blood products^1,2. If circumstances permit, the donor's T lymphocytes mount an immune attack against the recipient's tissues. The clinical manifestations of the disorder are fever, rash, hepatitis, diarrhea, bone marrow aplasia, pancytopenia, and infection³. Post-transfusion GVHD can usually be diagnosed clinically during its florid stage. However, in its early stage, it is not easily differentiated from toxic shock syndrome, drug reactions, or viral infections. Early diagnosis may allow more effective treatment of the disease.

Post-transfusion GVHD occurs when the blood donor is homozygous and the recipient heterozygous for certain HLA antigens^{4,5,6,7,8,9,10,11,12,13}. Therefore, HLA typing cannot identify the donor's lymphocytes in a patient with post-transfusion GVHD. Polymorphic markers other than HLA genes may thus be useful in diagnosing this disorder. In the past few years, reports have demonstrated the use of restriction-fragment-length polymorphisms (RFLPs) and DNA probes to document engraftment after bone marrow transplantation and to detect mixed hematopoietic and lymphoid chimeric states^14,15. DNA probes also revealed chimerism in a patient with post-transfusion GVHD¹⁶. However, these methods are cumbersome and often detect relatively uninformative variations in DNA sequences.

We describe a molecular method for the detection of donor DNA in patients with post-transfusion GVHD, based on an analysis of polymorphisms associated with variations in the length of dinucleotide or trinucleotide microsatellite repeats. These polymorphisms are highly informative as compared with conventional RFLPs and can be detected by amplification of the variable regions with use of the polymerase chain reaction (PCR) and electrophoresis of the products.

Case Reports

Patient 1

A 59-year-old man underwent a pharyngolaryngectomy for esophageal cancer in December 1992, during which he received four units of stored whole blood. His recovery was excellent until the eighth postoperative day (day 8), when diarrhea and fever occurred. His temperature rose to 39 °C every day until day 13, when an erythematous rash appeared on his face. The erythema spread over his body in a few days, forming an erythroderma-like eruption.

Liver dysfunction occurred on day 15 and worsened; the serum aspartate aminotransferase and alanine aminotransferase concentrations were 454 and 378 U per liter, respectively, on day 18. Leukopenia and thrombocytopenia developed on days 11 and 15, respectively. On day 18, the platelet count was 29,000 per cubic millimeter, the red-cell count 2,670,000 per cubic millimeter, and the white-cell count 1200 per cubic millimeter, with a differential count of 6 percent neutrophils, 50 percent lymphocytes, 36 percent atypical lymphocytes, and 8 percent other cells. Eighty percent of the mononuclear cells were CD8-positive. Bone marrow examination revealed a markedly hypocellular marrow. Pancytopenia persisted in spite of daily platelet transfusions and treatment with granulocyte colony-stimulating factor. Skin biopsy revealed satellite-cell necrosis and liquefaction of the basal layer of the epidermis. Histochemical staining showed an absence of Langerhans' cells and infiltration of the epidermis with CD8-positive cells. On day 28 hypotension and oliguria developed; a blood sample was taken for the DNA polymorphism analysis. The patient died on day 31.

Patient 2

A five-month-old girl underwent emergency cardiac surgery for anomalous common pulmonary venous return in March 1992. After the operation she received six units of fresh whole blood and two units of platelet concentrate. The postoperative course was uneventful until day 11, when a fever and rash developed. By day 16 pancytopenia was present; the serum aspartate and alanine aminotransferase concentrations were 568 and 147 U per liter, respectively. On day 18 the white-cell count was less than 100 per cubic millimeter, and the platelet count was 20,000 per cubic millimeter; the serum lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase concentrations were 4459, 352, and 136 U per liter, respectively. The patient died later that day. Blood and a skin-biopsy sample had been obtained on the same day for DNA polymorphism analysis.

Methods

Peripheral-blood samples were collected in heparin-treated tubes from Patient 1 before and after transfusion, as well as from the donor of the blood transfused during the operation. Peripheral blood and skin specimens were obtained from Patient 2 after transfusion; no samples of blood from the donor or pretransfusion samples from the patient were available.

Preparation of DNA

For the preparation of DNA, 20 microl of blood was mixed with 50 mM TRIS-hydrochloric acid (pH 8.8), 10 mM magnesium chloride, and 10 mM ammonium sulfate buffer and centrifuged. The cell pellet was suspended in 100 microl of distilled water and boiled for 15 minutes. After centrifugation, the supernatant was used as the template DNA solution for the PCR. DNA was extracted from skin by a phenol-chloroform method.

Primers

The oligonucleotide primers used to amplify polymorphic loci are shown in Table 1. The length of the allelic fragments (in base pairs [bp]) and the chromosomal localization of the markers are also summarized in Table 1.

View this table: [in this window] [in a new window]   Table 1. Microsatellite Primers Used for PCR Amplification.

 
Detection of Polymorphic Alleles

Genomic DNA was amplified in a 50-microl reaction mixture containing 10 microl of boiled blood sample or skin extract, PCR buffer (10 mM TRIS-hydrochloric acid [pH 8.3], 50 mM potassium chloride, 1.5 mM magnesium chloride, 0.5 percent polysorbate [Tween 20], and 5 percent formamide), four deoxynucleotide triphosphates (200 micro M each), 1 unit of Taq polymerase, and two primers (0.5 micro M each). The mixture was heated for 2 minutes at 94 °C; then, amplification was carried out for 35 cycles in a DNA thermal cycler (Perkin-Elmer-Cetus) according to a step program (for the primer sets TCFIID, D6S89, and D11S534, denaturation at 94 °C for 1 minute, annealing at 55 °C for 1 minute, extension at 72 °C for 1 minute, and finally extension at 72 °C for 10 minutes; for the primer sets INT and HGH, denaturation at 94 °C for 1 minute, annealing at 55 °C for 2 minutes, and finally extension at 72 °C for 2 minutes). The amplified DNA was initially subjected to electrophoresis in a 5 percent acrylamide gel. To detect the various polymorphic alleles, the amplified DNA fragments were loaded onto a nondenaturing 10 percent acrylamide gel that was 40 cm long and 0.5 mm thick in TBE buffer (44.5 mM TRIS-borate and 1 mM EDTA). The samples underwent electrophoresis for 12 hours at 400 V, and the gel was stained with silver (Daiichi Kagaku).

Results

To evaluate the sensitivity of the method, we mixed DNA samples from the leukocytes of two subjects in ratios ranging from 10:0 to 0:10. We could detect the presence of unshared DNA in 10-fold dilutions of the subjects' total DNA (Figure 1A). Then we tried to detect circulating donor lymphocytes by analyzing DNA polymorphisms in blood from four patients without post-transfusion GVHD. The first of these patients had received 200 ml of stored whole blood during surgery; the other three, who had acute myeloid leukemia, had received 20 units of filtered platelet concentrate from a single donor, 10 units of platelet concentrate from a single donor, and 10 units of platelet concentrate from 10 random donors, respectively. Blood for DNA analysis was obtained from the patients before transfusion and on the first six days after transfusion and from the corresponding donors. No bands of donor origin were found in any post-transfusion sample (Figure 1B).

View larger version (45K): [in this window] [in a new window]   Figure 1. Validation of DNA Polymorphism Analysis. To test the sensitivity of the method, DNA samples from peripheral-blood cells of two normal subjects were mixed in varying proportions and then examined by DNA polymorphism analysis after PCR with TCFIID primers (Panel A). Lane 1 shows the PCR products from the DNA of Subject 1 (solid triangles); lane 11, the products from the DNA of Subject 2 (open triangles); and lanes 2 through 10, those from the mixtures of DNA from both subjects in the ratios shown. The presence of the unshared DNA can be detected in the 9:1 and 1:9 mixtures (lanes 2 and 10). In a differential assessment of the circulating donor lymphocytes, DNA was extracted from blood samples of a patient without post-transfusion GVHD before and after transfusion of stored whole blood (200 ml), amplified with the primer set INT, and analyzed together with the sample from the patient's donor (Panel B). Donor DNA was not detected in any post-transfusion samples from the patient. Lane 1 represents the DNA size marker phiX174 digested with HaeIII; lane 2, the PCR products from the donor's peripheral-blood cells (with the donor's bands denoted by the solid arrowheads); lane 3, the PCR products from the patient's pretransfusion peripheral-blood cells (with the patient's bands denoted by the open arrowhead); and lanes 4 through 9, the PCR products from the patient's peripheral-blood cells on post-transfusion days 1, 2, 3, 4, 5, and 6, respectively.

 
Figure 2 shows the results of analysis for the highly polymorphic microsatellites in blood samples obtained before and after transfusion in Patient 1, who was thought to have post-transfusion GVHD, and his donor. Five primer sets were used in this study. Assays with each primer set revealed two band patterns, one representing the patient and the other the donor. Analysis of DNA from the post-transfusion samples revealed a mixed pattern of bands from the recipient and the donor, with donor-derived bands predominating at the TCFIID, D6S89, and D11S534 loci (Figure 2, lane 3). At the INT and HGH loci, only donor-derived bands were detected (Figure 2, lane 3). These results clearly indicated that chimerism had occurred in the patient because allogeneic cells from the donor had become engrafted.

View larger version (35K): [in this window] [in a new window]   Figure 2. Results of DNA Polymorphism Analysis in Patient 1 with Post-Transfusion GVHD. Five sets of microsatellite primers (INT, TCFIID, D6S89, D11S534, and HGH) were used for PCR amplification. Lane 1 shows the DNA size marker pBR322 digested with MspI; lane 2, the PCR products from the patient's pretransfusion peripheral-blood cells (with the patient's bands denoted by solid triangles); lane 3, the PCR products from the patient's post-transfusion peripheral-blood cells; and lane 4, the PCR products from the donor's peripheral-blood cells (with the donor's bands denoted by open triangles). The band patterns of the patient and the donor at the five loci were different. The patient's post-transfusion band patterns (lane 3) at the TCFIID, D6S89, and D11S534 loci showed band patterns from the patient (very faint) and the donor (predominant), whereas at the INT and HGH loci only donor-derived bands were seen.

 
DNA prepared from a post-transfusion skin specimen from Patient 2 was used to establish her pretransfusion genotype. The DNA polymorphic patterns of the post-transfusion blood sample clearly differed from those of the skin specimen (Figure 3). Because chimeric lymphocytes had infiltrated the skin specimen, a mixed pattern of patient-derived bands (predominant) and donor-derived bands (faint) was found at the TCFIID locus (Figure 3, lane 2); however, only bands from the patient were found at the HGH locus (Figure 3, lane 2). The DNA from the post-transfusion blood sample showed a mixed pattern at both the HGH and the TCFIID loci, with the donor-derived DNA predominant (Figure 3, HGH and TCFIID loci, lane 3).

View larger version (10K): [in this window] [in a new window]   Figure 3. Results of DNA Polymorphism Analysis in Patient 2 with Post-Transfusion GVHD. Lane 1 shows the DNA size marker pBR322 digested with MspI; lane 2, the PCR products from the patient's post-transfusion skin-biopsy specimen; and lane 3, the PCR products from the patient's peripheral-blood cells after blood transfusion. The patterns at the HGH and TCFIID loci show the patient's bands, denoted by solid triangles, and the donor's bands, denoted by open triangles. At the HGH locus, the post-transfusion skin sample contained only patient-derived bands (lane 2), although the patient's post-transfusion peripheral blood (lane 3) contained predominant donor-derived bands and faint patient-derived bands. At the TCFIID locus, both patient-derived bands (predominant) and donor-derived bands (very faint) were present in DNA from the post-transfusion skin specimen (lane 2), whereas the patient's post-transfusion peripheral blood showed a more evenly mixed pattern of both donor-derived and patient-derived bands, with the former predominating (lane 3).

 
Discussion

We used a new method of detecting DNA polymorphisms to study two patients thought to have post-transfusion GVHD. Blood from each patient was found to contain foreign DNA fragments that were derived from cells in the donor's blood. Post-transfusion blood samples from four patients who did not have post-transfusion GVHD contained only the patients' DNA polymorphisms. These results indicate that the two patients suspected of having post-transfusion GVHD did have that complication.

The presence of DNA-sequence polymorphisms in many regions of the human genome^{22,23,24,25,26,27} has facilitated the study of inherited disease^22,23,24,28. The five dinucleotide or trinucleotide repeat loci that we chose for analysis are highly polymorphic, each with seven or more alleles^{17,18,19,20,21}. The polymorphisms were examined by PCR amplification of the variable regions and electrophoresis of the products on nondenaturing acrylamide gels. Most microsatellite assays use radioactively labeled PCR products and denaturing acrylamide gels^29,30,31. Our method allowed direct analysis of PCR products on the silver-stained gels. This technique has the advantages of speed, sensitivity, and ease of analysis because it involves only two steps, amplification and electrophoresis.

We examined DNA in a skin specimen to identify the DNA polymorphisms in Patient 2 because no pretransfusion blood sample was available. The pattern of the skin DNA during GVHD at the TCFIID locus was a mixture of the patient's and the donor's polymorphisms, although the patient's type was dominant. This kind of mixed pattern may interfere with the verification of engraftment. Therefore, skin-biopsy specimens obtained after transfusion seem poorly suited to this diagnostic method. We also performed microsatellite analysis on an extract of fingernail. The patterns obtained were identical to those in peripheral blood (data not shown). For routine clinical diagnosis, the use of nail samples may be more useful than either skin specimens or blood samples.

In summary, we demonstrated that analysis of polymorphisms of dinucleotide or trinucleotide microsatellite repeats can identify donor DNA in patients with post-transfusion GVHD. Since this method is sensitive, easily applicable, and rapid, it should be useful for the diagnosis of GVHD. It may also be applicable to the documentation of bone marrow engraftment in recipients of allogeneic marrow transplants.

We are indebted to Drs. K. Tanaka, M. Onishi, R. Nagai, K. Shimizu, M. Handa, and T. Katougi for their valuable discussions and the patients' skin and blood samples.

Source Information

From the Department of Transfusion Medicine and Immunohematology (L.W., T.J., K. Tokunaga, K. Takahashi, S.K.), and the Department of Biochemistry and Nutrition (L.W., K. Takai), Faculty of Medicine, University of Tokyo, Bunkyo-ku; and the Japanese Red Cross Central Blood Center, Shibuya (K. Tokunaga, S.U., K. Tadokoro) -- both in Tokyo, Japan.

Address reprint requests to Ms. Wang at the Department of Research, Japanese Red Cross Central Blood Center, Hiroo 4-1-31, Shibuya-ku, Tokyo 150, Japan.

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