Development of flow cytometry for detection and quantitation of red cell bound immunoglobulin G in autoimmune hemolytic anemia with negative direct Coombs test.

About 2-10% of patients with warm-antibody autoimmune hemolytic anemia (WAIHA) exhibit a negative direct Coombs test (DAT), requiring more sensitive tests, including detection of RBC-bound immunoglobulins by flow cytometry, for diagnosis. In this study, the optimal conditions for detection and quantitation of RBC-bound IgG by flow cytometry were studied using blood samples from six patients with AIHA and two healthy individuals. Quantitation of RBC-bound IgG was performed using quantum simply cellular (QSC) beads coated with goat anti-mouse IgG antibodies. For detection of RBC bound IgG, a 60-minute incubation of all blood samples with 40 μl of 1:10 dilution of FITC-conjugated mouse anti-human IgG gave mean fluorescent intensity (MFI) values comparable to experiments using larger amounts or higher concentrations of the anti-human IgG. The acquired antibody binding capacity (ABC) values (or IgG molecules) for each QSC bead level, at 40 μl of 1:5 and 1:10 dilution of anti-human IgG for 60 minutes were close to the manufacturer-assigned ABC values. The IgG molecules per RBC in all six patients with positive DAT of 4+, 3+, 2+, 1+, trace and negative DAT were 31,725, 3,823, 1,753, 524, 260 and 88 respectively and in two healthy individuals with negative DAT they were 104 and 78.

Random amplified polymorphic DNA typing and phylogeny of Pythium insidiosum clinical isolates in Thailand.

Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.

Monosomy 7 in patients with aplastic anemia and paroxysmal nocturnal hemoglobinuria with evolution into acute myeloid leukemia.

We report two cases of Thai patients with aplastic anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH) who subsequently developed acute myeloid leukemia (AML) at their terminal phase. Monosomy 7 was demonstrated upon karyotypic analysis of bone marrow in both cases at the time leukemia developed The first patient was a 25-year-old man diagnosed with AA at age 14, recovered from AA at age 15, developed PNH at age 21 and turned into AML at age 25. The second patient was a 27-year-old man diagnosed with PNH at age 22, developed severe AA at age 25 and turned into AML at age 27. This latter patient received anti-lymphocyte globulin when he developed severe AA but did not respond well whereas the first patient fully recovered from AA with anabolic hormone treatment. Time to diagnosis of AML in the patient who received immunosuppressive therapy was strikingly shorter than that who received conventional androgen therapy (2 years vs 11 years after AA, respectively). The presence of monosomy 7 in leukemic cells of both patients emphasizes its central role in the development of AML from AA/PNH. However, other factors such as choice of AA/PNH therapy and patients response may modulate the time to emergence of monosomy 7-carrying AML clone and frank leukemia. Further studies into the biologic and genetic mechanisms involved in the development of leukemic clone arising from AA/PNH should be explored.

Quality analysis of comprehensive examination, faculty of medicine, Siriraj Hospital.

The quality of the tests and the relationship between clinical achievement and comprehensive exam scores were studied in the sixth year medical students of the Faculty of Medicine Siriraj Hospital during the academic years B.E. 2527-2534. The results were as follows: The content validity of the test used in each academic year was justified, which implied that such test was in accordance with the domain specifications defined in the Medical License Standard set by Thai Medical Council in the year B.E. 2527. However, the representativeness of the test was lacking because only 1-2 test items could not adequately sample such an extensive content area as listed in the table of specification. The tests showed a high degree of concurrent validity, implying that they could be used interchangeably with the 6-years cumulative grade point average to describe the student’s achievement. The reliability coefficients of the tests for eight academic years ranged from .77 to .90. The results of item analysis of the tests revealed the average difficulty with values from .54 to .59 and average index of discrimination with values from .10 to .13. The 4th and 5th year grade point averages were significantly correlated with the exam scores in each year (p < .01) and were able to predict the exam scores ranging from 50 to 66%.